A case study-based approach
to integrating QbD principles inCell-based Therapy CMC programs

Reviewers
Note: All chapters were reviewed, but individual reviewers did not review every chapter.		
Reviewers volunteered to provide editorial review for select chapters according to their expertise and interest.		

Houman Dehghani
Allogene

Rogelio Zamilpa
GenCureMalte Wittmann
Novo Nordisk

Jen Koevary
Avery Therapeutics

Christie Malone
GenCureKevin Okimura
OrcaBio

Becky Cap
BioBridge Global

Andy Lin
Genentech

Kevin Gordon
Ori Biotech

Emmanuel Casasola
BioBridge Global

Kathy Francissen
GenentechAnna Quach
Sartorius

Rachel Beddard
BioBridge Global

Vincent Leveque
Genentech

Kevin Kellner
Sartorius

Monica Nelson
Bristol Myers Squibb

Michael Mendicino
Hybrid Concepts
International

Vivienne MarshallSouth Texas Blood & TissueCenterJames Richardson
Interius Bio

Rob McCombie
Syncopation Life Sciences

Erinn Lanxon
Kite Pharma

Abigail Becker
Thermo Fisher Scientific

Max Tejada
Kite Pharma

Mohan Vemuri
Thermo Fisher Scientific

Alexis Cockroft
Lex Regulatory

Namritha Ravinder
Thermo Fisher Scientific

Karen Wolfrom
Lonza

Navjot Kaur
Thermo Fisher Scientific

Melissa DeFrancesco
Lyell Immunopharma

Dan Strange
TTP

Project Management
Josephine Lembong
Michael Lehmicke

EditorMarie Daghlian

Eugene Brandon
BrainXell Therapeutics
Don Yuen
Cellares
John Tomtishen
Cellares
Craig Hasilo
CellCANCarl Burke
Century Therapeutics
Joe Sondej
Century Therapeutics

Bruce Levine
University of Pennsylvania
School of Medicine
Merry Christie
Voisin Consulting Life Sciences
Tharmaja Inparaj
Voisin Consulting Life SciencesGaëlle Preiss
Voisin Consulting Life Sciences
Emmanuelle SabbahPetrover
Voisin Consulting Life Sciences
Anne Schlegel
Voisin Consulting Life Sciences
Robert Drape
WiCellSeth Taapken
WiCellTenneille Ludwig
WiCell

Production
Medical / Technical Writers
Amr Eissa
Lindsay Gasch

Graphic Design and Layout
Carol Collier
Graphic Design (select figures)Michaela Flatley

A-CELL✼ii

A-CELL | Contents
CHAPTER 1

Introduction....................................................................................... 1

CHAPTER 2

Regulatory Considerations, Comparability,
and Standards in Cell-Based Therapies............................................ 7

CHAPTER 3

Generation of Quality Target Product Profile................................. 55

CHAPTER 4

Risk Assessment and Critical Quality
Attribute Identification.................................................................... 65

CHAPTER 5

Cell Collection and Starting Materials Consideration.................... 81

CHAPTER 6

Ancillary Materials........................................................................... 98

CHAPTER 7

Lentiviral Vector Manufacturing Process....................................... 114

CHAPTER 8

Manufacturing of Cell-Based Therapies....................................... 145

CHAPTER 9

Analytical Technologies for Cell Therapies.................................. 182

CHAPTER 10 Control Strategy............................................................................ 199
CHAPTER 11

Manufacturing Strategy, Facility Design, andManufacturing Operations............................................................ 228

A-CELL✼iii

CHAPTER 1

Introduction
CHAPTER LEAD

Josephine Lembong, Alliance for Regenerative Medicine

Chapter 1 | Contents
Introductory Remarks....................................................... 2

Definitions.......................................................................... 4

Introduction to Cell Therapy............................................. 3

References.......................................................................... 6

Document Scope................................................................ 3

Introductory Remarks
The cell and gene therapy (CGT) field is rapidly developing due to its promise to provide significant long-term
health benefits to people suffering from a wide range of
diseases, from ophthalmological disorders to cancer.
Similar to other forms of new advanced therapies, CGT
products face the challenge of streamlined, cost effective
manufacturing due to a lack of standardized methodologies and training. Following success of A-Mab1 andrecently A-Gene,2 the Alliance for Regenerative Medicine(ARM) has decided to continue similar efforts applied tocell-based therapies.
In previous years, the broader life sciences industry
has encountered systemic barriers to the continued
development of promising technologies. In both themonoclonal antibody industry and the vaccine development world, the whirl of energy around scientificdiscovery was stalled by the realities of manufacturing.
While a small team of well-trained experts can produce
high-quality batches of drug product for use in processdevelopment and early clinical trials, it is inevitable that
this process will need to be scaled up dramatically, andthe process transferred to other parties for commercial
production. To help lower the barrier to this necessary
tech transfer, and to better prepare new entrants to theindustry, the leading developers of monoclonal antibodies and vaccines have produced a consolidated set of recommendations for implementation of Quality by Design(QbD). A-Mab and A-Vax, respective to each industry,
have played a significant role in elevating best practices
within their industries, and have been effective in continued workforce development efforts. More recently,
the Parenteral Drug Association (PDA) also publisheda similar effort on manufacturing control strategies for

Chapter 1: Introduction

a hypothetical chondrocyte product, A-Cet.3 Borrowing
from these models, the members of ARM have worked
to produce a similar document for use by the burgeoning
cell therapy sector. This follows the success of ARM’s
A-Gene project, completed in June of 2021 and now posted on ARM’s website, freely available to the regenerative
medicine community.4
This effort to catalog expertise in cell therapy development occurred in parallel with the approval of somecell-based therapies, which have shown the value andclinical relevance of such programs. As the field continues to develop, it has been recognized that the future of
cost-effective cell-based therapy relies on implementationof best practices, development of specialized technologies,
and uniformity of methods. Given the wide scope of innovation underway in each of these areas of focus, ARM
and the A-Cell team sought to bring in as much thought
leadership as possible to ensure that what was recorded
was truly a representation of best practices in the industry.
Reflecting on this, A-Cell is a truly collaborative effort thathas been crafted with contributions from more than 50
industry experts from more than 30 leading therapeutics
developers and service/technology providers.
A-Cell is not intended to represent a standard tobe rigidly applied. It is a hypothetical case study of thedevelopment of a cell-based therapy product. Therefore,
it is a snapshot in time of current best practices in a rapidly evolving field. The data cited in this document are
non-proprietary, and are intended for illustrative purposes only. While we have attempted to be as comprehensive
as possible, and have subjected the document to rigorous
review, it is not a “recipe book” for cell-based therapy
manufacture. Importantly, A-Cell is not an example of amock regulatory submission, nor should it be interpreted
as regulatory advice, or cited as regulatory guidance.

A-CELL✼2

Introduction to Cell Therapy
Cell therapy is a type of therapeutic where live cells aredelivered into patients to treat a disease. There are generally two approaches of cell therapies: (1) autologous,
where the cells originate from the patient themselves, and(2) allogeneic, where the cells originate from a healthy
donor.
The most common type of cell therapy is bloodtransfusion, and the transfusion of red blood cells, whiteblood cells, and platelets from a donor. Another common
cell therapy is the transplantation of hematopoietic stem
cells to create bone marrow. There is currently no formalclassification system for cell therapies. One way to classify
is by potency: pluripotent cells can transform into any
cell type in the body and multipotent cells can transform
into other cell types, however, multipotent cells are more
limited than pluripotent cells. Differentiated or primarycells are of a fixed type. Some examples of common celltherapies are:
• Immune cell therapy. These therapies use cells from
our immune system, such as T cells and natural killer(NK) cells to recognize and eliminate infected ordamaged cells due to diseases. These immune cells
can be removed from the body, isolated from a mixed
cell population, modified, and then expanded before
being returned to the body. Examples of immune celltherapies include Chimeric Antigen Receptor (CAR)
T cell therapy, Tumor-Infiltrating Lymphocyte (TIL)
therapy, engineered T Cell Receptor (TCR) therapy,
and NK cell therapy.
• Hematopoietic stem cells (HSCs) therapy. HSCs
are multipotent blood stem cells that give rise to alltypes of blood cells. HSCs can be found in adult bonemarrow, peripheral blood, and umbilical cord blood.
• Mesenchymal stem/stromal cells (MSCs) therapy.
MSCs are multipotent cells present in multiple tissues
including umbilical cord, bone marrow, and fat tissue,
which can differentiate to bone, cartilage, muscle, andadipocytes (fat cells).
• Embryonic stem cells (ESCs). ESCs are pluripotent
stem cells derived from embryos that retain the ability
to self-renew and to form any cell in the body. While

Chapter 1: Introduction

versatile, ESCs often raise ethical concerns due to theuse of embryos in the development of therapeutics.
• Induced pluripotent stem cells (iPSCs) therapy.
iPSCs are pluripotent cells that can be generated
directly from a somatic cell by transforming the adultcell with a cocktail of genes usually delivered via aviral vector. These cells offer the advantage of pluripotency but without the ethical concerns of embryonicstem cells. iPSCs may also be derived from the patient
and thus avoid the problem of immune rejection.

Document Scope
To make A-Cell an effective resource and reflective of
the ongoing innovation in the sector, we choose to focus
on immune cell therapy as the case study. This is basedon strong performance and the field’s enthusiasm of thecell-based immuno-oncology sector, as shown in thestrong financing in H1 2021 (raising $6.6B–for the firsttime outraising gene therapy companies), and a major
increase (>400%) in industry-sponsored clinical trials
worldwide in the past 5 years,5 accelerating scientific andclinical advances and bringing life-changing therapies topatients worldwide.
To further maximize the utility of A-Cell, we felt it wasnecessary to focus on one specific use case. We decided
to further focus this document on CAR-T cell therapy,
given the recent approvals and the number of available
products in the market. Although CAR-T therapy isregulated as an ex vivo gene therapy, the manufacturingof this cell-based modality shares a lot of the same paradigm as that of other cell therapies, thus it is the focusof this document. Most recently, the U.S. FDA approved
CARVYKTI™, the sixth CAR-T therapy in the U.S., andthe second targeting BCMA, for treatment of multiple
myeloma. The trial reported a 98% overall response rate
and an 78% stringent complete response rate.6 This trend
is expected to continue as developers pursue different
approaches for innovative CAR-T products to treat abroader range of liquid and solid tumors. In addition,
more data from CD19 CAR-T therapies are available tosuggest that CAR-T therapies may be used as an earlier
line of treatment. The primary case study example presented in this document is a lentiviral vector-modified

A-CELL✼3

CAR-T, as lentivirus is the predominantly used vector
for ex vivo gene modification of cell therapies.7
In CAR-T cell therapy, the patient’s own immune cells(T-cells) are modified to express a receptor on their surface that recognizes structures (antigens) on the surfaceof malignant cells. Once the receptor binds to a tumor
antigen, the T-cell is stimulated to attack the malignant
cells. So far, CAR-T therapies are only approved for hematologic liquid tumors such as B-cell lymphoma and relapsed or refractory multiple myeloma. While there are anumber of approved products out there showing positive

outcomes (complete or partial response) in adults andpediatric cases, a lot of learnings are still being collected
to assess the long-term success of these treatments, andnew strategies are being employed to manage the adverse
events that have been observed following treatment, suchas cytokine release syndrome and neurotoxicity.
While this document uses a hypothetical CAR-T
product as an example to illustrate various manufacturing and CMC principles, many of the concepts presented
here are applicable and beneficial to a broader profile ofcell therapy products.

Definitions
The following terms are concepts that are common in cell therapy development, and particularly CAR-T therapies
that will appear throughout the document.
Key terminology

Definition

Quality by Design (QbD)

A systematic approach to development that begins with predefined
objectives and emphasizes product and process understanding andprocess control, based on sound science and quality risk management.8

Quality Target Product
Profile (QTPP)

A prospective summary of the quality characteristics of a drug product(DP) that ideally will be achieved to ensure the desired quality, taking into
account safety and efficacy.8

Design SpaceThe multidimensional combination and interaction of input variables (e.g.,material attributes) and process parameters that have been demonstrated
to provide assurance of quality.8
Working within the design space is not considered as a change. Movementout of the design space is considered to be a change and would normally
initiate a regulatory post approval change process. Design space is
proposed by the applicant and is subject to regulatory assessment andapproval.8

Critical Quality Attribute(CQA)

Physical, chemical, biological, or microbiological properties orcharacteristics that should be within an appropriate limit, range, or
distribution to ensure the desired product quality.8
CQAs serve as the basis to identify critical process parameters (CPPs) andfacilitate development of the design space.9

Critical Process Parameter(CPP)

A process parameter whose variability has an impact on a CQA andtherefore should be monitored or controlled to ensure the process
produces the desired quality.8

Control Strategy

A planned set of controls, derived from current product and process
understanding that ensures process performance and product quality.
The controls can include parameters and attributes related to drug
substance and drug product materials and components, facility andequipment operating conditions, in-process controls, finished productspecifications, and the associated methods and frequency of monitoring
and control.10

continued on next page
Chapter 1: Introduction

A-CELL✼4

Definitions; continued from previous page
Risk assessment

Science-based process that can aid in identifying which material attributes
and process parameters potentially have an effect on product CQAs. Risk
assessment consists of the identification of hazards and the analysis andevaluation of risks associated with exposure to those hazards.11

Design of Experiment (DOE)

A structured, organized method for determining the relationship between
factors affecting a process and the output of that process.8
DOE is used to gain knowledge and estimate the best operating conditionsof a system, process, or product.

Ancillary materials

Materials that come into contact with the cellular therapeutic productduring the manufacturing process, but are not intended to be in the final
product, which can include culture media, growth factors, and otherbiological and non-biological components.12

Adventitious agentsContaminating microorganisms of the cell culture or source materials
including bacteria, fungi, mycoplasmas/spiroplasmas, mycobacteria,
Rickettsia, protozoa, parasites, transmissible spongiform encephalopathy(TSE) agents, and viruses that may have been unintentionally introduced
into the manufacturing process of a biological product.13

Current Good
Manufacturing Practice(CGMP)

A system that assures proper design, monitoring, and control ofmanufacturing processes and facilities to ensure a drug product (DP)meets the quality standards.14
This is done through establishing strong quality management systems,
obtaining appropriate quality raw materials, establishing robust operatingprocedures, detecting and investigating product quality deviations, andmaintaining reliable testing laboratories.

Chimeric Antigen Receptor(CAR)

A synthetic receptor designed to bind to certain proteins on cancer cells.
The CAR is then added to T cells to help the T cells find and kill cancer
cells that have the specific protein that the receptor is designed to bind.15

Cytokine Release Syndrome(CRS)

Systemic inflammatory response that is an adverse effect resulting
from rapid immune activation induced by CAR-T therapies. The clinicalsigns of CRS correlate with T cell activation and release of high levels of
inflammatory cytokines including Interleukin 6 (IL-6).16

Immune effector cellassociated neurotoxicity
syndrome (ICANS)

Common complication of CAR-T therapies in the form of acute neurologic
signs / symptoms (headache, confusion, delirium, language disturbance,
seizures, acute cerebral edema). It is associated with high levels ofcytokines in the blood and cerebrospinal fluid.17

CD19 (Cluster ofDifferentiation 19;
B-lymphocyte antigen CD19)

A member of the immunoglobulin superfamily of proteins expressed on
the surface of B cells, where it functions as a critical component of the B
cell receptor signaling complex.18
CD19 is an ideal target for CAR-directed therapies because it is expressedon most B cell malignancies­—chronic lymphocytic leukemia (CLL),
B-cell acute lymphoblastic leukemia (B-ALL), and many non-Hodgkin's
lymphomas (NHLs)­, it is not expressed on hematopoietic stem cells, andelimination of all CD19+ B cells in the body is a manageable on-target
treatment effect.19 The most mature CAR-T cell therapies to date are
directed against CD19.

B-cell maturation antigen(BCMA)

A member of the tumor necrosis factor receptor superfamily that is
highly expressed on mature B lymphocytes, with minimal expression on
hematopoietic stem cells or nonhematopoietic tissue.20
BCMA has become a novel treatment target for multiple myeloma (MM)due to its highly selective expression in malignant plasma cells.21

Chapter 1: Introduction

A-CELL✼5

References
1.

CMC Biotech Working Group. A-Mab: a Case Study inBioprocess Development. October 2009. https://ispe.org/
publications/guidance-documents/a-mab-case-study-in-bioprocess-development

12. International Organization for Standardization. Biotechnology—Ancillary materials present during the production of cellular therapeutic products. ISO/TS 20399-2:2018. November
2018

2.

Alliance for Regenerative Medicine. A-Gene: A case study
based approach to integrating QbD principles into genetherapy CMC programs. 2021. https://alliancerm.org/
manufacturing/a-gene-2021

13. World Health Organization. Annex 3: Recommendations
for the evaluation of animal cell cultures as substrates for the
manufacture of biological medicinal products and for thecharacterization of cell banks. WHO Technical Report Series,
No. 978. 2010. https://www.who.int/biologicals/vaccines/
TRS_978_Annex_3.pdf

3.

Parenteral Drug Association. Technical Report No. 81: CellBased Therapy Control Strategy. February 2019. https://www.
pda.org/bookstore/product-detail/4638-tr-81-control-strategy-for-cell-therapy

14. U.S. Food and Drug Administration. Facts About the Current
Good Manufacturing Practices (CGMPs). June 2021. https://www.fda.gov/drugs/pharmaceutical-quality-resources/
facts-about-current-good-manufacturing-practices-cgmps

4.

https://alliancerm.org/manufacturing/5.

Alliance for Regenerative Medicine. H1 2021 Sector Report.
https://alliancerm.org/wp-content/uploads/2021/08/ARMH1-2021-Report.pdf

15. NCI Dictionary of Cancer Terms. https://www.cancer.gov/
publications/dictionaries/cancer-terms/def/chimeric-antigen-receptor

6.

U.S. FDA Approves CARVYKTI™ (ciltacabtagene autoleucel),
Janssen’s First Cell Therapy, a BCMA-Directed CAR-T Immunotherapy for the Treatment of Patients with Relapsed or
Refractory Multiple Myeloma. February 2022. https://www.
jnj.com/u-s-fda-approves-carvykti-ciltacabtagene-autoleuceljanssens-first-cell-therapy-a-bcma-directed-car-t-immunotherapy-for-the-treatment-of-patients-with-relapsed-or-refractory-multiple-myeloma

7.

Milone, M.C., O’Doherty, U. Clinical use of lentiviral vectors. Leukemia 32, 1529–1541 (2018). doi: 10.1038/s41375018-0106-0

8.

International Conference on Harmonization (ICH) Q8(R2):
Pharmaceutical Development - Guidance for Industry. November 2009. https://www.fda.gov/regulatory-information/
search-fda-guidance-documents/q8r2-pharmaceutical-development

9.

Kelly M. Implementing the principles of quality by design forearly-stage gene therapy products. Presented at: WCBP 2012,
January 23-25, 2012

10. International Conference on Harmonization (ICH) Q10:
Pharmaceutical Quality System - Guidance for Industry.
April 2009. https://www.fda.gov/regulatory-information/
search-fda-guidance-documents/q10-pharmaceutical-quality-system

16. Frey N, Porter D. Cytokine Release Syndrome with Chimeric Antigen Receptor T Cell Therapy. Biol Blood Marrow Transplant. 2019 Apr;25(4):e123-e127. doi: 10.1016/j.
bbmt.2018.12.756
17. Gust J, Taraseviciute A, Turtle CJ. Neurotoxicity Associatedwith CD19-Targeted CAR-T Cell Therapies. CNS Drugs. 2018
Dec;32(12):1091-1101. doi: 10.1007/s40263-018-0582-9
18. Depoil D, Fleire S, Treanor BL, Weber M, Harwood NE,
Marchbank KL, Tybulewicz VL, Batista FD. CD19 is essential
for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.
Nat Immunol. 2008 Jan;9(1):63-72. doi: 10.1038/ni1547
19. Scheuermann RH, Racila E. CD19 antigen in leukemia andlymphoma diagnosis and immunotherapy. Leuk Lymphoma.
1995 Aug;18(5-6):385-97. doi: 10.3109/10428199509059636
20. Novak AJ, Darce JR, Arendt BK, Harder B, Henderson K,
Kindsvogel W, Gross JA, Greipp PR, Jelinek DF. Expression of BCMA, TACI, and BAFF-R in multiple myeloma:
a mechanism for growth and survival. Blood. 2004 Jan
15;103(2):689-94. doi: 10.1182/blood-2003-06-2043
21. Yu, B., Jiang, T. & Liu, D. BCMA-targeted immunotherapy
for multiple myeloma. J Hematol Oncol 13, 125 (2020). doi:
10.1186/s13045-020-00962-7

11. International Conference on Harmonization (ICH) Q9:
Quality Risk Management - Guidance for Industry. June 2016.
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q9-quality-risk-management

Chapter 1: Introduction

A-CELL✼6

CHAPTER 2

Regulatory Considerations,Comparability,
and Standardsin Cell-Based Therapies
CHAPTER LEADS

Debra Webster, BlueRock Therapeutics
Michael Mendicino, Hybrid Concepts International
CHAPTER CONTRIBUTORS

Merry Christie, Voisin Consulting Life Sciences
Mo Heidaran, GC Therapeutics
Dawn Henke, Standards Coordinating Body

Chapter 2 | Contents
Introductory Remarks............................................................9

Standards............................................................................. 48

Regulatory Considerations...................................................9

Benefits of Voluntary Consensus Standards in
Cell Therapy........................................................................ 48

Regulatory Framework.......................................................10
United States....................................................................10
Europe................................................................................11
Japan................................................................................. 12
Health Authorities and Sponsor Meetings....................... 12
FDA (US)............................................................................ 13
EMA and EU national agencies (Europe)......................... 17
EMA-FDA Programs......................................................... 20
MHRA (UK)........................................................................ 21
PMDA (Japan)....................................................................22
CMC Submission Content for Clinical Trials....................22
Electronic Common Technical Document......................22
IND Submission to the FDA............................................. 29

Current Landscape of Standards in Cell Therapy........... 49
Recent and Ongoing Efforts in Cell Therapy Standards
Development...................................................................... 49
1. Testing and characterization standards for cellulartherapeutic products (ISO 23033)................................. 49
2. Ancillary materials standard for production ofcell and gene therapy products (ISO/CD 20399)......... 50
3. Cell collection and apheresis standards(BSR/PDA 08-202x)......................................................... 50
4. Requirements for human and mouse pluripotent
stem cells (ISO/DIS 24603)............................................. 50
5. Cryopreservation standards for cell therapies,
gene therapies, and regenerative medicine
manufacturing (ANSI/PDA 02-2021)............................... 50
6. Determining and interpreting cell viability.................51

Clinical Trial Application (CTA) in the European
Economic Area (EEA)....................................................... 30

Needs and gaps for standards in cell therapy byfunctional area..................................................................... 51

Differences between IND and CTA................................. 33

Chain of Identity............................................................... 51

Submission of CMC-related amendments.................... 33

Data acquisition and management................................. 51

Requests for Out of Specification Exemptions............. 34

Implementing Standards in Cell-Based Therapy
Manufacturing................................................................................52

Life Cycle Management.................................................. 35
Designations to Expedite Development.......................... 36
US..................................................................................... 36
Europe..............................................................................40

FDA supported courses for standards
implementation........................................................ 52
Standards in Cell Therapy and Regulatory Approval......52

Japan................................................................................ 42

FDA Informal Recognition of Voluntary Consensus
Standards.................................................................... 52

Comparability...................................................................... 44

FDA Formal Recognition of Standards........................... 53

Comparability Studies for Cell-based Products............. 44

References........................................................................... 53

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼8

Introductory Remarks
Cell and gene therapies (CGTs) are transformative therapies addressing a wide range of conditions such as cancer
and genetic and infectious diseases. These modalities arestill in their early days, as reflected by the small numberof therapies that have been approved and commercialized
for patient use. Currently, there are pathways for regulatory review and approval for these therapies, which thischapter aims to cover.
For the purpose of the discussion in this chapter, it iscritical to understand the classifications of these advanced
therapies, which can be categorized into three main types:• Gene therapy medicines: these contain genes thatlead to a therapeutic, prophylactic, or diagnostic
effect. They work by inserting recombinant genes
into the body to treat a variety of diseases, including
genetic disorders, cancer, or long-term diseases.
Included in this category of medicines are:
- In vivo gene therapy, where the gene is administered directly to the patient. AAV-based genetherapy is an example of this therapy, which isthe focus of A-Gene.1 Gene editing can also be
performed directly in vivo.
- Ex vivo gene therapy, where the targeted cells,
which may be from healthy donors or removed
from the patients, are genetically modified (e.g.,viral, non-viral and/or gene editing-based) in vitro
before they are returned to the patient’s body. For
instance, autologous CAR-T therapies, which is thefocus of A-Cell, fall into this category.
• Cell therapy medicines: these contain cells or tissues
that have been manipulated (however not genetically modified, otherwise they are considered genetherapies) to change their biological characteristics,
or cells or tissues not intended to be used for thesame essential functions in the body. They can beused to cure, diagnose, or prevent diseases.
• Tissue-engineered medicines: these contain cells or
tissues that have been modified so they can be used to
repair, regenerate, or replace human tissue, which may
also contain a device component, such as a scaffold.

In the EU, these products belong to the overarchingclassification of Advanced Therapy Medicinal Products(ATMPs).
As prescribed in Chapter 1, this document focuses
on autologous CAR-T case study, which is classified
as an ex vivo gene therapy and therefore regulated as agene therapy.2 However, since this modality is cell-based,
and therefore shares a lot of the same regulatory considerations as other cell therapies, the term “cell-based
therapy” is used throughout this document where suchinclusiveness is appropriate, which also distinguishes thisdocument from A-Gene.
The approval of autologous CAR-T products has
shaped the regulatory thinking of these advanced therapies compared to other biologics. While this chapter uses
autologous CAR-T as an example to illustrate various
CMC principles, many of the concepts presented here
are applicable and beneficial to a broader profile ofcell-based products–both genetically modified and unmodified (e.g., mesenchymal stem/stromal cells (MSCs),
natural killer (NK) cells, induced Pluripotent Stem Cell
(iPSC)-derived products), and cell-based combination
products. As the field continues to pursue allogeneic
approaches, the regulatory landscape will take shape toaccommodate its specific considerations or requirements.
The authors acknowledge that regulations andstandards are constantly evolving and that this chapter
is written to capture a snapshot in time of current regulatory considerations and standard activities. Where theprinciples apply to both in vivo gene therapies and cellbased therapies, cross-references to the relevant A-Genesections are made throughout this chapter.

Regulatory Considerations
Currently, there are pathways for regulatory review andapproval for cell and gene therapies in numerous major
markets; three of the most advanced include: UnitedStates (US), Europe, and Japan. This section of the chapter covers the regulatory frameworks in these three major
markets, regulatory interactions including accelerated
regulatory pathways, information to be included in theelectronic Common Technical Document (eCTD), andsubmission content.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼

9

REGULATORY FRAMEWORKThe novel and diverse nature of cell-based therapies has
resulted in evolving regulatory frameworks to support
these products in the three major markets. As regulatory
agencies have increased experience with this family of
products, they will further define guidance to facilitate
therapeutic development to address patients’ unmet needs.

United States
In the US, regenerative medicine therapies are regulatedby the FDA Office of Tissue and Advanced Therapies(OTAT) within the Center for Biologics Evaluation andResearch (CBER). OTAT engages in extensive pre-submission communication with sponsors/applicants andconducts regulatory review of applications for investigational use and marketing approval, operating within the
Prescription Drug User Fee Act (PDUFA) timeframes.
OTAT also participates in the wider scientific community
by reviewing manuscripts and grant proposals, editing,and participating in and organizing scientific conferences.
Applications for cellular and gene therapy products are
first reviewed and evaluated by the Cellular, Tissue, andGene Therapies Advisory Committee (CTGTAC) within
OTAT. OTAT is comprised of 5 divisions in addition to
the Office of the Director:3• Division of Cellular and Gene Therapies (DCGT)• Division of Clinical Evaluation and Pharmacology/
Toxicology (DCEPT)• Division of Human Tissues (DHT)• Division of Plasma Protein Therapeutics (DPPT)• Division of Regulatory Project Management (DRPM)
DCGT, DCEPT, and DPPT constitute the main review disciplines of the regulatory review process: Product(specifically DCGT and DPPT), Preclinical (pharmacology/toxicology), and Clinical (specifically DCEPT).
References for the regulatory process of OTAT include
the following sources:• Cellular and Gene Therapy Guidance Documents• CBER-CDRH Cross-Center Guidance Documents• Cellular, Tissue, and Gene Therapies Advisory
Committee (CTGTAC)• Cellular and Gene Therapy Products

It is likely that OTAT will obtain independent
expert advice via an Advisory Committee meetingfor marketing applications for first-in-class cellbased therapies. The CTGTAC reviews and evaluatesavailable data relating to the safety, efficacy, andappropriate use of human cells (https://www.fda.gov/advisory-committees/blood-vaccines-and-other-biologics/
cellular-tissue-and-gene-therapies-advisory-committee).
OTAT has specific goals with respect to chemistry,
manufacturing, and controls (CMC), preclinical andclinical investigations, and safety issues related to humantissues. For CMC, the objectives of OTAT are aimed atenhancing quality, consistency, and performance ofadvanced therapeutics through development of strategies and methods for improved raw materials sourcing,
manufacturing, and product characterization (including
test methods, standards, identification of critical quality
attributes (CQAs), and pursuit of related biological investigations). For preclinical and clinical investigations,the goals of OTAT are aimed at enhancing safety andeffectiveness of advanced therapeutics through establishing in silico, in vitro, and in vivo preclinical models
and conducting analyses to increase understanding ofclinical trial design issues and patient characteristics that
determine outcomes. For safety issues related to humantissues, OTAT aims at enhancing safety and effectivenessof donor screening tests by contributing to researchon the development of devices and technologies usedin sourcing, manufacturing, processing, and testing oftissues and advanced therapeutics.
It should be noted that cell and gene therapy products
are regulated by both the Public Health Service Act withrespect to compliance with Good Tissue Practice (Donor
testing and screening; section 361) and with respect torequirements for a Biologic License (section 351) and regulation as drugs under the Food, Drug and Cosmetic Act.
Additional resources: CBER grants

CBER awards grants to support research projects aimed
at studying and recommending improvements for theadvanced manufacturing of biological products, including the investigation and development of innovative
monitoring and control techniques. The funded research
addresses knowledge and experience gaps identified for
emerging manufacturing and testing technologies andChapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼10

supports the development and adoption of such technologies in the biological product sector.
In 2018, as part of its commitment to achieving thegoals of the 21st Century Cures Act, a law enacted in 2016that allocates $500 million over 9 years to help accelerate
medical product development and bring new innovations
and advances to patients who need them faster and more
efficiently, CBER awarded nearly $3 million in grants to
support research at five institutions aimed at developing
more innovative, consistent, and dependable manufacturing of biological products. The agency continues tosupport efforts to develop the standards and policies
needed to foster the effective and efficient development
and adoption of new manufacturing platforms.
Particular consideration is given towards issuing
grants to study continuous manufacturing of drugs andbiological products (and similar innovative monitoring
and control techniques) as a counterpoint to “batch”
technology, a process that involves many stops andstarts in a series of manufacturing steps. The aim is toaddress inefficiencies, delays, and risks of introducing
human error into manufacturing processes that suchbreaks inherent to batch technology can cause. From theperspective of the FDA and CBER, continuous manufacturing has significant potential to improve the agility,
flexibility, cost, and robustness in the development of
manufacturing processes for a wide variety of therapeutic
products. Thus, expert FDA staff issue grants to enhanceknowledge of novel continuous manufacturing technologies and develop scientific standards and policies forarticulating how the FDA will evaluate these technologies
as part of regulatory submissions for new products.
The main grant application portal can be found athttps://www.grants.gov/. Grants awarded by the FDA(including those awarded specifically by CBER) arelisted, along with contact information for the grantmanagement specialists who are administrating the
grants and specific instructions for each grant proposal.
Applicants prepare and submit their applications throughthe https://www.grants.gov/ workspace and track theirsubmissions through the FDA eRA Commons system.

EuropeThe regulatory agency governing advanced therapies
medicinal product (ATMP) marketing applications

assessment in the European Union and European
Economic Area (EEA; the EU plus Lichtenstein, Iceland,and Norway) is the European Medicines Agency (EMA),
and within the Agency, the Committee for Medicinal
Products for Human Use (CHMP) and the Committee
for Advanced Therapy (CAT) are the main committees
evaluating these products. Others within the agency arealso involved in the review of applications, such as theCommittee for Orphan Medicinal Products (COMP; for
ODD), the Pediatric Committee (PDCO; for PIP), thePharmacovigilance Risk Assessment Committee (PRAC;
for MAA), or working parties, including the Scientific
Advice Working Party (SAWP; for Scientific Advices).
ATMP products are regulated by EU Regulation (EC)No. 1394/2007.
The EU, unlike the US, does not have an entirely
centralized system of approvals for market authorization,and sponsors may seek market authorization for somemedicinal products within individual EU member states.
However, all ATMPs must pursue approval under the
EMA’s centralized procedure because the evaluation ofthese products is considered to require broad and diverse
scientific expertise from across all states under EMA
jurisdiction. In addition, the scientific and technical requirements needed to demonstrate the quality, safety, andefficacy of ATMPs are particularly unique as comparedto other medicinal products.
If the ATMP will be used on a non-routine basis within a hospital environment and under the responsibilityof a medical practitioner, the ATMP may qualify to be
regulated under the “Hospital Exemption” scheme. It
will not be considered as a “medicinal product” and not
regulated under the scope of EU Directive 2001/83/EC
on medicinal products for human use. The preparationand manufacturing of such medicinal products will instead be regulated at the national level in the individual
member states.
In addition, and as with any other medicinal products,
if the ATMP is intended for treatment of a rare condition,
it may be eligible for the orphan drug designation.
Of note, in the EU, in addition to the EMA, all EU
member states have national competent authorities.
Germany, for example, has two agencies: one dedicatedto ATMPs and biologicals (the Paul Ehrlich Institute,
PEI) and another for all other products (the BfARM).

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼11

In addition, since 1 January 2021 and following
the UK’s decision to leave the EU, the Medicines and
Healthcare products Regulatory Agency (MHRA) is theUK’s standalone medicines and medical devices regulator.
ATMP classification

In order to get confirmation of the classification of a
medicinal product, developers of cell-based therapy
can request a formal ATMP classification from the
CAT (requests for ATMP classification should be sent
to advancedtherapies@ema.europa.eu). The procedureallows them to receive confirmation that their medicine
based on genes, cells, or tissues meets the scientific criteria that define ATMPs, and is of particular interest forborderline products. The CAT will deliver its scientificrecommendation after consultation with the European
Commission within 60 days of receiving the request. Cellbased medicinal products can be classified as somatic celltherapy medicinal product (sCTMP) or tissue-engineered
product (TEP), according to the primary mode of action.
While sCTMP action is through pharmacological activity(e.g., release of neutrophic factors), TEP is intended toregenerate, repair or replace a human tissue. Regarding
medicinal product containing genetically modified cells,
they can be classified either as gene therapy medicinal
products (GTMP) if the target genetic sequence provides
the therapeutic effect, or as sCTMP if the target genetic
sequence has another purpose (for example a manufacturing purpose for the generation of induced pluripotent
stem cells). Gene modified cellular products like CAR-T
cell therapies are classified as gene therapy medicinal
product (GTMP). Positioning a product in the ATMP
category gives developers an early opportunity to engage
with regulators, clarifies the applicability of downstream
regulatory processes, and opens the door to incentives
designed specifically for ATMPs.

Japan
Japan has two main regulatory authorities that are independent agencies with distinct roles during the drug approval
process: the Ministry of Health, Labor, and Welfare (MHLW)
is responsible for publishing regulatory guidelines, managing
advisory committees, and providing final authorizations forapplications; and the Pharmaceuticals and Medical Devices

Agency (PMDA), which is responsible for regulatory andscientific review (i.e., Office of Cellular and Tissue-based
Products), and Good Manufacturing Practice (GMP).
Following a major reform in 2013, Japan created two
regulatory paths to support and expedite the approval ofregenerative medicines. In the ‘drug’ track, regenerative
medicine products (RMPs) sponsored by companies are
regulated by the Pharmaceuticals and Medical Devices(PMD) Act, in which, after demonstration of safety andpresumed clinical benefit, PMDA issues a time-limited
market approval under the condition that the clinicalbenefit is demonstrated within seven years. As a result,the product becomes available for clinical use, and datacollected from that period can be used for final confirmation of safety and efficacy.
The ‘clinic’ track is regulated by the Regenerative
Medicine (RM) Act, and relates to regenerative products that are used in clinical research (other than clinical
trials) in an exploratory way, usually by physicians,
medical institutions, or outside companies with appropriate licenses. Clinical research under the RM Act has
different research and regulatory standards than clinical
trials under the PMD Act, and is usually a cheaper and aless stringent way to evaluate efficacy of a GTP product,
particularly one that is not ready for clinical trials. If thedata collected during this track are positive, a sponsor
may use the data to inform and initiate an application
for a clinical trial under the PMD Act track.
In addition to the three regions, other global regulatory agencies are listed in Table 2-1.
HEALTH AUTHORITIES AND SPONSOR MEETINGS

Development of innovative investigational products suchas cell-based therapies often introduce unique challenges
due to unknown safety profiles, complex manufacturing technologies, incorporation of innovative devices,and the use of cutting-edge testing methodologies. In
recognition of the complex nature of cell-based therapy
products, most health authorities (HAs) have introduced
informal consultations to allow sponsors to obtain feedback early in the product development (in addition to
the conventional meetings).

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼12

Table 2-1: List of global regulatory agencies
Country

Regulatory Agency

Website link

United States

Food and Drug Administration (FDA)

https://www.fda.gov/vaccines-blood-biologics/
cellular-gene-therapy-products

CanadaHealth Canada

https://www.canada.ca/en/health-canada/
services/drugs-health-products/biologicsradiopharmaceuticals-genetic-therapies/
applications-submissions.html

European Economic Area
(European Union + Iceland,
Lichtenstein and Norway)

European Medicines Agency (EMA)

https://www.ema.europa.eu/en

China

National Medical Products
Administration (NMPA)

https://www.emergobyul.com/resources/china/
china-food-drug-administration

Japan

Pharmaceuticals and Medical
Devices Agency PMDA

https://www.pmda.go.jp/english/

Korea

Pharmaceutical Affairs Act (PAA)
Ministry of Food and Drug Safety(MFDS)

https://www.mfds.go.kr/eng/index.do

India

Central Drugs Standard Control
Organization (CDSCO)

https://cdsco.gov.in/opencms/opencms/en/Home

Australia

Therapeutic Goods Administration(TGA)

https://www.tga.gov.au

United Kingdom

Medicines and Healthcare products
Regulatory Agency (MHRA)

https://www.gov.uk/government/organisations/medicines-and-healthcare-products-regulatoryagency

FDA (US)
Meetings between FDA and sponsors occur at criticaljunctures during the life cycle of product development
and are aimed at minimizing wasteful expenditures oftime and resources. These interactions can be in the formof informal and formal meetings, which are discussed insubsequent sections.
Informal meeting: INTERACT

Sponsors applying to the FDA can obtain a preliminary consultation with the FDA through the INitial
Targeted Engagement for Regulatory Advice on CBER
producTs (INTERACT) meeting prior to a pre-IND
meeting (in effect replacing the pre-pre-IND meeting).4
INTERACT meetings are proposed to become part of
the formal PDUFA meeting according to the PDUFA VII
Commitment Letter.5 The INTERACT meeting, available
only for cell and gene therapy (CGT) products, gives thesponsors the opportunity to access the FDA much earlier

and seek advice regarding CMC, nonclinical, and clinicalareas, particularly if innovative manufacturing or testingis used. Actual topics of discussion are at the discretionof FDA; therefore sponsors are encouraged to reach out
to FDA on whether they will receive clinical input. This
meeting is non-binding in nature, which means that asponsor is not bound to pursue a particular regulatory
pathway.
The INTERACT meeting can: 1) assist sponsors
conducting early product characterization and preclinical proof-of-concept studies; 2) initiate discussion for
new delivery devices; 3) inform sponsors about overall
early-phase clinical trial design elements; and 4) identify
critical issues or deficiencies for sponsors to address inthe development of innovative products.
An INTERACT meeting is not intended to take theplace of a pre-IND meeting, which occurs prior to thesubmission of an IND to discuss the scope and design ofplanned initial studies, design of animal studies needed

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼13to support human clinical testing, and the format for
the IND. Conversely, an INTERACT meeting also is
not a venue to provide advice to sponsors who have
yet to initiate any product development activities.
Prior to requesting an INTERACT meeting, a sponsor needs to have selected a specific investigational
product or a product-derivation strategy to evaluate
in a clinical study.
Optimizing the strategic value for the time of themeeting relative to product development might be thesponsor’s greatest challenge when seeking an INTERACT
meeting. Anecdotal evidence suggests that the meetingmight be declined if it is requested too early in the process at a point when a product candidate has not been
established, or when it is considered too late, after a Tox
program has already been developed/implemented.
Informal meeting: CATTThe CBER Advanced Technologies Team (CATT) wasestablished to create an interactive platform for prospective developers to seek advice and feedback fromCBER staff regarding advanced manufacturing andtesting technologies that fall under the scope of CBER
regulation. In addition, these technologies may haveregulatory implications or have a significant impact on
product development and CMC. Meetings with CATT
can be requested during the preclinical stage; however,inquiries can also be made to discuss new technologies
that are not part of a specific project.
The CATT will facilitate discussion of inquiries or
responses to meeting requests pertaining to advanced
manufacturing technologies that are intended to be
implemented in the development of products regulated by CBER. Inquiries or meeting requests submitted
to the CATT should focus on novel technologies that
can have a significant impact on product development,
manufacturing process, and control strategies, and may
also have regulatory implications. This includes manufacturing and analytical methods for those products
or classes of products for which the center has limited
experience with the manufacturing or development
process. Details on the process for submitting inquiries/meeting requests to CATT can be found here:
https://www.fda.gov/vaccines-blood-biologics/industry-biologics/cber-advanced-technologies-team-catt

Formal meetings

Formal milestone meetings under PDUFA include preIND, end-of-phase 1 (EOP1), EOP2, and pre-Biologics
License Application (pre-BLA) meetings. For most celland gene therapy manufacturers, the product undergoes
revision and evolution as more data are gathered anddeeper product knowledge is gained throughout productdevelopment. For example, changes to the manufacturing
process can occur, which may potentially impact the
product’s critical characteristics and therefore its clinicaloutcomes. It is therefore important for manufacturers to
perform risk assessment, report these changes, and seek
advice from health authorities during the product life
cycle to support a streamlined pathway to commercialization. Further discussion on managing changes during
manufacturing is provided in the Comparability section.
During the life cycle of drug development, sponsors
may seek advice from the FDA with regard to several topics, including (but not limited to) the following: regulatory
concerns, clinical/statistical concerns, safety concerns,
product quality concerns, clinical pharmacokinetics andpharmacology, nonclinical pharmacokinetics and pharmacology, and pediatrics. When soliciting feedback, the
FDA advises sponsors to keep in mind that FDA policy
positions are typically documented and described in FDA
guidances, Manual of Policies and Procedures (MAPPs),and Standard Operating Policy and Procedures (SOPPs).
Complex scientific and technical drug development questions should be directed to the FDA regulatory project
manager (RPM). General questions that cannot be answered by using existing resources can be directed to an
FDA RPM or to the designated enhanced communication
staff within each FDA center. The FDA will either respondto the questions or redirect the sponsor to an alternative
pathway for receiving a response (e.g., other FDA subject
matter experts or the formal meeting request process).
Details on the available meetings between the FDA andsponsors are described in the following sections and summarized in Figure 2-1. This figure simply provides a chronological guide; it is not meant to reflect actual timescale.
The FDA offers four types of meetings with requesters
seeking advice relating to the development and review of
INDs and biologics, and drug or biological product marketing applications under reauthorizations of the PDUFA:
Type A, Type B, Type B (end of phase [EOP]), and Type C.6

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼

14

Figure 2-1: Interactions with FDA for Regenerative Medicine
IND submitted

BLA submitted
RMAT/BT Meetings

ResearchpreclinicalInteract
Pre-IND
MeetingPhase I

Phase IIInteractive Review

Phase III
(if needed)

Marketing Authorization

Clinical Trials
IND Milestone Meetings(EOP1, EOP2, Pre-BLA)BLA Review: 10 months
from filing date
Priority Review: 6 months
from filing date2 month filing determination
period• Type A meetings are necessary for an otherwise
stalled product development program to proceed or
to address an important safety issue. It is importantto point out that Type A meetings are only granted
for stalled product development due to an action
taken by the FDA, and not for an issue from thedeveloper side. Topics that are often covered by TypeA meetings include dispute resolution as describedin 21 CFR 10.75, 312.48, and 314.103, clinical holds,
receipt of an FDA Nonagreement Special Protocol
Assessment letter, FDA regulatory action other thanan approval (must be requested within 3 months ofaction), and FDA issuance of a refuse-to-file letter.
• Type B meetings cover pre-investigational new
drug applications (pre-INDs), pre-biologics license
applications (pre-BLAs), pre-emergency use authorization, FDA regulatory actions other than approval(must be requested within 3 months of action), risk
evaluation and mitigation strategies, post-marketing
requirements outside the context of the review of amarketing application, and development programsfor products granted breakthrough therapy designation (BTD) and/or RMAT designation status.
• Type B Comprehensive Multidisciplinary RMAT
meeting7 is a comprehensive discussion of the drug
development program, including planned clinical
trials and plans for expediting the manufacturing
development strategy. The meeting must be held
within 6 months of designation granted andfollows the same PDUFA timelines as for any TypeBLA Milestone meetings(PMC/PMR)

Post-Marketing

Meetings Related toSupplements
PAS: 4 Months
Others: 6 months

B meeting. The objective of the meeting includes
agreement on Phase III clinical trial (or pivotal trialthat provides primary evidence of clinical efficacy for
BLA), potential surrogate or intermediate endpoints
in support of accelerated approval, agreement clinicaldevelopment plan, and plans for expediting the
manufacturing development strategy.
• Type B (EOP) meetings include certain end-of phase
I meetings of products considered for marketing
approval under 21 CFR part 312, subpart E, or 21 CFRpart 314, subpart H, or similar products and end-ofPhase II or pre-Phase III meetings (21 CFR 312.47).
• Type C meetings are any meeting other than Type A,
Type B, or Type B (EOP) meeting regarding the development and review of a product, including meetings to
discuss adequacy of facility design and establishment
issues, and to facilitate early consultations on the useof a biomarker as a new surrogate endpoint that has
never been previously used as the primary basis for
product approval in the proposed context of use.
There are three meeting formats: face to face, teleconference/videoconference, and written response only(WRO). In face-to-face meetings, the majority of attendees
participate in person at the FDA. WRO responses are sent
to requesters in lieu of meetings conducted in one of theother two formats. Most meetings shifted to WRO format
as a significantly reduced number of face-to-face meetings
were being granted, and FDA adjusts their resources for
efficiency. The FDA does permit follow-up questions/clarifications to its WRO feedback in many cases.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼15

Table 2-2: Summary of PDUFA meeting management procedural goals
MeetingType

FDA
Response
to Request

FDA Receipt
of Meeting
Package

FDA Preliminary
Responses toRequester (ifapplicable)

Requester
Response to
FDA Preliminary
Responses (ifapplicable)

FDA Scheduled
Meeting Date(days fromreceipt ofrequest)

FDA Meeting
Minutes toRequester (ifapplicable)A

14 daysWith meeting
requestNo later than
2 days before
meeting--Within 30 days

30 days after
meetingB

21 daysNo later than
30 days before
meeting

No later than
2 days before
meeting--Within 60 days

30 days after
meeting

B (EOP)*

14 daysNo later than
50 days before
meeting**

No later than
5 days before
meeting

No later than 3
Within 70 days
days after receipt
of preliminary
responses

30 days after
meetingC

21 daysNo later than
47 days before
meeting***

No later than
5 days before
meeting

No later than 3
Within 75 days
days after receipt
of preliminary
responses

30 days after
meeting* EOP=end of phase** If the scheduled date of a Type B (EOP) meeting is earlier than 70 days from FDA receipt of the meeting request, the requester’smeeting package will be due no sooner than 6 calendar days after FDA response time for issuing the letter granting the meeting.
*** If the scheduled date of a Type C meeting is earlier than 75 days from FDA receipt of the meeting request, the meeting package
will be due no sooner than 7 calendar days after the FDA response time for issuing the letter granting the meeting. Note that forType C meetings that are requested as early consultations on the use of a new surrogate endpoint to be used as the primary basis
for product approval in a proposed context of use, the meeting package is due at the time of the meeting request.

Pre-IND meetings

Pre-IND meetings are Type B meetings and are meant toinitiate dialogue regarding product development in its earlystages, with the aim of understanding the mechanism ofactions of the drug and possible study designs. They can
prevent clinical hold issues from arising and aid sponsors
in developing a complete IND. Pre-IND meetings are forcell-based products that are too mature for an INTERACT
program and permit a multi-disciplinary assessment of
CMC, pharmacology/toxicology, clinical, and other regulatory topics for a future IND sponsor to prepare for that IND.
Clinical Meetings — End of Phase

End of Phase (EOP) meetings serve to evaluate thenext clinical phase plan and protocols, the adequacy of
current studies and plans to assess safety and efficacy,
and the adequacy of manufacturing and testing plans to
support the next clinical phase studies.
EOP1 meetings are meant to review and reach agreementon the design of Phase II controlled clinical trials and to

discuss issues related to the proposed drug development
program. Because of limited resources, the FDA has
traditionally encouraged sponsors to request an EOP1
meeting only for drugs intended to treat life-threatening
and severely debilitating illnesses, particularly situations
where approval based on Phase II trials or accelerated
approval may be appropriate. Thus, EOP1 meetings
may be particularly useful for sponsors developing celltherapy products.
EOP2 meetings serve to evaluate the Phase III plan andprotocols, the adequacy of current studies and plans toassess safety and efficacy, evaluate the human factors
validation plan, and identify any additional informationnecessary to support a marketing application for the uses
under investigation, allowing for preparation for commercial manufacturing. The FDA encourages sponsors
to request an EOP2 meeting.
All EOP meetings are Type B meetings and subject todifferent timelines as summarized in Table 2-2. From a
CMC perspective, by the end of the Phase II clinical studies,

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼16

the sponsor should have a very robust knowledge of the
manufacturing process and have started preparing for thePhase III clinical materials that will be representative of thecommercial product. For cell-based therapy products, thistimeline is not straightforward. As previously discussed,
the clinical results in cell-based therapy products is often
ahead of the CMC development. Clinical Phase II (orpivotal) and Phase III (if needed) timelines are condensed
and, as a result, the CMC development must be expedited.
In preparation for the EOP2 meeting, the sponsor
should take the opportunity to seek advice from the FDA
that the current data package, in addition to the potential
planned studies, will be sufficient for a BLA submission.8
It is recommended that sponsors request a CMC-focused
EOP2 meeting to ensure that there is sufficient time dedicated to CMC discussions, while the pre-BLA meetingcan focus on other regulatory matters. Typical topics
discussed during the EOP2 meetings include, but are notlimited to: release specifications and justifications, overall
control strategy with definitions of critical quality attributes (CQAs) and critical process parameters (CPPs),
manufacturing process and analytical assay validationplans, and stability data to support product storage
and shelf-life. At such a meeting, sponsors should also
discuss readiness/plans for the device (used for productadministration) and/or companion diagnostics that will
be part of the marketed product.
Pre-BLA meetings are meant to familiarize FDA reviewers with the format and content of the planned marketing
application, including labeling and risk management
activities, presentation and organization of data, dataset
structure, acceptability of data for submission, and the
projected submission date of the marketing application.
They are also intended to uncover major issues, identify
studies intended to establish the drug’s safety and efficacy,
discuss the status of pediatric studies, and discuss statistical analysis methods and results. The FDA encourages
sponsors to request pre-BLA meetings for all plannedmarketing applications.

EMA and EU national agencies (Europe)
Like the FDA, the EMA and the EU national competentauthorities (NCA) offer a number of opportunities forsponsors to discuss scientific, technical, and regulatory

topics with the agency(ies) at any stage of the development. The choice of reaching out to national agencies
or to EMA is dictated by the stage of development,
the questions to be raised, and more importantly, theoverall objective of the advice (specific clinical study,
overall development, etc.), and should be determinedon a case-by-case basis. Of note, national agency
experts serve as members of the EMA’s scientific committees, working parties, or in assessment teams. A listof national competent authorities can be found here:
https://www.ema.europa.eu/en/partners-networks/
eu-partners/eu-member-states/national-competent-authorities-human#list-of-national-competent-authorities-in-the-eea-section.
Though meetings with the EMA provide opportunities for developers in industry to discuss productdevelopment strategies with the regulators and clarify
interpretations of the regulations, there are, in general,
relatively few guidance documents to help companies
prepare for meetings with the EMA compared with the
FDA. For developers who do not have the experience ofmeeting the EMA or submitting a briefing document,
there is the possibility to submit pre-submission meeting requests to clarify the content of the document (noscientific assessment performed at this stage).
Innovation Task Force and Innovation Network

In an effort to provide sponsors of emerging therapies
and technologies early access to the EMA and to ensure
coordination across all moving parts of the regulatory
process, the EMA offers the support of the Innovation
Task Force (ITF).9,10,11 ITF is comprised of experts fromscientific, regulatory, and legal sectors, and core members,
who are appointed based on competence, areas of interest,and commitment to participate in meetings relating totheir specialized area. ITF briefing meetings are informal
and non-binding and are intended to offer early feedback
on innovative medicinal products and technologies andtheir impact on regulatory and scientific proceedings.
Such meetings are intended to complement and reinforce
existing formal procedures (e.g., CHMP scientific advice,
ATMP classification and certification, and designationof orphan medicines) and prepare the sponsor for suchprocedures should they be necessary along the regulatory
track for the development program. The meetings are

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼17

free of charge and last 1.5 hours. After the meeting, thesponsor will provide meeting minutes to the ITF.
In addition, since 2015, the ITF has been supportedby the Innovation Network. The Innovation Network iscomprised of innovation offices from national competent authorities (NCAs) across the EU. The aim of the
network is to improve regulatory support for medicine
developers at national and EU levels and make this more
appealing to innovators.12
Scientific advice and protocol assistance from the EMA

Early engagement with the EMA is a key driver of faster,and more often, successful registration. The EMA provides developers scientific advice and protocol assistanceon the most appropriate way to generate robust evidence
on a medicinal product’s benefits and risks.13 Sponsors
can request meetings with the EMA for overall advice on
the topics of choice, and there is no limit to the number ofscientific advice meetings (or protocol assistance as it iscalled for products with orphan drug status) that can berequested during the development of a given cell therapy.
EMA scientific advice and protocol assistance are delivered by CHMP on the recommendation of the SAWP;
for cell-based medicinal products, the two rapporteurs
assigned to the procedure and in charge of preparing therecommendations are appointed from the CAT. This adviceis meant to support the timely and sound developmentof high-quality, effective, and safe medicines, and may besought at any stage of a product’s development program.
To request a scientific advice (or protocol assistance),
developers must submit a briefing document on the IRIS
portal hosted by the EMA, accompanied by information
to be filled in online.
Developers may ask questions relating to such topics as
manufacturing, chemical, pharmaceutical, and biologicalaspects of product development, clinical and nonclinicalaspects of development, and data analysis and statistics. In
addition, for products that have received the orphan drug
designation, developers may inquire about the demonstration of significant benefit of the medicinal product overexisting (and approved) treatments. Furthermore, the EMA
offers advice for post authorization safety studies (PASSs)
to improve the design of studies meant to collect further
information on a product’s safety once it is on the market.
With regard to timelines, once the request (including abriefing document) is submitted to the EMA, a validationof usually 3-4 weeks will take place. Once the briefing
document (with possible revisions as per the request of
the agency) is validated, the procedure (Day 0) will startwith the first SAWP meeting. During the second SAWP
meeting (one month after the first one), it will be decided
whether there is a need for a meeting with the Applicant.
If no meeting is required, the SAWP will finalize its recommendations and the final advice letter will be release by the
CHMP at Day 40 of the procedure. If a meeting is deemed
appropriate, it will take place at the third SAWP meeting(hence at Day 60) and followed by the approval of the final
advice letter by the CHMP at Day 70 of the procedure.
It should be noted that the EMA charges an administrative fee for advice (reduced fees are available if the developer has a small and medium enterprise (SME) status,
for ATMP, and orphan drugs). Importantly, patients (orpatients’ representatives) may be called upon to provide
comments or attend meetings with the EMA.
Scientific advice from EU national agencies

In addition to the EMA, most if not all EU national
agencies do offer scientific advice. Fees are variable
depending on the agency, topics, and number of questions. The procedure is highly similar: in most agencies, ameeting request should be submitted including proposed
meeting dates and questions. Some agencies do request
the briefing document at the same time (as EMA does).
Once the Agency validates the request, confirmation of
the meeting date will be sent to the developer as wellas the deadline to provide the briefing package and thepresentation. Indeed, all scientific advice at the national
level includes a meeting during which the questions are
discussed. Following the meeting, minutes and/or final
advice (written) will be sent to the developer.
Simultaneous national scientific advice

Developers can receive scientific advice nationally fromEU national competent authorities (NCAs) or from the
EMA. Depending on the overall clinical development,
when national advice is sought, it is often requested
from more than one NCA. To address this situation andaim towards more harmonization and optimization, in
February 2020, the Innovation Network started a pilot
program for simultaneous national scientific advice

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼

18

(SNSA) to provide an avenue for regulatory interactions
for developers in which scientific advice can be requested
from two NCAs simultaneously, optimizing the quality
and consistency of such advice.14 The initial phase wasa success and the SNSA has been extended until August2024.15 The participating NCAs for the pilot program
are from Austria, Belgium, Czech Republic, Finland,
Germany, Hungary, Italy, Norway, Poland, and Spain(participation has been kept optional for NCAs fromother member states as well). SNSA meetings and topics
for discussion are identical to single national scientificadvice currently offered by NCAs.
Developers can apply for a SNSA by sending an informal letter of interest to one of the two selected NCAs
or by using an existing application form. The applicant
should propose two preferred NCAs (i.e., based on thelist of NCAs volunteering for the SNSA pilot), after which
the NCAs accept or deny the request for advice. In case
one NCA is not able to join the SNSA, the applicant cansuggest an alternative NCA, keep the procedure as a standard national scientific advice request, or withdraw thewhole application. Assuming both NCAs can participate,
one NCA will take over the lead of the procedure (by mutual agreement with the other NCA) as the coordinating
agency and coordinate the advice procedure, acting as
the main contact point with the applicant and the other
NCA. The timeline of the SNSA will also be mutuallyagreed on by both NCAs, respecting the preferred datesof the applicant as far as possible. Briefing documents
and questions need to be sent to both NCAs separately,
considering special requirements with regard to submission timelines, template, scope, content, and extent of the
documents of each NCA; and assistance can be provided
by the coordinating NCA. The formal validation of thescope, focus of questions (and corresponding rationales)
contained in the briefing documents will be reviewed
independently by each NCA. In case of any queries (e.g.,
validation questions raised by one of the NCAs towards
the applicant), the coordinating NCA will get in touchwith the applicant. The applicant is not allowed to add
new questions or change questions or data during thecourse of the SNSA procedure. Both NCAs will be represented by the respective national experts equal to thenational procedures. The meeting minutes will be draftedby the applicant based on the common template provided

and sent to each NCA for review and comments. The
final document reflects the formal SNSA opinions fromboth NCAs, based on mutual agreement between them.
Potential requests for clarification from the applicant(e.g., on the scientific regulatory opinions provided in
the context of the formal SNSA) might be accepted andhandled in agreement between both NCAs and in compliance with their respective procedures, whereas newquestions from the applicant would be dealt with in afollow-up advice request. Fees will be based on the cost
regulations of each NCA involved.
PRIME scheme and meeting

The PRIME (PRIority MEdicines) scheme provides earlyand enhanced scientific and regulatory support to medicinal products that have the potential to significantly
address patients’ unmet medical needs. Any sponsor
engaged in the exploratory clinical trial phase of development (i.e., prior to reaching a pivotal/registration study
and at a stage at which the EMA advice can be used torefine the development) can submit an eligibility request
to enter the PRIME scheme. Sponsors should base theirrequest on the availability of preliminary clinical evidence
in patients indicating the promising activity of the medicinal product and its potential to significantly addressan unmet medical need (proof of concept). Applicantsfrom the academic sector and micro-, small-, and medium-sized enterprises may submit an eligibility request
at an earlier stage of development if compelling nonclinical data in a relevant model provides early evidence ofpotentially promising activity (proof of principle) andfirst-in-man studies indicate adequate exposure for thedesired pharmacotherapeutic effects and tolerability.
As one of the unique features of the PRIME scheme,
the sponsor has an opportunity to participate in the
PRIME kick-off meeting with the CHMP/CAT rapporteur and their multidisciplinary assessment team,including chairs of CAT, SAWP, COMP, PDCO, or PRAC,
as appropriate. The PRIME meeting usually takes place
two to three months after a product is accepted into
the PRIME scheme, and its purpose is to discuss future
steps in development support, including technical andscientific assistance.
In addition to the kick-off meeting, the agency will
provide scientific advice at key development milestones

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼19

and involve additional stakeholders such as health-technology-assessment bodies, in order to facilitate quicker
access for patients to the new medicine.
Parallel joint scientific consultation with regulatorsand health technology assessment bodies

In an effort to coordinate the advice from HTA andregulators, a pilot phase of joint advice was launched in
the EU, now under the format of a joint consultation.
There are strict eligibility criteria to apply and currently
8 meetings are granted per year. These meetings are 3
hours in duration and EMA as well as HTA may attend
on a voluntary basis.
It is not clear at the date of release of this chapter what
the future of such joint consultations will be.
Pre- submission interactions with the EMA andmeetings with (co)rapporteurs

Pre-submission interactions (PSI) with the EMA address
product-specific legal and regulatory issues, and areintended to facilitate the validation of the marketing
authorization application. For PSI, applicants should
submit a briefing package that includes questions raised
to the EMA (e.g., overall compliance of the intended
submission package with applicable regulatory requirements, possible gaps in knowledge that could be useful
to discuss). If a meeting is deemed appropriate, EMA willcontact the applicant.
Pre-submission meetings (PSM) with the (co)rapporteurs in charge of the evaluation of the MAA per se aremeant to discuss scientific aspects and to support applicants in submitting applications for smooth evaluation.
There is no specific timetable, though these meetings
should take place as soon as the (co) rapporteurs areappointed by the Agency (usually around 6 months prior
to the filing of the MAA).

EMA-FDA Programs

Parallel Scientific Advice

The EMA and FDA jointly offer the parallel scientific advice (PSA) program to provide a mechanism for staff fromboth the EMA and FDA to concurrently convey to sponsors their views on scientific issues during the development
phase of new medicinal products. These interactions aremeant to increase dialogue between the two agencies andsponsors from the beginning of the life cycle of a new
product, provide a deeper understanding of the basesof regulatory decisions, optimize product development,
and avoid unnecessary testing replication or unnecessary
diverse testing methodologies. The agencies conduct PSA
procedures according to the confidentiality arrangement
between the European Commission, EMA, and FDA.
PSA procedures usually occur at the request of thesponsor, though, in special circumstances, the EMA or
FDA may also initiate the PSA process in full cooperation
with the sponsor. PSA requests should focus on specificquestions or issues involving the development of a medicinal product for which the sponsor desires to have further
scientific input from both the EMA and FDA. The PSA
procedures should focus on sharing information andperspectives. Following PSA meetings, sponsors shouldhave a clearer understanding of the agencies’ respective
requirements and perspectives regarding the development
program discussed and, if divergent, the reasons for thedivergence. The FDA and EMA consider the best candidates for PSA to be important medicinal products being
developed for indications lacking development guidelines
or for those indications for which existing EMA and FDA
guidelines differ significantly.
Sponsors wishing to nominate a product for a PSA
should address a single “Request for PSA” letter to bothemainternational@ema.europa.eu and US-FDA-EUR@
fda.hhs.gov. In this letter, the sponsor should provide
the following information: the product in development;
why a discussion with EMA and FDA staff would be
beneficial to the product’s development; specific questions requiring clarification; the desired goals for the
meeting; and an explicit authorization for the agencies’
comprehensive exchange of all information relevant to
the product, including trade secret information. Anyfees applicable for scientific advice at both agencies areunaffected by the PSA status. If both agencies grant the
PSA request, the sponsor will receive an email from eachagency acknowledging the agreement and indicating the
primary contact person at each agency. The PSA process
generally corresponds to the 70-day timeline of SAWP
at the EMA and the timeline for a Type B meeting at the
FDA. The designated primary contact for each agency
will coordinate final meeting logistics with the sponsor, including timelines for submission of pre/meeting

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼20

background information to both agencies. The two agencies will conduct a pre-sponsor meeting teleconferenceor videoconference (usually around day 60 of the 70-day
timeframe) to discuss the sponsor’s questions prior
to the meeting. The two agencies may also conduct a
post-sponsor teleconference or videoconference if needed. The detailed timeline for this parallel review can befound here: https://www.ema.europa.eu/en/documents/
other/timeline-european-medicines-agency-fda-parallel-scientific-advice_en.pdf
If a sponsor’s request for a PSA is not granted, thesponsor is free to pursue a scientific advice procedure
with each agency individually, following each agency’s
normal procedural rules. Both agencies may also engage
in a Consultative Advice procedure, as described below.
Consultative Advice

The Consultative Advice procedure allows sponsors torequest scientific advice from one regulatory agency
and concurrently notify the other regulatory agencyof the request.16 At the invitation of the first agency,
the second will participate in the sponsor meetings or
teleconferences, as able. Unlike the parallel scientificadvice process, the second agency will be expected toonly engage on top level issues. The review and sponsor
meeting will follow the timelines of the regulatory agency
from whom the sponsor initially seeks scientific advice.
Only the initially contacted regulatory agency will provide written scientific advice in accordance with standard
agency meeting procedures.

MHRA (UK)

Scientific Advice

A sponsor can ask for scientific advice from MHRA at
any stage of the initial development before submitting
an application for a marketing authorization (MA), andduring the pre-submission period for a variation to an
existing MA.17 The types of advice a sponsor seeks fromMHRA can be on the topics of quality aspects (e.g., thechemical, pharmaceutical, and biological testing), nonclinical and clinical aspects, pharmacovigilance plans andpost-authorization safety study protocols, an application
for a variation or renewal, advice before publishingadvertising for a medicinal product, and changes tolabelling of packaging leaflets for medicinal products. It

may be possible to request scientific advice for a broader
scope that would cover a wide range of issues and would
not relate to only one development product. Broader
scope issues include any practical issues concerning
study design, risk management plans, and complex issuesof drug/device combination products.
The option to have a joint meeting with the MHRA
and the National Institute for Clinical Excellence (NICE)
is available. This option is useful if the sponsor would liketo have a discussion regarding study design that can caterto both regulatory and NICE requirements. The MHRA
does not have pre-defined meeting slots but the averagelead time between submission of a meeting request andthe date of the meeting is about 3-4 months.
To request a meeting, the process is similar to EU
national agencies, i.e., submission of a meeting request(MHRA form) that includes proposed dates, administrative information, and proposed questions.
Innovation Office (IO)

Although the ‘standard’ Scientific Advice process is certainly useful for ATMP/CGT, the Innovation Office (IO)
provides some more targeted options that are probably
more relevant for cutting-edge technologies.18 The MHRA
Innovation Office offers regulatory information, advice,and guidance to developers of innovative medicines whoseek early regulatory interactions in the UK. The IO is
open to all innovative queries, particularly those thatchallenge the current regulatory framework. Examples
of topics open for discussion with the Innovation Officeinclude medicines, medical devices, and manufacturing
processes. For queries about regenerative medicines inparticular, the IO offers a Regulatory Advice Service forRegenerative Medicine in the form of consolidated information and guidance, reviewed by four independent andUK-based agencies (Health Research Authority (HRA),
Human Fertilisation and Embryology Authority (HFEA),
Human Tissue Authority (HTA), National Institute for
Health and Care Excellence (NICE)) in addition to the
MHRA specialists.
MHRA request that interested parties submit theirquery and they will receive a single, consolidated, considered, and confidential response within 20 working
days (depending on the complexity of the query) withregulatory information, advice, and guidance to help

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼21

plan their project. In some cases, when experts consider
the issues to be complex or requiring clarification, a
regulatory advice meeting may be proposed to discuss
the details of the query in more depth.
Innovative Licensing and Access Pathway (ILAP)

ILAP aims to accelerate the time to market, facilitating
patient access to medicines. These medicines include
new chemical entities, biological medicines, new indications, and repurposed medicines. The ILAP is open
to both commercial and non-commercial developersof medicines (UK-based and or global). It comprises an Innovation Passport designation and a Target
Development Profile (TDP) and provides applicantswith access to a toolkit to support all stages of the design,development, and approvals process.19
The first step in the ILAP is the Innovation Passport(IP) application. The IP is the mandated entry point to
the ILAP and is open to developers at the preclinical trial
stage through to the mid-development program point.
Eligibility criteria for the IP include:• The condition is life-threatening or seriously debilitating, and there is a significant patient or publichealth need.
• The medicinal product fulfills one or more of thefollowing specific areas:a.) innovative medicine such as an ATMP or new
chemical or biological entity or novel drug device
combination;b.) medicines being developed in a clinically significant new indication for an approved medicine;c.) medicines for rare disease and/or other special
populations such as neonates and children,
elderly, and pregnant women; andd.) development aligning with the objectives for UK
public health priorities such as the Chief Medical
Officer, Department of Health and Social Care(DHSC), or Life Sciences Sector Deal (including
those in Devolved Administrations, whereappropriate)• The medicinal product has the potential to offer
benefits to patients.
Sponsors that wish to apply for an IP should complete
the submission form, after which they will be invited tomeet with the MHRA to discuss how their product fulfils
the three criteria (usually within 4-6 weeks following
receipt of the application form). Following the meeting,the partners (AWTTC, MHRA, NICE, and SMC) will
jointly consider if the criteria have been fulfilled andthe outcome will be informed within 4 weeks. Once theInnovation Passport has been acquired, then onward access to the core ILAP components (Target Development
Profile assistance, kick-off meeting, etc.) have additional
associated timelines.

PMDA (Japan)
The PMDA provides opportunities for meetings between
sponsors and the Agency to allow for feedback andguidance during clinical development.20 In clinical trial
consultations for new drugs, PMDA checks whether a
proposed clinical trial complies with the requirements
for regulatory submission, taking into consideration
the ethical and scientific aspects of the development
program, the reliability of the clinical trial, as well as thesafety of trial subjects. The PMDA also gives advice tofacilitate the improvement of the clinical trial.
Since 2009, PMDA started providing prior assessment
consultations, which add value to the development process through feedback from reviewers on CMC data, in
addition to efficacy and safety feedback on the product.
This consultation process constitutes part of the reviewof the product once the application is submitted.
For sponsors that are located outside of Japan, it
is recommended to appoint a Japanese Marketing
Authorization Holder (MAH). The sponsor can request
meetings with the PMDA through the MAH, who canalso assist with translation and interpretation, since allcommunications and submission forms are in Japanese.
CMC SUBMISSION CONTENT FOR CLINICAL TRIALSElectronic Common Technical Document
The electronic Common Technical Document (eCTD)
provides the backbone for providing information regarding CMCs. Modules 2 and 3 include sections anddefinitions for drug substance (DS) and drug product(DP). It should be noted that these data are expected to
evolve over time as the sponsor optimizes production
processes and formulation of the drug. Information to be

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼22

provided about the drug substance includes the proper
identification, quality, purity, and potency of the active
ingredient, with an emphasis on the identification andcontrol of raw materials and the new DS. Information
to be provided about the DP is similar to that whichis required for the DS section, with information about
the assays and acceptable results for assessing identity,
potency, quality, and purity. It is also necessary to provide information about stability (evidence on how thequality varies with time under the influence of a varietyof environmental factors, such as temperature) for atleast the duration of the clinical trial, with the purpose of
establishing the DP shelf life and recommended storage
conditions. Fitting information about cell-based therapy
products into the eCTD structure can be challenging due
to continuous manufacturing processes and the needfor clear separation between drug substance and drug
product. Currently, there is an effort to update the ICH
M4Q guidance to reflect the fact that the eCTD is not fit
for purpose for many modalities.
Recommendations on the CMC information to
be included in an original cell-based therapy investigational new drug (IND) are provided in the Content
and Review of Chemistry, Manufacturing, and Control(CMC) Information for Human Somatic Cell Therapy
Investigational New Drug Applications (INDs) guidance23
and Chemistry, Manufacturing, and Control (CMC)
Information for Human Gene Therapy Investigational
New Drug Applications (INDs)21 guidance (particularly
for genetically modified cell-based therapies). In addition
to providing guidance for sponsors, this guidance also
instructs FDA CMC reviewers about the information torecord and assess as part of an IND review, taking into
consideration the various manufacturing challenges forthese products. The recommended CMC information
covered in this guidance includes product manufacturing
and characterization information, product testing (including release and stability testing), product container
closure and labeling, as well as preclinical and clinicalstudies. Similarly, the EMA provides recommendationsin its draft Guideline on quality, nonclinical and clinicalrequirements for investigational advanced therapy medicinal products in clinical trials.
When filing an initial IND submission, details about
the following CMC information should be included: 1)

drug substance (DS); 2) drug product (DP); 3) placebo
formulation, if applicable; 4) labeling information for thelabeled products relevant to the investigational drug; and5) an environmental analysis for assessment of the effectsof the investigational new drug or biological product on
the environment (though many products qualify for an
exemption from this assessment). It should be noted that,in general, the amount of CMC information submitted
should be phase-appropriate, with more information
required to support later stages of development. Fora Phase I clinical trial, the primary focus of the CMC
information is on safety.
The contents of investigational new drug (IND)
application and clinical trial application (CTA) submission directly relating to CMC is to be submitted in
documentation structured according to the heading of
the corresponding sections of Module 3 of the eCTD. The
CMC content for the Module 3 will highly depend on thespecificities of the cell-based therapy product in terms of
the level of information provided. Table 2-4 and 2-5 summarize recommendations for the main Module 3 sections
for Drug Substance and Drug Product, respectively, on
how to provide sufficient CMC information required toassure safety, identity, quality, purity, and potency of theinvestigational product for IND/CTA submissions. The tables combine the recommendations from FDA and EMA.
Drug substance

Cells can be classified as being either drug substance(DS) or critical starting material, depending on whether
or not the cells undergo further modification. Unlike
in traditional biopharmaceuticals where the drug substance (DS) and drug product (DP) typically employ
separate manufacturing facilities, staff, and schedules
for manufacture, autologous cell-based therapies are
typically manufactured in a continuous process, where
there often are no intermediates or stopping points inthe manufacturing process and the same staff and facility
are used for the full process. Continuous manufacturingof an unmodified cell-based therapy product involves
expansion of therapeutic cells for treatment. Harvested
cells cannot maintain viability without storage in suitable
cryopreservation excipient(s). Therefore, harvested cells
are typically processed all the way through to formulation and filled into the final container. This is different

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼23

Table 2-3: Cell sources used in cell-based therapies
Autologous

Cells that are removed from a person, potentially expanded or manipulated, stored, and later given
back to that same person.

Allogeneic

Cells that are obtained from a healthy donor or tissue source, potentially expanded or manipulated,
stored, and administered to an unrelated person.

from processes used to produce classic chemical-based
pharmaceutical products. Most biologics that are active
pharmaceutical ingredients (APIs) are DSs and are stored
in the process container until materials are ready forformulation and filling into final containers.
For continuously manufactured cells, the DS sectionof the eCTD would describe the steps from harvesting
cells to just prior to formulation and filling.
For modified cells involving use of other critical components, each component can be controlled and released
as a DS, with a separate DS for each critical component(e.g., cells, plasmids, vectors, or gene editing tools. In
fact, the FDA considers, for example, a viral vector
carrying a CAR construct, as a DS). As an example,
in the case of leukapheresis-derived peripheral bloodmononuclear cells (PBMCs), cells are harvested prior toformulation and filling, but due to the continuous natureof the leukapheresis process, the DS is not actually stored.
Alternatively, these critical components could be described as starting materials in the appropriate sections,and their placement should be discussed with FDA at thepre-IND meeting.
Cells
Cells can be obtained from donors (allogeneic) or fromthe same patients for whom they are intended (autologous; Table 2-3). Cells can be simply expanded or canundergo further manufacturing by genetic modificationor selective culture conditions.
The FDA regulates establishments that manufacture
human cell and tissue products (HCT/Ps) to ensurecompliance with recommendations and requirements for
current Good Tissue Practices (CGTPs) as defined under
Title 21 Code of Federal Regulations, Part 1271 (21 CFRPart 1271, also referred to as the Food, Drug, and Cosmetic[FD&C] Act), Subpart D and requirements under Part
1271, Subpart E. Core CGTP requirements (§ 1271.150(b))
directly relate to preventing the introduction, transmission,or spread of communicable disease by HCT/Ps.

Establishments may request exemptions from CGTP
requirements such as Donor Eligibility from the FDA. The
request must be accompanied by all relevant valid scientific data and either information justifying the requested
exemption from the requirement or a description of a
proposed alternative method of meeting the requirement.
Requests should be submitted to the director of theappropriate FDA center. If the HCT/P is regulated solely under § 1271.10 as a 361 HCT/P, or as a biologicalproduct or a medical device regulated by CBER, requests
should be sent to the director of CBER. If the HCT/P isregulated as a medical device by the Center for Devices
and Radiological Health (CDRH), requests should besent to the director of CDRH. More information can befound at: https://www.fda.gov/vaccines-blood-biologics/
tissue-tissue-products/exemptions-and-alternatives
Establishments that manufacture human cells, tissues,and cellular and tissue-based products (HCT/Ps) regulated solely under section 361 of the Public Health Service(PHS) Act are required to register their establishmentswith the FDA and register and list their HCT/Ps within 5days of beginning operation. Registration must be updated annually in December. Establishments must amend
their HCT/P listing if a change described in §1271.25(c)(1) through (4) has occurred.
As of August 31, 2016, electronic submission of
HCT/P establishment and product listing informationis required under §1271.22, unless waived in certain
circumstances. HCT/P establishments that must registerand list electronically under 21 CFR Part 1271 should use
the electronic HCT/P establishment registration system(eHCTERS) to meet the requirement for electronic submission of establishment registration and product listing.
HLA matching
To establish histocompatibility for users of allogeneic-sourced HCT/Ps, human leukocyte antigen (HLA)
testing and confirmation may be required as part of the
product identity tests.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼24

Table 2-4: Summary of CMC information for Module 3 (Drug Substance)
Main Sections

Cell-Based Therapy Recommended Content

3.2.S.2.1
Manufacturers

Name, address, and responsibility of each manufacturer, including contractors, and each
proposed production site or facility involved in manufacturing and testing.

Description of the DS manufacturing process that represents the applicant’s commitment for the
3.2.S.2.2
manufacture of the DS. For developers of cell-based therapy, this includes the following:
Description of• Information to adequately describe the manufacturing process and process controls
Manufacturing
Process and Process • An explanation of the batch numbering system, including information regarding any pooling ofControls
harvests or intermediates and batch size or scale• A flow diagram (supplemented by written descriptions of each step) that illustrates the
manufacturing route from the original inoculum (e.g., cells contained in one or more vials of
the Working Cell Bank up to the last harvesting operation), with relevant information for all unit
operations and each stage, such as population doubling levels, cell concentration, volumes,
pH, cultivation times, holding times, temperature, culture media, equipment, process controls,
material transfer during process steps, and storage and shipping conditions• A flow diagram that illustrates the purification steps (i.e., unit operations) from the crude
harvests up to the step preceding filling of the DS including information about all steps andintermediates and relevant information for each stage (e.g., volumes, pH, critical processing
time, holding times, temperatures, elution profiles, selection of fraction, and storage ofintermediate)• Information about reprocessing procedures with criteria for reprocessing of any intermediateor the DS should be described, as given in 3.2.S.2.5• A description of the filling procedure for the DS, the container closure systems used for storageof the DS (details in 3.2.S.6.), and storage and shipping conditions for the drug.
3.2.S.2.3
Control of Materials

Quality and control of materials used in the manufacture of the DS (e.g., raw materials, starting
materials, solvents, reagents, catalysts); more information can be found in ICH Guidelines
Q6A and Q6B. For developers of cell-based therapy products, the following should also be
provided:• Summaries of viral safety information for biologically-sourced materials, including allogeneic
HCT/Ps (Details in 3.2.A.2)• Source, history, and generation of the cell substrate• Information on the source of the cell substrate and analysis of the expression constructused to genetically modify cells and incorporated in the initial cell clone used to develop the
Master Cell Bank, as described in Q5B and Q5D• Information about the cell banking system, quality control activities, and cell line stability
during production and storage.

3.2.S.2.4
Controls of
Critical Steps andIntermediates

Quality-related tests and acceptance criteria (with justification including experimental data)
performed at critical steps of the manufacturing process identified in 3.2.S.2.2, as well as
stability data supporting storage conditions (more information can be found in ICH Guidelines
Q6A, Q6B, and Q5C)

3.2.S.2.5
Process validation
and/or evaluation

Information about process validation and evaluation studies for aseptic processing andsterilization. Developers of cell-based therapy should also provide information on validationand evaluation studies to demonstrate that the manufacturing process (including reprocessing
steps) is suitable for its intended purpose and to substantiate selection of critical processcontrols and their limits for critical manufacturing steps (e.g., cell culture, harvesting,purification, and modification). The plan for conducting the study should be described andthe results, analysis, and conclusions from the executed studies should also be provided. For
manufacturing steps intended to remove or inactivate viral contaminants, the information from
evaluation studies should be provided in 3.2.A.2.

continued on next page

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼25

Table 2-4: Summary of CMC information for Module 3 (Drug Substance)
continued from previous page
Main Sections

Cell-Based Therapy Recommended Content

3.2.S.2.6
Manufacturing
Process
Development

Developmental history of the manufacturing process, as described in 3.2.S.2.2. The
significance of the changes made during the development process should be assessed byevaluating its potential to impact the quality of the DS. Reference should be made to the DSdata provided in section 3.2.S.4.4. More information can be found in ICH Guideline Q6B.

3.2.S.3
Characterization

Details about primary, secondary, and higher-order structure, post-translational forms,
biological activity, purity, and immunochemical properties (3.2.S.3.1 - Elucidation of Structure
and other Characteristics), as well as impurities (3.2.S.3.2). Further guidance on 3.2.S.3 can befound in ICH Guidelines Q3A, Q3C, Q5C, Q6A, and Q6B.

3.2.S.4
Control of Drug
Substance

Information about specification (3.2.S.4.1), analytical procedures (3.2.S.4.2), validation ofanalytical procedures (3.2.S.4.3), batch analyses (3.2.S.4.4), and justification of specification(3.2.S.4.5). Further guidance on 3.2.S.4 can be found in ICH Guidelines Q2A, Q3A, Q3C, Q6A,
and Q6B.

3.2.S.5
Reference
Standards orMaterials

Information on the reference standards or reference materials used for testing of the drug.
Further guidance on 3.2.S.5 can be found in ICH Guidelines Q6A and Q6B.

3.2.S.6
Container Closure
SystemDescription of the container closure systems, including the identity of materials of
construction of each primary packaging component, and their specifications.

3.2.S.7 Stability

Stability-related tests and protocols (3.2.S.7.1), information about post-approval stability
protocol and stability commitment (3.2.S.7.2), and overall stability data (3.2.S.7.3). Further
guidance on 3.2.S.7 in ICH Guidelines Q1A, Q1B, Q2A, Q2B, and Q5C.

The following need to be provided in marketing
applications for cord blood drug products:22• A description of the serologic and DNA-based
testing performed (serologic or DNA-based methodsfor HLA Class I (A and B) loci, and by DNA-based
methods for HLA Class II (DRB1) loci)• Sample types• The name and location of the CLIA certified andAmerican Society for Histocompatibility and
Immunogenetics accredited laboratory that performs
the initial and the confirmatory HLA typing of the
final product.
The recommended extent of HLA match between thedonor and recipient and the recommended cell therapy
product dose are important considerations for dosage andadministration specifications and, therefore, this information should be provided in the labeling for cord blood used
for reconstitution of the hematopoietic system.

Blood group (ABO) and Rh type must also be tested for and identified to meet CGTP requirements for
ensuring proper identification for these cord blood celltherapy products.
For all allogeneic cells or tissues (except those that meetthe exceptions in 21 CFR 1271.90(a)), in addition to thedonor screening and testing described above, addressing
other issues such as typing for polymorphisms and humanleukocyte antigen (HLA) matching, where appropriate,should be considered.23
Vector/Plasmid (for genetically modified cells)
Vectors are critical raw materials for the manufactureof genetically modified cell therapies as they represent
an important means of transferring and integratingtransgenes of interest into cells. For ex vivo cell therapies
such as CAR-T cell products, the vector is considereda DS. CAR-T therapies use lentiviral vectors (LVs) inthe manufacturing process, where third generation LVs
featuring a 4-plasmid system, comprised of 3 helper

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼26

plasmids24 and 1 self-inactivating gene transfer plasmid
are most commonly used. To that end, a description of the
LV manufacturing process and process controls must be
included in Module 3 of the eCTD. All components usedduring LV production should be sufficiently described in
the manufacturing summary included in an IND application, more specifically in the control of materials sectionof the DS section. This includes detailed descriptions of
the plasmid construct generation and the DNA sequenceof the entire plasmid(s).
Characterization studies will provide a comprehensive
picture and knowledge of the vector, including annotated sequence analysis for the vector in the original IND
submission and any additional sequence information
gathered during the course of product development in
subsequent submissions. Tests should be included toshow integrity and homogeneity of the recombinant viralgenome or plasmid and the genetic stability of the vector
and therapeutic sequence.
Detailed discussion on the manufacturing process andrecommended testing for LVs are discussed in Chapter 7of this document, and recommended testing for plasmids
used in vector manufacturing is covered in A-Gene chapter 5.1 We can expect these standards to continue evolving
as products are developed focused on in vivo modification.
Other gene editing techniques utilized to engineer
cell therapy products (e.g., CRISPR) may utilize recombinant proteins (e.g., Cas9), viral vectors (e.g., AAV or
LV), mRNA, or plasmids,25 each of which may need to
be described in its own DS section. Developers should
utilize the same principles outlined in A-Gene to assess
manufacturing controls needed to ensure the identity,
safety, purity, and stability of such gene editing agents.
Drug productFor cellular therapies, due to the nature of continuous
manufacturing, it is important to align with the FDA
the dividing line between DS and DP, and appropriate
placement in the Module 3 sections should be agreed
upon with the FDA in the pre-IND meeting.
Drug labeling
Product labels for autologous products should be madeon the primary container and secondary container (ifapplicable). Labeling information for an autologous

product should include patient ID (without compromising patient privacy in accordance with local/nationallaws), dose, cell density, volume, date of manufacturing,
expiry, storage condition, and manufacturer. Unique
Patient IDs are critical to ensure that autologous products administered to patients are traceable. Details on
required product labeling for autologous products, where
donor-eligibility determination or donor screening/
testing is not required, are presented in 21 CFR 1271.90.
Allogeneic product labelling should include lot andproduct ID to ensure product traceability to the donor,
and may also include information regarding HLAmatching from donor to recipient, as appropriate. For
investigational new drugs intended for human use, theimmediate package should be labeled “Caution: New
Drug, Limited by Federal Law to Investigation Use” asstated in 21 CFR 312.6(a).
Product Characterization
Robust product characterization is needed to demonstrate safety, identity, purity/impurity, quantity/strength,
and potency of the final cell products. The FDA regulates
the testing requirements for General Biological ProductsStandards in Title 21 of the CFR Part 610, which also
applies to cell therapy products. In addition, recommendations on potency assay measurements, design,and validation are presented in the FDA Guidance
Potency Tests for Cellular and Gene Therapy Products.26
For detailed discussion on the required product testing
and analytical assay development for characterization ofcell-based therapies, refer to Chapter 9. For autologous
cell therapy products, the starting cells from patients areoften heterogeneous. Sponsors should conduct productcharacterization as early as possible and throughout thedevelopment cycle in order better address variability, understand CQA and set phase appropriate specifications.
ATMPs and Medical Device Combinations

The combination of ATMPs with medical devices may
give rise to different regulatory scenarios from the perspective of the EMA. When a cell-based investigational
medicinal product (CBIMP) incorporates a medical
device as an integral part of the active substance, themedical device will be considered a starting material.
When an ATMP necessitates a medical device as part ofChapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼27

Table 2-5: Summary of CMC information for Module 3 (Drug Product)
Main Sections

Cell-Based Therapy Recommended Content

3.2.P.1
Drug Product
Description andComposition

Description of the DP and its composition (3.2.P.1). Further guidance can be found in ICH
Guidelines Q6A and Q6B).

3.2.P.2
Pharmaceutical
Development

Information about the development studies conducted to establish that the dosage
form, the formulation, manufacturing process, container closure system, microbiologicalattributes, and usage instructions, are appropriate for the purpose specified in theapplication. Further guidance on 3.2.P.2 can be found in ICH Guidelines Q6A and Q6B.

3.2.P.3
Manufacture

Information about the manufacturing of the DP. This includes the name, address, andresponsibility of each manufacturer, including contractors, and each proposed production
site or facility involved in manufacturing and testing (3.2.P.3.1), batch formula (3.2.P.3.2),
a description of manufacturing process and process controls in both flow chart andnarrative form (3.2.P.3.3), controls of critical steps identified in 3.2.P.3.3 and intermediates(3.2.P.3.4), and process validation and evaluation. Viral safety evaluation should be
provided in 3.2.A.2, if necessary. Further guidance on 3.2.P.3 can be found in ICH
Guidelines Q2A, Q2B, Q6A, and Q6B.

3.2.P.4
Control of Excipients

Information about the testing of excipients involved with the DP. For compendial
excipients, simply referencing the appropriate compendium(a) for each excipient issufficient. For novel excipients, additional information is expected, including specifications(3.2.P.4.1), analytical procedures (3.2.P.4.2), validation of analytical procedures (3.2.P.4.3)
and justification of specifications (3.2.P.4.4). If applicable, descriptions for any excipientsof human or animal origin (3.2.P.4.5) and novel excipients (3.2.P.4.6) should be provided
in the relevant sections. Further guidance on 3.2.P.4 can be found in ICH Guidelines Q2A,
Q2B, Q3C, Q5A, Q5C, Q5D, Q6B.

3.2.P.5
Control of Drug
Product

Information about the testing of the DP during manufacturing. This information includes
specification(s) (3.2.P.5.1), analytical procedures (3.2.P.5.2), validation of analyticalprocedures (3.2.P.5.3), batch analyses (3.2.P.5.4), characterization of impurities (3.2.P.5.5),
and justification of specification(s) (3.2.P.5.6). Further guidance on 3.2.P.5 can be found in
the ICH Guidelines Q2A, Q2B, Q3B, Q3C, Q5C, Q6A, and Q6B.

3.2.P.6
Reference Standards
or Materials

Information on the reference standards or reference materials used for testing of the DPshould be provided, if not previously provided in 3.2.S.5. Further guidance on 3.2.P.6 canbe found in ICH Guidelines Q6A and Q6B.

3.2.P.7
Container Closure
SystemDescription of the container closure systems, including the identity of materials of
construction of each primary packaging component and its specification.

3.2.P.8
Stability

Stability-related tests and protocols (3.2.P.8.1), information about post-approval stability
protocol and stability commitment (3.2.P.8.2), and overall stability data (3.2.P.8.3). Further
guidance on 3.2.S.7 in ICH Guidelines Q1A, Q1B, Q2A, Q2B, Q3B, Q5C, and Q6A.

3.2.A.1
Facilities andEquipment*

A summary of facility information, including a diagram of the manufacturing flow,
movement of raw materials, personnel, waste, and intermediates, description of productcontact equipment and its use, information on products manufactured or manipulated
in the same areas as the applicant’s product, and cleaning, sterilization, and storage ofequipment and materials.

3.2.A.2
Adventitious Agents
Safety Evaluation

Information assessing the risk of potential contamination with adventitious agents,
including avoidance and control of nonviral adventitious agents, detailed information fromviral safety evaluation studies, and assessment of viral clearance. Further guidance can befound in ICH Guidelines Q5A, Q5D, and Q6B.

*Section 3.2.A.1 is typically submitted for US INDs, but is not required for EU IMPDs.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼28

the final formulation, but the medical device is not an
integral part of the active substance, the medical device
will be considered an excipient. When the medical device
is used as the container closure system or is intended
to administer an ATMP and the administration device
and the ATMP are marketed as a single integral productand the device is not reusable, the combination will be
regulated under the medicines framework.

IND Submission to the FDA
Sponsors who wish to conduct a clinical trial with aninvestigational drug in the United States must, by law, do
so under an investigational new drug (IND) submission,
unless exempted under 21 CFR 312.2(b), 21 CFR 312.2(c),or 320.31(d), or unless a waiver is obtained under 21 CFR
312.10.17. This requirement is intended to ensure the safety of the subjects (patients and healthy volunteers) and toensure the adequacy of trials so that marketing/licensing
applications contain well-designed clinical investigations
that can support safety and efficacy. The sponsor is responsible for the nature and quality of all submissions in both
INDs and marketing/licensing applications. The FDA roleis carried out through reviews of submissions, responses
to questions posed by a sponsor, and meetings for further
discussion and communication. The FDA may also conduct inspections of sites and operations associated withan IND, including targeted inspections of select clinicalmanufacturing and clinical trial sites.
The contents of an IND and the scope of the FDA
regulatory authority with respect to an IND are described
extensively in 21 CFR 312.1 through 312.42. Because
sponsors may proceed with the submitted clinical trial
if they are not informed otherwise by the FDA within
the specified time period, the FDA must complete theinitial safety reviews and make decisions on whether the
proposed trial is safe by 30 calendar days after receipt of
the submission.
An IND is administratively assigned to the review
division with expertise in the relevant therapeutic area.
Since review of a new IND focuses primarily on safety,
FDA review divisions hold an internal IND safety meeting timed so that the division may provide requests toand receive responses from the sponsor for relatively
minor technical issues that, if not brought to attention,
might otherwise lead to a clinical hold.

The information submitted in a new IND mustensure safety for the proposed clinical trial before thesponsor can be allowed to proceed. If a clinical trial is notconsidered safe to proceed, it will be placed on clinicalhold, with appropriate notification to the sponsor of thereason for the hold and the information required to lift
the hold. Procedures for instituting a clinical hold andfor reviewing and acting on a response to a clinical holdare described in 21 CFR 312.1 through 312.42.
In certain situations, FDA review staff may recommend submission of a pre-IND meeting request to discuss
the sponsor’s development program. For example, sponsors submitting a new IND with clinical data obtained
from extensive development outside the United States anda later phase clinical trial protocol would benefit from a
pre-IND meeting. Similarly, it would be valuable to gain
FDA agreement via a pre-IND meeting prior to submission of a new IND with a complex clinical trial intended
to support a new indication for a marketed drug in a newpatient population where the risks and benefits of therapy
differ from the approved indication. Agreements madeduring the pre-IND meeting ensure that the sponsor andthe FDA are aligned on the scope of existing data that may
be used to support the application and the proposed later
phase development trials.
If a sponsor submits an IND without having previously
conducted a pre-IND meeting or if the IND contains
complex submission materials, FDA review staff are stillrequired to complete the safety review in 30 calendar days.
Given the complexity of cell-based products in general,
it is recommended to conduct a pre-IND meeting prior
to submitting an IND to mitigate risks of clinical hold.
With respect to manufacturing, the review of an IND
concentrates on determining if there are any reasons tobelieve the manufacturing or controls for the clinical trial
product present unreasonable health risks to the subjects
in the initial IND trials; as always, safety is the priority.
Such risks could arise in cases including (but not limited to):• A product made with unknown or impure components• A product possessing cellular phenotypes of known
or potential toxicity• A product with an impurity profile indicative of apotential health hazard or an impurity profile insufficiently defined to assess a potential health hazard• A poorly characterized master or working cell bank

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼29

If the FDA identifies too many unresolved CMCrelated safety issues in the IND, or if the FDA identifies
such issues arising during development, the FDA will
issue a clinical hold on the application. The FDA also
acknowledges there can be specific challenges for applications that have received (or are likely to receive) expedited
designations. These challenges include possible difficulties
in aligning CMC and clinical development and possible
difficulties in making risk/benefit assessments (with particular regard to patient benefit) in situations in which
there may be a relative paucity of CMC information.
Additional factors that can have an impact on the IND
submission process include CGMP compliance, and manufacturers’ licensing/registration. Upon receipt of a request
by an establishment physically located in the United Statesthat has been included as part of a marketing application
submitted to a foreign regulator, the FDA will issue a letterto an identified foreign regulator conveying the current
CGMP compliance status for the establishment. The FDA
will issue the response letter within 30 days of receipt of
the request. A CGMP declaration should only be requested if a foreign regulator does not accept a valid certificate
of pharmaceutical product (CPP) and wants additional
assurance of a facility’s CGMP status. Foreign regulators
can also find the CGMP status of an establishment bychecking the inspection classifications database for themost recent inspection classification.
Master FileIn an IND submission, sponsors can reference Master
Files (MFs) to provide information for products used in
manufacturing that the company does not own, such as
ancillary materials, packaging materials, or excipients.
Examples of components that can be covered by a Master
File include media, media components, cell banks, viral
vector platform, and manufacturing-enabling equipment.
While not required by statute or regulation, MFs are submitted directly to FDA by the manufacturers of said materials (the MF holder), which provide confidential, detailed
information about the facilities, processes, or articles
used in the manufacturing, processing, packaging, andstoring of these products.27 If the MF holder authorizes its
incorporation in writing, MFs allow sponsors to referencematerial without the MF holder having to disclose thecontents to the sponsors.28 MFs are neither approved nor

disapproved; instead, FDA reviews the technical contentsof MFs in connection with the review of the INDs thatreference them. The FDA can find an MF deficient, which
can have an impact on an IND that references it, including
the IND going on clinical hold. In Europe, MFs cannot
be used for biologic DS components but there may be
NCA-specific alternatives on a case-by-case basis.

Clinical Trial Application (CTA) in the European
Economic Area (EEA)
The regulatory environment for the conduct of clinical
trials in the EEA is currently evolving with the entry into
force, on 31 January 2022 of the new EU Clinical Trial
Regulation (Regulation (EU) N° 536/2014), repealing the
EU Clinical Trials Directive N°2001/20/EC.
The Clinical Trial Regulation is aimed at facilitating
clinical trials across the European Union by streamlining
the application procedure via a single-entry point andharmonizing the procedure for assessment of clinical trial
applications. The evaluation, authorization, and supervisionof clinical trials are the responsibilities of EU Member Statesand European Economic Area (EEA) countries.
Prior to the regulation, clinical trial sponsors had tosubmit clinical trial applications separately to national
competent authorities and ethics committees in each country to gain regulatory approval to run a clinical trial. The
Regulation now enables sponsors to submit one online application via a single online platform, known as the Clinical
Trials Information System (CTIS), for approval to run
a clinical trial in several European countries, making it more
efficient to carry out such multinational trials.
The CTIS system is centrally administrated by the EMA
and also allows transparency and access to information forany party interested in clinical trials conducted in the EEA
through a searchable public website.
A transition period applies to clinical trial submission
under the Regulation:• until 30 January 2023, clinical trial sponsors may use
CTIS to apply to run a clinical trial under the Clinical
Trials Regulation or may choose to apply to run a trial
under the Clinical Trials Directive;• from 31 January 2023, clinical trial sponsors will need to use
CTIS to apply to start a new clinical trial in the EU/EEA;• from 31 January 2025, any trials approved under theClinical Trials Directive that continue running will

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼30

need to comply with the Clinical Trials Regulation andtheir sponsors must have recorded information onthem in CTIS.
The content of the CTA under the CTR is described in
Annex 1 of the EU Clinical Trial Regulation (Regulation(EU) N° 536/2014) and is mainly composed of 2 parts:
Part I contains scientific and medicinal product documentation; Part II contains the national and patient-level
documentation.
Parts I and II will be assessed in parallel by the countries unless the sponsor has decided to submit only Part
I for assessment, and later submit Part II (within two
years, otherwise the Part I application in that Member
State will lapse).
Timelines for approval for a CTA submitted under the
CTR can vary from 60 days up to 105 days (+ 50 days for
ATMPs or biologics) versus 60 days under the Clinical Trial
Directive 2001/20/EC (which allows a delay of maximum
90 days for the review of clinical trial applications for
ATMPs), with a possible extension by a further 90 days.
Investigational Medicinal Product Documentation (IMPD)

The central document required for a CTA is the investigational medicinal product dossier (IMPD), which isincluded in Part I of a CTA (submitted through the CTR)
and is comprised of relevant information on quality,
preclinical studies, reference clinical studies, criticalanalyses of the nonclinical and clinical data related tothe potential risks and benefits of the proposed study,
any available previously generated human data, and anassessment of the overall risk/benefit. These data arepresented according to the heading structure of theeCTD, with the amount of information contained in eachsection depending on various factors such as producttype, indication, and development phase. Advanced therapy medicinal products (ATMPs) have additional datarequirements. It should be noted that CMC informationin the IMPD is subject to specifications not only issued
by the EMA, but also European Pharmacopoeia (Ph Eur)monographs and European Directorate of the Quality of
Medicines (EDQM) standard terms database.
The data on quality aspects of Investigational
Medicinal Products should be presented according
to the specified structure found in common technicaldocument (CTD) Module 3. Data requirements are
known to evolve as development progresses from exploratory to confirmatory clinical trials. As such, quality
data compiled in the IMPD are to reflect increasing
knowledge and experience during product development.
For the sake of the eventual marketing authorization, it
needs to be demonstrated that the medicinal product canbe produced consistently and with reproducible quality.
For example, acceptance criteria for test parameters/
in-process controls should be reviewed at later stagesof development. Also, the addition or removal of parameters and modification of analytical methods may
be necessary. In all cases, the suitability of the analyticalmethods used should be demonstrated. As trials progress
toward marketing authorizations, confirmatory clinical
trials for the product based on a mature manufacturing
process and specifications are expected.
The following Guidelines provide further informationon the content of the IMPDs for chemical, biological,
and ATMP IMPs:• Guideline on the requirements to the chemical andpharmaceutical quality documentation concerninginvestigational medicinal products in clinical trials• Guideline on the requirements for quality documentation concerning biological investigational
medicinal products in clinical trials• Guideline on quality, nonclinical and clinicalrequirements for investigation of advanced therapy
medicinal products in clinical trials
Cell-based investigational medicinal products(CBIMPs) often contain or consist of cell preparationsof limited size and many are intended to be used in apatient-specific manner. The traceability from the recipient of the product to the donor of the cells or tissues
should be ensured. The traceability system should bebidirectional (from donor to recipient and from recipient to donor). Data should be kept for 30 years after the
expiry date of the product unless a longer time period isrequired in the clinical trial authorization.
Specific case of GMOs

The Clinical Trial Regulation does not address local
GMO specific requirements, which have to be met
individually for each country in addition to the CTR.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼31

To facilitate this purpose, a Questions and Answers document has been published on the European Commission
website29 that describes the interplay of clinical trial
application and GMO application in the current situation, but also with a view on the new Clinical Trial
regulation No 536/2014. A prior authorization under the
GMO framework can no longer be a prerequisite for theclinical trial approval, and a CTA following Regulation(EU) No 536/2014 cannot be rejected due to lack of
GMO approval. However, once CTA approval has beenobtained, the GMO legislation still has to be respected,
so that a clinical trial cannot start until GMO approvalhas been issued on a country level.
Qualified persons and batch release

Each manufacturing site of ATMPs in the European
Economic Area (EEA) must have at least one Qualified
Person (QP). Two or more sites may have the same QP
if the QP can provide their services to each of the sites
in a continuous fashion. The QP’s main responsibility is
to verify and certify that each batch produced in the EU
has been manufactured and checked in accordance withrequirements for the marketing authorization/clinical
trial authorization, as well as relevant regulations governing the manufacture of medicinal products, including
GMP. If the product is being exported, the QP must also
verify that requirements are met for relevant productspecifications in the destination country.
Further information about QPs and batch release is provided in the ATMP GMP guideline
released by the European Commission: https://
ec.europa.eu/health/sites/health/files/files/eudralex/
vol-4/2017_11_22_guidelines_gmp_for_atmps.pdf.
GMP certification in the EU

Manufacturers must comply with EU GMP to obtain amanufacturing authorization. They can ensure that theymeet all their legal obligations by following the EU GMP
guidelines. In the case of developers of ATMPs, there are
specific guidelines for ATMP GMP. Importers of materials into the EU are responsible for ensuring that thethird-country exporter is in compliance with EU GMP.
Manufacturers of active substances intended for the
manufacture of human medicines for the EU market mustregister with the NCA of the Member State in which the
manufacturing site is located. Manufacturers of finishedproducts must also ensure that the active substances usedhave been manufactured in compliance with GMP. Importersof active substances intended for the EU market are also
required to register. In addition, the competent authority of
the country in which the consignment is produced needs toconfirm that the consignment conforms to GMP standards
equivalent to those in the EU, unless a waiver applies.
In the EU, NCAs are responsible for inspecting
manufacturing sites located within their own borders.
Manufacturing sites outside the EU are inspected by theNCA of the MS where the EU importer is located, unless amutual recognition agreement (MRA) is in place between
the EU and the country concerned. If an MRA applies, theauthorities mutually rely on each other’s inspections andthe requirement for batch testing of products on entry into
their territories may be waived. If products are imported
directly into more than one Member State from a manufacturing site outside the EU, there may be more than one
NCA responsible for inspecting the site. The EMA facilitates
cooperation between the involved authorities. Inspections
are carried out according to a risk-based approach and areespecially important if there is suspicion of non-compliance. After inspecting a manufacturing site, EU competentauthorities issue a GMP certificate or a non-compliancestatement, which is entered in the EudraGMDP database.
For products derived from blood or blood plasma, the
EMA is responsible for coordinating inspections of theestablishments in which collection, testing, processing, storage, and distribution is carried out under the plasma master
file (PMF) certification procedure. More information on
the PMF certification procedure can be found at: https://www.ema.europa.eu/en/human-regulatory/overview/plasma-master-file-pmf-certification.

Differences between IND and CTA
INDs and CTAs are composed of overlapping yet
non-identical components. The IND consists of several
forms specific to the FDA, all nonclinical study reports(including validation reports of bioanalytical methods),
nonclinical summaries, CMC information, the clinicalprotocol, and the IB. Once an IND has been cleared by the
FDA, multiple studies can be conducted under the same
IND, as per the FDA legal requirements as described in
21 CFR 312.22. These studies usually must use the sameinvestigational drug and be used in patients with the same

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼32

disease (i.e., the same indication), but there are recent
Guidances that speak to multiple versions of a productand those involving umbrella or basket trials that can also
be discussed with FDA. For a CTA, the main documents
are the protocol, informed consent form, IB, and IMPD,
as well as several EU-specific and ethics committee-related documents; the range of nonclinical data requiredin an IND is not submitted. However, there are specific
GMP-related documents that are required for a CTA
that are not required for an IND. Additionally, in the EU,
each interventional clinical study requires a new CTA, in
contrast to a cleared IND.
INDs have set clearance timelines of 30 days, whereas
CTAs have variable timelines ranging from 60 to 106
days, depending upon whether the applicant is asked
to provide additional information through Requests
for Information (RFIs). If an applicant fails to respondto RFIs within the deadline, the application will lapse;
therefore, it can be said that deadlines are stricter for a
CTA. CTAs are not subject to clinical holds like INDs;
the CTA is either approved (perhaps with mandatorychanges) or rejected. However, in certain circumstances
CTA approval could be revoked by the country.
An IND Sponsor can amend an IND with changes to aclinical protocol, but FDA is not obligated to respond on a
PDUFA time frame. However, with CTAs each substantial
protocol amendment requires CTA approval.
Cell-based therapy product CMC information for
IND versus CTA

In the EU, CTAs are in the remit of NCAs even for
ATMPs that will later be approved under the centralized procedure. In addition, since there is a regulatory
framework that includes regulations and directives, withthe latter being recommendations to be implemented bynational law, CTAs are evaluated using criteria derived
from a wider range of regulatory bodies and legal source
documents than is the case for FDA-reviewed INDs.
Consequently, there can be challenges when fillingin CMC information for the respective applications.
Though the content follows the same eCTD structural
guidelines in both IND applications and CTAs, CMC
information in an IMPD, in general, has different (and insome cases more) specifications than CMC informationin an initial IND (pre-Phase I). In particular, submission

contents of an IND often fail to meet or lack data withrespect to requirements and recommendations that aredelineated in applicable EMA guidelines and European
Pharmacopoeia (Ph Eur) monographs. Thus, applicants
based in the United States can be relatively prone to receiving grounds for non-acceptance upon review of the
CTA, even if their initial IND applications do not resultin a clinical hold (especially taking into account agency
response deadlines), and can, subsequently, be forced towithdraw the CTA or receive a rejection.
For example, for 3.2.P.3.4 (Control of critical steps andintermediates), US-based sponsors may fail to take into
account the acceptance criterion for acceptable bioburden prior to sterilization by filtration that is in line with
EU requirements. If submitted in an IMPD, this might
be questioned by EU regulatory authorities because theacceptance criterion will not match expectations.

Submission of amendments
Changes in the manufacturing of cell therapy products
can be categorized as minor, moderate, or major changes.
Depending on the potential impact of the change on the
product quality, amendments to an IND might be required.
FDA

After an IND is opened, a number of supporting documents may be submitted to the IND during its life
cycle. The types of IND submissions and amendments
are separated into four categories: submissions with a
specific regulatory-mandated timeline; safety-related
submissions; product development submissions without regulatory-mandated timelines where communication to the sponsor is often critical and recommended;and other submissions that may overlap any of thepreceding three categories and where communication
with the sponsor may be needed (e.g., general correspondence, final reports, annual reports, drug quality
amendments). In general, the FDA considers review ofsafety-related amendments to be the top priority. Other
high-priority amendment submissions are those that apply to new Phase II and Phase III adaptive trial designs,
new Phase III protocols, and post-marketing requirement protocols. Lastly, IND amendments for products
granted breakthrough therapy designation are treatedwith high priority as well, with intense involvement

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼33

of senior managers and experienced review staff in aproactive collaborative, cross-disciplinary review. It
should be noted that the administrative Form FDA 1571
document should accompany most IND submissions
to indicate the content and purpose of the submission
and that all IND submissions should include summaries
that allow FDA staff to understand the regulatory anddevelopmental context of the submissions.
CMC-related changes are classified as information
amendments (as opposed to protocol amendments),
which are reported by the developers, routed to therelevant FDA staff, and assigned to reviewers as needed. Typical amendments include minor changes inmanufacturing process, batch size change or updated
batch data, and new labeling. The most importantamendments are those that could possibly affect thesafety of the product. These include (but are not limitedto): 1) changes in the container closure system affecting
product quality; 2) changes resulting in different impurity profiles; and 3) changes from synthetic to biologicalsources (human or animal) of a drug substance.
Information amendments to an IND are submitted through the same electronic portal as forthe original IND, the Electronic Submissions
Gateway (ESG): http://www.fda.gov/ForIndustry/
ElectronicSubmissionsGateway/default.htm.
CTA substantial amendments (EEA)

Under the new CTR, substantial amendments to a CTA andaccompanying documentation must be submitted for approval to the concerned countries via CTIS. Modifications
to a trial are regarded as ‘substantial’ when they are likely
to have a significant impact on the safety or rights of thesubjects and/or the reliability and robustness of the data
generated in the clinical trial.
It is, in principle, the responsibility of the sponsor toassess whether a modification is to be regarded as ‘substantial’. This assessment is to be made on a case-by-case basis
in view of the above criteria. In case of doubt, sponsors areencouraged to contact the relevant countries.
The sponsor should also assess whether a substantial
modification (or the combination of a number of substantial modifications) leads to changes in the clinical trial toan extent that it has to be considered as a completely newclinical trial, which would require an application for a newtrial authorization. For example, unplanned introductionof a new IMP, a change of the main objective, primary
endpoint of the clinical trial in all phases, or an unplanned
and unjustified addition of a trial arm or placebo group are
considered as resulting in a new clinical trial and would
therefore require a new trial authorization.
Information about changes in the CMC or quality aspects of the IMPD, can be found in the EMA:
Guideline on quality, nonclinical and clinical requirements for investigational advanced therapy medicinal
products in clinical trials.
The following changes to the manufacturing processof the medicinal product have a high likelihood of being
substantial:• Major changes to the product formulation• Changes to storage conditions• Changes to test procedures of the DS or DP• Changes to test procedures of non-pharmacopoeial
excipients• Changes to the manufacturing process of the DS or DP• Changes to the release or shelf-life specifications of the
DS or DP (widening or deletion of acceptance criteria,
addition of specification for safety/quality concerns)• Changes to the immediate packaging material
Once the application is validated and assessed, awritten notice is sent to the applicant within 49 to 95
days (99 to 145 days for ATMPs) after receipt.

Requests for Out of Specification Exemptions
In some cases, a cell therapy product does not meetthe quality requirements as specified in the regulatory
submission (whether IND or CTA). In such a case, theproduct is considered to be out of specification (OOS)
and is ordinarily not eligible for delivery to patients inthe clinical trial. For example, a given instance of a celltherapy product may contain fewer cells than is specified
in the CMC section of the regulatory submission and is,therefore, OOS. However, OOS products can, in selectcases, still be delivered if a positive benefit/risk assessment is made with respect to the patient’s status. The
process for requesting OOS exemptions differs between
regulatory agencies and is described below for the FDA
and the EMA.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL

✼

34

FDA

The process for requesting OOS exemptions under
FDA regulatory jurisdiction is to email the RPM/CMC
reviewer with a description of the OOS and the status of
the patient and request a teleconference for discussion.
The purpose of the teleconference is to arrive at a conclusive risk/benefit assessment with respect to safety forthe patient. The PM/CMC reviewer will follow-up with
an OOS-granted or OOS-rejected email. All correspondence regarding the OOS is then submitted as an IND
CMC information amendment.
EEA

In EEA, there is no common rule for OOS exemption
requests and this aspect is not regulated under the CTR.
Instead, the sponsor, physician, and individual countries
are invited to communicate when delivery of an OOS
product is required. After a request from the treating
physician is received, the manufacturer should provide
the treating physician with its evaluation of the risks andnotify the physician that the OOS product is being supplied
to the physician at their request. Subsequently, the sponsor
should inform the relevant national competent authority.
As an example, in the Netherlands, the process forreporting to the relevant competent authority is to send a
notification of an OOS ATMP to the Central Committee
for Research Involving Human Subjects (CCMO), an
office within the Medicines Evaluations Board (MEB),
by e-mail to tc@ccmo.nl within 48 hours after delivery of
the product, with the subject stating ‘Notification of OOS
ATMP’ and the relevant file number (NL number). The
notification to the CCMO must consist of the following:a description of the OOS with a risk analysis that takes
into account impact on product quality, safety, and efficacy; a statement from the investigating physician; anda statement from the sponsor as to whether or not the
product has been administered.
Further details can be found in:
https://www.ccmo.nl/onderzoekers/
standaardonderzoeksdossier/d-productinformatieLife Cycle Management
Both the FDA and the EMA adhere to life cycle management principles defined by ICH. The ICH Quality
Guidelines ICH Q8, Q9, Q10, and Q11 define anddiscuss concepts important for the assessment of CMC
changes across the product life cycle. These documents
detail a harmonized approach to technical and regulatory
considerations for life cycle management with the aim of
benefiting patients, industry, and regulatory authorities
by promoting innovation and continual improvementin the biopharmaceutical sector, strengthening quality
assurance, and improving supply of medicinal products.
Additional goals include the facilitation of the management of post-approval CMC changes in a more predictable and efficient manner and the demonstration of how
increased product and process knowledge can contributeto a reduction in the number of regulatory submissions.
The latest ICH Quality Guideline, ICH Q12, addresses thecommercial phase of the product life cycle (as describedin ICH Q10). In addition, in line with prior ICH Quality
Guidelines, ICH Q12 provides a framework to facilitate
the management of post-approval CMC changes in amore predictable and efficient manner and is intended
to demonstrate how increased product and process
knowledge can contribute to a reduction in the numberof regulatory submissions. In certain ICH regions, thecurrent ICH Q12 guideline is not fully compatible withthe established legal framework with regard to the use of
explicit established conditions (ECs) and with the productlife cycle management (PLCM) concept. These concepts
will, however, be considered when the legal frameworks
are reviewed and, in the interim, to the extent possible
under the existing regulation in these ICH regions.
The concept of ECs provides a clear understanding
between the MAH and regulatory authorities regarding
the necessary elements to assure product quality andidentify the elements that require a regulatory submission,
if changed.
The Post-Approval Change Management Protocol(PACMP) is a regulatory tool that provides predictability
regarding the information required to support a CMC
change and the type of regulatory submission basedon prior agreement between the MAH and regulatory
authority. Such a mechanism enables planning and implementation of future changes to ECs in an efficient andpredictable manner.
The PLCM document serves as a central repository for
the ECs and the associated reporting category for changes
made to ECs. The document also captures how a product

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼35

will be managed during the commercial phase of the life
cycle including relevant post-approval CMC commitments
and PACMPs.
An effective Pharmaceutical Quality System (PQS) as
described in ICH Q10 and compliance with regional GMPs
are necessary for implementation of ICH Q12. In particular,
management of manufacturing changes across the supply
chain is an essential part of an effective change management
system. ICH Q12 provides recommendations for robustchange management across multiple entities involved in
the manufacture of a pharmaceutical product. It outlines
the complementary roles of regulatory assessment andinspection, and how communication between assessors andinspectors facilitates the use of the tools included herein.
ICH Q12 outlines approaches to facilitate changes tomarketed products and provides detailed guidance to enablechanges to analytical methods to be made with immediateor other post-implementation notification. ICH Q12 also
describes science- and risk-based approaches for stability
studies in support of the evaluation of CMC changes.
In summary, the tools and concepts covered by ICH
Q12 are complementary and are intended to link together
different phases of the product life cycle. Pharmaceuticaldevelopment activities result in an appropriate control
strategy, the elements of which are considered to be ECs.
All changes to an approved product are managed througha firm’s PQS; changes to ECs must also be reported to theregulatory authority. Where the regulatory system provides
for categorization of post-approval CMC changes for reporting according to risk, the MAH may propose reporting
categories for changes to ECs based on risk and knowledge
gained through enhanced pharmaceutical development. A
system with risk-based reporting categories also facilitates
the use of post-approval change management protocols,
which provide predictability regarding planning for futurechanges to ECs. The PLM document is a summary thattransparently conveys to the regulatory authority how the
MAH plans to manage post-approval CMC changes.
DESIGNATIONS TO EXPEDITE DEVELOPMENTThe United States, European Union, and Japan haveestablished expedited pathways to support accelerated
development and regulatory approval for medicinal
products that have the potential to address unmet
medical needs. These pathways provide opportunities

for developers to engage with regulators during the development process and participate in accelerated review
programs within each agency.

USRegenerative medicine therapies to treat, modify, reverse,
or cure serious conditions may be eligible for several
FDA expedited programs.30 The FDA has developed five
expedited pathways that are relevant for cell-based therapies: Fast Track (FT) designation, Breakthrough Therapy
designation (BTD), Regenerative Medicine Advanced
Therapy (RMAT) designation, priority review designation,
and accelerated approval. Cell-based therapy products,
including those that received FT, BTD, or RMAT designation, may also be eligible for priority review designation
and accelerated approval. Sponsors may apply for andreceive more than one designation for a given product,but sponsors should apply for each designation separately.
Information that supports more than one designation may
be submitted in each separate designation request.
Manufacturing and product quality are importantfactors to consider when filing requests for expedited
programs. The sponsor of a product that receives an expedited drug development designation should be preparedto pursue a more rapid manufacturing development program to accommodate the accelerated pace of the clinicalprogram. The Type B multidisciplinary meeting (e.g., after
an IND sponsor is granted a BTD or RMAT) is an opportunity to discuss this with the FDA. If sponsors receive an
expedited designation, they should be prepared to propose
a commercial manufacturing program that will ensure
availability of quality product at the time of approval. The
proposal should consider estimated market demand, thecommercial manufacturing development plan, manufacturing facilities, and a life cycle approach to process validation.
Additionally, the proposal should include a timeline fordevelopment of the manufacturing capabilities with goalsaligned with the clinical development program. Sponsors
of such products should allow for an earlier submission of
the CMC section (including product quality information)
for timely review, and, critically, for inspection activities.
Coordination with the sponsor and contract manufacturers
may be necessary to ensure that manufacturing facilities
and equipment are ready for inspection during review of
the clinical section of the application.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼36

Requests for expedited programs may be indicatedin the relevant submission. The Office of Tissues andAdvanced Therapies (OTAT) will notify the sponsor nolater than 60 calendar days after receipt of the designation
request as to whether the requested designation has been
granted. If OTAT determines that the designation request
was incomplete or that the drug development program
does not meet the criteria designation, OTAT will include
a written description of the rationale for such determination. If a designation has been granted but, later indevelopment, the product no longer meets the qualifying
criteria, then CBER may rescind the designation.
The criteria and key features of each expedited pathway is summarized in Table 2-6 below. Further guidanceon accelerated pathways, including the specifications
for requests for each designation, can be found in the
Expedited Programs for Regenerative Medicine Therapies
for Serious Conditions guidance document.31

regard to one or more clinically significant endpoints. This
designation is available for all treatment modalities, including cell-based therapies, and incorporates all the benefits offast track designation and more, including intensive FDA
guidance on efficient drug development, and an organizational commitment to involve senior management infacilitating the product’s development program.
The request for a breakthrough therapy designation
can be made either concurrently with submission of an
IND application or as an amendment to an existing IND.
A request should ideally be made no later than the EOP2
meeting. Because preliminary clinical data are requiredfor granting the breakthrough therapy designation,
the FDA expects that, in the majority of cases, a breakthrough therapy designation request will be submitted
as an amendment to an ongoing IND. The breakthroughtherapy designation will not be granted if an IND is on
hold or is placed on hold during the designation review.

Fast track designation

RMAT designation

As specified and described in Section 506(b) of the
FD&C Act (as added by section 112 of the Food andDrug Administration Modernization Act of 1997
[FDAMA] and amended by section 901 of the Food andDrug Administration Safety and Innovation Act of 2012
[FDASIA]), an IND can receive fast track designation if it
is intended to treat a serious condition, and if nonclinicalor clinical data demonstrate its potential to address anunmet medical need in patients with such a condition. The
fast track designation provides advantages for facilitating
development and expediting review of the product. The
request for a fast track designation must be made either
concurrently with submission of an IND application or as
an amendment to an existing IND and should ideally bemade no later than the pre-BLA meeting. The fast track
designation will not be granted if an IND is on hold or isplaced on hold during the designation review.

The RMAT designation is the only expedited pathwaythat is specifically relevant to regenerative medicine therapies, such as cell therapies, gene therapies, therapeutic
tissue engineering products, and human cell and tissue
product. The designation was introduced in December
2016 as part of the 21st Century Cures Act.32 A combination product can also be eligible for RMAT designation when the biological product constituent part is aregenerative medicine therapy and provides the greatest
contribution to the overall intended therapeutic effectsof the combination product (i.e., the primary mode ofaction of the combination product is conveyed by the
biological product constituent part).
When determining whether preliminary clinicalevidence is sufficient to support the RMAT designation,
CBER considers factors, including but not limited to, therigor of data collection, the consistency and persuasiveness of the outcomes, the number of patients or subjects
and the number of sites contributing to the data, and theseverity, rarity, or prevalence of the condition. In addition, CBER considers the potential that bias (e.g., bias
in the study design, treatment assignment, or outcome
assessment) may be a factor in the evidence provided insupport of the RMAT designation.
The request for an RMAT designation must be madeBreakthrough therapy designation

As specified and described in section 506(a) of the FD&C
Act (as added by section 902 of FDASIA), an IND may
qualify for breakthrough therapy designation (BTD) if it
is intended to treat a serious condition, and if preliminary
clinical data indicate that the product may demonstrate
substantial improvement over available therapies with

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼37

Table 2-6: Summary of Expedited Pathways in US
Fast Track (FT)BreakthroughTherapy (BTD)

Regenerative
Medicine
Advanced
Therapy (RMAT)

Priority Review

Accelerated
ApprovalYear
1997
established

2012

2017

1992

1992

Qualifying
criteria

Chemical,
Biological andRegenerative
Medicines

Meets thedefinition ofregenerative
medicine therapy

Must treat a serious
condition

Must treat a serious
condition

Must treat
seriouscondition
Clinical or
nonclinical data
demonstrates
that the therapy
has thepotential
to address
unmet
medical needs
for such diseaseor condition.
OR
Must bedesignatedas a qualified
infectious
disease productKey
programfeaturesMust treat
seriouscondition
Preliminary
clinical evidence
indicates thatthe therapy may
demonstrate
substantial
improvement
on a clinicallysignificant
endpoint(s)over available
therapies.

Frequent written Same as FT,
communication plus: Early andmore frequent
Actions tocommunications
expeditewith FDA during
development and development.
review
Rolling
submission andRolling review
reviewPriority Review

Must provide
a significant
Must treat,
improvement in safety
modify, reverse,
or effectivenessor cure a seriousORor life-threatening Any supplement thatdisease/proposes a labelingconditionchange pursuant to areport on a pediatricPreliminary
study under 505A
clinical evidence OR
indicates that
An application for a
the therapy has
drug that has been
the potential todesignated as aaddress unmet
qualified infectious
medical needs for disease productsuch disease orOR
condition.
Any application or
supplement for adrug submitted with apriority review voucher

Provides ameaningfuladvantage overavailable therapies

Same as BTD, plus:
Early discussionof potential
surrogate orintermediate
clinical endpoint

Approval basedon the effect on asurrogate endpointor an intermediate
clinical endpoint

Shorter review ofmarketing application(6 months comparedwith the 10-monthstandard review)

Demonstrates aneffect on a surrogate
endpoint that isreasonably likely
to predict clinicalbenefit or on aclinical endpoint thatcan be measured
earlier thanirreversible morbidity
or mortality (IMM)
that is reasonably
likely to predict aneffect on IMM orother clinical benefit(i.e., an intermediate
clinical endpoint).

Post-approval
advantages
for those withaccelerated
approval

either concurrently with submission of an IND application or as an amendment to an existing IND. The request
should contain a concise summary of information that
supports the RMAT designation, as described in the
Expedited Programs for Regenerative Medicine Therapies
guidance.34 As opposed to breakthrough therapy designation, the RMAT designation does not require evidence toindicate that the drug may offer a substantial improvement

over available therapies. An RMAT designation is not thesame as an approval and does not change the statutory
standards for demonstration of safety and effectivenessneeded for marketing approval. The application successrate for RMAT designations from 2017-2022 is 38%.33
Both BTD and RMAT can be rescinded based on
substantial changes to product/CMC without showing
comparability during clinical development.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼38

Table 2-7: Comparison of benefits and requirements for Breakthrough Therapy and RMAT designation34
BreakthroughTherapy
Designation(BTD)

Regenerative
Medicine
Advanced
Therapy (RMAT)

✓
✗
✓

✓
✗
✓

Show substantial improvement on clinically significant endpoint(s) over available
therapy

✓
✓
✗
✓

✓
✓
✓
✗

Early interactions with FDA to discuss and determine potential surrogate orintermediate endpoints in support of accelerated approval✗✓

Regulatory interactions with FDA should be more focused around manufacturing issues✗✓

Use of preliminary clinical evidence to support designation
Use of preliminary nonclinical evidence to support filing
Frequent interactions with FDA for discussions e.g. study design, extent of safety datarequired to support approval, dose-response concerns, and use of biomarkers
Eligible for priority review
Eligible for accelerated approval
Restricted to regenerative medicines

Priority review

Accelerated approval

As specified and described in the Prescription Drug
User Fee Act of 1992, products (including those thatreceived the fast track, breakthrough therapy, or RMAT
designation) may be eligible for priority review if theymeet the criteria for priority review at the time the marketing application is submitted. Sponsors of regenerative
medicine therapies should consider discussing eligibility
for priority review at the time of a pre-biologics license
application (pre-BLA) meeting with CBER.
A regenerative medicine therapy may receive priority
review if it treats a serious condition, and, if approved,
would provide a significant improvement in the safety
or effectiveness of the treatment for the condition. A
decision about granting priority review is made within
60 calendar days of receipt of the marketing application
or efficacy supplement. If the priority review designation
is granted, CBER has a 6-month goal (reduced from 10
months for standard review) for reviewing the BLA or
efficacy supplement.
The request for a priority review designation must be
made with the original BLA or efficacy supplement.

Under the accelerated approval program, the FDA may
consider accelerated approval for products for serious conditions that fill an unmet medical need (to the extent thatthe product provides a meaningful therapeutic benefit overexisting treatments) based on whether the product has aneffect on a surrogate or an intermediate clinical endpoint.
A surrogate endpoint used for accelerated approval is amarker (including laboratory measurements, radiographic
images, physical signs, or other measures) that is thought
to predict clinical benefit, but is not itself a measure ofclinical benefit. For the purposes of accelerated approval,
an intermediate clinical endpoint is a measurement of atherapeutic effect that can be measured earlier than aneffect on irreversible morbidity and mortality (IMM)
and is considered reasonably likely to predict the drug’s
effect on IMM or other clinical benefit. The FDA bases its
decision on whether to accept the proposed surrogate orintermediate clinical endpoint on the scientific support forthat endpoint. Studies that demonstrate a drug’s effect ona surrogate or intermediate clinical endpoint must be “adequate and well controlled” as required by the FD&C Act.
The FDA has published examples of surrogate endpoints

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL

✼39

that have been used in approved drug development programs under the basis of accelerated approval.35
In general, the accelerated approval pathway has beenused primarily in settings in which the disease course islong and an extended period of time would be required tomeasure the intended clinical benefit of a drug. Accelerated
approval is also potentially useful in acute disease settings
where the intended clinical benefit can be demonstrated
only in a very large study because the clinical event thatwould need to be evaluated to demonstrate clinical benefit
occurs rarely. The FDA encourages sponsors to communicate with the agency early in development concerning
the potential eligibility of a drug for accelerated approval,
proposed surrogate endpoints or intermediate clinicalendpoints, clinical trial designs, and planning and conductof confirmatory trials. It should be noted that a sponsor
seeking accelerated approval may also need to prepare for amore rapid pace for other aspects of the drug development,such as manufacturing. Drugs approved under the accelerated approval program still need to be tested in clinical trials
using endpoints that demonstrate clinical benefit, in trials
known as Phase IV confirmatory trials. If the drug later
proves unable to demonstrate clinical benefit to patients,
the FDA may withdraw approval.
If the FDA determines that there are grounds forwithdrawal, the agency may ask the applicant to request
withdrawal of approval under § 314.150(d) or notify theapplicant of an FDA proposal to withdraw approval in a
notice of opportunity for hearing (NOOH). The NOOH
generally will state the proposed grounds for withdrawalof approval. Upon receipt of a NOOH, an applicant has 15days to file a written request for a hearing. If an applicant
does not request a hearing within 15 days, the applicant
waives its opportunity for a hearing. An applicant may also,of its own volition, request the agency to withdraw approvalof an application approved under accelerated approval.

EuropeSimilar to the U.S., Europe (more specifically the EEA) has
established expedited pathways to support accelerated development and regulatory approval for medicinal products thathave the potential to address unmet medical needs. Thesepathways (Table 2-8) provide opportunities for developers
to engage with regulators during the development process
and participate in accelerated review programs.

PRIME scheme

The PRIority MEdicines (PRIME) scheme supports accelerated development of clinical programs to facilitate earlier
patient access for unmet, serious medical needs. The PRIME
scheme leverages on existing procedures and tools provided
by the EMA with a commitment to engage more closely.
This scheme broadly corresponds to the FDA breakthroughtherapy designation. Between May 2016 and 2022, the success rate for PRIME applications for ATMP was 24%.36
Accelerated assessment

Sponsors may apply to the EMA for accelerated assessment
for submissions. The accelerated assessment procedure isprovided for by recital 33 and Article 14(9) of Regulation(EC) No 726/2004. Accelerated assessment reduces thetimeframe for CHMP to review a marketing authorization
application under the centralized procedure from 210 days
to 150 days. Applications may be eligible for accelerated
assessment if the CHMP decides the product is of majorinterest for public health and therapeutic innovation.
Requests for accelerated assessment should be made atleast two to three months before submitting the marketing
authorization application. Applicants must also prepare
justifications for why the product is expected to be ofmajor public health interest, particularly from the pointof view of therapeutic innovation.
The EMA also recommends that applicants request
a pre-submission meeting six to seven months before
submission to prepare for evaluation under accelerated
assessment. In this meeting, applicants can discuss their
proposal for accelerated assessment with EMA staff
from CHMP, PRAC, and CAT, as needed. Requests forpre-submission meetings should be sent electronically to
the EMA together with supporting documentation via
the EMA Service Desk; required forms can be foundat https://www.ema.europa.eu/en/human-regulatory/
marketing-authorisation/accelerated-assessment. The
CHMP decisions will be communicated to the applicant
and the reasons for accepting or rejecting the request will
be summarized in the CHMP assessment report.
Conditional Marketing Authorization (CMA)

Introduced in 2006 through Regulation (EC) No.
507/2006 according to Regulation N°726/2004, CMA
is a mechanism by which an innovative medicine

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼40

Table 2-8: Summary of Expedited Pathways in Europe
PRIME

Accelerated
Assessment

Conditional Marketing
Authorization

Authorization
under Exceptional
CircumstancesYearestablished

2016

2004

2004

2004

Qualifying
criteria

Address unmet
medical need

Request should bemade at least twoto three months
before submitting
a marketingauthorization
application

Filling an unmet
medical need

Applicants are notable to provide clinicaldata comprehensively
because of rarity of thedisease, for example

Important in termsof public health andinnovation

Company must be able
to provide clinical datacomprehensively

Fulfills an unmet need

Positive benefit/riskbalance

Provide a major
therapeutic advantage
over existingtreatments
Based on early clinicaldata

Pertaining to lifethreatening, serious,or emergency disease,or orphan products

Strong evidenceKey
programfeatures

Enhancedinteraction and earlycommunication withsponsors

Reduce the timeframe
for marketing
authorization to 150
days

Accelerated
assessment andscientific advice

addressing an unmet medical need can be made available for market supply as early as a positive benefit-riskbalance supported by a sufficient body of clinical data
can be demonstrated. The sponsor must then renew
the conditional approval on an annual basis to ensurethat the benefit-risk balance is monitored while further
clinical trials are performed such that data are obtained
to enable the conditional approval to be converted to a
standard approval later. CMA is, therefore, a strategic
way of providing therapies to patients who may lackaccess to treatment options that can be administered ina timely manner.
Distinction from authorization under exceptional
circumstances

EMA may also grant a marketing authorization in absence of comprehensive clinical data under exceptional
circumstances. Unlike CMA, where marketing approval

Active for one year
only with an annual
renewal of theapproval until the
EMA converts theapproval to standard
authorization

Applicable to lifethreatening or serious
diseaseApplicants do notneed to submit
comprehensive data

Enables early approval
while confirmatoryis granted in the likelihood that the sponsor will provide
such data within an agreed timeframe, EMA can grantauthorization under exceptional circumstances whencomprehensive data cannot be obtained even after
authorization. This route normally does not lead to a
standard marketing authorization. For more informationand comparison between these authorization routes,
see: https://www.ema.europa.eu/en/human-regulatory/
marketing-authorisation/pre-authorisation-guidanceJapan
In Japan, expedited pathways that are relevant for genetherapies are: Priority Review, Conditional and TermLimited Approval, Conditional Approval, and Sakigake(Table 2-9). Priority Review lessens the target review
date to nine months and is available for medications
that fulfill an unmet need. The Conditional and TermLimited Approval pathway is for regenerative medicines

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL

✼41

that show promising early phase results. The Conditional
Approval pathway is targeted for highly useful andeffective drugs treating serious diseases. The Sakigake
early access scheme was introduced in 2014 to expedite
innovative assets and was implemented in 2015.
Conditional approval

Under the PMD Act, an RMP can obtain conditional approval on the basis of safety and predicted probable efficacy
demonstrated in early clinical trials. Conditional approval
for an RMP under the PMD Act lasts for a maximumof 7 years, during which time the applicant is required
to perform the later-stage trials that will be required for
subsequent full approval. It should be noted that this time

roughly corresponds to Phase II and Phase III clinical trials
under most circumstances. If these trials are not performed
or if the data obtained from them are considered inadequate to support full approval, the product must be withdrawn from the market at the end of the 7-year conditional
approval period. Additionally, PMDA recommends thatthe following factors should be kept in mind with respect
to conditional approval: 1) evaluation frequently needs tobe done based on a small number of study participants
due to patient scarcity; 2) controlled clinical trials are usually difficult to perform; 3) cellular heterogeneity makes
it difficult to evaluate based on a fixed/limited number ofstudy participants; and 4) there are frequently cases when

Table 2-9: Summary of Expedited Pathways in Japan
Priority Review

Conditional and TermLimited Approval

Conditional Approval

SakigakeYearestablished

2004

2004

2004

2016

Qualifying
criteria

No standard
existing
therapy orsuperior clinicalusefulness ascompared withthe existingproducts in termsof quality oflife of patients,
efficacy, or safety

Promising resultsof early-Phase I/II
registration trials interms of efficacy andsafetyNo standard therapy
exists or superior clinicalusefulness is demonstrated
as compared with theexisting products in termsof quality of life of patients,
efficacy, or safety

Products for diseases indire need of innovative
therapy

Applicable toserious disease

Key program
features

Target total
review time isnine months

Sponsors must
conduct postmarketing clinicalstudies and so on toconfirm the efficacy
and safety andresubmit applications
for regularapproval within apredetermined period

Applicable to serious
diseaseIt is difficult or would take
too long to conduct a
confirmatory study

Applied for approval
firstly or simultaneously(defined as submissions
within 30 days of eachother) in Japan
Prominent effectivenesscan be expected basedon nonclinical and earlyphase trials

Exploratory clinical studies
show efficacy and safety

Only for regenerative
medicines

Surveillance or clinicalstudies must be conducted
as a post-marketing
requirement.

Valid for no more thanseven years

Conditional approval for
drugs
Priority Review

Prioritized consultation
Prioritized review
Review partner
Substantial post-marketing
safety measures
Rolling submission andreview

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼42

it is difficult to conduct rigorous statistical analysis. While
items 1, 2, and 4 in the preceding list are considerations
that are important for medicinal products in general, item
3 is unique to cell therapy products.

months, after submission of the briefing documents• Scope for extensive consultation prior to submission of
the marketing application• Accelerated review of the eventual marketing application, targeting review within 6 months rather than 12
months• Enabling submission of Phase III study data after
submission of the marketing application• Assignment of a PMDA concierge to facilitate anefficient development program and marketing application process• Implementation of specific post-authorization safetymeasures, including extended follow-up (over 10 years)

Sakigake designation

The sakigake (the meaning of which is “pioneer”) designation was introduced in 2015 as the PMDA priority
review system for innovative therapies targeting an unmet medical need. This designation roughly corresponds
to the EMA PRIME scheme and the FDA breakthroughtherapy designation. To qualify for the sakigake designation, a medicinal product must meet the following
conditions: 1) be initially developed and submitted forauthorization in Japan; 2) must be an innovative therapy
for a disease or condition with an urgent unmet need;and 3) must demonstrate high efficacy in early stage
clinical trials. To be considered an innovative therapy
for an urgent unmet need, the product should possessa new and different mechanism of action compared tocurrently authorized products and treat either a serious
life-threatening disease or a chronic disease that leads tothe deterioration of patient quality of life.
The benefits of the sakigake designation include thefollowing:• Prioritized PMDA consultation (scientific advice),with the meeting taking place 1 month, rather than 2

Overall comparison of accelerated pathways toother agencies

Figure 2-2 shows a comparison of Japanese accelerated
regulatory pathways to those of agencies in the UnitedStates and Europe.

Comparability
This section focuses on providing supplementary
guidelines about the best practices to establish productcomparability for cell-based products. These include
unmodified cellular products (i.e., ex vivo expanded
NK, T cells, or MSCs) manufactured from autologous

Relationship to existing treatments

Figure 2-2: Comparison of PMDA, FDA, and EMA accelerated regulatory pathways

Other

Superior
to existingtreatments

RMAT
(FDA)

&

CMA*
(EMA)

Conditional/term-limited approval(PMDA)

Fast Track designation(PMDA)SAKIGAKE designation(PMDA)No alternative
treatments
Life-threatening

Serious

Chronic

Other

Indication type

Source: FDA, EMA, PMDA web sites*Can also be used in emergency situations of if designated as an orphan medicine

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼43

and allogeneic source materials, gene modified cellular
products including CAR-T, gene edited cellular products,
and iPSC-derived cell-based products. Most elements ofcomparability as presented in Chapter 8 of A-Gene1 areapplicable to cell-based therapies (Table 2-10). To better
highlight major differences in comparability approaches
in this chapter, we will focus on some key differences andtwo relevant case studies.
COMPARABILITY STUDIES FOR CELL-BASEDPRODUCTS

For cell-based products, establishing the comparability
study design could be impacted by the nature of product,i.e., autologous versus allogeneic product. Althoughcomparability of the DP is essential in both cases, forallogeneic-derived products there could be a need forestablishing comparability of the DS (not shown), and if
manufactured, the MCB and WCB as well (Figure 2-3).
For cell-based products, donor-to-donor variability
is considered to be the primary source of complexity
associated with establishing product comparability,

whether one is comparing two different manufacturing
processes or two different facilities. This variability iscommonly addressed using either healthy donor material
or preferably patient materials that are split for establishing product comparability that are manufactured bydifferent processes (Figure 2-4A), or the same productmanufactured by the same process at two different facilities (Figure 2-4B).
Another consideration is related to the choice of
healthy donor material versus material obtained frompatients in the autologously sourced setting. Although
it is recommended that for establishing comparability(when process change is introduced, or additional facility
is added) the most optimal starting biological material
is patient-derived, sometimes it is not feasible to obtainpatient materials. For this reason, FDA has shown flexibility historically with regards to the use of healthy donor
materials as a substitute for patient-derived materials.
However, manufacturers are expected to demonstratethat the product can be reliably manufactured frompatient-sourced material post-comparability studies by

Table 2-10: Applicable sections and page number from A-Gene Comparability chapter
Section

Page number in A-Gene

Regulatory Requirements for Managing Manufacturing Changes

194-197

Introduction and Background

201

Description of Change and Rationale for Introducing Change

201

Categorization of Changes

201

Comparison of CQAs: potency, purity, strength, identity, and safety202-203

Predefined Approach to Establishing Product Comparability

203

Well-Defined Acceptance Criteria to Establish Analytical Comparability

203

Detailed Analytical Procedure203

Sampling Plan and Statistical Analysis

204

Statistical Strategy for Comparability Assessment

204-205

Overview of Commonly Used Statistical Approaches and Applicability to Process
Comparability Assessment

205-209

Applying SPI as the Predetermined Acceptance Criteria

209

Process Validation for New Processes

211

Stability of Products Manufactured Using a New Process212

Comparability Study Conclusion

213

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼44

performing in the future, for example, during PPQ runs.
There are additional considerations that could impact
the strategy for establishing comparability for autologous
versus allogeneic off-the-shelf products. For allogeneic
products, it is prudent to test more than one, preferably
three donor sources, in order to establish comparability
in a setting where donors are continuously introduced
into the manufacturing process. For changes in facility,
split starting materials can be useful.
Regardless of autologous or allogeneic, examples ofchanges include change of facility, change of process,change of ancillary materials, change in manufacturing-enabling equipment, and/or change of starting
materials. In some cases, however, it is not feasible toperform a side-by-side comparison., for example when
the starting materials are changed from MSC-derived
to iPSC-derived (or even two different iPSC induction
processes), when moving away from a facility that is nolonger operational where the legacy product was manufactured, and/or the reagents or material for the oldprocess are no longer available. In such cases, the use ofhistorical lot data could be valuable for a comparativebasis. In the event that side by side comparison is not feasible, it is also possible to compare the quality attributesof the product after certain changes are introduced using
post-change materials as well as pre-change materials
retained and stored appropriately. The availability ofsuch samples for clinical batches may allow for limited
comparative studies for cell-based products, as long as
they are supported with stability data.
In all cases, where comparison of CQA for the DS/DP
is performed, it is highly recommended that the materials are tested using assays that are adequately qualified.

Figure 2-3: Potential differences in productintermediates for autologous vs allogeneic products

In cases where the assays have changed, the validity ofhistorical data could be further complemented with theuse of reference materials generated from product manufactured previously. In the situation where the assay is
performed at two different sites of manufacturing, FDA’s
expectation is to demonstrate that assays are properlytransferred and deemed to be equivalent in terms ofsensitivity, accuracy, linearity, and precision.
Another critical aspect of comparability study isavailability of highly qualified potency assay for the
drug product. Potency assay development is describedin Chapter 9, but it is important to emphasize that themore reflective the potency assay is of bioactivity (orsurrogate therein) of the product in vivo and in patients,
the better. With this, the more readily available analytical
data can be used to support product comparability. The
use of surrogate markers that are not shown to reflect the

Figure 2-4: Example starting material split strategy for establishing CAR-T product comparability(A) Process 1 versus Process 2 in same facility, and (B) Process 1 in one facility versus Process 1 in another facility

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼45

Figure 2-5: Phase-dependent, risk-based approach to establishing product comparability

biological activity of the product is not recommended asa basis for establishing product comparability in the latephase of the product development cycle.
Although donor eligibility for off-the-shelf products is
not necessarily considered one of the parameters to be used
for establishing product comparability, it is important that
donor eligibility and testing for relevant adventitious agentsshould be part of mandatory safety testing performed by
IND Holders, as appropriate for the donor source. If thebiological starting material, e.g., Leukopaks, are sourced
from different vendors, and/or they are stored differently(i.e., fresh vs. cryopreserved), this can impact comparability
and should be included as part of the comparability exercise.
Another important consideration is the phase-appropriateness of the comparability exercise. Regardless of thephase of development, it is recommended for sponsors tohave early interaction with the FDA on their comparability
plans, and provide a draft comparability protocol to the FDA
in advance of execution, in order to take into account any
advice the Agency may have on the protocol itself. Figure
2-5 shows a phase-dependent, risk-based approach to establishing product comparability during development. In
general, the earlier major CMC changes can be implemented, the better (such as between the first-in-human study
and pivotal trial), with comparability addressed (with FDA
concurrence) prior to initiation of the pivotal trial.

Table 2-11 provides some examples of these changes andtheir categorization as minor (not requiring comparability)or major (requiring comparability). In some cases, a change
in the type of donor material from one tissue to another may
represent a change that requires a separate IND submission,in which case the FDA should be consulted for confirmation
on impact.
Below we provide case studies of cell-based therapy
products where manufacturing changes have been introduced: an autologous CAR-T that has been gene-modified
via lentiviral vector in order to express the CAR transgene(Table 2-12), and an allogeneic iPSC-derived MSC (Table
2-13). First, the most relevant attributes to the study should
be determined, as well as the methods and procedures thatshould be used to measure these attributes. A risk- andscience-based approach allows prioritization of the relevant
attributes, which may include biological activity, potency,
identity, and purity (refer to Chapter 4 for discussion onrisk assessment tools). These attributes are key elements ofcomparability studies along with standard safety tests suchas sterility and endotoxin.
Based on the risk assessment and assay considerations,
it is possible to select a subset of release tests to evaluate
comparability with the highest relevance to product quality
and effectiveness. These tests must be performed on the
final DS/DP, but it is also important to monitor the process

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼46

Table 2-11: Examples of changes and Associated Risk Categories
Type of Change

Change Category

Process changes re: cell washing

Minor

Note

Process changes re: multiple changes

Minor to Major
depending ontype of changes

Cumulative minor changes could
potentially require establishment ofcomparability

Process changes re: starting material from cord blood tobone marrow

Major

Potentially may require a new IND;
consult the appropriate review division

Process change: change of manufacturing from adherent
to suspension cultures

Moderate to
Major

Change of facility 1 to facility 2 of the same process

Major

Change of MCB for manufacturing of iPSC or MSC products

Major

by conducting additional in-process testing (which could
be a subset of the tests described here). Statistical considerations are covered in A-Gene as stated in Table 2-10.
Table 2-12 summarizes an example of a list of attributes
to be measured and the test methods for a CAR-T cell
product. These attributes need to be prioritized, some ofwhich are considered critical, such as purity and potency.
Not all attributes are listed; process-related impurities are
one example of other attributes that should be assessedand prioritized.

For MSC products derived from iPSCs, perhaps themost challenging exercise in establishing product comparability is when the MCB for MSC or iPSC is changed fora variety of reasons, which is outside of scope of this casestudy. To illustrate unique challenges in establishing comparability we will focus on a hypothetical product, MSC
derived from differentiated iPSCs. The change involves
major changes in manufacturing and using a different
donor material. Table 2-13 summarizes a list of attributes
to be measured in the comparability study and the test

Table 2-12: Relevant list of product attributes and test methods for an autologous CAR-T cell product
Potential changes include process/CMC changes within the same facility (Figure 2-4A), or change in facilities (include
splitting source materials between two sites; performing same analytics at two sites) (Figure 2-4B).
Attributes

Test

Method Procedure

Cell counts

Measure total number of cells

Dye-based automated cell counter

Cell viability

Measure live cells/total cells ratio

Dye-based automated cell counter

Safety - Sterility

Measure of microbial contamination

USP <71>

Safety - Endotoxin

Measure of endotoxin

LAL (USP <85>)

Safety - Mycoplasma

Measure of mycoplasma

USP <63>

Safety - RCL

Replication Competent Lentivirus

qPCR / ddPCR

Safety - VCN

Measure vector integration into host cell
genomeqPCR

Identity

CD3; CAR expression

Flow cytometry

Potency

Target cell killing

Cell based assay; ELISA (forsurrogate marker if data supports)

Safety-in vitro proliferation

Measure IL-2 independent expansion

Cell-based assay

Purity - Contaminating cell types

Measure other cell types (B cells, NK cells)

Flow cytometry

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼47

Table 2-13: Relevant list of product attributes and test methods for an iPSC-derived MSC productAttributes

Test

Method Procedure

Cell counts

Measure total number of cells

Dye-based automated cell counter

Cell viability

Measure live cells/total cells ratio

Dye-based automated cell counter

Safety - Sterility

Measure of microbial contamination

USP <71>

Safety - Endotoxin

Measure of endotoxin

LAL (USP <85>)

Safety - Mycoplasma

Measure of mycoplasma

USP <63>

Safety - Tumorigenicity

Aberrant cell growth

Cell-based

Identity MSC cell surface Marker

Relevant cell surface CD markers

Flow cytometry

Potency

Coculture immunosuppression assay

Cell based assay

Purity - Cell types and composition

Relevant cell surface CD markers;
Measure cell types

Flow cytometry

Safety - Undifferentiated iPSC

Residual iPSCs

Flow Cytometry

Genome stability

Measure changes in genome structuralor point mutation and / or deletions

STTR (qPCR), optical imaging
method, FISH, karyotype (G-banding)methods for this hypothetical MSC product. These attributes need to be prioritized, some of which are considered
critical, such as purity and potency. Not all attributes arelisted; process-related impurities are one example of otherattributes that should be assessed and prioritized.

Standards
Cell and gene therapy is a rapidly developing field of medicine, with many promising products in development that
could help manage and potentially cure a wide range of
diseases that are intractable, chronic, and even terminal.
Given that the cell therapy field is currently at a tipping
point, with disruptive innovation pushing the boundaries
of science and a number of products poised for commercialization (with a few already on the market), robust standards that can support developers in ensuring the safety,
efficacy, and quality of products must be established. Thissection will provide an overview of the current landscapeof standards development in cell therapy.
BENEFITS OF VOLUNTARY CONSENSUS
STANDARDS IN CELL THERAPYStandards are considered to be voluntary rules, conditions, characteristics, or physical materials that an organization can adopt to make a process safer, more eﬃcient,or better aligned with the practices of other organizationsin their industry. In general, standards can be considered
to be either documentary or non-documentary.
Cell-based medicinal products are one of the key
modalities of newly emerging regenerative therapeutics.
The number and diversity of the product platforms
in current use has exploded over the last ten years.
However, given that relatively little experience has accumulated in the years that cell therapy has been a partof the therapeutic landscape, there are relatively few
ways for developers across the research and industrial
sectors to achieve consistency in areas such as protocol development, process infrastructure, and productquality testing and assurance. This lack of consistency
and experience has made it challenging for stakeholders
involved in cell therapy development and manufacturing to operate with a sense of certainty and provide
patients in need of these essential and groundbreaking
therapies with the confidence that products are of the
maximum possible quality. The ongoing absence ofstandards to support safe and eﬃcient practices that
reduce the burden on companies seeking regulatory
approval for their products may result in promisingtherapies being unable to successfully transition fromthe clinical development phase to being commerciallyavailable for the benefit of patients. The appropriately

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL

✼48

targeted, field-wide development of standards will help
to address the previously tolerated shortcomings.
Specifically, standardizing equipment, methodologies,
and testing methods will result in a number of benefits
for industry stakeholders. Standards establish a base of
legitimacy on which patients, regulators, and investors
can rely—standards development will instill the publicwith confidence that development of cell therapy is adequately informed by thoroughly researched best practices
and is, therefore, a safe and effective option for treating awide range of diseases. In addition, from an operational
standpoint, standardization can help industry stakeholders streamline business practices by allowing for more
eﬃcient coordination of efforts throughout the entire
supply chain, as well as improving the predictability of
costs and resource management. In effect, barriers toentry into the clinical development space may be lowered
for smaller companies or academic researchers, thus facilitating the delivery of therapies to patients. Standards
can assure regulators that the fundamental processes
underlying the development of a new therapy are sound.
This assurance, in turn, allows regulators to review a
product more rapidly. A smoother, less uncertain regulatory review process increases industry stability, lowers
perceived risk to investors, and accelerates market availability of products, all of which serve to increase accessand options for patients. Lastly, standards can greatly aid
the regenerative medicine therapy community as a wholeby enhancing the ability of developers to collaborate andshare knowledge with others. This can reduce the potential for redundant efforts to be undertaken and serve topatch up the relatively fragmented state of knowledge
that characterizes the emerging state of this field.
CURRENT LANDSCAPE OF STANDARDS IN
CELL THERAPY

Currently, additional research is needed to assess thesafety and efficacy of cell therapies for commercial use,which is becoming more widespread with the regulatory
approval of several products in recent years. Furthermore,factors such as variations in manufacturing, measurement, and analytical techniques across developers ofexperimental cell therapy products make it diﬃcult toevaluate product quality and safety, and to assess the impact of manufacturing changes or innovations intended

to improve product safety and eﬃcacy. A common setof standards in cell therapy will advance development oftreatments for a number of diseases.
Standards Developing Organizations (SDOs) play a
critical role in the publishing of consensus standards thatare universally recognized. There is a big overlap between
SDOs involved in cell therapy and gene therapy. A list ofthese organizations and the scope of their standards development efforts are presented in the Standards chapter ofthe A-Gene document.1 In addition, organizations like theStandards Coordinating Body (SCB) helps accelerate such
efforts by facilitating the use and development of standards
in response to the diverse needs of stakeholder groups,
including government and regulatory agencies, productdevelopers, raw materials providers, and clinicians andhealthcare professionals. An interactive database of thecurrent regenerative medicine standards landscape isavailable at https://portal.standardscoordinatingbody.org/.
RECENT AND ONGOING EFFORTS IN CELLTHERAPY STANDARDS DEVELOPMENT

Various organizations are developing and publishingtechnical standards using a consensus-based development process to benefit the broad regenerative medicine
community. The examples below show a few standards
that were recently published and are currently in development in the cell-based therapy field.

1. Testing and characterization standards forcellular therapeutic products (ISO 23033)
This effort (ISO 23033:2021 Biotechnology—Analyticalmethods—General requirements and considerations forthe testing and characterization of cellular therapeutic
products), led by The International Organization forStandardization (ISO), aims to define terms related tocharacterization of human cells for therapeutic applications, as well as provides general requirements forthe testing of cellular therapeutic products intended
for human use. It covers considerations for the characterization and testing of cellular therapeutic products,
including approaches to select and design analyticalmethods that are fit-for-purpose and considerations
for setting specifications for the analytical methods.
Such considerations are critical to establishing CQAs
for cell therapy products. Aspects of this document

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼49

are applicable to starting materials (including those fortissue-engineered products) and intermediates of cellulartherapeutic products. This effort has been published andinformation relating to the effort can be found at: https://www.iso.org/standard/74367.html.

2. Ancillary materials standard for production ofcell and gene therapy products (ISO/CD 20399)
The quality of ancillary materials used in the production ofcell-based medicinal products will determine the safety andefficacy of the final cell therapy products. This document(ISO/CD 20399 Biotechnology—Ancillary materials present during the production of cellular therapeutic products
and gene therapy products) provides guidance to suppliers
and users of ancillary materials to improve the consistency
and quality of ancillary materials of biological (human andanimal) and chemical origin through an appropriate level of
documented lot-to-lot consistency in the aspects of identity,
purity, storage and stability, biosafety, and performance.
This standard will combine ISO/TS 20399-1, ISO/TS 20399-2, and ISO/TS 20399-3 into an international
standard while adding further information on the relationship between ancillary material suppliers and users
and quality considerations.

3. Cell collection and apheresis standards (BSR/
PDA 08-202x)
The Parenteral Drug Association (PDA) is leading thedevelopment of a cell collection and apheresis standard,
which is intended to minimize the variation during theapheresis collection process. This effort will result increation of a standard leukapheresis manual/standard
operating procedure (SOP) template to help align theformat of cell collection requirements for product manufacturers and sponsors. This will aid both manufactures
and apheresis sites in minimizing mistakes and increasing
quality of starting materials for cell therapy. This standard
is intended to clarify and minimize the variation in basic
steps of the apheresis process while allowing for flexibility
for manufacturing processes when needed.

4. Requirements for human and mouse pluripotent
stem cells (ISO/DIS 24603)
Pluripotent stem cell (PSC) lines hold unique characteristics and behavior due to their capability for bothself-renewal and differentiation into multiple cell types.
However, the stem cell phenotype can be changed bysuboptimal cell culture technique, prolonged passage,
or changing the culture conditions. Accordingly, mouse
PSCs have been used to establish our fundamental understanding of PSC biology, and these discoveries havebeen translated into human PSC research to drive thedevelopment of new human cell-based in vitro assays andpotential regenerative medicines.
Mouse PSCs and human PSCs have become widely
studied in this field, and many significant scientificadvances have been made by using these PSCs, giving
clues to the development of cell therapies. It is therefore
critical to ensure that cell lines used in this field havebeen prepared and documented appropriately and havethe correct identity and characteristics to help assure
reproducibility in PSC-based studies.
This standard (ISO/DIS 24603 Biotechnology—
Biobanking—Requirements for human and mouse pluripotent stem cells) specifies establishment, expansion,preservation, maintenance, characterization, quality
control, storage, and distribution requirements for thebiobanking of mouse and human PSCs. This document
is applicable to all organizations performing biobanking
with mouse and human PSCs used for research and development, excluding cell lines used for in vivo applicationin humans, clinical applications, or therapeutic use. This
document aims to meet the current demand for the
standardized PSC procedures of biobanks and builds oninternational consensus agreed by PSC resource centers(International Stem Cell Banking Initiative [ISCBI], 2009).
This effort is currently underway and information
relating to the effort can be found at: https://www.iso.
org/standard/79046.html.

5. Cryopreservation Standards for Cell Therapies,
Gene Therapies, and Regenerative Medicine
Manufacturing (ANSI/PDA 02-2021)
Cryopreservation is a key step in the manufacturing ofcell therapies as many process parameters could affectcell recovery, viability, and function. This standard aims
to present a best practices approach on how to prepare,cryopreserve, and recover cells, cell lines, and cell-based
tissue products. It provides general, non-cell type specificguidance and considerations when developing a robust

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼50

protocol for freezing, storage, and recovery of primarycells or cell lines in research, development, and manufacturing of cell and gene therapy products, as well as
potential sources of variability and mitigation strategies.
The best practices and guidance in this document provide general procedural support for cryopreservation ofcell-based products during both early and late phases of
product development. This document provides guidanceon how to ensure that your fit-for-purpose cryopreservation process is effective and validated appropriately
based on the needs of your manufacturing process.
This document has been published and more information can be found at https://www.pda.org/bookstore/
product-detail/6498-pda-standard-02-2021.

6. Determining and interpreting cell viability
Being able to measure cell health and response to different stimuli is critical to understanding a cell therapy’s quality, safety, and efficacy. With various available
methods to measure cell viability, it is often challenging
to identify the most appropriate method or assay for agiven therapy or cell type. In addition, test methods canbe difficult to interpret due to lack of understanding ofwhat the assays measure and how measured parameters
correlate with cell viability.
The cell therapy field has identified the need for thisanalytics and testing standard, with the goal being todevelop a flexible, non-protocol-based standard aroundguidelines and considerations in designing and using
cost-effective assays that yield accurate and precise cell
viability results. These efforts will guide the fit-for-purpose decision-making process for developing a cell viability assay, with potential to develop a reference standard
for cell viability assays. Possible areas of standardization
include data type (e.g., number of living/dead cells), assay
parameters (e.g., incubation time), stock cultures andtesting environments, criteria for methods selection, cellsampling time during culture/manufacturing process,data recording, documentation, and interpretation, as
well as assessing impact of patient variability.
A new effort in ISO to address this topic has begun
development. This is being led by NIST and other U.S.
experts. It will address how to assess cell viability forcellular therapeutic manufacturing.

NEEDS AND GAPS FOR STANDARDS IN CELLTHERAPY BY FUNCTIONAL AREA

Variations in manufacturing, measurement, and analytical techniques across developers of cell therapy products
often cause difficulties for evaluating product quality
and safety and addressing the impact of manufacturingchanges or innovations. A common set of standards incell therapy will advance development of treatments
beyond the realm of clinical trials, to safe approved treatments for genetic diseases and syndromes. The following
list shows a few examples of needs and gaps for standards
development in the cell therapy sector.

Chain of Identity
Chain of custody (COC) and chain of identity (COI) arerecords used in the regenerative medicine therapy manufacturing process to ensure product safety and quality.
COI is a record associated with a single patient, including
health records both before and after treatment. COC
records all points of transfer and control for a product,
including product starting material and all in-process
manipulations through the point of delivery.
COC and COI currently lack standardized definitions
and strategies. Because of this, stakeholders throughout
the regenerative medicine supply chain often use different
tracking systems or formats, which can potentially resultin inaccurate or incompatible records. Such errors createa risk of administering the wrong product to a patientor the inability to administer a product due to delays.
Standardization of this area would ease communication
between the manufacturers and apheresis sites while also
minimizing burden on the apheresis sites.

Data acquisition and management
The data generated throughout the research, development,
and application of regenerative medicine therapies can inform efficient innovation of therapeutic products and help
demonstrate the efficacy, safety, or regulatory compliance ofa product. This data needs to be organized and formatted.
The quality, accuracy, and completeness of the existing body of data varies. Forms to be organized exist for
acquiring and recording data, but there is no standard toencourage their use, which makes it difficult to compare
similar data. Additionally, companies do not have a
streamlined method to share their data while retaining

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼51

their intellectual property (IP) and maintaining patient/donor privacy.
Standardization of guidelines and protocols to increase
data quality, make data easier to share, and establish mechanisms to safeguard IP and patient privacy would help
advance cell therapy and ease life cycle management andcommunication. There are many potential areas to standardize that could address this, such as data management
and storage plans, data registry/database characteristics,
data elements (form fields), application programming
interfaces (APIs), methods to study long-term effects oftherapies in patients, and management of metadata.
IMPLEMENTING STANDARDS IN CELL-BASEDTHERAPY MANUFACTURING

The SCB has teamed up with the American National
Standards Institute (ANSI) to create standards packages
tailored to specific regenerative medicine application areas.
The SCB has so far curated three standards packages: 1)
Tissue Engineering Standards Addressing Product Quality
and Characterization Package, 2) Cell Therapy Standards
Addressing Product Quality and Characterization
Package, and 3) Tissue Engineering Standards Addressing
Analytical and Testing Methodologies Package, with moreplanned for other application areas.
The packages aim to make it faster and easier forregenerative medicine product developers to identify
standards that can help stakeholders. These packages
save time and money by offering insight into efficientmethodologies and practices, and improve the safety,
consistency, and comparability of regenerative medicine
products. These packages can also facilitate a smootherregulatory approval process for new therapies.
The SCB plans to continue developing more standards
packages in collaboration with ANSI. In addition to thelarge, broad packages that cover several methods withvarying applications, the SCB also plans to create smaller,
more targeted packages, focused on specific applications
to help guide users through an entire process.

FDA-supported courses for standards
implementation
There is a need for guidance in implementing current regenerative medicine standards. Currently the
SCB is working on developing courses for standards

implementation. These would be focused on helping
stakeholders interpret and understand how standards canbe put into place in their manufacturing processes. These
courses will each focus on individual critical standards
for the regenerative medicine field. The first course will
focus on standards for cell counting (ISO 20391-1:2018
Biotechnology—Cell counting—Part 1: General guidance on cell counting methods and ISO 20391-2:2019
Biotechnology—Cell counting—Part 2: Experimental
design and statistical analysis to quantify counting
method performance). Currently the SCB is working todevelop courses for nucleic acid synthesis (ISO 20395)
and ancillary materials (ISO 20399). Further courses for
additional standards are also planned.
STANDARDS IN CELL THERAPY AND REGULATORYAPPROVALThe FDA evaluates consensus standards for appropriateness for the review of medical device safety and performance by formally recognizing, all or partially, or notrecognizing consensus standards. Manufacturers may
submit declarations of conformity to FDA-recognized
consensus standards to demonstrate they have met
relevant requirements in the Federal Food, Drug, andCosmetic Act (FD&C Act). When used appropriately,
this may reduce the amount of supporting testing documentation typically needed in a premarket submission.
The informal and formal recognition processes are
discussed below.

FDA Informal Recognition of Voluntary Consensus
Standards
Voluntary consensus standards can be defined as standards developed by voluntary consensus standards bodies.
The Food and Drug Administration Modernization Act
of 1997 (FDAMA) (Pub. L. No. 105-115) and the 21stCentury Cures Act of 2016 (Pub. L. No. 114-255) amended
section 514(c) of the Federal Food, Drug, and Cosmetic
Act (FDC Act) to require FDA recognition of voluntary
consensus standards. The FDA has used such standards
to develop and evaluate performance characteristics ofdosage forms, testing methodologies, manufacturing
practices, product standards, scientific protocols, compliance criteria, ingredient specifications, labeling of drug
products, and other technical or policy criteria.

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼52

In addition, the FDA Center for Drug Evaluation andResearch (CDER) has drafted a document titled CDER’s
Program for the Recognition of Voluntary Consensus
Standards Related to Pharmaceutical Quality Guidancefor Industry. This guidance describes a proposed
program at CDER to make public a comprehensive
listing of informally recognized voluntary consensus
standards related to pharmaceutical quality. The
program, once established, will facilitate submissions
by external stakeholders and CDER staﬀ proposing
voluntary consensus standards related to pharmaceutical quality for informal recognition. CDER believes
that this informal program, which is diﬀerent than
the formal recognition standards program in the FDA
Center for Devices and Radiological Health, will help
promote innovation in pharmaceutical development
and manufacturing and streamline the compilation
and assessment of marketing applications for products
regulated by CDER. It should be noted, however, thateven if an applicant decides to use one of the CDER
informally recognized voluntary standards, CDER
may request that the applicant provide additional
information to support an IND application or a marketing application. In addition, the applicant’s use of
an informally recognized consensus standard will bestrictly voluntary. CDER has issued this draft guidance
to obtain public comments on the proposed program.
After CDER considers submitted comments, CDER
will establish this program and describe it by publishing a final guidance. Thus, though not yet formalized,
this draft guidance, when finalized, will comprehensively represent the current thinking of the FDA.

FDA Formal Recognition of Standards

In addition to informal recognition, the FDA also issues
formal recognition of standards. The FDA recognizes
consensus standards are standards that the FDA has vetted and determined are appropriate to support clearance
or approval of a device.37 The purpose of the FDA formal
recognition of consensus standards is to streamline thepremarket review process. This formal recognition allows
companies to submit a declaration of conformity with arecognized standard in a premarket application, rather
than submit complete data and test reports demonstrating conformity with a standard.
The FDA maintains a formal database of recognized
consensus standards. This database consists of national
and international standards recognized by the FDA towhich manufacturers can declare conformity and is acomponent of the information that regulators can use
to make an appropriate decision regarding the clearance
or approval of a submission.
In the January 2020, FDA Chemistry, Manufacturing,and Control (CMC) Information for Human Gene
Therapy Investigational New Drug Applications(INDs)—Guidance for Industry, the FDA recognizes
three types of reference standards: 1) certified reference
standards (e.g., USP compendial standards); 2) commercially supplied reference standards obtained from a
reputable commercial source; and 3) other materials of
documented purity, custom-synthesized by an analyticallaboratory or other noncommercial establishment.

References
1

Alliance for Regenerative Medicine. Project A-Gene: A casestudy-based approach to integrating QbD principles in Gene
Therapy CMC programs. 2021. https://alliancerm.org/
manufacturing/a-gene-2021

4

SOPP 8214: INTERACT Meetings with Sponsors for Drugs
and Biological Products. FDA Center for Biologics Evaluation
and Research. December 11, 2020. https://www.fda.gov/
vaccines-blood-biologics/industry-biologics/interact-meetings

2

US Food and Drug Administration. Considerations for theDevelopment of Chimeric Antigen Receptor (CAR) T Cell
Products, Draft Guidance for Industry. March 2022. https://www.fda.gov/media/156896/download

5

PDUFA Reauthorization Performance Goals and Procedures,
Fiscal years 2023 through 2027. https://www.fda.gov/industry/
prescription-drug-user-fee-amendments/pdufa-vii-fiscalyears-2023-2027

3

References for the Regulatory Process for the Office of Tissues andAdvanced Therapies. https://www.fda.gov/vaccines-blood-biologics/
other-recommendations-biologics-manufacturers/referencesregulatory-process-office-tissues-and-advanced-therapies

6

Formal Meetings Between the FDA and Sponsors or Applicantsof PDUFA Products. FDA Guidance for Industry, December
2017. https://www.fda.gov/regulatory-information/searchfda-guidance-documents/formal-meetings-between-fda-andsponsors-or-applicants-pdufa-products-guidance-industry

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼53

Referencecs, continued
7

SOPP 8212, Appendix A: Discussion Topics for the Initial
Comprehensive Multidisciplinary Breakthrough Therapy TypeB Meeting with the Sponsor

24

Dull T, Zufferey R, Kelly M, et al. A third-generation
lentivirus vector with a conditional packaging system. J Virol.
1998;72(11):8463-8471. doi:10.1128/jvi.72.11.8463-8471.1998

8

U.S. Food and Drug Administration. Questions to Ask FDA
during Formal Meetings. https://www.accessdata.fda.gov/cder/sb-navigate/topic3/topic3/da_01_03_0180.htm

25

9

https://www.ema.europa.eu/en/human-regulatory/researchdevelopment/innovation-medicines

US Food and Drug Administration. Human Gene Therapy
Products Incorporating Human Genome Editing, Draft
Guidance for Industry. March 2022. https://www.fda.gov/
media/156894/download

26

10

The EMEA Innovation Task Force. https://www.ema.europa.
eu/en/documents/presentation/emea-innovation-task-forceconstantinos-ziogas_en.pdf

Potency Tests for Cellular and Gene Therapy Products. FDA
Guidance for Industry, January 2011. https://www.fda.gov/
media/79856/download

27

11

Mandate of the EMA Innovation Task Force (ITF). 6 August
2014. https://www.ema.europa.eu/en/documents/other/mandate-european-medcines-agency-innovation-task-forceitf_en.pdf

Drug Master Files. FDA Draft guidance for Industry, October
2019. https://www.fda.gov/media/131861/download

28

Code of Federal Regulations: 21CFR314.420. Drug Master
Files. April 1, 202012https://www.ema.europa.eu/en/documents/other/mandateeuropean-innovation-network_en.pdf

2913

https://www.ema.europa.eu/en/human-regulatory/researchdevelopment/scientific-advice-protocol-assistance

Medicinal Products for Human Use Containing or Consistingof GMOs: Interplay between the EU Legislation on Medicinal
Products and GMOs, Frequently Asked Questions. https://
health.ec.europa.eu/system/files/2019-10/gmcells_qa_en_0.pdf

30

14Guidance for applicants: pilot project for simultaneous national
scientific advice (SNSA). https://www.famhp.be/sites/default/
files/FAMHP_SNSA_guidance_v2.1_DEF.pdf

15

Extension of the EU pilot project on simultaneous national
scientific advice. https://www.famhp.be/sites/default/files/
Extension_SNSA_pilot_project_DEF_EN.pdf

Expedited Programs for Serious Conditions – Drugs andBiologics. FDA Guidance for Industry, May 2014. https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/expedited-programs-serious-conditions-drugsand-biologics

31

16

GENERAL PRINCIPLES: EMA-FDA PARALLEL SCIENTIFIC
ADVICE (HUMAN MEDICINAL PRODUCTS). July 2021.
https://www.fda.gov/media/105211/download

US Food and Drug Administration. Expedited Programs forRegenerative Medicine Therapies for Serious Conditions,
Guidance for Industry. https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/expeditedprograms-regenerative-medicine-therapies-serious-conditions

17

https://www.gov.uk/guidance/medicines-get-scientific-advicefrom-mhra

32

https://www.fda.gov/regulatory-information/selectedamendments-fdc-act/21st-century-cures-act

18

https://www.gov.uk/government/groups/mhra-innovation-office33

19

https://www.gov.uk/guidance/innovative-licensing-and-accesspathway

https://www.fda.gov/vaccines-blood-biologics/cellular-genetherapy-products/cumulative-cber-regenerative-medicineadvanced-therapy-rmat-designation-requests-received-fiscal

20

https://www.pmda.go.jp/english/review-services/
consultations/0002.html

34

21

Chemistry, Manufacturing, and Control (CMC) Informationfor Human Gene Therapy Investigational New Drug
Applications (INDs). Guidance for Industry. January 2020.
https://www.fda.gov/media/113760/download

RMAT Designation: Impact on Regenerative Medicine
Sector & How it Compares with Other Accelerated Approval
Programs. Alliance for Regenerative Medicine Webinar, June
2018. https://alliancerm.org/wp-content/uploads/2018/06/
ARM-RMAT-Webinar-Slides-PDF.pdf

35

https://www.fda.gov/drugs/development-resources/tablesurrogate-endpoints-were-basis-drug-approval-or-licensure

36

European Medicines Agency, PRIME: Analysis of the first 5
years’ experience. https://www.ema.europa.eu/en/humanregulatory/research-development/prime-priority-medicines#5year-review-(2016-2021)-section

37

https://www.fda.gov/medical-devices/premarket-submissionsselecting-and-preparing-correct-submission/standards-andconformity-assessment-program#process

22

Biologics License Applications for Minimally Manipulated,
Unrelated Allogeneic Placental/Umbilical Cord Blood Intendedfor Hematopoietic and Immunologic Reconstitution in Patientswith Disorders Affecting the Hematopoietic System. FDA
Guidance for Industry, March 201423

Content and Review of Chemistry, Manufacturing, andControl (CMC) Information for Human Somatic Cell Therapy
Investigational New Drug Applications (INDs), Guidance for
FDA Reviewers and Sponsors. April 2008. https://www.fda.
gov/regulatory-information/search-fda-guidance-documents/
content-and-review-chemistry-manufacturing-and-controlcmc-information-human-somatic-cell-therapy

Chapter 2: Regulatory Considerations, Comparability, and Standards in Cell-Based Therapies

A-CELL✼54

CHAPTER 3

Generation of Quality
Target Product Profile
CHAPTER LEAD

Bruce Thompson, Resilience
CHAPTER CONTRIBUTORS

Taylor Zhang, Sana Biotechnology
Neil Leatherbury, Precision BioSciences

Chapter 3 | Contents
Concept of QTTP and its role in the development
process...........................................................................56

Purity.........................................................................60

Use of the quality target product profile...................... 57

Stability..................................................................... 61

QTPP for cell-based therapy..........................................58
Safety.........................................................................59
hPSCs.....................................................................59
CAR-T......................................................................59
Potency......................................................................60

Concept of QTTP and its role in thedevelopment process
The Quality Target Product Profile (QTPP) provides
developers of cell-based therapy products with a powerful tool for considering and assessing all elements ofthe production process that could have an impact on
the quality, safety, and efficacy of the final product, andcan help developers to focus resources and efforts on themost critical aspects of production. As first defined in ICH
guideline Q8 (R2), the QTPP is a “prospective summaryof the quality characteristics of a drug product that ideallywill be achieved to ensure the desired quality, taking into
account safety and efficacy of the drug product.”1 The concept of the QTPP follows directly from approaches taken
for the target product profile (TPP), in which developers
establish the goals for the drug development program (e.g.,
the target indication, target vein-to-vein time) and define
safety- and efficacy-related information for labeled use.
The QTPP focuses the concepts established in the TPP
to guide chemistry, manufacturing, and controls (CMC)
development, and is meant to identify, define, and justify
quality characteristics to ensure safety and efficacy expectations established by the TPP.
The QTPP is an integral element of a Quality by Design(QbD) approach, in which quality of the product is considered at the earliest possible stage of development prior
to clinical manufacturing. As part of this framework, theChapter 3: Generation of Quality Target Product Profile

Identity...................................................................... 61
Evolution of the QTTP during development................. 61
QTPP for allogeneic cell-based therapy products....... 62
Conclusion.....................................................................64
References.......................................................................64

information contained in the QTPP should contain the
Critical Quality Attributes (CQAs), defined to be “physical, chemical, biological, or microbiological properties orcharacteristics that should be within an appropriate limit,
range, or distribution to ensure the desired product quality”,1 which are identified through risk assessment (Chapter
4). Upon CQA identification, a series of development
studies should be performed to quantify the relationshipbetween process parameters and CQAs, resulting in theestablishment of the critical process parameters (CPPs),
defined as process parameters that have been proven tohave a direct impact on the CQAs of the manufactured
product. Additionally, the CQAs captured in the QTPP
should provide the information required to define a design
space and control strategy. Cumulatively, the QTPP serves
as a link between the TPP and process validation steps
necessary for ensuring the required quality standard.
The QTPP should be established at the researchphase and be updated regularly throughout all phasesof the product life cycle, up to the Biologics License
Application (BLA) phase. A successful QTTP provides
an understanding of the factors that will ensure the quality, safety, and efficacy of a specific product, as well as acomprehensive overview of the entire CMC module of
the BLA. Once the product is approved, the QTPP serves
as the product standard-bearer that grounds continuous
process improvement and, therefore, is essential to theentire product life cycle.

A-CELL✼56

Figure 3-1 depicts the path of QTPP maturation throughthe development process, as well as the relationship of the
QTTP to the major stages of product development.

Figure 3-1: QTPP maturation through productdevelopment

Use of the quality target product profile
When initiating development of a pharmaceutical product, developers should begin drafting a list of desired
characteristics targeted for the new product. This target
product profile (TPP) is an instrument intended to facilitate a common vision across all disciplines to guide thedevelopment, conduct, and analysis of clinical trials by
focusing on strategic product label claims.
The TPP encompasses a broad range of attributes, someof which are likely to evolve as development progresses
and product knowledge becomes refined. Throughout aproduct’s life cycle, the TPP provides the focus among allareas involved, forming the foundation through all stagesof development and all disciplines that contribute to thedevelopment program of a medicinal product, including
research, nonclinical, clinical, manufacturing, quality,
and regulatory teams. It allows all medical, technical,
and scientific information to be consolidated within one
development tool or document.
Because it links safety and efficacy requirements to thefinal product, the TPP is considered the starting point forthe development of a control strategy (Chapter 10). The
TPP serves as the cornerstone to defining the QTPP and
derived CQAs that should be monitored in order to assess
the consistency of the manufacturing process. For a Celland Gene therapy (CGT) product, as for any product,
the TPP is a dynamic summary of the CGT product development that changes as knowledge increases, steering
toward the goal of creating a commercialized product.
Once the initial TPP is agreed upon, a QTPP canbe developed that focuses on the desired product characteristics and sets development goals. The QTPP is atactical implementation of the strategic vision outlinedin the TPP. As with the TPP, the QTPP may also definetargets for each stage of development. It establishes thecharacteristics that a product must possess to safely andreproducibly deliver the therapeutic benefit promised on
the final product label.
The QTPP should capture the entire body of knowledge about the product. This may include research

Chapter 3: Generation of Quality Target Product Profile

1. Define
QTPP

4. Devise
controlstrategy

2.
Identify
CQAs
3.
Establish
CPPs

experiences, available literature, small-scale development
activities, risk assessment and mitigation plans, regulatory requirements, and regulatory guidance. The QTPP
embodies the refined list of CQAs that enable productquality and consistency, and as the body of knowledge
expands, the QTPP should evolve, as clinical experienceand additional process understanding progresses andthe TPP matures. QTPP updates typically consider any
preclinical studies, first in-human (FIH) studies, Phase
I/IIa trials, and subsequent clinical trial data, as well as
product and process design changes, manufacturability,
route of administration, dosage form, key market-specificrequirements, and data from similar platforms.
Typical contents of the QTPP can be categorized into:• Drug Product Design Attributes: broadly defined as
those attributes that illustrate the physical descriptionof the drug product• Drug Product Quality Attributes: broadly defined as
those attributes that relate specifically to drug productsafety, identity, strength, purity, potency, or quality(SISPQ)
Table 3-1 lists the examples to consider for inclusion in
the QTPP.

A-CELL✼57

Drug product Quality Attributes

General Property

Table 3-1: Attributes to consider for a cell-based therapy product QTPP.
Category of Attributes

ExamplesTherapeutic Indication

Relapsed or Refractory Diffuse Large B-Cell Lymphoma, r/r Acute
Lymphoblastic Leukemia

Shelf life

years

Storage Conditions

2-8°C, cryopreserved in vapor phase LN2 <-130°C

Container Closure System

Bag, vial, sterile-sealed

Dosage Form

Liquid suspension, tissue equivalent, cryopreserved, fresh

Dose Regimen

Daily, monthly, single infusion

Delivery volume per dosemL, mL/kg

Method of administration

IV administration

General attributes

pH, osmolality

Appearance

Color, opalescence, visible particulates

SafetyMicrobial testing that, depending on the nature of the product, is likely to be
based on a multidimensional approach encompassing in-process and finalproduct testing

Identity

Tests to distinguish the specified cells through physical or chemical
characteristics of the cell line (i.e., phenotype, genotype, or other markers;
qPCR of transgene; tissue-specific gene expression)

Content

Total cell number, cell concentration (cells/ml), active (transduced) cells/ml

Purity and impurities

Tests to assess product purity, considering the product (e.g., live cells, deadcells, cellular impurities, residual vector, process-related impurities such as
residual media components, DMSO, anticoagulant)

Potency

Measure of the relevant product biological functions.
Methods to assess product biological activity are based on the different elements
involved with the mechanism of action (MoA), often multiple tests evolving from
specific markers in early stage to more functional assays at later stage.

QTPP for Cell-based Therapy
The starting materials and manufacturing processes ofa drug development program can have fundamental
effects on the quality characteristics of the final product.
Therefore, one of the purposes of the QTPP is to provide
information that readers can use to identify places andvariables in the production process where quality can becompromised or drift from the required quality standard.
Considering this, developers should aim to identify thepotential CQAs of the product early in the development
lifecycle. CQAs and, consequently, CPPs for the manufacturing process are product-specific and should be definedon a case-by-case basis; any available published data on
similar products should also be considered. Acceptancecriteria for CQAs and CPPs are defined through process

Chapter 3: Generation of Quality Target Product Profile

characterization. As the process is validated and moreclinical experience is gained, refinements in these
specifications can be made. Most production processes
for cell-based therapy products are still in early days ofdevelopment, but several key areas should be captured or
addressed by the QTPP.
Of note, cell-based therapy products are subject toconsiderable inherent variability of the cellular starting
material, which may have a further impact on the consistency of the manufacturing process and final product.
As a result, these potential inconsistencies could causea misalignment between quality standards and clinicalresults (safety and efficacy). It is important to clearly
establish a link between quality and the clinical performance of the product. Drug product quality attributes
for cell-based therapy products should consider safety-,

A-CELL✼58

potency-, purity-, identity-, and functionality-relatedparameters, which will be discussed in depth below.
Note that the details of the associated testing for these
attributes are discussed in Chapter 9 of this document.
SAFETY

Cell-based therapy products, being composed in part orin whole of cells, present several safety concerns based on
factors unique to cells, such as differentiation and proliferative potential, as well as the potential for local and systemic toxicities that could arise thereof. Additionally, cells
are subject to infection by adventitious agents, immune
response, variability based on formulation, and improperquality assessment due to potential deficiencies in donor
testing, particularly if used in an allogeneic application.
To address these risks, a safety profile that captures safetyparameters through categories such as microbial assurance, cellular impurities, non-viral and viral integrationsites, genomic instability, as well as manufacturing residuals, is an important component of the QTTP.
It should be noted that safety assessment for cell-based
therapy products can be challenging because of factors
such as inconsistent approaches to product characterization, inadequately defined product parameters that anticipate adverse events, and a lack of standardized approachesin evaluating in vivo host responses. These could result
from a poorly defined TPP or QTPP, which impacts CQA
and overall analytical strategy (control, characterization).
In particular, sterility of the final product is difficult, if not
impossible, to fully guarantee. This is because cell-based
therapy products cannot be terminally sterilized due totheir biologically active state; therefore, aseptic controls
and processing at the relevant stages of the manufacturing
pipeline are needed to maintain sterility. As a result, regulatory authorities such as FDA and EMA recommend theuse of closed manufacturing systems whenever possible.
One potential sterility assurance strategy is to perform
in-process sterility testing (of both media and drug substance/product intermediates) and only release products
for administration to the patient if pre-harvest testing
results are all negative. If in-process testing is difficult
or not possible, surrogate testing may also be performed(for example, testing the last liquid with which the cells
came into contact). Another solution is to use rapid assay
methods, such as gram stains, which, while not highly

Chapter 3: Generation of Quality Target Product Profile

sensitive, are quick and can detect gross contamination.
Lastly, since batch sizes can vary and, in some cases, yieldsmall amounts of materials for analysis, sterility testingthat matches regulatory guidelines in terms of sample size
volume is often not possible; in these cases, in-process or
surrogate testing should be performed.
Furthermore, the specific type of cell-based therapy
products can pose their own unique safety-related challenges. To illustrate some of the specific complications
that can arise when determining the safety profile, it canbe useful to consider the cases of human pluripotent stem
cells (hPSCs) and chimeric antigen receptor (CAR)-T
products. Both of these cases are briefly described below.

hPSCs
It is known that the contamination of a differentiatedcell therapy product with undifferentiated stem cells,
incompletely differentiated progenitor/precursor cells,
or functionally mature cells, all pose potential safetyconcerns for recipients of the final product. In thecase of hPSCs, expression characteristics of both stem
cell-derived and functionally differentiated cell-derived
products are present. Therefore, it can be seen that asingle cell therapy substance can pose the full rangeof safety risks that exist for a given safety parameter, acharacteristic that does not apply to many traditional
pharmaceutical or biologic products.

CAR-T
Given that CAR-T cell manufacturing hinges on theintentional incubation of patient-derived T cells (purified
from apheresis material) with viral vectors to deliver,
integrate, and express the transgene of interest, there
are several safety-related concerns during the manufacturing process. Among these are the potential forrecombination events leading to off target integration,
insertional mutagenesis, and the presence or creation of
replication-competent vectors.
With regard to the choice of viral vector, both gamma
retroviral vectors (γRVV) and lentiviral vectors (LVV)
deliver RNA that is reverse-transcribed into DNA.
However, their safety profiles are different, primarily
due to the ability of LVV to transduce non-proliferating
cells. Nonetheless, third-generation platforms offer thebest safety profile and commercial viability by ensuring

A-CELL✼59

efficient gene transfer and absence of replication-competent virus. For both viral vectors, the main safety concern
is in regard to potential insertional mutagenesis. Because
viral vectors mediate the integration of the genetic payload into the host cell genome, insertional mutagenesis
is a possible occurrence in all cases involving integrating
vectors. Insertional mutagenesis can, in turn, cause
oncogenicity, depending on the site of insertion. It has
been shown that lentiviral integration patterns favorsites away from risk-prone cellular promoters, while
gammaretroviral integration more frequently occurs
near transcriptional start sites. Therefore, the use of
lentiviral vectors can mitigate the potential for insertional mutagenesis. However, it should be noted thatthe potential for insertional mutagenesis also appears to
depend on the transgene, the promoter, and the target
cell type. Lastly, oncogenic potential may be increased
if replication-competent vectors are present in the final
vector process materials (all vectors to be used should be
replication-defective). Assays for the presence of replication-competent vectors (RCR or RCL) among productcells and media should be performed to ensure that the
final CAR-T product is safe for infusion into patients.
Additionally, it should be noted that, due to the mechanism of attack on cancer cells implemented via CAR-T cells,“bystander” cells may become targeted by CAR-T cell therapy products as an unintended consequence of treatment. In
particular, CAR-T cells may attack healthy B-cells, thereby
exposing some patients to increased risk for infections.
POTENCY

According to 21 CFR 600.3, potency is defined as the
specific ability or capacity of the product, as indicated byappropriate laboratory tests or by adequately controlled
clinical data obtained through the administration of the
product in the manner intended, to effect a given result.
Tests for potency consist of either in vitro or in vivo tests(or both) that have been specifically designed for eachproduct to indicate its functional activity (potency).
Potency assays include tests to indicate and confirm thebiological activities of the product and tests to measureactivity of all ingredients considered necessary for activity of the product. These tests can be direct measurements of biological activity (in vitro or in vivo depending
on product-specific attributes), surrogate measures

Chapter 3: Generation of Quality Target Product Profile(immunochemical, molecular, biochemical) that are
correlated to but not direct measurements of biologicalactivity, and assay matrix approaches (combination of
assays based on product-specific attributes).
The matrixed approaches to potency that are recommended for protein-based therapeutics are particularly
important for cell-based therapies. While there arefunctional assays in place for CAR-T products, thecomplex nature of the mechanism of action (MOA) for
these therapies presents a limitation to potency assay
development. Current cytokine release and cytotoxicity assays are often not predictive of the product’s
clinical outcome. It is therefore necessary to generate
and monitor data throughout the clinical life cycle
and use statistical methods and correlative analysis todetermine relationships between the product CQAs
and the clinical outcomes. This exercise allows for abetter understanding of the manufacturing variables
that impact product quality and clinical outcomes,
which enables the appropriate control strategies to be
implemented in the commercial setting.
PURITY

Provisions to remove possible contaminants or impurities
should be made early on in CMC development. As CMC
development progresses, an emphasis should be placedon validating manufacturing processes that either remove or introduce contaminants. Contaminants should
be removed or prevented in accordance with limits set
based on safety data from relevant preclinical and/or clinical studies. Common contaminants in final cell-based
therapy products include product-related impurities(which originate from the heterogeneous nature of thestarting cellular material for cell-based therapies) suchas of non-T cells, residual tumor cells, non-viable andnon-transduced T cells, and extracellular vesicles; as wellas process-related impurities such as cell culture media(especially in cases in which animal-derived supplements
such as fetal bovine serum are used), matrix components,
residual beads, residual reagents (e.g. DMSO), cellular
DNA, antibodies, product isoforms, helper viruses, viral
vectors or associated components (including empty capsids), and leachables and extractables from manufacturing components. Additionally, because of contingencies
such as batch mix-up or the accidental transduction of

A-CELL✼60

other cells, unintended cell populations also pose majorcontamination risks for both allogeneic and autologous
products. There are also risks posed by process substances such as contaminated reagents, cesium chloride(CsCl), and chromatography media.
IDENTITY

Protocols to test final product identity should be establishedearly in the CMC development process. Tests for identity
should be able to identify the product as designated on
the final container and package label. Tests should also beable to distinguish the product from any other productbeing produced and/or processed in the same facility. Somemethods for identity-testing cell-based therapy products
include (but are not limited to): flow cytometry, PCR, andgenome sequencing-based assays (such as those to identify
short tandem repeats (STRs)). The appropriate applicationof these methods will vary according to the particularitiesof the manufacturing context at hand.
STABILITY

Tests for stability should demonstrate that the productis within acceptable quality limits for the duration of theplanned clinical study. Stability testing should be donein the context of planned storage conditions, planned
formulation, manufacturing holding steps, stressed
conditions, shipping/transport, and clinical in-use holdsteps, and thus should prioritize testing the attributes
that could potentially change over time in cryostorage.
Acceptable limits and ranges for stability should beestablished incrementally through the stages of CMC
development, with data from preclinical lots in Phase I
informing initial limits, and further refinement during
Phase II and Phase III (especially with ongoing datacollection for setting the expiry date of the product andrequired/recommended storage conditions).

Evolution of the QTTP during
development
Over the course of product development, changes tomanufacturing or processing stages are likely to occur.
While it is impossible to know the full range of changes
that could occur, the QTPP should aim to highlight atleast some of the most likely areas at which changes could

Chapter 3: Generation of Quality Target Product Profile

occur and establish possible steps that could be taken to
maintain product quality. Elements of the cell-based
therapy product development process subject to possible
change include: ancillary materials (cytokines, antibodies,
media, etc.), vector supplier, manufacturing site, formulation/concentration, analytical procedures, and prospectsof scale-up. When assessing changes in manufacturing,
establishing knowledge/understanding of the impact ofspecific process steps or unit operations on a particular
CQA is critical because even small changes could resultin unpredictable effects on safety, efficacy, stability, andother aspects of cell-based therapy product quality.
Cells often vary greatly in different parameters that
could impact target product quality. These parameters
include (but are not limited to): growth potential, cellsample composition, genetic stability, and phenotype. It isparticularly important for developers of cell-based therapy
products to be able to define CQAs early in the development process so that the relevant factors can be identified
and addressed when/if they appear during the course of
production. To this end, developers of cell-based therapy
products should consider factors such as morphology,
phenotypic markers, potency, and genetic stability. It is also
important to define the maximum population doublings(PDs), both for autologous and allogeneic cell-based therapy products, because any degree of cell-count variability inthe final product may result in variability in the nonclinicaland clinical results. A major aim with regard to developing
an effective QTPP should be to monitor these factors as
the development program evolves. Diligent tracking ofthese factors will, in turn, allow the developer to update
the QTPP according to new findings if necessary.
Some other factors that warrant specific commentarywith respect to the in-process evolution of cell-based
therapy product development programs are processand product-related impurities in autologous cell-based
therapy products and changes in target and non-target
cells during expansion. Impurities can present special
challenges for developers of autologous cell-based therapy products when defining CQAs that can be used as
standards for updating the QTPP because, though it iscommonly assumed that there are no cellular impurities
in the autologous cell-based therapy product (because thesource donors and patients are identical), the administration of the cells does not always follow the physiological

A-CELL

✼61

place and magnitude; higher cell doses may be given to
unconventional places where the presence of certain cell
types may be harmful in terms of safety or may even lower
efficacy. Therefore, clinical experience must be used to
inform and update the CQAs that define purity-related
quality standards. With respect to changes during expansion, cells are especially subject to change during expansion
when growth factors and cytokines are used to drive highproliferation. In order to address this tendency, developers
should establish cell selection and isolation steps in the
production process (particularly if the starting material isheterogeneous and the final cell composition is critical).
More generally, optimization and process validation
studies should always be used to provide a clear rationale
behind all changes made to processing and manufacturing
steps. Differences in processing and manipulation conditions should be evaluated and validated with commercial
production in mind. Additionally, when the manufacturing
process is changed, the risk assessment must be repeatedin order to identify whether the given change is a minor or
major one and to consider the possible impact on the quality, safety, and efficacy of the final product. In many cases,
it may be sufficient to demonstrate that the product has notchanged on a quality level as defined in the QTPP. For therequired comparability studies, one should utilize not only
release test methods, but also extended characterizationand comparison of in-process controls. It is also importantthat the changes do not impact the stability of the product.
If the quality of the product does, in fact, change, additional
clinical studies may be required to demonstrate unchanged
safety and efficacy. As new data accumulate throughout
the development process and new CQAs are identified,
the QTPP should be updated in order to assess the needfor comparability studies and to evaluate possible gapsthat may need to be filled before further clinical studies
or submission of the BLA. The QTPP enables a proactive
approach to product design as any process changes would
need to result in targets defined within the design space
documented in the QTPP, ensuring process modifications
are implemented based on no changes to product quality.
An example of an early, pre-IND QTPP for an autologous CAR-T product is presented in Table 3-2. Note thatthe values are demonstrated by units of measurements, but
no targets are prescribed as these are heavily dependent oneach product’s individual modality and target indication.

Chapter 3: Generation of Quality Target Product Profile

During later stages of development when sufficient
clinical data has been gathered, correlation between
CQAs and clinical efficacy and safety can be made. This
correlative analysis exercise can be the basis of justification
for widening the product specification acceptance criteria,
which can then be used to update the QTPP throughout
the product life cycle. As discussed earlier, product impurities (e.g., non-viable cells, presence of other cell types)
can present challenges for cell-based therapy products
since their presence may impact the product safety or
lower efficacy. Using clinical experience to inform andupdate the CQAs that define purity-related quality standards is therefore critical. For example, in tisagenlecleucel,
cell viability that is below the specification (80%) has beenshown to not compromise its clinical safety or efficacy inpatients with acute lymphoblastic leukemia, non-Hodgkin lymphoma,3 or diffuse large B-cell lymphoma.4 By
expanding this specification to the broader range observedin the clinical data, delay of product release caused byunnecessarily stringent specifications, and consequently
delivery to patients, can be prevented. The QTPP should
then be updated to reflect this new design space of the
product based on no changes to product quality.

QTPP for allogeneic cell-based
therapy products
The QTPP for an allogeneic product can differ substantially from that of an autologous product as allogeneic
cell therapy products possess a few unique additional
attributes. Such additional attributes can be safety-related as allogeneic products in general pose more safetyconcerns than autologous products, particularly withregard to presence of adventitious agents that may bepresent in the donor material. To that end, the QTPP ofan allogeneic product may include attributes reflecting
the presence of Relevant Communicable Diseases andDisease Agents (RCDAD), such as HIV 1 and 2, Hepatitis
B virus (HBV), Hepatitis C virus (HCV), and Humantransmissible spongiform encephalopathy (TSE), among
others. Consequently, in addition to robust control andtesting strategy, the allogeneic approach requires additional factors to ensure safe administration to patients,
such as donor screening and testing. Additionally,
genomic stability is important to monitor in allogeneic

A-CELL✼62

Table 3-2: Example QTPP for autologous CAR-T (pre-IND).
General Properties
Property

TargetIndication

Hematologic malignancies

Geographic scope

Global: test to USP, EP, JP compendia where possible

Shelf life

12 months at intended storage conditions

Storage conditions≤-120°C (vapor phase liquid nitrogen)Container closure

Selected to support product compatibility and stability and to ensure sterility (cryobag)

Delivery volume

Variable based on patient weight

Method ofadministration

IV administration
Product Attributes

Attribute Category

AttributeTargetGeneral attributes

pH

Appropriate for the formulation
to support product stability andcompatible with the route ofadministration

Osmolality

Appropriate for the formulation
to support product stability andcompatible with the route ofadministration

Color / opalescence

Colorless to yellow

Visible foreign particles

Negative

Dose range

XX CD3+ transduced cells perweight- or XX ml/kg body weight

Cell growth profile andtransduction efficiency
ranges to be determinedfrom dose needs

Cell concentration

XX cells/ml

Automated cell counter

Product / container
volumeXX ml

Volume to be determined tosupport the dose

In-use stability< XX minutes

Stable at room temperature
before infusion

Sterility

Negative

USP <71> / Eur Ph 2.6.27

Mycoplasma< XX

USP <63> / Eur Ph 2.6.7

Endotoxin< XX

USP <85> / Eur Ph 2.6.14

Bacterial contamination

Negative

Gram staining

Replication competent
lentivirus (RCL)

NegativeqPCR / ddPCR

Vector copy number (VCN)

< XX

qPCR

% CAR+ CD 3+> XX

Flow cytometry

Appearance

Content / strength

SafetyIdentity

Method / Justification

continued on next page
Chapter 3: Generation of Quality Target Product Profile

A-CELL✼63

Table 3-2: Example QTPP for autologous CAR-T (pre-IND)
continued from previous page
PurityPotency

Residual beads< XX beads/mL

Microscope imaging

Residual BSA / serum< XX pg/ml

ELISA

Cell viability> 70%

Automated cell counter% B cells< XX

Flow cytometry

Product related impurities(% NK cells, % monocytes,
etc.)

< XX

Flow cytometry

Cytotoxicity> XX% CD19+ cell apoptosis

Cell killing

IFN-γ cytokine release

Report result

ELISA

cell-based therapies as long-term culture of cells canalter the genomic composition and lead to chromosomal
abnormalities, which in turn can affect protein expression, cell function, safety, and purity. The QTPP for an
allogeneic product can therefore include characterizationof changes in genome structural or point mutation and/or deletions. Overall, a risk-based approach should betaken for selection of attributes to characterize for aparticular product to best inform the product development strategy. For discussion on qualification and testingof starting material and components, donor eligibility
determination, as well as appropriate product testing
and characterization for allogeneic cell-based therapy
products, refer to Chapter 5. Additionally, to establish
histocompatibility for users of allogeneic products, human leukocyte antigen (HLA) testing may be performed
as part of the product identity tests. Discussion on HLA
matching is presented in Chapter 2. The methods andtimepoints for genomic characterization in allogeneic
product manufacturing are discussed in Chapter 9.

Conclusion
In closing, as many cell-based therapy products proceed
toward late-stage development and BLA-enabling activities, it is going to be increasingly important to takelessons from biologics and vaccines in applying a Quality
by Design approach in product development. One of themost important things when taking and implementing
QbD principles in cell-based therapy is to establish the
QTPP early during development. The importance of a
QTPP cannot be overemphasized as it, as well as the next
step—CQA identification, dictates the rest of the product

Chapter 3: Generation of Quality Target Product Profile2

development activities. Being able to define the QTPPand CQA early enables product developers to manage
comparability across products and process changes.
Various risk-based approaches are used throughout
the product development to establish manufacturing
processing operations and define the control of criticalquality attributes, the control of raw materials, and theareas for further investigation. Such assessments are
performed by a cross-functional team of subject matter
experts to ensure that the process delivers a productthat satisfies the QTPP in a reproducible manner.

References
1

International Conference on Harmonization (ICH) Q8(R2):
Pharmaceutical Development - Guidance for Industry. November 2009. https://www.fda.gov/regulatory-information/
search-fda-guidance-documents/q8r2-pharmaceutical-development

2

FDA Guidance: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic
Cell Therapy Investigational New Drug Applications (INDs).
Section IV,E,2. April 2008.

3

Pasquini MC, Hu ZH, Curran K, et al. Real-world evidenceof tisagenlecleucel for pediatric acute lymphoblastic leukemia
and non-Hodgkin lymphoma. Blood Adv. 2020;4(21):54145424. doi:10.1182/bloodadvances.2020003092

4

Chong EA, Levine BL, Grupp SA, et al. CAR T cell viability release testing and clinical outcomes: is there a lower limit?. Blood. 2019;134(21):1873-1875. doi:10.1182/
blood.2019002258

A-CELL✼64

CHAPTER 4

Risk Assessment andCritical Quality Attribute
Identification
CHAPTER LEAD

Pinar Cicalese, Immatics
Laure Rittié, GlaxoSmithKline
CHAPTER CONTRIBUTORS

Nolan Polson, GlaxoSmithKline
Max Tejada, Kite PharmaChapter 4 | Contents
Definition of a Critical Quality Attribute....................... 66

CQA Life Cycle Management......................................... 73

Definition of a quality attribute................................. 68

Phase appropriate CQA determination.................... 74

Types of quality attributes....................................... 68

Correlation of CQAs to clinical outcomes................ 75

Defining measurable quality attributes.................. 68

Risk assessment throughout the productlife cycle...................................................................... 75

Risk Assessment as Basis of Criticality
Determination.............................................................. 69

Mapping CQAs to CPPs.................................................. 76

Definition of risk......................................................... 70

Identifying and setting acceptable ranges
for CPPs................................................................................. 77

Tools for risk assessment........................................... 70
Risk ranking.............................................................. 70
Preliminary hazards assessment (PHA).................... 71
Examples of uncertainty and severity
determination based on prior knowledge.................... 72
Cell extrinsic expansion rate................................... 72
Residual beads process impurities......................... 72

Case Study: Risk Assessment and CQA
Identification for Autologous CAR-T Product.............. 78
The use of risk assessment tools
to establish CQAs................................................. 78
Conclusion....................................................................... 78
References....................................................................... 79

Media supplement impurities................................. 73
Due to similarities in risk and criticality assessment approaches
across various types of cell-based therapy products, some of theoverall content and flow of this chapter was based on, but adaptedfrom when applicable, Technical Report No. 81: Cell-Based Therapy
Control Strategy, a document published by the Parenteral Drug
Association (PDA).

The authors of this A-Cell chapter acknowledge the work of theauthors and editors in constructing the PDA document.
For further details on the PDA document, please refer to:
Parenteral Drug Association. Technical Report No. 81: Cell-Based
Therapy Control Strategy. https://www.pda.org/bookstore/productdetail/4638-tr-81-control-strategy-for-cell-therapy. February 2019.

Definition of a Critical Quality Attributeare important to generate clinical data, demonstrate lotto-lot consistency and in-process attributes, determine
relationships between product quality attributes andsafety and efficacy, support meaningful specifications
for release, and show product comparability after
manufacturing process changes. Developing a goodunderstanding of the inter-relationships between CQAs
and clinical outcomes and between CQAs and criticalprocess parameters (CPPs) is fundamental to the implementation of an effective control strategy. Ideally thiswill result in predictable process performance, lot to lot
consistency, and knowledge of the potential impact ofany planned changes.
As discussed in Chapter 3, CQAs are defined during
the drug development process. Initially, a quality target
product profile (QTPP) is drafted that describes thedesign criteria for the product, and from this an initial

Cell therapy product manufacturers must ensure that thequality of their products are acceptable with respect topatient safety and efficacy (depending on stage of development) and are consistent with regulatory requirements
for the drug product (DP) and drug substance (DS).
Critical quality attributes (CQAs) define the desired
product quality of the manufactured product and aredefined in International Conference on Harmonization(ICH) Guideline Q8(R2) as a physical, chemical, biological, or microbiological property or characteristicthat should be within an appropriate limit, range, or
distribution to ensure the desired product quality.1
CQAs are related to resulting patient safety and product efficacy and are therefore used by manufacturers toassure suitability of each product batch for use. CQAs

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼66

Figure 4-1: CQA considerations during development

Identify quality attributes that impact
safety, potency, purity, identity

Control Strategy:• Process parameter controls• Material attribute controls• Procedural controls• Testing controls

CQAs

Process
Characterization

Understanding functional relationships
between process and CQA provides foran enhanced approach

list of quality attributes is defined. Quality attributes
are identified and scored in relation to their impact on
efficacy and/or patient safety. This is typically performed
by a cross-functional team through a risk assessment todefine and rank the quality attributes as critical (CQA),
potential (pCQA), or non-critical (non-CQA). Risk assessments are also used to link material attributes and processparameters to the drug product CQA. Along with processcharacterization studies, the accumulated knowledge canbe used to identify critical process parameters (CPPs),
define an initial control strategy, and confirm its suitability
through Process Performance Qualification (PPQ).
Understanding product CQAs is perhaps the mostcritical aspect for establishing a suitable manufacturing
process as well as establishing process controls for assuring
product quality and consistency. CQAs serve as benchmarks to enable the selection of operational ranges. Most
importantly, control of CQAs is used to ensure that every
batch of final product is as safe and effective as possible.
Figure 4-1 summarizes the key considerations related
to defining CQAs with respect to the overall development process. To define CQAs, first all quality attributes
that could affect safety, potency, purity, and identity
are identified. It is important to establish these earlyin development as this is critical to managing changes
during development. For example, it is very common
to start a manufacturing process with a research-based
process up to Phase I, and upon realizing the need for

scaling, transition to a different manufacturing platform.
As CQAs relate to patient safety and efficacy, detrimental
impacts to CQAs can predict a detrimental impact toclinical outcomes. Changes to CQAs could also resultin the conclusion that batches of product post-change
are not comparable to batches pre-change and therefore
the inability to group clinical data. Therefore, it is highly
advisable to create the QTPP and initially identify CQAsprior to first-in-human studies, and to test these CQAs
from an early stage of clinical development.
Various risk-based approaches are used throughout the
manufacturing process to establish processing operations
and define the control of CQAs, the control of raw materials (including critical material attributes), and the areas
for further investigation. Risk assessments are performed
using scientific understanding and experience and productknowledge to identify areas that pose a risk of failure todeliver a quality product that satisfies the QTPP. Risk
assessment will inform a prospective process characterization plan, i.e., the high-risk parameters and materials
that should be investigated in process characterization.
Process parameters are defined as critical or non-critical based on study data and additional operational
information. These will then be followed by an initial
control strategy derived from current product and process understanding, which includes process parameter
controls, material attribute controls, procedural controls,
and testing controls, as discussed in Chapter 10.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼67

DEFINITION OF A QUALITY ATTRIBUTE

DEFINING MEASURABLE QUALITY ATTRIBUTES

A quality attribute is a distinctive characteristic that
relates to the safety, identity, purity, potency, or stabilityof a product. Refer to Chapter 3 for a discussion about
these product attributes.
Cell-based therapy products are generally more complex than biopharmaceutical products, and therefore,
potentially have more quality attributes that must be
controlled to minimize their potential impact on safety
and assure efficacy. It is not feasible to perform a comprehensive assessment of all possible attributes; therefore,
selecting the right attributes to control during manufacturing is of paramount importance to developing a viable
and effective control strategy.2 To define which attributes
are critical and necessary to the control strategy, all relevant quality attributes should be considered and ranked
during risk assessment. The identification of a CQA does
not indicate that a test will be performed to assess that
CQA. Some CQAs may either be highly challenging totest analytically and therefore only feasible to study using
non-clinical and/or clinical studies (e.g., stem cell engraftment), or it may be unnecessary to test the CQA since
the manufacturing process design will always control the
CQA. Manufacturers must grade their product CQAs byranking the attributes that have the most influence on thesafety and efficacy of the product first.

Identification of CQAs is a multidisciplinary effort
utilizing all available sources of relevant information,
including scientific publications and pre-clinicaland clinical data, in conjunction with formal risk
assessments.2
Identifying potential CQAs is an integral part ofmanufacturing process development. With progressive clinical development and correlational analysis
of quality attributes, measures of product safety andefficacy ultimately provide evidence as to whether aproduct attribute affects the clinical outcome.2 Due tothe complexity of cell and gene therapy and the lowpatient numbers for some rare diseases, this might
only be achieved during commercial use of a product;
therefore, use of a risk-based analysis is recommended to identify CQAs.2 Analyzing the criticality of thequality attributes early in the development cycle will
enable the development of controlled manufacturing
processes, defining the control strategy for clinicalmanufacturing, and testing for process development
and comparability studies.2
It is recommended to have the QTPP defined so its
content can serve as the basis for establishing potential
CQAs.2 As a starting point for a systematic approach,product attributes are grouped into categories based onclassification. A comprehensive list of quality attributes
should be generated.2 Examples for these categories
for cell therapies and genetically modified cell-based
therapy products are presented in Table 4-1.
After grouping, each quality attribute (e.g., percent
cell viability, percentage of cells with the desired phenotype, residual cell culture media components such as
bovine serum albumin [BSA]) should be assessed for
its risk (including severity and uncertainty) to productquality, patient safety, and intended mode of action (efficacy). This assessment should be supported by reliable
scientific data. If experimental data is unavailable or
unreliable, coherent scientific reasoning that addresses
the potential impact of a given attribute (i.e., examining
worst-case outcomes) must be provided.2

TYPES OF QUALITY ATTRIBUTESThe QTPP serves as the starting point for establishing CQAs. Manufacturers start by grouping productattributes into categories based on classification (e.g.,
appearance, identity, safety, strength, purity, potency) togenerate a comprehensive list of quality attributes. CQAs
are distinct from tests; there may be tests that are capableof assessing multiple CQAs, and conversely, multiple tests
may be required for a single CQA. However, the name for
the CQA and test may be interchangeable, e.g., sterility,
bacterial endotoxins.
With the variety of DPs, each with their unique characteristics, it is not easy to provide a simple solution for
defining and measuring quality attribute that will fit allcases. Refer to Chapter 9 for more information on someof the tools available to measure quality attributes forcell-based therapy products.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼68

Table 4-1: Example attribute categories for cell-based therapy products
Category

Attributes for cell therapy product

Additional attributes for geneticallymodified cell-based product

SafetyBacterial endotoxins, sterility, mycoplasma,adventitious viruses

Replication competent lentivirus (RCL),
vector copy number (VCN), off-target edits

Identity

Expression of surface markers specific to theintended cell phenotype (e.g., T-cell marker CD3+)

Confirmation of genetic modification (e.g.,
CAR expression)

Purity andimpurities

Cellular impurities, dead cells/cell viability, processrelated impurities (e.g., residual media components,
DMSO, anticoagulant)% untransduced cells, residual vector

Potency

Functional response, biological activity

General attributes pH, osmolality
Appearance

Color, opalescence, visible foreign particles

Content (Quantity, Total cell number, cell concentration, viability
strength)

Risk Assessment as Basis of
Criticality Determination
Risk assessments serve as the basis for defining controls
and developing a strategy for managing risk. Assessing
risk follows a systematic approach to support risk decisions using accurate, analyzable, and well-organized
information. Whether part of a formal risk management
strategy or not, risk assessments are also integral to anorganization, because they help increase the scope andaccuracy of its institutional knowledge of the risks andhazards within its own processes, systems, and operational business models. Organizations should maintainformal documentation describing their quality risk
management processes at the level determined throughtheir risk assessment.
Managing risk as it relates to quality is generally basedon a structured set of processes that allow manufacturers
to assess and control risks throughout the life cycle of a
product.
The first step to assessing risk involves identifying all
possible points of failure that exist in the current manufacturing process. Manufacturers should use all factors
available to them, such as historical data, theoretical

Quantity of genetically modified cells

analysis, and the informed opinions of experts.
After identifying possible sources of risk, manufacturers must perform a risk analysis using qualitative andquantitative methods to estimate the amount of risk due
to each source. Associations can be established for the
probability that a harmful event occurs, the severity ofsaid event, and the ease of detecting the harmful event.
If data are scarce or if the project is in early or preclinicalstage, efficacy and safety risk can be identified based
mainly on severity/detectability only. In these cases, it isrecommended to add an uncertainty rating as these risks
may be theoretical.
The final step to assessing risk is to perform a risk evaluation where the results of the identification and analysis
steps are compared, and proper context is added to eachrisk with respect to the complete manufacturing process.
Risk assessment is an ever-evolving process that starts
with bench research, develops through the clinical stages,
continues through product approval, and finishes withcommercial manufacturing. With a wide array of entities
involved throughout the risk assessment process, ongoing communication related to the existence, probability,
severity, acceptability, and detectability of risks withothers in the industry and regulators is vital.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼69

DEFINITION OF RISKIn cellular therapy, as in other biopharmaceutical manufacturing contexts, patient safety is a primary concern.
Risk is the metric used to assess the potential amountof danger a situation, variable, or product may pose to apatient, with a focus on safety as a chief concern. The goalof risk assessment is to minimize the potential for harm
to patients while maximizing the therapeutic benefits. It
should be noted that some safety attributes of the cell-based
therapy such as sterility, endotoxin, and mycoplasma canbe considered obligatory and therefore do not need to berisk assessed. Efficacy risks must also be considered, although their severity is often lower than that of safety risks.
Attributing risk to any given situation may not be
straightforward when factoring in business- and operations-related matters. One such factor is the multitudeof stakeholders involved in decisions that shape the
manufacturing program development, as each stakeholder bringing their own subjectivity can lead to varying
degrees of uncertainty and severity. These diverse influences may result in inaccurate or disproportionate risk
estimates, potentially exposing patients to side effects orineffective therapies.
TOOLS FOR RISK ASSESSMENT

Risk assessment for identification of CQAs follows theprinciples outlined in ICH Q9, which defines risk analysis as “the qualitative or quantitative process of linking
the likelihood of occurrence and severity of harms.” It

is often unclear whether an attribute listed in the early/
pre-IND QTPP is critical; risk assessment can informwhether data generation is needed to determine thatcriticality. Several scoring methods can be used to assess
the criticality of a particular attribute.

Risk ranking
Criticality assessments can utilize qualitative or semiquantitative measurements. A quantitative risk assessment requires describing and ranking the risk. Several
scoring scales exist to assess the criticality of a particularattribute. For products where no previous knowledge
or understanding is available, a simple system can be
implemented.
Severity x Uncertainty scoring

Severity refers to how significantly the attribute would
impact product safety or efficacy. Severity scoring is particularly helpful in situations where the range of risks andthe potential consequences to be managed are diverse anddifficult to compare using other methods. Severity scoring
has the advantage of allowing for both quantitative andqualitative assessment of risks within the same organizational framework. If severity scoring is properly appliedat key points throughout the product life cycle, starting at
the pre-IND phase and through to licensure and post-approval, developers will be able to identify the attributes
that pose the highest levels of risk, and therefore will beable to implement effective precautionary measures andTable 4-2: Example criteria for impact (severity) scoring of product attributes2
Severity

Value

ImpactLow

1

Variability in attribute has minor or negligible potential for decreased safety/efficacy.
Marginal patient impact or minor transient adverse effects are expected based on historicalexperience.

Medium3

Variability in attribute may have moderate potential for decreased safety or efficacy within
the clinical history of the product. Attribute may result in manageable adverse effect, but
significant patient impact is improbable.

High

10

Variability in attribute may have potential for severe effect on patient. Potential significant
change in safety/efficacy or risk/benefit profiles. May result in a serious (reversible orirreversible) adverse effect.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼70

Table 4-3: Example criteria for uncertainty scoring of product attributes
Uncertainty

Value

Prior Knowledge

Pre-Clinical Studies

Clinical Studies

Low

1

Well characterized effect based on extensive inhouse data (in vitro, in vivo, or clinical). Large bodyof knowledge in the literature for a similar class of
products.

Demonstrated
relevance of animalmodel results.
Extensive in vitro andin vivo studies for thisproduct.

Significant
clinical
experience
with thisproduct.

Medium2

External published literature available on similarclass of products. Well characterized effect known.
Some available internal data (in vitro, in vivo, or
clinical) from this or similar class products.

Only limited in vitro orin vivo data available
for this product.

Only limited
clinical
experience
with thisproduct.

High

3

Limited scientific understanding, no publishedexternal scientific literature and no internal datafrom this or similar class products.

No data available forthis product.

No dataavailable forthis product.

mitigation strategies. Descriptions used to define severity
are shown in Table 4-2. Severity may be scored as low (1),medium (3), or high (10).
Uncertainty refers to the level of confidence in assessing the criticality of an attribute. As cell and gene therapy
products (CGTPs) are a novel class of therapy, and theliterature and in-house experience of the sponsors areconsequently small when compared to the large body ofknowledge available for pharmaceutical proteins, uncertainty levels are expected to trend higher.2 Uncertainty
may be scored as low, medium, or high. Those attributes
for which there is limited knowledge (i.e., high uncertainty) should be the subject of characterization studies
or a relevant clinical study.2 Descriptions used to define
uncertainty as it relates to the level of confidence inunderstanding an attribute are shown in Table 4-3.
After assigning severity and uncertainty scores, thetwo are multiplied together to assign an overall attributecriticality, or risk score. Table 4-4 shows an example CQA
classification matrix. Note that there are multiple ways
to assign an attribute as critical versus potentially critical
based on this example matrix. Risk must be determinedby the organization, depending on the level of risk it iswilling to accept.2
The resulting list of attributes classified as CQAs andpCQAs (potential CQAs) is used during early development
and reviewed and revised periodically during accumulation of the product knowledge based on the availability ofclinical and product characterization studies across the life
cycle of the product.2

Preliminary hazards assessment (PHA)
As defined by ICH Q9, the preliminary hazards assessment
tool is a risk assessment approach based on applying priorexperience or knowledge of a hazard or failure to identify
future hazards, hazardous situations, and events that might

Table 4-4: Example product attribute criticality assessment outcome

Severity

Uncertainty
1 (Low)

2 (Medium)

3 (High)

1 (Low)

1 (non CQA)

2 (non CQA)

3 (pCQA)

3 (Medium)3 (pCQA)

6 (pCQA)

9 (pCQA)

10 (High)

10 (CQA)

20 (CQA)

30 (CQA)

Table adapted from PDA Technical Report No. 81; Cell-Based Therapy Control Strategy, with permission of PDA, Inc.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼71

cause harm, as well as to estimate their probability of occurrence for a given activity, facility, product, or system.3
The PHA consists of: 1) identifying the possibility that therisk event will happen, 2) the qualitative evaluation of theextent of possible injury or damage to health, 3) a relative
ranking of the hazard using a combination of severity
and likelihood of occurrence, and 4) the identification of
possible remedial measures.
Similar to risk ranking, PHA is based in part on severity,
but unlike risk ranking, PHA uses likelihood as the otherparameter instead of uncertainty. Likelihood refers to theprobability that, should a quality attribute stray outside of
accepted ranges based on the most recent understanding
and knowledge about the attribute (drawn from literature,clinical, and non-clinical studies relevant to the product inquestion or similar products), the occurrence will affect thesafety or efficacy of the product. When limited clinical data
are available for a particular quality attribute, likelihood is
to be assessed conservatively. In a similar way to how risk
ranking scores are determined, the risk priority number(RPN) of PHA is calculated as Severity x Likelihood. The
direness of the risk posed by the attribute in question canthen be determined based on its relative placement along
the priority scale compared to the other attributes being
assessed. Because the assessment of likelihood depends onprior knowledge, PHA is particularly useful when performing risk assessment in existing systems.
PHA is most commonly used early in the developmentof a project, at a time when there is little information on
design details or operating procedures. Thus, results of PHA
can inform process and facility design, as well as serve asa pointer for further study of quality attributes using otherrisk management tools.
Other risk assessment tools are available, such as the
Failure Mode and Effects Analysis (FMEA) as referred
to in ICH Q9. FMEA provides a method to evaluate potential failure modes for processes and their likely effecton outcomes or product performance and is calculated as
Severity × Occurrence × Detection. Since it requires scoringfor occurrence and detectability, FMEA relies on productand process understanding and is typically performed inlater stages of development (e.g., during the control strategy
stage) where data is available to predict occurrence anddetectability. FMEA can be applied to equipment andfacilities and might be used to analyze a manufacturing

unit operation and its effect on product or process. It
identifies elements/operations within the system that renderit vulnerable. The output/results of FMEA can be used asa basis for design or further analysis or to guide resource
deployment. PDA Technical Reports Nos. 44, 54, 54-4, and60, which discuss quality risk management and productlife cycle management, also offer examples of suitable risk
assessment tools and explain their use.2
EXAMPLES OF UNCERTAINTY AND SEVERITY
DETERMINATION BASED ON PRIOR KNOWLEDGE

Cell extrinsic expansion rate
For autologous CAR-T cells, each batch corresponds toa different donor, and is therefore characterized with cell
growth rate that is patient-dependent and may greatly
vary between batches. Cell expansion rates will ultimately
influence the cell yield and viability of the final product,both being CQAs. The quantity (dose) of active substance(CAR-T cells) needed for a positive clinical outcome andto minimize risks to patient safety will require an understanding of multiple factors including the mechanismsof action and intended patient population.
To achieve a target cell number and meet the dose
size requirements, different cell seeding densities may be
implemented during the CAR-T therapy manufacturing
process. However, there may be an associated high levelof uncertainty due to limited prior knowledge of the correlation between various cell culture parameters that may
influence cell expansion rate and the actual cell growth.
Given this uncertainty, setting too tight of a productspecification on final cell yield and viability poses a riskof the product not meeting the specification.4
The decision on seeding density may rely on priorknowledge on cell size, cell composition (% of each celltype), and patient health and prior therapy.5 Developing
prior knowledge linking culture conditions to cell type
behavior in culture is key to developing process controls
and predictability for the CQAs.6

Residual beads process impurities
During the manufacture of some cell-based therapies,
such as CAR-T cells, reagent-coated beads are commonlyused (refer to Chapter 8 for details on the use of beads inthe activation step of CAR-T cell manufacturing). These

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼72

beads are considered ancillary materials used in the cell
production process. Thus, sufficient bead removal or
reduction has to be demonstrated prior to administrationof the final product to patients. Residual beads could
elicit a pharmacological or toxicological effect in thepatient, e.g., due to unwanted T-cell activation in vivo,7thus posing a risk to patient safety. Therefore, limits areoften set for residual bead counts as part of the control
strategy.8
While there are no standard acceptance criteria forresidual bead impurity in the final DP due to the various
types of beads (and reagents), broad range of indications,and method of delivery, studies for CAR-T production
typically report >5-log depletion of beads throughout the
manufacturing process to establish proof of safety.9,10 To
determine the criticality of residual bead impurity to the
product CQA, an uncertainty score of 2 can be assigned
due to the presence of some data in the literature. Due toreported acute toxicity of residual beads in intravenous
safety studies in animal models,11 a severity scoring of
10 can be assigned.

Media supplement impurities
Effective cell therapies are dependent on optimal cell
culture conditions, including the culture media selection.
Choosing an appropriate medium that is conducive tothe activation and expansion of T cells, as well as transduction with the CAR construct, is key to maintaining
cell health and producing efficacious CAR-T products.
Traditionally, media supplemented with serum (fromanimal or human origin) are widely used in the manufacturing process, but these supplements come with a rangeof challenges. Serum can vary in quality (critical material
attributes) between batches due to its complex composition of a large number of constituents, and possibly resultin inconsistency of the final CAR-T product efficacy.12
In some cases, high concentrations of serum have beenshown to inhibit cell growth.13 Additionally, componentsof serum may also introduce adventitious agents into the
production process, inhibit genetic modification such astransduction, resulting in adverse effects in the patientsuch as hypersensitivity reactions.
In CAR-T therapies, it has been shown that infusionof CAR T cells with less differentiated phenotypes (e.g.,naive-like or central memory T-cells) is associated withprolonged in vivo persistence and superior anti-tumor
effects, at least for liquid tumors.14,15,16,17,18 Several studies
have indicated that such anti-tumor function is dependent on the serum composition in the media. In one
study where three media supplements were evaluated,i.e., fetal bovine serum (FBS), human AB serum (ABS),and human platelet lysate (hPL), CAR-T cells cultured in
hPL-supplemented media exhibited the least differentiated T cell phenotype and displayed superior proliferative
and anti-tumor effects in vitro and in a mouse xenograftmodel.19 Another study compared human serum to axeno-free concentrated growth factor from whole blood
fractions, and reported that cells expanded in the latter
showed enhancement of transduction in activated T
cells, as well as superior engraftment in a neuroblastoma
model and potency in vivo.11
These studies highlight the potential criticality ofserum concentration in culture media on overall potencyof CAR-T therapies. In this example, due to the presenceof some data in the literature, an uncertainly score of 2
can be assigned, and due to the potential risk to patient
safety from serum impurities, a severity scoring of 10
can be assigned. If the potency assay and metric used inthese published studies are not relevant to the product indevelopment, additional data can be generated to properly evaluate the effect of serum concentration on theintended potency using a Design of Experiment (DOE)
approach.

CQA Life Cycle Management
It is critical to establish the product CQAs as early as possible since this is the foundation for a rational, risk-based
approach to product development and product life cycle
management. Early determination of CQAs allows earlydata collection. This approach should provide knowledge
and experience for product understanding and the ability
to adopt a risk-based approach to planned and unplannedchanges (comparability assessments) essential for efficient
life cycle management. The expectations of regulatory
agencies for the content of clinical trial applications,marketing applications, and post-submission changes
are consistent with this understanding of risk.
CQAs are dynamic and should be continuously reevaluated and revised throughout development,20 as driven

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼73

Figure 4-2: Systematic and iterative approach to identifying clinically relevant CQAs

Figure adapted from PDA Technical Report No. 81; Cell-Based Therapy Control Strategy, with permission of PDA, Inc.

by increased product knowledge. The cycle of knowledge
starts with identifying potential critical quality attributes(pCQAs) and subsequently defining these as either criticalor non-critical as experience is gained. A pCQA that was
initially not proven to be critical can later be reevaluatedbased on further information or drug product experience.
If found to not have any direct impact on the quality of
the final drug product, these can be dropped out during
development. This is also pertinent to the re-assignmentof criticality to process parameters.
PHASE APPROPRIATE CQA DETERMINATION

Throughout development, knowledge of what is a CQA
will evolve based on development history and increased
product understanding as more robust characterization is
implemented. The FDA recognizes that identifying product-specific and clinically relevant CQAs can be challenging.
Accordingly, the agency encourages a systematic approach
involving several steps starting with the identification ofseveral candidate CQAs for each product and the development of qualified assays to measure such candidate CQAs.
Achieving confidence in CQA measurement is critical,

as CQAs are affected by the robustness of the analyticalmethod that measures them.21,22 It is important to notethat not every CQA will have a qualified method; some
CQAs can be assessed within early phase with minimallydeveloped methods or feasibility testing. The knowledge
gathered during the product development cycle forms ascientific basis for establishing meaningful specifications.
In addition, there should be a systematic approach tocorrelate potential CQAs (pCQAs) with product quality.
Clinical outcomes form the basis for establishing which
pCQAs are in fact CQAs.
Figure 4-2 shows a holistic integrated strategy toidentifying CQAs through product understanding,
process understanding, and control strategy. Thishighly systematic and iterative process allows the identification of pCQAs by addressing all essential parts ofthe manufacturing process. Development phases willrefine pCQAs based on increased product and processcharacterization.23 For attributes identified during thepre-clinical phase (prior to first-in-human studies andplatform knowledge), in vitro metrics are often foundto be not predictive of in vivo activity. Further pCQA

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼74

characterization will be guided and influenced by clinical data as they become available, by long-term safety
profiles as they become established, and by increased
understanding of patient heterogeneity and variability.
This further characterization will confirm the true criticality of various quality attributes and allow refinementof pCQAs. Attributes that are found to correlate withclinical outcomes will become confirmed CQAs.24
CORRELATION OF CQAS TO CLINICAL OUTCOMES

To explore potential associations between quality attributes (e.g., purity, potency) and clinical outcomes (e.g.,
adverse reactions, efficacy), correlative analysis is often
used. By using statistical methods, subject-matter reviews,
and advanced analytics infrastructures, these analyses
can inform the true criticality of quality attributes aswell as defining targets and ranges for manufacture andbatch release. Correlative analyses also facilitate a better
understanding of how variation within quality attributes
impact both product performance and clinical outcome.
The result may enable streamlining the manufacturing
process by pointing out where flexibility lies in the processor the product.
Examples of clinical factors and CQAs examinedin correlative analysis for CAR-T cell products arepresented in Chapter 10, where numerous genotypic,
phenotypic, and functional CQAs and patient-specificcharacteristics can be evaluated for their relationship toclinical outcomes of interest. This chapter also covers theuse of correlative relationships to set or widen acceptancecriteria for certain CQAs based on the impact of variability within the particular CQA, e.g., a suitable acceptancecriteria for cell viability, since it has been shown that cell
viability below the lower limit originally defined did notcompromise the clinical safety or efficacy of tisagenlecleucel in patients with acute lymphoblastic leukemia, or
non-Hodgkin lymphoma.25 This approach uses knowledge from clinical outcome to revise the control strategy
to focus on preventing the use of batches that would
be predicted to result in suboptimal efficacy and/or a
detrimental impact to patient safety. Additionally, thispractice can minimize delays in drug product release andsubsequently, delivery to patients due to unnecessarily
stringent specifications.

RISK ASSESSMENT THROUGHOUT THE PRODUCTLIFE CYCLE

The progression from the initial QTPP to the commercial
product includes an iterative learning process, where theinitial envisioned cell-based therapy product often undergoes substantial revision and evolution as it translates
from the scientific bench to the patient. Ideally, productand process changes will be deliberate and planned.
Changes made to the manufacturing process may
potentially impact the product’s critical characteristics(CQAs and pCQAs) and therefore clinical outcomes.
Failure to detect the potential impact of these changes
during late-phase clinical trials or post-approval could
potentially affect product quality, effectiveness, and ultimately, commercial success of the product. It is therefore
important to reevaluate risks associated with the processchanges throughout the product life cycle.
While manufacturers are encouraged to introduce
major manufacturing changes early in the process whenpossible, product developers often introduce major
manufacturing changes late in the product development
life cycle. A common example is a change in manufacturing platform as manufacturers scale up their processfrom small-scale flask systems to scalable suspensionbioreactors when the yield of the initial manufacturing
process is not sufficient to support later phase trials andpredicted commercial demand. It is therefore importantfor manufacturers to develop a comparability plan as earlyas possible in product development, preferably before aPhase I trial. A comparability exercise is used to assess
whether pre- and post-change drug products are comparable. In this context, comparable implies that the CQAs
are highly (or sufficiently) similar such that patient safety(and efficacy if relevant) is not detrimentally impacted,
and importantly, clinical data from pre- and post-change
products can be considered to come from the “same product”. If risk assessment indicates that one or more CQAs
are likely to be adversely impacted by planned changes,
then it is highly advisable to re-think these changes, andthere may be limited value in a comparability study.
Risk factors that affect product comparability are
dependent on the complexity of the manufacturingchanges, the impact on product quality attributes, andthe timing during the product development life cycle.
Multiple simultaneous process changes, limited product

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL

✼75

knowledge, and later phases of clinical trials are associated with increased risk that pre- and post-change productare either not comparable or cannot be concluded ascomparable without further non-clinical or clinical data.
More details on product comparability throughout
its life cycle are provided in Chapter 2, which includes
discussion on potential risks, reporting requirements forchanges during the IND and post-approval phases, tools
for establishing comparability, and essential elements of acomparability study to support a streamlined pathway fortranslation of research into later-stage product development and commercialization. Comparability case studies
for hypothetical CAR-T products are also presented toillustrate common scenarios and problems related to
manufacturing control and comparability of pre- andpost-changes in products.

Mapping CQAs to CPPs
During product development, CQAs and CPPs are usedhand-in-hand and are essential aspects of the control
strategy to ensure product quality and manufacturingconsistency (Figure 4-3). To identify the process parameters that must be controlled to assure the control
of CQAs, product developers can work backwards fromidentifying: 1) what critical properties of the productneed to be controlled to achieve the desired safety andpotential efficacy (CQAs), 2) what process step is linked
to said attribute, and 3) what parameters in that process
impact or may impact the attribute (CPPs or potential
CPPs). This identification exercise is followed by processcharacterization, identification/confirmation of CPPs
and implementation of a control strategy.
Process characterization begins with process risk
assessments, which can be divided into Process Attribute
Matrix and Process Capability assessments. A processattribute matrix is a map containing the attributes andthe unit operation the attribute is associated with. Once
this is determined, process capability assessment entails
evaluation of how well process parameters are controlled
by comparing the performance of a process against its
specifications. For example, in a CAR-T cell manufacturing process, final cell yield and viability have been
determined to be CQAs since these relate to the quantityof active substance and therefore, efficacy. In the process

Figure 4-3: Linking CPPs To CQAs are critical toensuring product quality and manufacturing
process consistency
Action limits for
specific steps

Final productrelease criteria

Criteria forintermediates

CQA
Equipment
performance

CPP
In-process
criteria

Process
limitsAdapted from FDA presentation “Early-Stage Manufacturing
Considerations for Cell Therapy Products.”26

attribute matrix, both attributes are affected by multiple
steps in various unit operations, from cell collection toinfusion. Example parameters within these process steps
that have been found to influence the final cell number
and viability include temperature during transport of theapheresis product, hold time between cell collection andstart of manufacturing, volume / concentration of DMSO
in the final product, and time between DMSO additionand start of cryopreservation. Quantification of the effectof these process parameters on established CQAs will
drive the identification of the CPPs.
In developing a control strategy, targets and/or ranges
for CPPs are defined, including: 1) normal acceptable
ranges (NORs), which are the targets and intended
ranges used for routine manufacture, and 2) proven acceptable ranges (PARs), which are wider than NORs andfor which operation within this range should result in
producing a material that meets the relevant quality criteria based on accumulated knowledge and experience.
More detailed discussion on product control strategy,
including further discussion on how to define NORs andPARs for a process, is provided in Chapter 10.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼76

IDENTIFYING AND SETTING ACCEPTABLE RANGESFOR CPPS

Identification of CPPs in a manufacturing process is an
important step for any process development program.
By performing development studies to quantify theeffects of each process parameter on the CQAs, CPPs
can be defined as those that strongly impact the CQAs.
An example of the DOE study approach to identify CPPs
for a T-cell expansion process is presented in Chapter
10. Prioritization of these characterization studies isimportant as it is impossible to evaluate the effect of
the hundreds of interconnected parameters within acell and gene therapy (CGT) manufacturing process.
Prioritization could be based on prior knowledge fromprevious experience in other systems, existing product/
process development experimental data, published literature, and quantifiable impacts via an FMEA.
Several guidelines and examples on identification of
CPPs in cell-based therapy manufacturing have been
published.27,28 The next step is to set a target (NOR) andacceptable range (PAR) for each parameter based on
experimental data. Process parameters have boundaries
within which a given process yields an expected resultthat is defined in terms of CQAs. In most process steps,
the process parameter acts as an input that directly affects
the CQA; for example, centrifuge speed and/or duration(input) has been shown to affect cell recovery (yield) in
different cell types.29,30 Therefore, a range of speeds andtime needs to be established to concentrate cells during
downstream processing prior to fill-finish that will not
impact the cell recovery and viability. A target value canbe picked in the middle of the identified range. For example, if the study suggests that 300-500 × g centrifuge speed
for 10-15 minutes can recover >85% of cells with >80%
viability (i.e., the established CQA metric), a target of 400
× g for 12 minutes can be selected, with an acceptable
range of 300-500 × g and 10-15 minutes. As better process
understanding is gained, this range can be broadened until a failure point is hit, i.e., a centrifuge speed that resultsin drop in recovery or viability below the specification.
Broadening the range allows higher process tolerance
without changing the manufacturing process.26

Case Study: Risk Assessment andCQA Identification for Autologous
CAR-T Cell ProductIn this case study, a risk assessment is performed todetermine which quality attributes are critical to guidethe process characterization plan and design processvalidation studies. The purpose of this risk assessment
is to identify and summarize the CQAs for a generic
autologous CAR-T cell product introduced in Chapter
3. The ICH guideline Q8(R2) requires the identificationof potential CQAs, including those related to DS, DP,and excipients, so that any characteristics that may havean impact on the desired product quality can be studied
and controlled.
THE USE OF RISK ASSESSMENT TOOLSTO ESTABLISH CQAS

The risk ranking approach outlined in earlier sections is
adopted for identification and assignment of CQAs. Eachquality attribute is evaluated for criticality by assessing
its potential impact and uncertainty as it relates to theefficacy and safety of the product. For examples of rationale for determining the uncertainty and severity scoring,refer to the earlier section titled Examples of uncertainty
and severity determination based on prior knowledge.
During stage I process validation activities, CQAs will be
used to identify the CPPs for the proposed commercial
manufacturing process via a risk assessment, observations
from historical experience, and findings from processcharacterization experiments.
The identification and justification of CQAs are being
performed in accordance with the Quality by Design(QbD) principles and recommendations made in ICH
Q8(R2), Pharmaceutical Development and ICH Q9,
Quality Risk Management, as well as the generally accepted approach described in guidance documents related toprocess validation. The criticality determination example
presented in Table 4-5 is based on the attributes identified
in the QTPP of a generic autologous CAR-T cell productintroduced in Chapter 3.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL✼77

Table 4-5: Example of criticality determination using risk ranking for an autologous CAR-T productQuality
Attribute
Category

Quality Attribute

SafetyContent /strength

Identity

PurityPotency

Severity
Score

Uncertainty
Score

Overall
Ranking

Criticality

Sterility

10

1

10

CQA

Mycoplasma

10

1

10

CQA

Endotoxin

10

1

10

CQA

Bacterial contamination (Gram stain)

10

1

10

CQA

Replication competent lentivirus (RCL)

10

2

20

CQA

Vector copy number (VCN)

10

2

20

CQA

Dose range (# CD3+ transduced cells perweight or XX ml / kg body weight)

10

1

10

CQA

Cell concentration (cells/ml)

10

1

10

CQA

% CAR+ CD 3+

10

1

10

CQA

% CD 4+ / CD 8+ ratio

3

1

3

pCQA

Residual plasmid DNA

3

1

3

pCQA

Residual beads

10

2

20

CQA

Residual BSA / serum

10

2

20

CQA

Viability

3

1

3

pCQA

% B cells

3

2

6

pCQA

% NK cells

3

2

6

pCQA

Cytotoxicity / cell killing

10

3

30

CQA

IFN­-γ cytokine release

10

3

30

CQA

Conclusion
A prospectively planned, systematic, risk-based approachto product development is key to facilitating efficient
clinical development and marketing authorization.
Lessons can be learned from the development of other,more established biologics such as vaccines. The risk andcriticality assessment tools presented in this chapter canbe used to identify and confirm CQAs, to understandcriticality of materials and process parameters, andinform a robust control strategy to provide consistentprocess performance and batch consistency. Risk assessment approaches allow a large pool of attributes and/or
parameters to be narrowed down to a smaller number

deemed important for further characterization, therefore
allowing greater focus on aspects of the process that
present the highest amount of risk to the overall qualityof the product. An integrated and iterative strategy thatincorporates product and process understanding, control
strategy, and correlative analyses with clinical outcome
should provide increasing CQA knowledge and experience throughout the product life cycle, leading to greater
assurance of product quality.

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL

✼78

References
1

International Conference on Harmonization. ICH HarmonizedTripartite Guideline: Pharmaceutical Development Q8(R2),
Current Step 4 version. August 2009. https://database.ich.org/
sites/default/files/Q8_R2_Guideline.pdf

13

Carson BP, Patel B, Amigo-Benavent M, et al. Regulation ofmuscle protein synthesis in an in vitro cell model using ex vivo
human serum. Exp Physiol. 2018; 103: 783– 789. doi:10.1113/
EP086860

2

Parenteral Drug Association. Technical Report No. 81: CellBased Therapy Control Strategy. https://www.pda.org/
bookstore/product-detail/4638-tr-81-control-strategy-for-celltherapy. February 2019.

14

Porter DL, et al. Chimeric antigen receptor T cells persist
and induce sustained remissions in relapsed refractorychronic lymphocytic leukemia. Sci Transl Med. 2015 Sep
2;7(303):303ra139. doi: 10.1126/scitranslmed.aac5415

3International Conference on Harmonization (ICH) Q9, Quality
Risk Management - Guidance for Industry. June 2016. https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/q9-quality-risk-management

15

Maude SL, et al. Chimeric antigen receptor T cells for sustained
remissions in leukemia. N Engl J Med. 2014 Oct 16;371(16):150717. doi: 10.1056/NEJMoa1407222

4

Pagliarulo N. Novartis still hasn’t solved its CAR-T
manufacturing issues. BioPharma Dive. https://www.
biopharmadive.com/news/novartis-kymriah-car-tmanufacturing-difficulties-cell-viability/568830/

16

Sommermeyer D, Hudecek M, Kosasih P, et al. Chimericantigen receptor-modified T cells derived from defined
CD8+ and CD4+ subsets confer superior antitumor reactivity
in vivo. Leukemia 30, 492–500 (2016). https://doi.org/10.1038/
leu.2015.247

5National Academies of Sciences, Engineering, and Medicine;
Health and Medicine Division; Board on Health Sciences Policy;
Forum on Regenerative Medicine. Navigating the Manufacturing
Process and Ensuring the Quality of Regenerative Medicine
Therapies: Proceedings of a Workshop. Washington (DC):National Academies Press (US); October 26, 2017.

17

Xu Y, Zhang M, Ramos CA, et al. Closely related T-memory stem
cells correlate with in vivo expansion of CAR.CD19-T cells andare preserved by IL-7 and IL-15. Blood. 2014;123(24):3750-3759.
doi:10.1182/blood-2014-01-552174

18

Zarling S. et al. The Survival of Memory CD8 T Cells That Is
Mediated by IL-15 Correlates with Sustained Protection Against
Malaria. The Journal of Immunology. May 15, 2013, 190 (10)5128-5141; doi: 10.4049/jimmunol.1203396

19

Torres Chavez A, McKenna MK, Canestrari E, et al. Expanding
CAR T cells in human platelet lysate renders T cells with in vivo
longevity. Journal for ImmunoTherapy of Cancer 2019;7:330. doi:
10.1186/s40425-019-0804-9

20

Medical Trend. The evolution of cell and gene therapy fromdiscovery to commercialization. June 2021. https://medicaltrend.
org/2021/06/09/the-evolution-of-cell-and-gene-therapy-fromdiscovery-to-commercialization/

6

7

Garcia-Aponte OF, Herwig C, Kozma B. Lymphocyte
expansion in bioreactors: upgrading adoptive cell therapy. J
Biol Eng. 2021;15(1):13. Published 2021 Apr 13. doi: 10.1186/
s13036-021-00264-7
van Schalkwyk, M.C.I. et al. Development and Validation ofa Good Manufacturing Process for IL-4-Driven Expansion of
Chimeric Cytokine Receptor-Expressing CAR T-Cells. Cells 2021,
10, 1797. doi: 10.3390/cells10071797

8

Summary Basis for Regulatory Action – KYMRIAH. August 30,
2017. BLA/ STN#: 125646/0

9Hollyman D, et al. Manufacturing Validation of Biologically
Functional T Cells Targeted to CD19 Antigen for Autologous
Adoptive Cell Therapy, Journal of Immunotherapy 2009,32(2):169-180. doi: 10.1097/CJI.0b013e318194a6e8

21

Myers M. CQAs for C&GT Products to Enable Comparability
Assessment: Case Studies. Slide 9: Impact of Assay Robustness.
Presentation from ISCT Cell Therapy Liaison Meeting. October
2016.

10

Thompson JA, et al. A phase I trial of CD3/CD28-activatedT cells (Xcellerated T cells) and interleukin-2 in patients withmetastatic renal cell carcinoma. Clin Cancer Res. 2003, 1;9(10 Pt
1):3562-70

2211White RD, Glosson JA, Gordon DE, Northup SJ. Intravenous
Safety Study in Rats Given Paramagnetic, Polystyrene Beads with
Covalently Bound Sheep Anti-Mouse Immunoglobulin G (IgG).
Journal of the American College of Toxicology. 1995, 14(4):251265. doi:10.3109/10915819509008701

National Academies of Sciences, Engineering, and Medicine;
Health and Medicine Division; Board on Health Sciences Policy;
Forum on Regenerative Medicine. Navigating the Manufacturing
Process and Ensuring the Quality of Regenerative Medicine
Therapies: Proceedings of a Workshop. Washington (DC):National Academies Press (US); 2017 Oct 26. 3, Identifying and
Measuring Critical Quality Attributes. https://www.ncbi.nlm.
nih.gov/books/NBK475684/

23

12

Ghassemi S, et al. Enhancing Chimeric Antigen Receptor T
Cell Anti-tumor Function through Advanced Media Design.
Mol. Ther. Methods Clin. Dev. 2020, 18:P595-606. doi:10.1016/j.
omtm.2020.07.008

Polson N. Presentation: Autologous Cell Therapy Phase
Appropriate Control Strategies from Early Clinical Development
to Commercialization. Jun 2021. https://www.casss.org/
docs/default-source/cgtp/2021-cgtp-speaker-presentations/speaker-presentation-polson-nolan-bristol-myers-squibbcompany-2021.pdf?sfvrsn=379064b2_3

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL

✼79

References, continued
24

25

26

27

Polson N. Presentation: Stability Considerations and Challenges
in Autologous Cell Therapy. Jan 2020. https://www.casss.org/
detail-pages/resource/stability-considerations-and-challengesin-autologous-cell-therapy
Pasquini MC, et al. Real-world evidence of tisagenlecleucelfor pediatric acute lymphoblastic leukemia and non-Hodgkin
lymphoma. Blood Adv. 2020 Nov 10;4(21):5414-5424. doi: 10.1182/
bloodadvances.2020003092
Finn T. “Early Stage Manufacturing Considerations for Cell
Therapy Products.” CASSS. Cell and Gene Therapy Products. 10
July 2018. https://cdn.ymaws.com/www.casss.org/resource/
resmgr/cell&gene_therapy/cgtp_slides/2018_CGTP_
FinnThomas.pdf
Identify & Define Your Cell Therapy’s Biomanufacturing Approachfor Critical Process Parameters (CPPs). https://www.roosterbio.

com/blog/identify-define-your-cell-therapys-biomanufacturingapproach-for-critical-process-parameters-cpps/
28 Campbell A, Brieva T, Raviv L, et al. Concise Review: Process
Development Considerations for Cell Therapy. Stem Cells Transl
Med. 2015 Oct;4(10):1155-63. doi: 10.5966/sctm.2014-0294
29 Hønge BL, Petersen MS, Olesen R, Møller BK, Erikstrup
C. Optimizing recovery of frozen human peripheral bloodmononuclear cells for flow cytometry. PLoS One. 2017 Nov
1;12(11):e0187440. doi:10.1371/journal.pone.0187440.
30 Wong JW. Centrifugal recovery of embryonic stem cells forregenerative medicine bioprocessing. University College
London, September 2008. https://discovery.ucl.ac.uk/id/
eprint/16358/1/16358.pdf

Chapter 4: Risk Assessment and Critical Quality Attribute Identification

A-CELL

✼

80

	


CHAPTER 5

Cell Collection andStarting Materials
Consideration
CHAPTER LEADSErin Goodhue, Terumo Blood and Cell Technologies
Dalip Sethi, Terumo Blood and Cell Technologies
CHAPTER CONTRIBUTOR

Elizabeth Valdez, Novartis

Chapter 5 | Contents
Introduction................................................................... 82
Apheresis Instrumentation Technical
Considerations.............................................................. 83

Donor considerations for allogeneic cell therapy
products...........................................................................91
Adventitious agents..................................................91

Separation of blood components............................. 83

Starting material qualification..................................91

Extracorporeal volume.............................................. 84

Donor screening and testing.................................. 92

Mitigation strategies................................................ 85

Donor screening................................................... 92

Anticoagulation.......................................................... 85

Donor testing........................................................ 92

Contamination reduction.......................................... 86

Considerations for other transmissible agents........ 93

Methods.......................................................................... 87

Final cell product release testing............................ 94

Optimization techniques........................................... 88

Perspectives on HHV testing................................. 94

Processing volume.................................................. 88

Regulatory considerations.......................................... 95

Prompt response to alarms..................................... 88

Conclusions.................................................................... 95

AC management...................................................... 89

References......................................................................96

Patient Preparation/Management............................. 89
AC infusion management.......................................... 89
Blood counts...............................................................90
Vascular access..........................................................90

Introduction
Quality cell-based therapeutic products start with quality raw materials, whether those materials are a set ofchemical or biologic reagents, or the living cells that form
the basis of a cell-based therapy. The use of cell-based
therapies, including CAR-T therapies, in the treatment
landscape for some forms of cancer has shined a spotlighton the relatively unknown sub-specialty of apheresis, a cellcollection process that lays the foundation for these therapies, whether the cellular starting material originates fromthe patients themselves (autologous) or healthy donors(allogeneic). Apheresis, Greek for “a taking away,” is theprocess of separating a donor’s or a patient’s whole bloodby centrifugation into its respective components (plasma,platelets, white cells, and red cells), and “taking away”
the desired component. In the case of CAR-T therapies,
the cell type of interest, i.e., white blood cells (commonlyreferred to as the “buffy coat” or leukopak during theChapter 5: Cell Collection and Starting Materials Consideration

apheresis process), is collected into a bag and retained,
while the other blood components are returned to thepatient. For autologous cell therapies, the manufacturing
process begins with apheresis collection from a patient andends with release of the therapeutic product for imminent
use in that same patient. For allogeneic therapies, apheresis
is performed on a donor, and the final therapeutic productis intended for off-the-shelf use in many patients.
Apheresis device selection and collection protocols
can vary by application and clinical need. The process
involves many variables that require detailed consideration to ensure the best possible outcome of the cellcollection while minimizing negative effects on thedonor, who frequently is also the intended recipient of
the manufactured therapeutic. Implementation of donor
eligibility criteria as well as technical patient considerations (e.g., blood volume and count, recent therapies
that may depress lymphocyte count or function) ensure
safe administration to patients and successful collection

A-CELL

✼82

Figure 5-1: Overview of therapeutic apheresis process
Patient
Patient

Anticoagulant
Anticoagulant
Blood pump

Blood

Blood

component
Component

Substitution
substitution

Blood
BloodPump
pump

Blood
component
waste

Pressure
SensorPressure
sensorBlood

Blood Component
component
Separator

separator

Pressure
Pressure sensor
SensorBlood pump
Pump
Blood

Adapted from: Asahi Kasei Medical; www.asahi-kasei.co.jp/medical/en/apheresis/product/plasma/about/cure.html

procedure, mitigati ng the risks in the therapeutic productmanufacturing. In addition, the industry overall is taking
steps to ensure consistent, robust apheresis processes in
the clinical manufacturing context through standardization of apheresis procedures and collection targets1
to help align cell collection requirements for productmanufacturers and sponsors.
This chapter aims to cover various aspects of theapheresis process, including instrument technical operation, optimization techniques, patient preparationand monitoring, donor screening and testing, and clinic
operation considerations. In many sections, the authors
present considerations that are specific to one of the morecommonly used apheresis systems, the Spectra Optia®.2

Apheresis Instrumentation Technical
Considerations
The use of apheresis instrumentation for cell collections
is a well-established methodology for collecting mononuclear cells (MNC) destined for curative cancer therapies.
Key topics related to apheresis instrumentation include theconcept of separation of blood components, extracorporeal

Chapter 5: Cell Collection and Starting Materials Consideration

volume, anticoagulation management, barriers to prevent
contamination of the collected product, and instrument
optimization techniques. A graphical overview of theapheresis process is presented in Figure 5-1.
SEPARATION OF BLOOD COMPONENTS

Most apheresis instruments used for cell collection
procedures utilize continuous centrifugation methods
to separate whole blood into its components based on
density or specific gravity. Taking advantage of the density
characteristics of whole blood cells allows the instrument
to separate cells from whole blood, collect the MNC layer,
and return remaining components (RBC, plasma, platelets) to the person undergoing apheresis, whether it is thepatient or a donor.
When whole blood enters the apheresis instrument
centrifuge it is exposed to gravitational force throughcentrifugal rotation that separates the whole blood into
its components based on specific gravity. Red cells, being
the densest cells of whole blood, collect at the bottom of
the disposable kit, with the MNC layer stacking on top of
the RBCs, followed by the platelets on top of the MNCs,
and then the plasma floating to the top (Figure 5-2).

A-CELL✼83

Figure 5-2: Separation of blood components
Separation of the cellular components from wholeby centrifugation
blood is dependent on two main variables: centrifuge
speed (measured in RPMs) and the duration of the applied
centrifugal force in the separation chamber. When applied
to whole blood, centrifuge speed affects how tightly thecells are packed in the chamber. The faster the centrifuge
speed, the more compressed the cellular portion of whole
blood is (high packing factor). Conversely, the slower thecentrifuge speed, the less compressed the cellular portionof whole blood is (low packing factor), as illustrated in
Figure 5-3. Different packing factors are utilized based on
Plasmathe target blood component. Aside from these variables,
55% total
the overall separation ultimately depends on the patient/volumedonor status and vascular access options, which will be
discussed in subsequent sections of this chapter.
PackedUnderstanding what influences where MNCs will rered cells
side in the collection chamber allows the designers of the
45% total
disposable tubing sets to engineer the placement of thevolumecollection port within the tubing set collection chamber
to actively collect the target cells into a collection bag.
Buffy coat
Another variable that affects the quality of cell sepa<1% total volume
ration, which dictates the purity and recovery of target
cells in the final isolated fraction, is the rate at which thewhole blood enters the centrifugal field, and thus, therate at which the separated components exit the centrifSource: Terumo Blood and Cell Technologies
ugal field. This is referred to as dwell time. Operators ofthese devices have some control over how fast cells are
A person’s vasculature has a finite amount of cirintroduced into the centrifugal field but rather limited
culating whole blood. Apheresis instruments allow forcapacity to impact large changes to the speed of theblood separation by routing whole blood through thecentrifuge. Whole blood requires sufficient exposure tocentrifugal field via a sterile disposable kit loaded onto
the gravitational force in the centrifugal field to allow for
the instrument. During the cell collection process, acell separation. If the whole blood does not have enoughsmall portion of red cells are inherently retained in thedwell time to allow for the separation to occur, the result Source:
disposable
retention of red cells in the
Terumo tubing
Blood andkit.
CellTheTechnologies
may be a less than optimal cell collection procedure.
disposable tubing set can affect the ability of the donor/
Another variable is vascular access, which often limits
patient to maintain hemodynamic stability. Prior to
overall whole blood flow through the device. Poor vascucollection, the apheresis clinical team will need to assesslar access can contribute to inadequate cell separation in
the patient’s total blood volume/clinical status within thethe centrifugal field resulting in instrument malfunction
defined parameters of the device’s extracorporeal volume.
up toFigure
and including
loss of the
To ensure hemodynamic stability during the col5-2: Separation
ofcollection.
blood components by centrifugation
A-CELL ✼ Chapter x: xxxxxxx
lection, it is important to know the required volume
Source: Terumo Blood and Cell Technologies
EXTRACORPOREAL VOLUMEof blood in the disposable kit compared to the donor’s/
Extracorporeal volume (ECV) refers to the volume ofpatient’s blood volume. The ECV of the disposable kit isblood thatis flowing
throughthe disposable tubing set at
generally included in the manufacturer’s operating manA-CELL✼ Chapterx: xxxxxxx
8
any given time during the apheresis procedure. ECV canual. For most adult sized (~70kg) donors/patients with ahave a profound effect on the person undergoing apheresis.
healthy presentation, hemodynamic stability should be

Chapter 5: Cell Collection and Starting Materials Consideration

A-CELL✼84

Figure 5-3: Schematic of low (left), medium (middle), and high (right) packing factor

Platelets

Lymphocytes
Monocytes
Granulocytes

Red blood cells

Adapted from “Principles of Blood Separation and Apheresis Instrumentation”3maintained if the ECV is less than or equal to 15% of
total blood volume (TBV). Of course, this is dependent
on their individual clinical presentation. For smaller
adults or pediatric subjects with low TBV, or patientswith a compromised clinical condition including a low
hematocrit, interventions may be required to ensure theirsafety during the collection. Ultimately, the clinical andapheresis teams need to evaluate how blood volume
shifts, including how concomitant red cell loss will affectpatients/donors based on clinical disposition, and planfor potential mitigation strategies.

who would not be able to tolerate the volume of red cellretention in the disposable kit.
The blood prime procedure is used to offset the ECV
requirement and promote patient tolerance. This procedure displaces the saline used to prime the disposable
kit dead space with packed red blood cells. When thepatient is attached to the instrument tubing, the packed
red blood cells are delivered to the patient while theirwhole blood is removed into the disposable kit, thus
ensuring proper volume replacement while maintaining
oxygenation to promote hemodynamic stability.

Mitigation strategies

ANTICOAGULATION

A blood prime procedure is a solution offered by the
instrument manufacturers to overcome the ECV requirements of the disposable kit when clinically appropriate.
Most instruments will display a warning screen to theoperator if the ECV of the disposable tubing kit exceeds
10-15% of the patient’s TBV and offer a recommendation to perform a blood prime of the disposable kit. A
blood prime may also be necessary for anemic patients

Effective anticoagulation of the extracorporeal circuit is a
critical consideration of the apheresis procedure. Free flowing whole blood into the extracorporeal circuit vastly affects
the instrument’s ability to separate and collect target cells.
Acid citrate dextrose formula A (ACD‐A) is the most widely used anticoagulant (AC) for cell collection procedures.
The acid component of ACD-A lowers the pH ofwhole blood to prevent platelets from aggregating andChapter 5: Cell Collection and Starting Materials Consideration

A-CELL✼85

keeps them in suspension. The citrate component of
ACD-A binds ionized calcium in the extracorporeal
circuit, which prevents platelet aggregation and theactivation of calcium‐dependent plasma coagulation
factors.4 As a consequence of citrate delivery to theextracorporeal circuit, the patient will receive citrate ata prescribed rate with the components of blood that are
returned to the patient. Citrate reactions constitute oneof the top adverse events patients or donors experiencefrom an apheresis procedure4 and need to be monitored
carefully by the apheresis team.
Citrate is actively metabolized by the liver and does
not serve as an anticoagulant of the blood circulating in
the patient or donor, but the patient can have a rangeof side effects from the citrate they receive, which canbe as minor as circumoral tingling, or hand and feettingling, to tetany reactions.5 However, hypocalcemia
can also manifest more severely as tetany, laryngospasm,
cardiac conductional abnormalities, or even seizures.4
Close monitoring of the patient or donor for symptoms
of citrate reactions is critical, and the patient or donor(or family member) needs to be made aware of the signs
and symptoms and given instructions about triggers forseeking medical intervention. Patients with immatureorgan function (e.g. liver) or at increased risk mighteven need cardiac monitoring.4 As citrate reactions canprogress quickly, early intervention is key.
Clinicians should consider serially monitoring electrolyte levels of the patient and could consider providing
replacement infusions of calcium or magnesium to offset
the binding of these electrolytes when applicable. Special
considerations should also be made for patients withimmature liver function or liver dysfunction to prevent
citrate lock.6 Many institutions will, at minimum, monitor
electrolyte panels of the patient/donor at the beginningand end of the procedure, and these times can be adjusted
if a strong reaction to citrate is suspected. Pediatric institutions may adapt a more aggressive monitoring regimen
given the age and communication ability of the patients.
In the Spectra Optia system, the inlet:AC ratio is thecontrol that ensures adequate anticoagulation of theextracorporeal circuit. The inlet:AC ratio is the amountof AC present in the extracorporeal circuit compared tothe amount of whole blood. This mixture determines the

Chapter 5: Cell Collection and Starting Materials Consideration

concentration of AC in the extracorporeal circuit and thedegree to which blood in the system is anticoagulated.
It is expressed as parts of whole blood to one part ofanticoagulant. For example, an inlet:AC ratio of 12:1
means there are eleven parts whole blood to one part
anticoagulant. Think of this parameter as how anticoagulated the disposable circuit is. The higher the AC ratio,
the more whole blood the one part of anticoagulant must
prevent from coagulating, and thus the greater potential
for clumping to occur. The lower the AC ratio, the lesswhole blood to one part of anticoagulant, and thus theless whole blood that one part of anticoagulant must
contend with.
The other anticoagulant control on the Spectra Optia
system is the AC infusion rate. AC infusion rate should
not be confused with inlet:AC ratio. The two controls
have a distinct difference for which aspect of AC management they control. Simply put, the inlet:AC ratio controls
the degree that the extracorporeal circuit is anticoagulated. The AC infusion is the dose of anticoagulant thepatient receives. The AC infusion rate is defined as thedose the patient receives in mL/min/LTBV (liters of total
blood volume) processed.
We will discuss AC infusion management in moredetail in the Donor/Patient Preparation/Management
section as AC infusion rate determines the delivery rateof citrate to the donor/patient, so the concept is more
relevant to understand in the context of how it affects
the donor/patient.
CONTAMINATION REDUCTION

Disposable tubing systems on most apheresis devices
are considered functionally closed systems, which aid inmaintaining the integrity and sterility of the collected
cells. Manufacturers of the tubing systems have developed engineering controls to prevent the contaminationof collected cell products. Some of the contamination
mitigation features are described below.
• Sterile barrier filters. Tubing that control the flow of
fluid used to prime and anticoagulate the disposable kits
are equipped with filters to filter out any particulates.
Sterile filters are on the final product bag for adding
anticoagulant to the collected product if needed.

A-CELL✼86

Figure 5-4: Schematic of cell collection procedures­
Continuous Mononuclear Cell Collection (CMNC)
2.
Red blood cells
flow passively
to the reservoir

1.
Whole
blood enters
disposable

3.
Plasma is pumped into thereservoir or collected
4. White blood cells
are continuously
pumped into thecollection bag

White arrows indicate direction of blood flow in separator

Mononuclear Cell Collection (MNC)
5. Chamber
contents
periodicallycollected

4.
MNCS
accumulate
In chamber

2.
Red bloodcells areremoved1.
Wholeblood enters
disposable

3.
Plasma isremovedWhite arrows indicate direction of blood flow in separator

Source: Terumo Blood and Cell Technologies

Methods• Sampling port. A sample bulb system is on the
final product bag to allow for aseptic samples to beobtained for product analysis.
Spectra Optia has two cell collection procedures available• Pre-attached venous access. Some disposable kits
for customer use. The first, Continuous Mononuclear Cell
come with a pre-attached venous access device,
Collection (CMNC), is a single staged cell separation proor sample diversion pouch, on the inlet line of thecedure. Whole blood is separated by specific gravity using
disposable kit to collect the first 15-30 mL of blood
centrifugation and the resulting white blood cell layer isand the skin plug artifact created by the needlestick.
continuously pumped in mL/min to the collection bag.
Skin plugs are a common contamination source. The A-CELL ✼The
second,Mononuclear Cell Collection (MNC) is
Chapterx: xxxxxxx
donor arm prep procedure is known to be effective,
a dual staged cell collection procedure. The whole blood
however issues may arise in frequent donors whereis separated by specific gravity first in the disposable
repeated needlesticks have occurred and where theconnector, removing RBCs and plasma, then the MNC
needlestick site may be difficult to clean effectively.
layer is routed to a collection chamber where the MNCDiverting the initial collection volume and potential
are exposed to elutriation and separated by size. Thisskin contaminants can limit microbial access to therefined MNC layer is then pumped to the product bag
disposable tubing and the collection.
in batches when the system has determined the collec• Heat sealers. Heat sealers come with most devices
tion chamber is full of refined MNCs. The resulting cellto allow for aseptic sealing of the product bag oncecollection efforts are not seen in the product bag until thethe product is ready to be transported to the cellsystem triggers a collection cycle. A schematic of the twoprocessing lab.
collection procedures is presented in Figure 5-4.

Chapter 5: Cell Collection and Starting Materials Consideration

A-CELL ✼ Chapter x: xxxxxxx

A-CELL

5✼

87

Another important difference between the two collection
protocols is the ECV required by the respective disposabletubing systems: 253-297 mL for CMNC and 147-191 mL for
an MNC collection.7 This means that the volume of bloodrequired to be outside of the patient’s body for the durationof the procedure is a key consideration and should be used
to help determine a patient’s tolerance to the procedure and,
when appropriate, plan mitigation strategies.
There are minimal head-to-head studies comparing the
CMNC and MNC collection methods for the non-mobilized donor. Of the three studies, eight data points weregathered: procedure run time, collection volume, productmononuclear collection efficiency, percentage of lymphocytes, monocytes, granulocytes and hematocrit in
the product bag, and platelet loss to the patient. The only
consistently replicated difference between the three studies
was the significant difference in overall run time between
the two procedures, with CNMC runtime being shorter.8,9,10
Overall, both procedures have similar outcomes of thecell types collected in the product bag. The target cell layercollection percentage was reasonably consistent between
the two cell collection procedures in these three studies
with CMNC showing superiority in one study regarding
the percentage of lymphocytes in the product bag. Choiceof protocol may be more influenced by patient tolerance(e.g., MNC collection might be preferable for well mobilized pediatric patients due to the lower ECV), operator
protocol competency, and the legacy cell collection methods of the individual collection site.
OPTIMIZATION TECHNIQUES

Apheresis instruments have been developed with the operator in mind. Human factors testing is quite extensive
and considered by the FDA to be an important aspect of
instrument development. While these instruments monitor
for conditions and situations that require operator intervention, it is always best practice that operators understandhow they might tailor instrument configurations to patient
conditions that may impact the ability of the instrument tocollect cells efficiently.

Processing volume
Ensuring that the instrument processes adequate blood
volume to obtain target cell yield is one of the most important aspects of optimizing collection procedures. The

Chapter 5: Cell Collection and Starting Materials Consideration

use of processing prediction algorithms adopted from site
specific data is one way to take the guess work out of how
much blood should be processed to obtain target cell yield.
Processing prediction algorithms can utilize pre-collection and mid-collection patient cell counts obtained
from flow cytometry, whole blood processed, instrument
collection efficiency, and final product cell yield to calculate the volume of blood in liters to process for future
procedures. To adequately predict the processing volume
and the likelihood of obtaining the cell yield within thespecified processing volume, data needs to be collectedfrom a minimum set of collection procedures. After the
initial data is obtained, a modified processing prediction
algorithm can be developed that can accurately predictcell yield based on the apheresis center processes.
Unfortunately, limits on availability of flow cytometry
at apheresis collection centers may limit use of theseprocessing prediction algorithms.
That said, implementing this processing prediction algorithm is beneficial from the perspective of both patient/donor safety and operational cost. Apheresis procedures
are relatively safe; however, the risk cannot be totally
removed. Using the processing prediction algorithm
to ensure the patient/donor are on the instrument for
only the time needed to obtain the necessary target cell
yield can potentially reduce safety risks related to patient
tolerance, i.e., overcollection. Additionally, by preventing
overcollection, this algorithm could impact storage capabilities in apheresis clinics. For clinics that juggle multiple
types of procedures, using the processing prediction algorithm could affect their staffing models and capabilities,
potentially affecting their return on investment.

Prompt response to alarms
Responding to apheresis instrument alarms is anotherimportant optimization technique. Every time an alarm
condition is initiated, the pumps on the instrument are
paused and the separation of cells is lost. The prevention
and mitigation of potential alarm conditions become important to maintaining consistent cell separation. It is important to note that for most alarm conditions the pumps
pause on the instrument. When the system restarts thepumps, it takes time to re-establish cell separation andthus decrease the time dedicated to pumping separated
MNCs from the disposable tubing set to the final product

A-CELL✼88bag. This can often impact collection efficiency, andconsequently product quality due to undesired blood
components that can be collected during the pause,
as well as lengthen the duration of the procedure. The
clinical team needs to carefully evaluate pauses/delays
in procedures and their impact on patient/donor safety.
For example, the top alarm received on apheresis instruments are inlet access or return access alarms. These
alarm conditions are directly related to the vascular access device used for the procedure. Promptly addressing
this alarm condition by flushing the access point, using
repositioning techniques, or ensuring proper flow prior
to attaching the vascular device to the disposable tubing,can help improve the overall efficiency of the collection
procedure by minimizing pump pauses.

AC management
Monitoring for cell aggregation in the circuit is a key
optimization technique to consider when conducting cellcollection procedures. Monitoring the disposable tubing
set for signs of platelet clumping will prevent clogging
and, consequently, potential decreases in cell collection
yield. Cell clumping prevents free flow of the blood and,
consequently, the ability of the centrifuge to separate thecells properly is stunted.
During operation, the instrument will default to aset Inlet:AC ratio. There are times when these default
values do not adequately maintain anticoagulant effects
while the cells are in the plastic disposable tubing kit.
When clumping is seen in the circuit, most manufacturers will instruct operators to decrease the Inlet:AC
Ratio for a specific inlet volume processed to add more
anticoagulant into the disposable tubing kit. In addition,
an operator can re-evaluate the disposable connector or
tubing after a specific inlet volume has been processed toassess whether the clump has been broken up. Operators
always monitor patients or donors carefully for any side
effects associated with machine adjustment.

Patient Preparation/Management
Considerations for patient preparation and procedural
patient management are equally as important as technicalinstrument considerations. Developing strategies for preparing patients for a cell collection procedure, determining

Chapter 5: Cell Collection and Starting Materials Consideration

optimal timing for cell collections, vascular access needs,
and monitoring the patient during the procedure are equally important and can vastly impact the collection outcome.
Patient preparation for a cell procedure and management of the patient or donor during the procedure are instrumental for a safe patient/donor-centric cell collection.
Developing a process whereby the patient or donor cancome see the apheresis clinic may help reduce anxiety thatis experienced by patients/donors, particularly in pediatricpatients. Many patients have not heard of apheresis and the
instrument can be overwhelming for them. Some institutions have the patient or donor come to the apheresis clinic
to be consented for the procedure. This gives apheresis
physicians, nurses, or technicians an opportunity to discuss
the collection risks and benefits, potential side effects, andhow the donor or patient can prepare for the procedure.
Patient/donor preparation should focus on setting
expectations for the procedure and equipping themwith management tactics to help the procedure go moresmoothly. Good information to communicate includes
approximately how long they will be in the clinic for thecollection, the need for proper diet and hydration the
day before collection, type of anticoagulant used andpotential complications, and preparation for the type ofvascular access they will need to complete the procedure.
Of course, this is all part of the consenting process, but
meeting the collection center staff will aid the patient anddonor in making a connection to mentally prepare for theprocedure. Ultimately, the approach to preparing the patient for apheresis collection is multidisciplinary, involving the clinical team and apheresis team collaboratively.
AC INFUSION MANAGEMENT

The AC infusion rate, which is the rate at which the patient
receives AC from the disposable circuit, is another criticalconcept in the Spectra Optia anticoagulation management. The AC infusion rate is the dose the patient receives
in mL/min/liter of TBV processed. There is a safety rangeof 0.8–1.2mL/min imposed by the system. Operators canoverride the safety range of the instrument until they reachan ultimate hard stop range, but the instrument will alert
the operator that they are operating out of the establishedsafe ranges. This is called running in caution status.
The patient’s hematocrit (HCT) is a crucial data input.
In some instruments the HCT data is used to determine

A-CELL✼89

the patient’s red cell volume and plasma volume. These
volumes feed into calculations that inform the setup of
the interface in the separation connector. Based on the
HCT, the device will determine the speed of the plasmapump controlling where the MNC layer resides in theconnector, and thus how it aligns with the collection porton the tubing system. The white blood cell layer shouldalign with the collection port in order to be removed
from the connector to the product collection bag.
BLOOD COUNTS

The target cell count during collection varies between
stem cell collections and immune effector cell collections. The target cells for a stem cell collection are thehematopoietic stem cells or CD34+ cells. These cells
circulate in the peripheral blood at a low steady state,
but their numbers can be increased dramatically withthe use of cytokine stimulation (e.g., granulocyte colony-stimulating factor) to mobilize CD34+ cells into
circulation. Additionally, plerixafor (Mozobil®, Genzyme
Corporation) can also be used, most commonly forpoorly mobilized autologous stem cell collections. The
patient’s or donor’s pre-collection CD34+ cells canreasonably predict the quantity of stem cells collected.
Typically, an efficient stem cell collection can be obtained
if the patient’s/donor’s pre-collection CD34+ count is
20 cells/μL.11 For most stem cell transplants, the minimum number of cells needed is commonly 2.0×106/kg,
however a dose of 5×106 CD34+ cells/kg is associatedwith faster recovery of platelets and granulocytes.12,13
Collection of peripheral blood lymphocytes or CD3+
cells for chimeric antigen receptor processing does not
require cytokine or medicinal mobilization. For CAR-T
cell product manufacturing, collections could target 2.0
×109 total nucleated cells (TNC) and 1.0×109 CD3+ T
cells.14 An overview of the different CAR-T cell products,
the varying requirements and procedures set by the manufacturers regarding leukapheresis product, apheresis,
and product application are summarized in Korell et al.14
VASCULAR ACCESS

Vascular access is often a rate limiting step for apheresis procedures including both stem cell and immune
effector cell collection. Adequate vascular access is key
to maintaining the high venous blood flow throughChapter 5: Cell Collection and Starting Materials Consideration

the apheresis device necessary for cellular collection.
Blood flow through apheresis collection devices canrange from 10 to 120 mL/minute and needs to be
maintained potentially for several hours for efficient cellular collection procedures. It is important to know the
projected inlet rate of the apheresis device so a catheterwith appropriate maximum flow rate may be chosen. A
flexible 22-gauge catheter can sustain maximum flow
rates of up to 35 mL/minute while an 18-gauge can do
60 mL/minute, whereas a steel 17/18 gauge needle cansustain higher rates of approximately 75 mL/minute.
Several factors to consider when choosing a venous
access approach are: 1) type and duration of apheresis
therapy; 2) the potential need for additional intravenousinterventions (medications, chemotherapy, radiologic
studies requiring dye injection); 3) patient age/size;and 4) diagnosis. Ultimately patient (and family)
understanding, comfort, and preference are the mostimportant considerations.
Each venous access approach has its own set of risks,benefits, and considerations. Stem cell and immune effector cell collections typically do not require long term
vascular access solutions such as tunneled catheters andsubcutaneous ports. Ideally peripheral access should beconsidered first for non-emergent, short-term procedures when patient clinical status and preference allow.
Hospitals worldwide have tried to decrease reliance on
central venous lines (CVLs) in healthy allogeneic blood
donors undergoing peripheral blood stem cell collection. Ghazi et al. were able to decrease the rate of CVL
usage for these donors at their institution from 72% to
0% over the course of a 16-month quality improvement
program.15 Autologous patients, however, represent a
different population where two points of peripheral
access may not be possible due to clinical status. If acentral venous catheter is needed for a temporary situation like stem cell collection or immune effector cellcollection, a non-tunneled line should be considered
and can often be placed expediently. Care needs to be
used as they do not have a cuff and are sutured in place.
Length of use for temporary catheters should be lessthan 2 weeks due to higher infection rates as comparedto tunneled lines. It is essential the patient, clinical team,and apheresis team collaboratively assess the vascularaccess options and chose the most effective approach.

A-CELL✼90

Allogeneic therapies, including allogeneic CAR-T, aregaining traction due to several advantages over autologous
products: immediate availability to patients, reduced cost,and more standardized product. However, this approach
can be associated with safety risks to the patients, such as
infection from adventitious agents in the donor blood, graft
versus host disease (GvHD), and rejection of allogeneic
cells, in addition to cytokine release syndrome and CARrelated gene modifications often observed with autologous
products.
While a well-defined manufacturing process anda robust control and testing strategy are critical for allcell-based therapy product development, the allogeneic
approach requires additional factors to ensure safe administration to patients. Donor screening and testing are
critical factors that must be taken into account during the
product manufacturing. It should be noted that even in
an autologous setting where there are no regulatory requirements on donor eligibility-based human viral testing,contract development and manufacturing organizations(CDMOs) often require it to minimize the risk of bringing viruses into their GMP facilities. This section of thechapter discusses the principles and available guidance forsuitable qualification and testing of starting material andcomponents, donor eligibility determination, as well as appropriate product testing and characterization for humancells, tissues, and cellular and tissue-based products (HCT/
Ps). Additionally, to establish histocompatibility for users
of allogeneic-sourced HCT/Ps, human leukocyte antigen(HLA) testing and confirmation may be performed as partof the product identity tests. Discussion on HLA matchingis presented in Chapter 2.

in the manufacturing process described in the approved
biologics license application.” Most importantly, suchcontamination could result in detrimental clinical consequences for the eventual patients. Therefore, it is criticalthat developers establish thorough testing methods andprotocols to ensure that manufacturing process-produced
batches are free of adventitious agent contamination.
Possible sources for contamination include the patientthat was the source of the initial agent isolate, the cell substrates involved in the manufacturing process, other raw
materials used during manufacturing that were of biologicalorigin, personnel handling, and improper maintenance of
manufacturing equipment. Adventitious agent testing should
utilize a combination of methods and strategies to provide
assurance (to the extent possible) that products are free fromadventitious agents and should be performed at various
stages during the manufacturing process to maximize thechance of detecting contaminants. Tests for contaminants ofcell-based products include electron microscopy, assays for
retroviral reverse transcriptase, and detection of contaminants in cell culture and animal host systems. Contaminantdetection in vitro can be achieved by observing for cytopathic
effect, hemagglutination, or hemadsorption. In vivo assays
involve the inoculation of animal species and subsequent observations for mortality and testing of tissue for the presenceof hemagglutinins and production of antibodies. Polymerase
chain reaction (PCR) is also a powerful tool that can be used
to detect the presence of contaminant genomes. Tests should
comply with the International Council for Harmonization(ICH) Q2 (R1) guideline. Further guidance is provided
in FDA Guidance for Industry, Characterization andQualification of Cell Substrates and Other Biological Materials
Used in the Production of Viral Vaccines for Infectious Disease
Indications.16

Adventitious agents

Starting material qualification

Adventitious agents are considered to be viruses, bacteria, fungi, mycoplasma/spiroplasma, mycobacteria,
rickettsia, protozoa, parasites, and transmissible spongiform encephalopathy (TSE) agents. A major concern
for developers of cell-based therapy products is the riskof contamination of cells by adventitious agents during
the manufacturing process. This is reflected in 21 CFR
610.13, which states the following: “Products shall be freeof extraneous material except that which is unavoidable

In the November 2020 workshop on Allogeneic Adoptive
Cell Therapies Donor Cell Test Requirements, held by
the Alliance for Regenerative Medicine,17 the FDA CBER
recommended that starting material qualification for
allogeneic CAR-T therapies include the following:• Safety testing: sterility, mycoplasma, adventitious
agents, as well as other relevant human pathogens
not included in donor eligibility testing (discussed insubsequent sections);

DONOR CONSIDERATIONS FOR ALLOGENEIC CELLTHERAPY PRODUCTS

Chapter 5: Cell Collection and Starting Materials Consideration

A-CELL✼91

• Established acceptance criteria, e.g., minimum cellnumber, % CD3+, viability;• Additional characterization studies such as phenotypic
analysis (e.g., % of CD4+ and CD8+ cells, % NK cells,% monocytes, % B cells) as these might inform theneed for cell selection during process development.

screening above, medical records of donors of viable,
leukocyte-rich cells must be screened for risk factors forand clinical evidence of relevant cell-associated communicable disease agents and diseases, including Human
T-lymphotropic virus.

Donor screening and testing

Donor testing for RCDADs must be performed as described in 21 CFR § 1271.80 and § 1271.85.19 Testing
must be performed in an FDA licensed, CLIA certified laboratory (Clinical Laboratory Improvement
Amendments of 1988) or equivalent as determined by
the Centers for Medicare and Medicaid Services (CMS),
using appropriate FDA-licensed, approved, or cleared
donor screening tests, according to the manufacturer’s
instructions for use (IFU). It should be noted that for
diseases or disease agents where no FDA-approved tests
are available, a risk-based approach should be taken toqualify the donor cells to ensure that the final cell-based
product safety is not compromised.
According to 21 CFR 1271.80(b), general requirements for donor testing are as follows:• Testing must be done for relevant communicablediseases. In the case of a donor one month of age or
younger, a specimen from the birth mother must be
tested instead of a specimen from the donor.
• In general, donor specimens must be collected fortesting at the time of recovery of cells or tissue fromthe donor or up to 7 days before or after recovery.
However, specimens from donors of peripheral blood
stem/progenitor cells, bone marrow (if not excepted
under Title 21 CFR, Parts 1271(d)(4)), or oocytes may
be collected for testing up to 30 days before recovery.
• Appropriate FDA-licensed, approved, or cleared
donor screening tests, in accordance with the
manufacturer’s instructions, must be conductedto adequately and appropriately reduce the risk oftransmission of relevant communicable diseaseagents or diseases; however, until such time asappropriate FDA-licensed, approved, or cleared
donor screening tests for Chlamydia trachomatis andfor Neisseria gonorrhea are available, FDA-licensed,
approved, or cleared tests labeled for the detection of
those organisms in an asymptomatic, low-prevalence
population must be used.

The FDA provides guidance on appropriate standards forcell donor screening and donor testing. In general, the
FDA requires donor screening and testing for humancells, tissues, or cellular or tissue-based products (HCT/Ps) when source material is collected from allogeneic
human donors (21 CFR 1271).18 Donors are defined as
individuals to be used as sources for HCT/Ps intended
for implantation, transplantation, infusion, or transfer
into a human recipient. In general, FDA guidelines fordonor testing and donor screening are meant to address
the following three areas:• Limiting the risk of transmission of communicabledisease from donors to recipients;• Establishing manufacturing practices that minimize
the risk of contamination;• Requiring an appropriate demonstration of safety
and effectiveness for cells and tissues that present
greater risks due to their processing or use.
Donor screening

Donor screening determines whether a donor is either
eligible or ineligible to donate cells or tissues to be usedin HCT/Ps. According to 21 CFR, Parts 1270 and 1271,
all donors of cells and tissue must be screened for thefollowing medical conditions:• Risk factors for, and clinical evidence of, RelevantCommunicable Diseases and Disease Agents(RCDAD), including:-Human immunodeficiency virus (HIV), types 1 and 2-Hepatitis B virus (HBV)-Hepatitis C virus (HCV)-Human transmissible spongiform encephalopathy(TSE), including Creutzfeldt-Jakob disease (CJD)-Treponema pallidum (syphilis)• Communicable disease risks associated withxenotransplantation
In addition to the general requirements for donor

Chapter 5: Cell Collection and Starting Materials Consideration

Donor testing

A-CELL✼92

Donors must be deemed ineligible if found to be anyof the following:• A donor whose specimen tests reactive on ascreening test for a communicable disease agent
in accordance with §1271.85, except for a donor
whose specimen tests reactive on a non-treponemal
screening test for syphilis and negative on a specifictreponemal confirmatory test.
• A donor in whom plasma dilution sufficient toaffect the results of communicable disease testingis suspected unless: a) the specimen taken from thedonor is tested before transfusion or infusion and istested up to 7 days before recovery of cells or tissue,or b) an appropriate algorithm designed to evaluate
volumes administered in the 48 hours before specimen collection is used, and the algorithm showsthat plasma dilution sufficient to affect the results ofcommunicable disease testing has not occurred.
• Clinical situations in which plasma dilution issuspected to be sufficient to affect the results ofcommunicable disease testing including, but notlimited to the following: a) blood loss is known
or suspected in a donor over 12 years of age, andthe donor has received a transfusion or infusionof more than 2,000 milliliters (mL) of blood (e.g.,whole blood, red blood cells) or colloids within
48 hours before death or specimen collection(whichever occurred earlier) or more than 2,000mL of crystalloids within 1 hour before death or
specimen collection (whichever occurred earlier),or b) regardless of the presence or absence of blood
loss, the donor is 12 years of age or younger and hasreceived a transfusion or infusion of any amount ofblood (e.g., whole blood, red blood cells) or colloids
within 48 hours before death or specimen collection(whichever occurred earlier) or crystalloids within 1
hour before death or specimen collection (whichever occurred earlier).
Specimens from all donors must be tested for evidence of infection due to RCDADs listed in the Donor
Screening section above. In addition to the general
requirements for donor testing, testing requirements for
specimens from donors of viable, leukocyte-rich cells or
tissue are as follows:

Chapter 5: Cell Collection and Starting Materials Consideration• Specimens from donors of viable, leukocyte-rich cells
or tissue must be tested to adequately and appropriately reduce the risk of transmission of relevant
cell-associated communicable diseases, including
Human T-lymphotropic virus, type I and Human
T-lymphotropic virus, type II.
• Specimens from donors of viable, leukocyte-richcells or tissue must be tested for evidence of infection
due to cytomegalovirus (CMV) to adequately andappropriately reduce the risk of transmission. A
standard operating procedure must be established andmaintained for governing the release of an HCT/P
from a donor whose specimen tests reactive for CMV.
It is important to note that donor eligibility screening and testing requirements can differ by country. For
example, other countries may not use FDA licensed
test kits, CLIA certified labs, or require all the nucleic
acid and antibody-based testing. This is especially
important in the case of allogeneic therapies that are
being expanded for use by many patients.
Considerations for other transmissible agents

The COVID-19 pandemic has highlighted the necessity
for some testing considerations for specific transmissible agents that are not covered in the publishedguidance. These can include infections that may beregionally endemic, associated with specific donor populations, or occur during a particular period of time.
The FDA determines whether certain communicabledisease agents and diseases are relevant based on therisk of transmission, severity of effect, and availability of
appropriate screening measures or tests. For example,
West Nile Virus (WNV), Sepsis, and Vaccinia, whichwere not specifically listed under 21 CFR 1271.3(r)(1),
were determined to be relevant under 21 CFR 1271.3(r)(2).20 Additionally, the Zika virus (ZIKV), which wasrecognized as a relevant transfusion-transmitted infection in August 2016 under 12 CFR Part 630, is nolonger considered relevant.21
The general perspective is that manufacturers
should perform risk assessment that identifies, evaluates, and mitigates factors that may allow for transmission of such disease agents through their product.

A-CELL✼93

Additionally, when appropriate, the FDA publishes andupdates disease-specific guidance for the industry. The
recent COVID-19 pandemic shaped the FDA’s thinking
and expectations for cell and gene therapy productmanufacturers as outlined in the 2021 Guidance forIndustry.22 At the time of the writing of this chapter,
FDA does not recommend using laboratory tests toscreen asymptomatic HCT/P donors for SARS-CoV-223
because SARS-CoV-2 has not been declared a relevant
communicable disease agent and disease under 21 CFR
1271.3(r). However, if a manufacturer is considering
incorporating donor testing for SARS-CoV-2 as a risk
mitigation strategy for manufacturing a CGT product, then viral tests (molecular or antigen) approved,
cleared, or authorized by the FDA should be used todiagnose current infection.

Final cell product release testing
Release testing of cell-based therapy products, including
CAR-T, entails performing a series of assays that demonstrate product safety, identity, purity/impurity, quantity/
strength, and potency. For detailed discussions on thedevelopment of these assays, recommended tests forautologous therapies, and challenges related to characterization for cell-based therapies, refer to Chapter 9.
In addition to the tests outlined in Chapter 9, allogeneic therapies require additional tests for adventitious
virus. Similar considerations/recommendations apply
to these tests, including:• Final product must be tested using qualified tests
with establish limit of detection (LOD)/limit ofquantification (LOQ).
• Acceptance criteria must be set for lot release withdetailed justification.
• Presence of active viral replication needs to beevaluated if virus is detected.
• Testing of incoming donor material and in-processsamples are recommended to determine if there
is correlation with DP test results and if there is
amplification during the manufacturing process if avirus is present.
• Retain samples are recommended in case additional
testing is necessary.

Chapter 5: Cell Collection and Starting Materials Consideration

Perspectives on HHV TestingIn addition to the common virus testing listed in various
guidelines,24,25,26 FDA also recommends monitoring of
HHV-6 and HHV-7 during the manufacturing process
and in the final product. HHV is ubiquitous and latent
infection in the general population is expected. Althoughlatency is typically asymptomatic, HHV-6 reactivates in> 50% of allogeneic hematopoietic stem cell transplantation (HSCT) recipients and can produce clinically significant manifestations, including encephalitis, delayedengraftment, and an increased rate of graft-versus-hostdisease, substantially increasing mortality.27,28.29 In organtransplants, horizontal transmission of HHV-6 in liver
has been shown to result in a fatal outcome.30
In patients receiving CAR-T therapies, the riskof potential HHV reactivation is also inherent. 31,32
Unfortunately, HHV-associated encephalitis can be difficult to distinguish from neurotoxicity, a common adverse
effect of CAR-T therapy. The FDA acknowledges that additional information is needed in the clinical trial setting
to determine whether and to what extend the productcontributed to HHV reactivation. Samples can be banked
until additional information from CMC related productassays mature to initiate testing of CAR-T recipients.
Considerations for appropriate testing of banked samples include next-generation sequencing (genotyping) todistinguish product-related vs. reactivation effects, andserological testing to understand the prevalence of baseline rates of latent infection, asymptomatic rising titersof Ab and qPCRs, and contribution of rising titers toclinical infection. In addition, real time assessments forrising titers and high DNA copy numbers may be neededin recipients experiencing neurotoxicity consistent withthe practice of medicine to diagnose HHV infection.
Given the unknown risks from products, difficultyof differential diagnosis, and serious nature of HHV
infection, the FDA currently recommends the following
in regard to allogeneic CAR-T products:17• Negative lots be released first;• Positive lots be released in sequence (least to mostpositive) after sponsor submits preliminary efficacy andsafety data in 3-5 subjects who receive negative lots;• Protocols should mandate assessment and treatment of
HHV infections with the onset of neurotoxicity symptoms.

A-CELL✼94

Regulatory considerations

Conclusions

The regulatory guidance surrounding the identificationof appropriate donors, collection of cells, and manipulation and preservation of cells varies around the world.
In the U.S., the FDA mandates the requirements of not
only donor selection and collection but also processing
and storage. Particular to controls and quality management around cell collection, the FDA Code of Federal
Regulations requires collection sites to establish andmaintain procedures to control all documents required
for the entire cellular therapy process. This includes identifying the appropriate individuals to review procedures
for accuracy, approvals with dates/signatures, ensuringcertain documents are available at the appropriate pointof use, and removing obsolete procedures and procedureversions from the system. A well-established protocol for
an effective document control is also critical, as this is
the foundation of quality management systems.
Additional comprehensive evidence-based standards
in cellular therapy for voluntary accreditation are published by the Foundation for the Accreditation of Cellular
Therapy (FACT) and JACIE, the Joint Accreditation
Committee of ISCT and EBMT (the European Society
for Blood and Marrow Transplantation.)These standards
on cellular therapy product collection, processing, andadministration apply to hematopoietic progenitor celltransplantation/therapies as well as immune effector celltherapies and are accepted in many countries worldwide. Overall, it is important to review the regulatory
and accreditation guidelines for the country where thecell collection process takes place. Refer to Chapter 2
for an overview of the regulatory landscape related tocell-based therapies.

Recent commercialization and ongoing developmentof cell-based therapies emphasize the criticality ofhigh-quality starting cell material. For cell-based therapies, this starts with the apheresis process, which involves
many variables that require detailed consideration toensure the best possible outcome for the cell collection
procedure and, ultimately, the patients.
This chapter covers various technical aspects of thecell collection process, clinical operation considerations
including patient preparation and monitoring, as well asavailable regulatory guidance on donor screening andtesting. As the industry evolves, efforts are constantly
made on ensuring consistent, robust apheresis processes
for clinical manufacturing through standardization ofapheresis center training, equipment, and protocols tominimize variation and increase the quality of starting
materials, as well as access to a large, diverse, and reliable donor network. Through process standardizationand comprehensive quality management programs, celltherapy manufacturers can help ensure consistent qualityof cellular starting material that optimizes and maintains
product safety and efficacy, without sacrificing the safety
and comfort of the donor and/or patient.

Chapter 5: Cell Collection and Starting Materials Consideration

A-CELL✼95

References
1

Cell Collection Standards for Cell and Gene Therapies.
https://www.standardscoordinatingbody.org/project-cellcollection-standards-for-cell-therapies

2

https://www.terumobct.com/spectra-optia

3

Kiprov D. Principles of Blood Separation and Apheresis
Instrumentation. https://cdn.ymaws.com/www.apheresis.
org/resource/collection/E759EDA3-9E20-4A23-A02BA22C602AEC42/15.05.06-0830-LonestarBC-Kiprov-v2.pdf

4

Lee G, Arepally GM. Anticoagulation techniques inapheresis: from heparin to citrate and beyond. J Clin Apher.
2012;27(3):117-125. doi:10.1002/jca.21222

5

Philip J, Sarkar RS, Pathak A. Adverse events associatedwith apheresis procedures: Incidence and relative frequency.
Asian J Transfus Sci. 2013;7(1):37-41. doi:10.4103/09736247.106730

6

Bouchard J, Madore F. Role of citrate and other methodsof anticoagulation in patients with severe liver failure
requiring continuous renal replacement therapy. NDT Plus.
2009;2(1):11-19. doi:10.1093/ndtplus/sfn184

7

TerumoBCT Spectra Optia® Apheresis System Operator’s
Manual.

8

Preslar AM, Homsombath AA, Hsieh FF, Roig J, Boyer
M, Couriel DR, Reems J. Comparison of the Continuous
Mononuclear Cell and Mononuclear Cell Protocols on theSpectra Optia Apheresis System for Collecting Autologous
and Allogeneic Peripheral Blood Stem Cells (PBSCs). March2018. Biology of blood and marrow transplantation: journal of
the American Society for Blood and Marrow Transplantation
24(3):S177

9

Putensen, D, Smith, R, Pilcher, L, Trandafir, G. Comparisonof the CMNC and MNC apheresis protocol for the collectionof T-cells showed comparable outcome: An observational
study in a single centre. J Clin Apher. 2018; 33: 349– 356.
https://doi.org/10.1002/jca.21618

10

Punzel M, Kozlova A, Quade A, Schmidt AH, Smith R.
Evolution of MNC and lymphocyte collection settings
employing different Spectra Optia® Leukapheresis systems.
Vox Sang. 2017 Aug;112(6):586-594. doi: 10.1111/vox.12540.

11

Armitage S, Hargreaves R, Samson D, et al. CD34 counts
to predict the adequate collection of peripheral blood
progenitor cells. Bone Marrow Transplant 1997;20:587–591

12

Keating GM. Plerixafor: a review of its use in stem-cell
mobilization in patients with lymphoma or multiple
myeloma. Drugs. 2011 Aug 20;71(12):1623-47. doi:
10.2165/11206040-000000000-00000

13

Shimura Y, Kuroda J, Uchiyama H, Kawata-Iida E,
Tsutsumi Y, Nagoshi H, Mizutani S, Yamamoto-Sugitani M,
Matsumoto Y, Kobayashi T, Horiike S, Shimazaki C, Kaneko
H, Kobayashi Y, Taniwaki M. Hematopoietic progenitor
cell mobilization using low-dose cyclophosphamide andChapter 5: Cell Collection and Starting Materials Consideration

granulocyte colony-stimulating factor for multiple myeloma.
J Clin Apher. 2013 Oct;28(5):368-73. doi: 10.1002/jca.21283
14

Korell F, Laier S, Sauer S, Veelken K, Hennemann H,
Schubert M-L, Sauer T, Pavel P, Mueller-Tidow C, Dreger
P, Schmitt M, Schmitt A. Current Challenges in Providing
Good Leukapheresis Products for Manufacturing of CAR-T
Cells for Patients with Relapsed/Refractory NHL or ALL.
Cells. 2020; 9(5):1225

15

Ghazi et al. Reducing Central Venous Catheter Use in
Peripheral Blood Stem Cell Donation: Quality Improvement
Report. BMJ Qual Improv Rep. 2017; 6(1): u211975.w4817.
doi: 10.1136/bmjquality.u211975.w4817

16

Characterization and Qualification of Cell Substrates andOther Biological Materials Used in the Production of Viral
Vaccines for Infectious Disease Indications. Guidance forIndustry. FEBRUARY 2010. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/characterization-and-qualification-cell-substrates-and-other-biological-materials-used-production

17

Allogeneic Adoptive Cell Therapies Donor Cell Test
Requirements. Alliance for Regenerative Medicine virtual
workshop, November 17, 2020.

18

21CFR1271. Code of Federal Regulations PART 1271-- HUMAN CELLS, TISSUES, AND CELLULAR AND
TISSUE-BASED PRODUCTS, Subpart C - Donor Eligibility.

19

Testing Donors of Human Cells, Tissues, and Cellular andTissue-Based Products (HCT/P): Specific Requirements.
https://www.fda.gov/vaccines-blood-biologics/safetyavailability-biologics/testing-donors-human-cells-tissuesand-cellular-and-tissue-based-products-hctp-specificrequirements

20

https://www.fda.gov/regulatory-information/search-fdaguidance-documents/eligibility-determination-donorshuman-cells-tissues-and-cellular-and-tissue-based-products

21

https://www.fda.gov/vaccines-blood-biologics/blood-bloodproducts/information-blood-establishments-regarding-fdasdetermination-zika-virus-no-longer-relevant

22

Manufacturing Considerations for Licensed andInvestigational Cellular and Gene Therapy Products
During COVID-19 Public Health Emergency. FDA
Guidance for Industry, January 2021. https://www.fda.gov/
regulatory-information/search-fda-guidance-documents/
manufacturing-considerations-licensed-and-investigationalcellular-and-gene-therapy-products-during

23

Updated Information for Human Cell, Tissue, or Cellular or
Tissue-based Product (HCT/P) Establishments Regarding
the COVID-19 Pandemic. https://www.fda.gov/vaccinesblood-biologics/safety-availability-biologics/updatedinformation-human-cell-tissue-or-cellular-or-tissue-basedproduct-hctp-establishments

A-CELL✼96

References, continued
24

21CFR1271. Code of Federal Regulations PART 1271-- HUMAN CELLS, TISSUES, AND CELLULAR AND
TISSUE-BASED PRODUCTS

25

Points to Consider in the Characterization of Cell Lines Usedto Produce Biologicals (1993). CBER Memorandum 84N0154.

26

ICH Q5A “Viral Safety Evaluation of Biotechnology Products
Derived from Cell Lines of Human or Animal Origin,” FDA1996-D-0065, September 1998

27

P.J. de Pagter, Rob Schuurman, Ellen Meijer, Debbie van
Baarle, E.A.M. Sanders, J.J. Boelens. 2008. Human herpesvirus type 6 reactivation after haematopoietic stem celltransplantation. Journal of Clinical Virology 43(4), 361-366.
doi: 10.1016/j.jcv.2008.08.008

28

P.J. Anne de Pagter, Rob Schuurman, Henk Visscher, Machiel
de Vos, Marc Bierings, Anton M. van Loon, Cuno S.P.M.
Uiterwaal, Debbie van Baarle, Elisabeth A.M. Sanders,
JaapJan Boelens. 2008. Human Herpes Virus 6 Plasma DNA
Positivity after Hematopoietic Stem Cell Transplantation inChildren: an Important Risk Factor for Clinical Outcome.
Biol Blood Marrow Transplant 14:831-839. doi: 10.1016/j.
bbmt.2008.04.016

Chapter 5: Cell Collection and Starting Materials Consideration

29

Brian C. Betts, Jo-Anne H. Young, Celalettin Ustun,
Qing Cao, Daniel J. Weisdorf. 2011. Human Herpesvirus
6 Infection after Hematopoietic Cell Transplantation:
Is Routine Surveillance Necessary? Biol Blood Marrow
Transplant 17:1546-1568. doi: 10.1016/j.bbmt.2011.04.004

30

P. Bonnafous, J. Marlet, D. Bouvet, E. Salamé, A.-C. Tellier,
S. Guyetant, A. Goudeau, H. Agut, A. Gautheret-Dejean,
C. Gaudy-Graffin. 2018. Fatal outcome after reactivation of
inherited chromosomally integrated HHV-6A (iciHHV-6A)
transmitted through liver transplantation. Am. J. Transplant.
18(6): 1548-1551. doi: 10.1111/ajt.14657

31

Melanie T Rebechi, Jacqueline T Bork, David J Riedel. 2021.
HHV-6 Encephalitis After Chimeric Antigen Receptor T-cell
Therapy (CAR-T): 2 Case Reports and a Brief Review of
the Literature. Open Forum Infectious Diseases 8(11): doi:
10.1093/ofid/ofab470

32

Guy Handley, Rodrigo Hasbun, Pablo Okhuysen. 2021.
Human herpesvirus 6 and central nervous system disease inoncology patients: A retrospective case series and literature
review. Journal of Clinical Virology 136. doi: 10.1016/j.
jcv.2021.104740.

A-CELL✼97

CHAPTER 6

Ancillary Materials
CHAPTER LEADSClaudia Zylberberg, Akron Biotech
Ramon Montero, Akron Biotech
Allen Parsons, Akron Biotech

Chapter 6 | Contents
Introduction.....................................................................99

Reagents Considerations............................................. 105

Principles of Ancillary Materials................................. 100

Safety, efficacy, consistency, and stability............ 105

International........................................................... 102

Case studies to support AM standardization......... 106

Australia................................................................. 102

Case study 1: Research-grade vs. CGMP-grade
substrates and scaffolds........................................ 106

Japan...................................................................... 102
Europe.................................................................... 102
USA......................................................................... 102
Risks Associated with Ancillary Materials................. 103
Risk management..................................................... 103
Risk assessment..................................................... 103
Risk classification for ancillary materials.............. 104
Risk control............................................................ 104

Case study 2: Enhancing the safety ofvirus-inactivated ancillary materials throughneutralizing antibodies...........................................107
Case study 3: Variability in the industry­—
recombinant protein example.............................. 108
Conclusions..................................................................... 111
References...................................................................... 112

Current selection criteria and qualification
program outline........................................................ 105

Introduction
At the cutting edge of technology in medicine, we can findmany products that involve the utilization of additional
materials not intended to remain in the final consumabletherapeutic. These materials are referred to as Ancillary
Materials (AMs), which are defined as “material thatcomes into contact with the cell or tissue product duringcell-processing, but is not intended to be part of the final
product formulation.”1 The final therapeutic products
commonly referred to in the literature include those thatfall under broader categories such as advanced therapy
medical products (ATMP), cell and gene therapies (CGT),and tissue engineered medicinal products (TEMP).
Throughout this chapter we will commonly refer to alltherapies herein mentioned as CGTs.
In the rapidly changing landscape of CGTs, a lack ofcommon terminology leads to a lack of global regulatory
oversight for AMs. For example, the European Union(EU) commonly refers to these materials as “raw materials,” a term that could easily be misconstrued within thisdocument’s context. To make matters worse, CGTs typically use many unique and diverse AMs with different
levels of quality control and availability (refer to Table 6-1
for common definitions of AMs). This diversity of AMs,

Chapter 6: Ancillary Materials

along with their unique and complex characteristics,
makes them difficult to qualify. Generally, the AM qualification process involves identification, selection, andsuitability for use in manufacturing, characterization,
vendor qualification, and quality assurance and control;
however, it depends on many factors, including the typeof CGT product being manufactured and the stage ofmanufacture in which the AM is used. As a result, there
is no “one-size-fits-all” qualification program, and many,
if not all, regulatory concerns fall within AM users andsuppliers. Hence, there is a clear need to globally standardize terminology and regulations to ensure higher
quality and consistent CGT products.
In this chapter, we give the reader an in-depth
understanding of the landscape by defining the many
nomenclatures utilized, review different regulatory
bodies by country of origin, and explore specific AMs
commonly used during CGT manufacturing. Where appropriate, principles and examples specific to cell-based
therapies are presented. In this document, the authors
follow the general definitions and principles of AMs
presented in the ISO/TS 20399 standards, which exclude
non-biological consumables and plasticware (e.g., tissue
culture flasks, bags, tubing, pipettes, needles) and feeder
cells, but include consumables that can have a biological

A-CELL✼99

component (e.g., coated dishes or beads). Finally, we introduce some case studies to further strengthen the casefor AM standardization, which aims to drive improvement to the consistency and quality of AMs through anappropriate level of documented lot-to-lot consistency
and best practices for AM suppliers and users.

Principles of Ancillary Materials
During the manufacturing process, AMs come into contact with the cellular therapeutic product, however are
not intended to be in the final product. Examples of AMs
used in CGT applications include, but are not limited to:• Monoclonal antibodies: used in cell selection/depletion• Cytokines, growth factors, and other supplements:
used to regulate/activate/differentiate cells in culture• Antibiotics, serum culture media, and enzymes: used
to passage cells• Substrates/scaffolds (e.g., Matrigel): used for cell
expansion and differentiation
These materials come in direct contact with cells
destined for clinical use and require the utmost safety,
potency, and purity. As a result, AM manufacturers utilize labels such as “For Research Use Only,” “Laboratory
Grade,” “CGMP,” and “GMP” amongst others in an
attempt to categorize and distinguish their productswith little to no global guidance. The most common
nomenclature found in product labels are presented inTable 6-1.
So, the question arises, what exactly is the difference
between one label and another? To answer this question,
we must understand the current state of global regulationin the AM space as well as regional regulatory agencies andhow they regulate or lack regulation of these imperative
materials utilized in the manufacturing process of CGTs.

Table 6-1: Definitions for commonly used nomenclature by AM manufacturers in product labels
Term

Meaning

Alternative Terms

Animal Derived
Component Free
(ADCF)

A term to describe products or materials that do not contain an ingredientthat is an animal tissue or body fluid, or is isolated or purified from animal
tissue or body fluid.
May contain recombinant proteins produced in animal cell lines or byfermentation processes.
Does not necessarily limit the use of animal-derived components used in the
manufacture of AM raw materials (secondary materials) or materials usedfurther downstream unless indicated.

Animal Component
Free (ACF)
Animal Origin Free
(AOF)

ChemicallyDefinedA term to describe products or materials that have known chemical structures(defined by a chemical formula) and high purity, for example, small molecules,
salts, carbohydrates, amino acids, fatty acids, and steroids (cholesterol,dexamethasone). This is evaluated on a case-by-case basis.
Does not contain proteins or other ingredients with a complex structure.
Ingredients may be synthetic or biologically derived (e.g., cholesterol fromsheep’s wool grease).

ACF
Serum Free

Clinical GradeA term to describe products or materials that are suitable for clinical use,for example, injectable grade. Material shown to be safe and efficacious
for human use through appropriate clinical trials and regulatory approvals.
Usually, clinical-grade products are approved as drugs by regulators, andlabeling or product documentation should state sterility and safety profile.
Suitable for clinical use for a specific intended use only. Clinical-grade AM fora specific and approved intended use does not mean that the AM is approvedfor other “off-label” processing uses without qualification and approval fromthe appropriate regulatory agency.

Pharmacopeialgrade
Infusible-grade
Activepharmaceuticalingredient (API)grade
Approved forhuman use

The authors acknowledge that some terms listed under “Alternative Terms” are not direct synonyms of the terminology, however represent
common use of these definitions in the industry.

continued on next page

Chapter 6: Ancillary Materials

A-CELL✼100

Table 6-1: Definitions for commonly used nomenclature by AM manufacturers in product labels
continued from previous page
Term

Meaning

Alternative Terms

Good
Manufacturing
Practice (GMP)

Refers to the current GMP (CGMP) regulations requiring manufacturers,
processors, and packagers to take the necessary steps to ensure that theirproducts are traceable, safe, pure, and effective.
When associated with a product or material, this term denotes that the
product has been prepared under some or all of the CGMP requirements
to ensure proper design, monitoring, and control of the manufacturing
processes, facilities, and the final product. The CGMP regulations can beconsidered under a phased approach, depending on the phase and intended
use of a product within a clinical application.2
All users should review intended use statements on product documentation.
Intended uses of GMP products vary and are not necessarily qualified orintended for applications outside of “research use only.”
Regional differences exist, where some national regulatory agencies provide
GMP certificates to manufacturers of GMP AMs and others do not provide them.
Term is universally recognized by regulatory authorities.

CGMP
Manufactured
under GMP
GMP-compliant
GMP-grade
Compliant to 21
CFR 210, 211, 820

Laboratory
Grade

A term to describe products or materials that are intended to be usedwithin the laboratory or for research use and not intended for food, drug, or
medicinal/clinical use.

Research-gradeResearch use only
Laboratory use
only
Non-pharmaceutical–
grade

Research Use
Only (RUO)

A term to describe the intended use of materials generally limited for use
in research or preclinical applications only and not for clinical trials unlessqualified appropriately and approved for use in a given application by
applicable regulatory authorities.
RUO products may be manufactured under various quality management
systems, including ISO-certified or GMP.

Laboratory use only
Research-grade
Non-pharmaceuticalgrade
Laboratory-grade

Serum-free

A term to describe products or materials that do not contain serum or plasma
as an ingredient.
May contain processed or derived ingredients from blood, serum, or plasma
such as albumin, transferrin, low-density lipids, hormones, and platelet lysate.
May contain other undefined ingredients that are not serum or plasma (e.g.,
tissue extracts such as bovine pituitary extract, platelet lysate, growth factors,
hormones, and carrier proteins).
Serum-free media (SFM) allows researchers to grow specific cell types or
perform specific applications in the absence of serum.

Defined media

Xeno-free

A term to describe products or materials that do not contain components
derived from other species.
May contain serum or serum-derived components from the native species(e.g., human cell lines can be cultured using human-derived components
such as human serum).
Xeno-free media (XFM) allows researchers to grow specific human cell
types in the absence of animal serum (e.g., fetal bovine serum).

ACF

TSE/BSE freeDeclaration that products or materials are fully chemically synthesized or freefrom transmissible spongiform encephalopathy (TSE) and bovine spongiform
encephalopathy (BSE) risk materials.
Country of origin of material or product is essential in assessing BSE/TSE
risk, because some countries are categorized by the World Organization for
Animal Health (OIE) as BSE-negligible risk or controlled for BSE risk. In the U.S.,
USDA lists and updates list of country at risk.
Testing for BSE/TSE on raw materials does not currently exist.
The term is universally recognized by regulatory authorities.

Chapter 6: Ancillary Materials

A-CELL✼101

There is currently no AM-specific legislation within
the United States or the European Union that provides aclear legal framework to guide AM qualification practices.
Compliance requirements are not made explicit, and regulators depend on the judgement of product developers
to justify and validate the use of each AM within eachspecific application.3,4,5,6 Despite the dearth of legislative
text specifically addressing AM use, with the number
and breadth of AMs used in CGT, regulators across theglobe approve and generally prefer the use of standards
in AMs. Guidelines are available from various national
and international organizations to guide best practices inqualifying AMs, including from the United States Foodand Drug Administration (FDA) through the UnitedStates Pharmacopeia (USP), the International Council for
Harmonization (ICH),7 the International Organization forStandardization (ISO),8 the European Medicines Agency(EMA),9 and European Pharmacopoeia (Ph. Eur.).10 This
chapter draws on U.S.- and EU-based sources and otherwell-established regulatory agencies11,12,13,14 with the understanding that regulators across these jurisdictions share acommon understanding of AM qualification through theirsubscription to ICH principles. The following is a list of themore relevant ones.

• ICH Q8 (R2): Pharmaceutical development• ICH Q9: Quality risk management• ICH Q10: Pharmaceutical quality system

Australia• Australian Regulatory Guidelines for Biologicals(ARGB)• Therapeutic Goods Administration (TGA)Japan• Pharmaceuticals and Medical Devices Agency(PMDA) and Ministry of Health, Labor and Welfare(MHLW) Public Notice No. 210, 2003 - Standard for
Biological Ingredients• Standards for manufacturing and quality control
for medical device and in vitro diagnostic reagents(MHLW No. 169, 2004)• General principles for the handling and use of cellular/tissue-based products (MHLW No. 266, 2001)• Guidelines on ensuring quality and safety of products
derived from processed human stem cells (MHLW
No. 1314, 2000)• Points to consider on manufacturing and quality
control (MHLW notification No. 0327025, 2008)

International

Europe• World Health Organization (WHO) GMP for
Biological Products• ISO/TS 20399-1:2018 Biotechnology — Ancillarymaterials present during the production of cellulartherapeutic products• ISO 9001: Quality Management Systems:
requirements• ISO 13485: Medical Device Quality Management
System: requirements for regulatory purposes• ISO/TC 276• ICH Q5A/D Quality of biotechnological products• ICH Q5E: Comparability of biotechnological/
biological products subject to changes in their
manufacturing process• ICH Q6B Specifications: Test procedures andacceptance criteria for biotechnological/biologicalproducts• ICH Q7: GMP Guide for Active Pharmaceutical
Ingredients• ATMP Regulation (EC) No. 1394/2007• Ph. Eur 5.2.12 Raw Materials of Biological Origin
for the Production of Cell-Based and Gene Therapy
Medicinal Product• EudraLex Volume 4 GMP guidelines

Chapter 6: Ancillary Materials

USA• USP <1043> Ancillary Materials for Cells, Gene andTissue-Engineered Products• USP <92> Growth Factors and Cytokines used inCell Therapy Manufacturing (limited to rh-IL4)• FDA Chemistry, Manufacturing, and Controls(CMC) guidance• 21 CFR 1271.210 - Current Good Tissue Practice(GTP)• Part 211 CFR Part 11 subpart E - Control of Components
and Drug Product Containers and Closures

A-CELL✼102

In an IND submission, sponsors can reference
Drug Master Files (DMFs) to provide information on
the ancillary materials used in their product manufacturing. DMFs are submitted directly to FDA by the
AM manufacturers, and provide confidential, detailed
information on the AM that the agency can review in
an Electronic Common Technical Document (eCTD)
format, including the facilities, processes, or articles usedin the manufacturing, processing, packaging, and storingof these AMs. There are 4 types of DMFs; AMs generally
use Type II DMFs. DMFs allow sponsors to referencematerial without the MF holder (the AM manufacturer)
having to disclose the contents to the sponsors.

Risks Associated with Ancillary
Materials

Risk can be defined as a metric for the amount of danger
posed by a given situation or variable. With respect toquality-related risk in CGT product manufacturing, as
is the case for other biopharmaceutical manufacturingcontexts, risk should always be assessed with the potential for diminishment of safety to the patient as theprimary concern. To that end, regulatory guidelinesemphasize the need for a risk-based approach in CGT
manufacturing,15 which includes the selection and implementation of AMs. During early phase discovery anddevelopment, it may be acceptable to assume higher risk
and use research-grade AMs with lower requirementsfor quality testing and documentation. This will allow
AMs to be used early that are less costly, available from
more manufacturers, and not restricted to the highest
quality standard. Furthermore, this will also allow morecomprehensive screening of sources for critical reagentsthat may not have been thoroughly evaluated in theprocess. As development progresses into preclinicaland early phase clinical trials, a CGT manufacturer
may select intermediate grade AMs produced using
additional manufacturing and testing processes included in the product quality documentation. In late phaseclinical trials and commercial manufacturing, CGMP
grade AMs are preferred to reduce risk by ensuring theAMs are traceable, consistent, safe, pure, and effective.
In the absence of products used for manufacturing
ATMPs that follow current regulations on AM quality

Chapter 6: Ancillary Materials

from either ISO or Pharmacopeia, a scheme to bringAMs into CGMP manufacturing can be implemented,
where each subsequent step in that path is associatedwith an increasingly stringent set of quality principles,
culminating in the production of an AM that is suitablefor commercial use.16
RISK MANAGEMENT

The general approach for mitigating risk is to follow a
strategic framework endorsed by all ICH harmonizedcountries. These principles underpin GMP concepts
implemented through a quality management system(QMS). The latter is defined as a set of well-documented
business practices and procedures responsible for assuring quality policies. While ISO 9001:2015 provides an
exemplar QMS that AM manufacturers can follow andbe certified against, it does not define the QMS, hence
other models may be implemented. More specific forthe manufacture of medical devices is ISO 12485:2016,
which is generally considered a higher level of quality
compliance than ISO 9001. Risk management strategies
for AM users are covered in ISO/TS 20399-1:2018 Part 3.
Ancillary Materials for Cell, Gene, and Tissue-Engineered
Products (USP <1043>) describes the application of thesame risk-based approach to AM qualification and sharesmuch of the same risk mitigation as Ph. Eur. 5.2.12 Rawmaterials of biological origin for the production of cellbased and gene therapy medicinal products.

Risk assessment
Risk management’s first stage is risk assessment, wherein
individual hazards are identified, analyzed, and evaluated. Recommended modalities for risk assessments
are failure mode effects analysis (FMEA), or hazard
analysis and critical control point. In the context of AM
qualification, a number of different grades of AMs, certificates, terminologies, and compliance with QMS can beapplied or used, potentially posing different levels of risk/hazard. Hazards to the product specification are mostapparent, but patient safety issues that are not directly
covered by the product specification are also paramount.
Irrespective of the AM grade or quality standard that isclaimed by the AM supplier, a number of factors should
be considered by AM users when evaluating a biologicalmaterial for its suitability in the manufacture of cellular

A-CELL✼103

therapeutic product.17 Examples of hazards to identify arethose that threaten identity, functionality, purity, sterility,
and other specification components; supply chain risks
such as continuity of supply should also be considered.
Each identified hazard should be analyzed to estimate
the likelihood of occurrence and severity of harm. A risk
evaluation is then performed to determine the strengthof evidence for each of the risk assessment stages (identification and evaluation), an essential step to understanding the risk assessment output quality. A quantitative,
semi-quantitative, or qualitative output can be produced
depending on the exact risk assessment method used.
The output can then provide guidance on how far down
the process streams these AMs are acceptable for use,
and/or guide risk control activities (e.g., the appropriate
levels of testing depending on the point of use in theprocess) by prioritizing those that score highest.
It should be noted that changes in the AM manufacturing can occur, potentially causing changes to the composition of the biological AMs, which can impact the quality of
the final cellular therapeutic product. It is important that
the AM user is aware of these changes prior to them being
effected. Additionally, the user should identify and qualify
an alternative AM supplier in the case that the preferredsupplier ceases its manufacture. As such, it is critical toestablish a Quality Agreement mandating the supplier toprovide sufficient notice for any change, and include aclause relating to the continuity of supply.18
From the sponsor’s perspective, a risk assessment
should be performed on the impact of such change, andcomparability studies might be warranted to evaluate
the product quality pre- and post-change. Principles ofcomparability studies are covered in Chapter 2.

is a licensed biologic, an approved drug, an approvedor cleared medical device, or it is intended for use as animplantable biomaterial. Generally, these components ormaterials are obtained as a sterile packaging system ordosage form intended for their label use but are instead
utilized “off label” in the manufacturing process for thecell, gene, or tissue-engineered product.
Tier 2—AMs are low-risk, well-characterized materials that are well-suited for use in manufacturing. Theirintended use is for drug, biologic, or medical device
manufacture, including cell, gene, and tissue-engineered
products as AMs, and they are produced under relevant
CGMPs. Most animal-derived materials are excluded
from this category.
Tier 3—AMs are moderate-risk materials that requirea higher level of qualification than previous tier materials. Frequently, these materials are produced for in vitro
diagnostic use and are not intended for use in the production of cell, gene, or tissue-engineered products. In
some cases, an upgrade of AM manufacturing processes
may be necessary to employ the AM in manufacturing
these products (e.g., modification of the production
process for a diagnostic grade monoclonal antibody toinclude robust viral removal steps in purification).
Tier 4—Highest risk level for AMs and extensive
qualification is necessary prior to use in manufacturing.
The materials are not produced in compliance with
CGMP and are not intended for use in the productionof cell, gene, or tissue-engineered products. This risk level
includes highly toxic substances with known biologicalmechanisms of action, and also includes most complex,
animal-derived fluid materials not subjected to adventitious viral removal or inactivation procedures.

Risk classification for ancillary materials

Risk control

In an attempt to aid manufacturers and developers indesigning their qualification programs for a variety ofAMs, tiers of sample risk categories are presented below.
Nonetheless, risk also depends on the amount and thestage that AMs are introduced into the manufacturing
process. The following sample tiers do not address theimpact on quantity or time of use, which is left up to both
the AM supplier and the CGT manufacturer.
Tier 1—AMs are low-risk, highly qualified materials
that are well-suited for use in manufacturing. The AM

After risks have been assessed and stipulated, steps canbe implemented to manage them appropriately. AMs
that are particularly important to the manufacturing
process or are not sufficiently qualified by the supplier
may require additional in-house testing before they canbe used within an acceptable risk tier. AMs that do notmeet the required specification may need to be removed
or replaced, or an alternative supplier may be found thatis able to provide the AM with a suitable quality. If process modifications are implemented, the risk assessment

Chapter 6: Ancillary Materials

A-CELL✼104

procedure may require revisiting to assess the new practices. For each hazard, the acceptable level of risk should
be decided to guide the degree of risk reduction measuresrequired (i.e., risk acceptance).
CURRENT SELECTION CRITERIA AND QUALIFICATION PROGRAM OUTLINEAM qualification is defined by USP <1043> as “the processof acquiring and evaluating data to establish the source,
identity, purity, biological safety, and overall suitabilityof a specific AM.”19 AM qualification is, in effect, a risk
control measure that sits within the risk management planof a product supply chain and life cycle. A product’s QMS
should consider in its design the specific application ofeach AM to the production process; the qualification
activities of any given AM may therefore be different
across two instances of use. A well-designed qualification
framework becomes more comprehensive throughout
clinical development and should be constantly reviewed
and updated as new scientific understandings and analytical capabilities become available.
In general, as a good starting point for qualifying AMs
for use in the production of CGTs, we recommend thefollowing list of action items:• Verify the vendor’s Certificate of Analysis to ensurematerial meets acceptance criteria• Perform audits of suppliers and partners• Verify that purification of material does not use
untested components or components that can risk
the end-user• Verify adequate viral inactivation/removal techniques are implemented to ensure material is free ofadventitious agents• Verify the Certificate of Origin to determine humanor animal-derived components and confirm BSE-freecountries of origin for the materials• Analyze and verify identity, purity, and performanceof crucial AMs in manufacturing procedures

Reagents Considerations
Reagents that fall under the AM classification include
well-characterized chemicals, complex compounds (e.g.,
antibiotics, anticoagulants, density gradients, toxins),
multi-component mixtures (e.g., buffers, culture media),

Chapter 6: Ancillary Materials

and complex biological compounds or mixtures (e.g.,
enzymes; blood-, plasma-, or serum-derived products;
biological extracts; cytokines; antibodies; and conditioned media from cultured cells). These components
pose an augmented risk to the end-user if removal isinadequate or incomplete.
SAFETY, EFFICACY, CONSISTENCY, AND STABILITY

Reagents are defined as materials used for cell growth,differentiation, selection, purification, or other manufacturing steps but are not intended to remain in the
final product. Reagents have the potential to affect thesafety, potency, and purity of the final cell-based therapy
product, and therefore the quality of reagents should betested for these attributes prior to their introduction into
the cell therapy manufacturing process. The variabilityof reagents should be controlled by implementing vendor qualification strategies built into the process. It is
recommended to utilize FDA-approved or FDA-cleared
reagents, or clinical grade reagents when possible. It is
common during the early stages of development to opt
for research-grade reagents; however, if therapies use
research grade materials due to the inability to procure or
unavailability of clinical grade reagents, the FDA recommends including a certificate of analysis provided by the
supplier and verified in-house if possible. Additional tests
may be needed to verify safety if the reagent contains
human-derived or animal-derived material. Testing forthese attributes should also be done on the cell therapy product during various stages of manufacturing tocontinuously ensure high quality of the final product.
Example testing timepoints are presented in Chapter 9.
As the cell therapy manufacturer gains experience during
development, there should be increasing understandingof the reagent attributes that could impact activity, purity,and stability of the cell product.
Quality parameters and critical product attributes of
the reagent must be identified to ensure lot-to-lot consistency and the desired clinical effect of the final cell
product. Therefore, to ensure safety before final producttesting, AMs should include assessments for sterility, mycoplasma, and adventitious viral agents. Microbiologicaltesting should be performed on starting raw materials,
ancillary materials, in-process intermediates, and final
products as appropriate depending on the clinical phase.

A-CELL✼105

Microbiological testing should include sterility testingfor bacterial and fungal contamination as well as mycoplasma testing as described in 21 CFR 610.12 and in the
United States Pharmacopoeia (USP) <71>.20,21 In-process
sterility testing is recommended at critical points during
manufacturing, such as during purification, ex vivo
gene modification, extended culture periods, and othertimepoints as identified by the sponsor to ensure safety.
A negative or no-growth result is an accepted criterion
to move forward with the utilization of tested materials
or intermediate after primary processing. Additionally,
when animal- and/or human-derived biological materials are used in the production or formulation of AM, anddepending on the risk assessment of the AM’s exposure
to a cellular therapeutic product, step(s) for removal
or inactivation of viruses should be included.22 These
processes require validation studies and documentation
so that they are available to the AM user.
Sponsors need to appropriately perform and describe
adventitious agent testing as specified in Points to Consider
in the Characterization of Cell Lines Used to Produce
Biological Products23 and ICH guidance Q5A: Guidance on
Viral Safety Evaluation of Biotechnology Products Derived
from Cell Lines of Human or Animal Origin.24 Viral testingis recommended on all cell banks, viral banks, and final
vector products. A number of well-stipulated and effective
assays have been developed to detect many known virusesin ancillary materials, such as polymerase chain reaction(PCR), quantitative PCR, antibody detection, and others.
If human- and animal-derived materials (cells, blood
derivatives, etc.) are used in the manufacturing process,donor testing for human pathogens and human viral agentsshould also be performed, as described in any global regulatory guidance.25
In addition, assessments of other product characteristics such as identity, purity (including endotoxin), andpotency are needed. It is recommended to perform thesetests throughout the manufacturing process to evaluate
manufacturing and ensure the quality and consistency ofAMs utilized in the production of the CGTs. It is criticalto determine the specifications used for intermediate
acceptance criteria and AM acceptance beforehand.
Specifications are the quality standards or tests, analytical procedures, and acceptance criteria that confirm
the quality of AM products, product components, andChapter 6: Ancillary Materials

other materials used in the production of the final therapy. Specifications should be appropriate to the productdevelopment stage and should be refined and tightened
as product development progresses.
Purity testing is important to understand the safety
risks associated with impurities that are either product-related or process-related components and theability of the process to remove such impurities. Purity
testing should include assays for pyrogenicity/endotoxin,
residual proteins, DNA, RNA, and others as determinedby the producer. Purity testing should also include assays
for solvents used during production and purification, andreagents used during manufacture. For further detailed
guidance please refer to ICH Q3 on Impurities.26,27,28
CASE STUDIES TO SUPPORT AM STANDARDIZATIONIn this section, we introduce a few case studies involving
various types of AMs. These examples are meant to reflect best practices considerations for AM suppliers andusers for successful implementation of AMs in cellulartherapeutic products.

Case study 1: Research-grade vs. CGMP-grade
substrates and scaffolds
Cell/tissue-based products rely on cell-substrate interactions to properly integrate in vivo. This interaction, which
entails cell adhesion to the substrate, is influenced by thesubstrate mechanics, as shown by the effect of substraterigidity on the stability of adhesion complexes and motility.29,30,31 Other cellular processes, such as proliferation,stem cell differentiation, cell growth, and apoptosis
are also governed by the mechanical and geometricalproperties of the cells and their environments.32,33,34 It istherefore crucial to engineer the substrate to allow for
proper cell functionality.
This case study presents an example of a product developed using research-grade raw materials that failed totranslate into CGMP raw materials. The project consistedof creating a hydrogel containing nanofibrous structures
to give the injectable gel structural integrity and optimize
cellular infiltration and propagation. The raw material inquestion was polycaprolactone (PCL) synthetic polymer
at 80K molecular weight. The project required electrospinning PCL nanofibers in a random deposition patternof a specific fiber diameter (Figure 6-1). The constraints

A-CELL✼106

Figure 6-1: (Left) Nanofibrous construct electrospun with recipe A, research-grade PCL;
(Right) Construct electrospun with recipe A, CGMP-grade PCL

included tight fiber diameter distribution, lack of beadformation, and no webbing or fusion.
The fibers were expected to be as smooth as possible(i.e., no pores or any other imperfections that would
render roughness on the fiber surfaces). The contractdevelopment and manufacturing organization (CDMO)
was well aware of the driving parameters that require closemonitoring to ensure a successful electrospinning process
to produce the final nanofibrous product. Close attention
and monitoring were performed on voltage potential,
needle-to-collector distance, rotating collection mandrel
speed (RPM) based on mandrel outer diameter, polymer
extrusion flow rates, polymeric solution concentration,and environmental condition (temperature and humidity).
The CDMO advised the client to utilize the final CGMPgrade material even during development, especially forthe electrospinning process, knowing that while highly
reproducible, subtle changes in the parameters mentioned
above could have profound effects on the final product.
Despite the CDMO recommendation, the client elected to use research-grade PCL during development. The
CDMO evaluated the PCL raw material and attempted toget as much information as possible from the vendor. Not
surprisingly, as research-grade material, there was very
little documentation aside from the standard safety data
sheet (SDS) and a bare Certificate of Analysis (CoA). Per
the customer’s instructions, the CDMO herein moved
forward, developing an electrospinning recipe to produce
the customer-specified fiber product. While the CDMO
successfully procured a recipe, the final fibers did not

Chapter 6: Ancillary Materials

meet the user-specified criteria when the polymer grade
switched to CGMP grade.

Case study 2: Enhancing the safety of virusinactivated ancillary materials throughneutralizing antibodies
The presence of adventitious agents in biologics manufacturing, particularly viruses, can impose severe consequences on patient safety. Cell therapy manufacturers minimize
the risk of virus presence in their final product by selecting
starting and raw materials with low risk of containing
adventitious virus, and performing in-process testing at
appropriate timepoints during the manufacturing process
to ensure the material is free from detectable viruses.
To that end, the use of virus-inactivated AMs in
CGT manufacturing helps produce optimal end drug
products while overcoming regulatory hurdles in thepathway towards commercialization. AM manufacturers
can incorporate steps to remove and inactivate potential
viral contaminants; for example, there are a few providers
in the advanced therapy market with a virus-inactivatedhuman serum to be used as a safe and effective media
supplement. 35 This virus inactivation effect can beachieved through immune neutralization, which is oneof the three main functions of antibodies.36
Immune neutralization

Immune neutralization results when a bound antibody
interferes with an essential function of its target antigen
molecule, neutralizing the normal effect of the target.

A-CELL✼107

It has been a powerful ally for humans attempting tounderstand and fight infectious diseases. Antiserums
containing neutralizing antibodies against viruses andbacteria have been used since the early twentieth century
and are still being used today (e.g., against the Ebola37
and SARS-CoV-238 viruses). After someone has been
infected and recovers from a pathogen, their body fluids(blood plasma) will contain antibodies specific to that
pathogen. This convalescent plasma can be collected
and infused into another person, sometimes creating
what is known as passive immunity. It is possible forthe neutralizing antibodies present in this antiserum toprovide both effective prophylaxis against their target
and a therapeutic intervention. This same neutralization
phenomenon is responsible for what has been achieved
via purified antibodies and monoclonal antibody production technology.39
In the age of purposefully engineered immune
cells, we find immune neutralization working to ourbenefit within some of the ancillary materials currently available that support these advanced cellulartherapies. There are a few providers in the advanced
therapy market with a virus inactivated human serum
to be used as a safe and effective media supplement,
helping to produce optimal end drug products while
overcoming regulatory hurdles in the pathway towards
commercialization.40 Several products undergo a robust
solvent detergent treatment and benefit from immune
neutralization through the pooling of numerous single
donor units. This results in a significant reduction ofenveloped and non-enveloped viruses.
Since the validated virus and pathogen reduction
methods have already been approved by the FDA, drug
and biologic manufacturers can move forward withconfidence knowing the material they incorporate into
their manufacturing process is maximizing the ultimate
safety profile for the end user. The consistency achievedby pooling so many donor units enables greater control
over a therapy’s manufacturing process compared toconventional human-derived media supplements.
Furthering this consistency is immune neutralizationand dilution of harmful interfering biomolecules. Thisis evident in pooled plasma products that minimize therisk of a leading cause of transfusion-related deaths in the
U.S.—Transfusion-related acute lung injury (TRALI).41

Chapter 6: Ancillary Materials

A large number of TRALI cases have been shown to beconnected to the harmful antibodies Anti-HLA andAnti-HNA.42,43 Despite comprising both male and female
donations, this reduction in immunization risk is also
seen with the absence of anti-D immunization and thetotal lack of inhibitor formation during clinical trials.44

Case study 3: Variability in the industry –recombinant protein example
Ensuring the quality and consistency of ancillary materials used in the manufacture of cell and gene therapies iscritical to both controlling costs and ensuring the integrityof the therapeutic product. It is the responsibility of theancillary material manufacturers to reduce the variability
in the biomanufacturing of recombinant proteins intended
for further manufacturing of therapeutics. Because theseproducts are biologics in and of themselves (in addition
to the therapeutic products they enable), they represent
critical materials that must be controlled and released in a
way that ensures consistency. The primary means by which
recombinant protein manufacturers can ensure consistency in therapy developers’ manufacturing processes is by
ensuring a reproducible process and a reproducible meansof measuring and reporting specific and biological activity.
Activity values

Cell therapy companies often rely on the lot-specificactivity values (reported in International Units) on
their supplier’s CoA to measure the amount of material
being used in their manufacturing processes (Figure6-2). However, it is critical to realize that the values
reported by different CGMP recombinant cytokine
manufacturers cannot be directly compared withconfidence if they do not adopt the same approach
to measuring activity and analyzing the results. As
an industry, it is important to align around standard
Figure 6-2: Example of lot-specific activity
values reported on a CoA
Test

ResultSpecific Activity

14.38 MIU/mg

Biological Activity

13.23 MIU/vial

A-CELL✼108

reporting backed by an accurate and reproducible
consensus-based process for analyzing and reporting
activity values. This limits variability and ultimately increases the consistency of the final therapeutic product.
Specific activity: Expressed in IU/mg. Specific activity
is inherent to the molecule and does not change, regardless of the concentration of the protein in the solution.
Biological activity: Expressed in IU/ml or IU/vial.
Biological activity changes when the concentration of
the protein in the solution changes.
Relative potency and parallel line assay

Relative potency is defined as the specific activity value (IU/
mg) obtained for a test sample as a relative value compared
to a reference standard’s reported specific activity (IU/mg),by directly comparing the difference in concentrationneeded by the reference standard and the test sample toresult in the same biological response for both.
To measure and report activity on Recombinant
Human Interleukin-2 (rHu IL-2), one manufacturer
utilizes a parallel line concentration-response model toestimate a relative potency compared to the National
Institute for Biological Standards and Control (NIBSC
86/500) reference, applying the principles outlined
in USP <1032> Design and Development of Biological
Assays and USP <1034> Analysis of Biological Assays.

In comparison, other CGMP recombinant cytokine
manufacturers take an alternative approach when calculating activity values. This, along with other differencesin assay methodology, can result in reported values thatare not directly comparable. We caution against using reported activity values to measure and dilute samples forcomparability assays between different manufacturers.
It is important for the industry to align with bestpractices around the determination and reporting ofcytokine activity. Successfully identifying best practices and implementing standards can streamline theraw material qualification process, resulting in a morerobust and resilient supply base, ensuring that therapy
developers can assess suppliers accurately, and onboard
secondary suppliers without the need for extensive andcostly comparability assessments.
It is worth noting that the World Health Organization
also chose to use a parallel-line approach when determining potency for the current International Standard
for IL-2 (NIBSC 86/500).45 Using this method, the reported rHu IL-2 specific activity levels are comparable tothe specific activity of the current international standard(NIBSC 86/500), which is known to be approximately
13.73 MIU/mg.46
See Figure 6-3 for a simplified example of the model
the authors recommend to compare relative potency byplotting biological response vs. concentration (log scale)for a test sample and a reference standard. The horizontal

Figure 6-3: Example of concentration-response model showing region applied in parallel line assay (left);and relative potency displayed within linear portion of parallel line model per USP<1034> (right)

Parallelregiononly

Assay response

Biological response(cell proliferation)

Max responseStandard
Baseline

Test
Concentration (log scale)

Chapter 6: Ancillary Materials

log concentration

A-CELL✼109

Figure 6-4: Example tests of statistical validity for data points used in parallel line model.
Test of regression (F-test)

Fregression

454.249

This test passes if Fregression > Fcritical

Fcritical (95.0%)

4.600

Test Passed

Fnon-linearity

1.065

This test passes if Fnon-linearity < Fcritical

Fcritical (95.0%)

3.112

Test Passed

Fnon-parallelism

2.663

This test passes if Fnon-parallelism < Fcritical

Fcritical (95.0%)

4.600

Test Passed

Test of linearity (F-test)

Test of parallelism (F-test)

distance between these graphs represents the differencein concentration needed by the reference standard andthe test sample to result in the same biological response
for both. This defines relative potency.
The parallel, linear regions for the test sample and reference standard in this model are compared to estimate
a relative potency if, and only if, they first pass statistical
validity tests for regression, linearity, and parallelism(Figure 6-4). Per USP <1032>, all biologically similar

compounds will produce statistically similar results, butjust because something has statistically similar results,
does not ensure that it is biologically similar. Failure
to produce statistically similar results, however, can betaken as evidence that they are not biologically similar.47
Once found to be statistically valid, we assume biological similarity and parallelism, and a relative potency is
calculated by comparing the concentration-response
function of the test sample to the concentration-response

Figure 6-5: Relative potency determination using parallel line model via direct relationshipbetween test sample’s and reference sample’s concentration-response functions

0.25

□

El

0.12
Response

I

□

0.50

/j•

FT(2) = FR(p2)p = relative potencyc

NIBSC Standard:FR(z) =FR(2)αR =+ aR
βlog(z)+ 13109(2)NIBSC Standard
FR(z) =FR(2)αR =+ aRβx+ l3x•

0.06

FR(z) = concentration-response function for Reference (NIBSC)
FR(2) = concentration-responsefunction
function forfor
Reference(NIBSC)
FT(z) = concentration-response
Test Sample
FT(2)=concentration-response
functionfor
Test
SampleFT(z) = FR(ρz)ρ = relative potency
z=concentration+ l3lo9(p2)IL-2 test sampleFT(z) =FT(2)IL-2 test sample:
αR += aR
βlog(ρz)
FT(2) = aR + l3lo9(p) + 13109(2)FT(z) =FT(2)αR += aT
βlog(ρ)+ βlog(z)+ l3x
FT(z) = αT + βx

0.03
0.02

If parallel:aT = aR + l3lo9(p)If parallel:
αT = αRβlog(ρ)relative potency
p =+antilog[ (aT - aR) / 13]
relative potency: ρ = antilog [ (αT - αR) / β]0.01
I 1 1 I 11(11

I

0.18

I

I I I 11111

I

I 11

111111

1.00

5.66

Log
Concentration
Log
concentration
10 10

Chapter 6: Ancillary Materials

A-CELL✼110

Alternative method for calculating activity levels

The commonly employed alternative method for calculating activity levels, used by many cytokine manufacturers,
is to determine the ED50 using the entire concentration-response graph and convert that directly into specificactivity as shown in the graph and equation in Figure 6-6.
ED50 is the concentration (ng/mL) that results in
50% of the maximum biological response. By applying
the equation, one can calculate a specific activity reported in U/mg. This then needs to be normalized against areference with known potency so that specific activity
in IU/mg can be reported, which is supposed to be auniversally comparable value.
In our experience, for cytokine activity, there is abigger error involved when calculating a relative specific activity using the ED50 value rather than using aparallel-line model. This is supported by the following
guidance in USP <1032> regarding variance:“Simple analysis of quantitative bioassay datarequires that the data be approximately normally
distributed with near constant variance across therange of the data.47“In practice, many bioassays have relatively large
variation in log EC50 (compared to the variation inlog relative potency) among assays (and sometimes
among blocks within assay). If not addressed in thevariance model, this variation in log EC50 induces
what appears to be large variation in response near
the mean log EC50…”47
If manufacturers use different methods to measure
and calculate specific activity, there will be completely
different sources of error, resulting in activity values thatshould not be directly compared. Relative activity values
can only be compared relatively, which means they must
result from the same comparability testing protocol anddata analysis methodology.
One simple option available for companies looking
to compare cytokines from different manufacturers via
comparability assays is to measure and dilute the test

Chapter 6: Ancillary Materials

Figure 6-6: Concentration-response modelshowing example ED50 comparison andequation used to convert ED50 value directly
into specific activity (U/mg)
Max response
Biological response (cell proliferation)function of the reference standard, as shown in Figure
6-5, adapted from USP <1034>.48

1/2 of total
response
standard
test

ED50

ED50
Baseline

Concentration (log scale)Specific activity (U/mg) = 1x106 / ED50 (ng/ml)

samples according to mass concentration, rather than thereported specific activity. If a consensus-based reference
standard is included during this comparison, the relative
specific activities of the test samples can be recalculated
and compared using these results.

Conclusions
The authors have long been at the forefront of the efforts tocoordinate and accelerate the development and diffusion ofindustry standards. Through the Standards Coordinating
Body (SCB)’s working group, the authors co-lead astandards advancement project on Ancillary Materials,
resulting in a three-part series of documentary standards
on ancillary materials: ISO/TS 20399: Ancillary materials
present during the production of cellular therapeutic products. It is in our interests, as a community of manufacturers
seeking to accelerate the development and commercialization of cell and gene therapies, to standardize the assaysthat most directly impact our customers’ manufacturing
processes and ultimately the end patient experience. Only
through greater standardization can we hope to achieve
the resilient supply chains, cost of goods reductions, andmanufacturing robustness that define a mature industry.

A-CELL✼111

References
1

ISO/TS 20399-1:2018 Biotechnology — Ancillary materials
present during the production of cellular therapeutic products —Part 1: General requirements

2

U.S. Food and Drug Administration. Guidance for Industry:
CGMP for Phase 1 Investigational Drugs. July 2008.

3

FDA 21 CFR 210 Current Good Manufacturing Practice (Drugs).
2009.

4

FDA 21 CFR 211 Current Good Manufacturing Practice (Drugs).
2009.

5

FDA 21 CFR 610.15: General Biological Products Standards;
Constituent materials. 2009.

6

FDA 21 CFR 820 Quality System Regulation (Devices). 2009.

7

International Council for Harmonization. Guideline E17:
General Principles for Planning and Design of Multi-Regional
Clinical Trials. November 16, 2017.

8

ISO/TS 20399-1:2018 Biotechnology — Ancillary materials
present during the production of cellular therapeutic products

9European Commission. EudraLex – Volume 4 – Good
Manufacturing Practice (GMP) guidelines. https://ec.europa.eu/
health/documents/eudralex/vol-4_en.

19

U.S. Pharmacopeia. USP <1043> Ancillary Materials for Cell,
Gene, and Tissue-Engineered Products. 2019.

20

U.S. Food and Drug Administration. Code of Federal Regulations
Title 21. April 2020. http://www.accessdata.fda.gov/scripts/cdrh/
cfdocs/cfcfr/cfrsearch.cfm?fr=610.12.

21

U.S. Pharmacopoeia. USP <71> Sterility Tests. 23rd ed. 1995.

22

ISO/TS 20399-1:2018 Biotechnology — Ancillary materials
present during the production of cellular therapeutic products —Part 1: General requirements

23

U.S. Food and Drug Administration. Memorandum: Points toConsider in the Characterization of Cell Lines Used to Produce
Biologicals. July 1993. https://www.fda.gov/media/76255/
download.

24

U.S. Food and Drug Administration. Guidance: Q5A Viral Safety
Evaluation of Biotechnology Products Derived from Cell Linesof Human or Animal Origin. September 1998. https://www.fda.
gov/regulatory-information/search-fda-guidance-documents/
q5a-viral-safety-evaluation-biotechnology-products-derivedcell-lines-human-or-animal-origin.

25

U.S. Food and Drug Administration. Guidance for Industry:
Eligibility Determination for Donors of Human Cells, Tissues,
and Cellular and Tissue-Based Products (HCT/Ps). August 2007.
https://www.fda.gov/media/73072/download.

10

Ph Eur 5.2.12 Raw Materials of Biological Origin for the
Production of Cell-Based and Gene Therapy Medicinal Products.

11

Australia Therapeutic Goods Administration. Australiancode of good manufacturing practice for human blood andblood components, human tissues and human cellular therapy
products: quality control. 2013.

26

12

Health Canada. Guidance Document: Preparation of Clinical
Trial Applications for use of Cell Therapy Products in Humans.
2014.

U.S. Food and Drug Administration; ICH. Guidance forIndustry: Q3A Impurities in New Drug Substances. June 2008.
https://www.fda.gov/regulatory-information/search-fdaguidance-documents/q3ar-impurities-new-drug-substances.

27

13

ISO. Biological evaluation of medical devices — Part 1:
Evaluation and testing within a risk management process. ISO
10993-1:2009.

U.S. Food and Drug Administration; ICH. Guidance forIndustry: Q3B(R3) Impurities in New Drug Products. August
2006. https://www.fda.gov/regulatory-information/search-fdaguidance-documents/q3br-impurities-new-drug-productsrevision-3.

14

Culme-Seymour EJ, Mason C. ‘The Little Purple Book’, 2ndedition: Cell therapy and regenerative medicine glossary. Regen
Med. 2012;7(3):263-264. doi:10.2217/rme.12.26.

28

U.S. Food and Drug Administration; ICH. Guidance forIndustry: Q3C Impurities: Residual Solvents. December 1997.
https://www.fda.gov/regulatory-information/search-fdaguidance-documents/q3c-impurities-residual-solvents_2011.

15

International Conference on Harmonization (ICH) Q9, Quality
Risk Management - Guidance for Industry. June 2016. https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/q9-quality-risk-management

29

Discher DE, Janmey P& Wang Y-L. 2005Tissue cells feel andrespond to the stiffness of their substrate. Science 310, 1139–
1143. (doi:10.1126/science.1116995).

16

Ball, O & Zylberberg, C. 2019. Towards a common frameworkfor defining ancillary material quality across the development
spectrum. Cytotherapy 21(12), P1234-1245. https://doi.
org/10.1016/j.jcyt.2019.10.007

30

Pelham RJ& Wang Y-L. 1997Cell locomotion and focal adhesions
are regulated by substrate flexibility. Proc. Natl Acad. Sci. USA 94,
13 661–13 665. (doi:10.1073/pnas.94.25.13661).

31

Engler A, Bacakova L, Newman C, Hategan A, Griffin M&
Discher D. 2004 Substrate compliance versus ligand density
in cell on gel responses. Biophys. J. 86, 617–628. (doi:10.1016/
S0006-3495(04)74140-5).

32

Banerjee A, Arha M, Choudhary S, Ashton RS, Bhatia SR,
Schaffer DV& Kane RS. 2009The influence of hydrogel modulus
on the proliferation and differentiation of encapsulated
neural stem cells. Biomaterials 30, 4695–4699. (doi:10.1016/j.
biomaterials.2009.05.050).

17

ISO/TS 20399-1:2018 Biotechnology — Ancillary materials
present during the production of cellular therapeutic products —Part 3: Best practice guidance for ancillary material users

18

ISO/TS 20399-1:2018 Biotechnology — Ancillary materials
present during the production of cellular therapeutic products —Part 3: Best practice guidance for ancillary material users

Chapter 6: Ancillary Materials

A-CELL

✼112

References, continued
33

Chen CS, Mrksich M, Huang S, Whitesides GM& Ingber DE.
1997 Geometric control of cell life and death. Science 276,
1425–1428. (doi:10.1126/science.276.5317.1425).

41

Bertolini J, Gross N, Curling J, eds. Production of Plasma
Proteins for Therapeutic Use. Hoboken, NJ; John Wiley & Sons;
2013: 345-351.

34

Engler AJ, Sen S, Sweeney HL& Discher DE. 2006 Matrix
elasticity directs stem cell lineage specification. Cell 126,
677–689. (doi:10.1016/j.cell.2006.06.044).

42

Sachs UJH, Wasel W, Bayat B, et al. Mechanism of transfusionrelated acute lung injury induced by HLA class II antibodies.
Blood. 2011 Jan 13;117(2):669–77.

35

Akron Biotech signs exclusive global agreement with
Octapharma to produce virally inactivated human AB serum
derived from Octaplas® for the cell therapy market. Octapharma
Press Release. 2021 Jan 20 https://www.octapharma.com/news/
press-release/2021/akron-biotech-signs-exclusive-globalagreement-with-octapharma.

43

Curtis BR, McFarland JG. Mechanisms of transfusion-related
acute lung injury (TRALI): Anti-leukocyte antibodies: Crit CareMed. 2006 May;34(Suppl):S118–23.

44

36

Huber M, Trkola A. Humoral immunity to HIV-1: neutralization
and beyond. J Intern Med. 2007 Jul;262(1):5–25.

U.S. Food and Drug Administration, Blood Products Advisory
Committee. Pooled Plasma (Human), Solvent/Detergent Treated(Octaplas). BLA 125416/0. 2012 Sep 20.

45

37

World Health Organization. Use of convalescent whole bloodor plasma collected from patients recovered from Ebola virus
disease. September 2014. WHO/HIS/SDS/2014.8.

Wadhwa M, Bird C, Heath A, Thorpe R. Report on a
Collaborative study for proposed 2nd International standard
for Interleukin -2 (IL-2). Expert Committee on BiologicalStandardization WHO/BS/2012.2194.

38

COVID-19 Treatment Guidelines Panel. Coronavirus Disease2019 (COVID-19) Treatment Guidelines. National Institutes of
Health. Available at https://www.covid19treatmentguidelines.
nih.gov/.

46

World Health Organization. International Standard: 2nd
International Standard for INTERLEUKIN 2 (Human, rDNA
derived). NIBSC code: 86/500. Instructions for Use. (Version 1.0,
Dated 23/04/2013).

39

Salazar G, Zhang N, Fu T-M, An Z. Antibody therapies for theprevention and treatment of viral infections. Npj Vaccines. 2017
Dec;2(1):19.

47

U.S. Pharmacopoeia. USP <1032> Design and Development of
Biological Assays. USP-NF.

48

U.S. Pharmacopoeia. USP <1034> Analysis of Biological Assays.
USP-NF.

40

Akron Biotech signs exclusive global agreement with
Octapharma to produce virally inactivated human AB serum
derived from Octaplas® for the cell therapy market. Octapharma
Press Release. 2021 Jan 20 https://www.octapharma.com/news/
press-release/2021/akron-biotech-signs-exclusive-globalagreement-with-octapharma.

Chapter 6: Ancillary Materials

A-CELL✼113

CHAPTER 7

Lentiviral Vector
Manufacturing Process
CHAPTER LEAD

Kelly Zinn, Atara Biotherapeutics
CHAPTER CONTRIBUTORS

Jeffrey Hung, Vigene Biosciences
Pratima Cherukuri, Genezen Labs
Balwant Patel, Charles River Laboratories
James Brown, Agathos Biologics
Sebastian Almeida, Akron Biotech

Chapter 7 | Contents
Introduction.................................................................... 116

Critical quality attributes of LV..................................129

Lentiviral Vector Manufacturing.................................. 118

Release testing........................................................... 130

Starting materials......................................................118

Assay qualification and validation............................ 130

Considerations for viral contamination andclearance.................................................................... 119

Identity testing............................................................132

Upstream manufacturing processes........................ 120

RCL testing................................................................ 133

Transient transfection vs stable cell line.................. 120
Adherent vs suspension production......................... 121
Serum vs serum-free production..............................122
Downstream manufacturing processes....................123
Clarification/cell removal...........................................124
Chromatography........................................................124
Concentration............................................................125
Final formulation........................................................125
Sterile filtration or aseptic processing......................125
Final vector product container closure.....................126
Process Characterization........................................... 127
Process Qualification.................................................. 127
Comparability.............................................................128
Internal Versus External Manufacturing and Testing.......128
Transfer of Manufacturing Process and Analytical
Methods.......................................................................129

Safety testing..............................................................132
SARS-CoV-2 testing.................................................. 134
Purity testing.............................................................. 134
Residuals.................................................................... 134
Aggregates/Particulates........................................... 134
Potency testing...........................................................135
Multiplicity of infection..............................................135
Sterility testing........................................................... 136
Stability.......................................................................... 136
Vector bulks.................................................................137
Finished vector........................................................... 138
Stressed and Accelerated Stability Studies............ 138
Container closure system......................................... 138
Extractables and leachables..................................... 139
References..................................................................... 140

Technology transfer between sponsor and CDMO..129
Assay qualification and validation.............................129
Proprietary processes................................................129
Product Testing and Characterization.........................129

Lentiviral Vector Manufacturing Process

A-CELL

✼115

Introduction
Lentiviral vectors (LVs) represent an important meansof transferring and integrating transgenes of interest into
cells. They are capable of transducing dividing and nondividing cells including T-cells, making them particularly
suitable for the ex vivo generation of CAR T-cell therapies.
In fact, LVs are being used to manufacture several recently
approved CAR T-cell therapies (Kymriah®,1 Breyanzi®,2and Abecma®3), and at least 50 ongoing or planned clinical
trials of CAR T-cell therapies involve the use of LVs according to ClinicalTrials.gov.4 Importantly, LVs can also be
used as drug products for in vivo gene therapy. Given theirbroad applications, the LV production market is expected
to grow to $800 million by 2026.5
Lentiviral vectors are typically based on HIV-1, awell-studied virus pathogenic to humans. 6 Different
generations of LV systems have been developed, withearly generations being fine-tuned to remove unnecessary or safety-concerning elements, while ensuring both
efficient gene transfer and absence of replication-competent lentivirus (RCL). Currently, third generation LVs
are most widely used for clinical applications. They are
typically produced using a 4-plasmid transient transfection system comprised of 3 packaging plasmids7 and
1 self-inactivating gene transfer plasmid (Figure 7-1).

The transfer plasmid consists of the lentiviral backbone
containing the transgene expression cassette flanked
by HIV-1 long-terminal repeats essential for packaging the viral RNA genome into viral particles, reverse
transcription, and integration into the recipient cell.8,9The 3 packaging plasmids contain gag-pol (encoding forstructural proteins and viral enzymes), rev (encoding fora post-transcriptional regulator), and env which encodes
the pseudotype of the viral vector (typically the vesicularstomatitis virus [VSV]-G envelope gene is used).6,7,10
Though LVs can be pseudotyped with different envelope
glycoproteins, the VSV-G glycoprotein enhances stability
during downstream processing and features a large transduction spectrum due to its interaction with low-density
lipoprotein (LDL) receptors that are ubiquitous among
most cell types.6,10,11 However, resting lymphocytes used
for CAR T-cell therapies have low expression of the LDL
receptor, leading to inefficient transduction in the absenceof cell activation.12 Though VSV-G is the most common
envelope protein pseudotyped onto LV particles, otherenvelope glycoproteins such as RD114 (derived from a feline endogenous retrovirus) have found application in thetransduction of lymphocytes for CAR T-cell therapies.13,14
Lentiviral vectors can be viewed as either drug substance (DS) or drug product (DP) depending on theirapplication (Figure 7-2). When used in vivo to directly

Figure 7-1: Plasmids used in traditional third-generation LV production

Transgene ofinterest

VSV-G (or other
Env)

GagPolRev”Helper” packaging plasmids

Expression plasmid
Co-transfection

Lentiviral vector
Adapted from: Brown J. Supporting AAV and lentiviral vector development and commercialization. Pharma’s Almanac. May 24, 2019.
https://www.pharmasalmanac.com/articles/supporting-aav-and-lentiviral-vector-development-and-commercialization15

Lentiviral Vector Manufacturing Process

A-CELL

✼116

Figure 7-2: Vector can represent drug substance or drug product depending on the application

1. Genetic material
is inserted into aviral vector.
genetic
material

Ex vivo:
2. Cells are
removed fromthe patient

Viral vector

In vivo:
2. Vector loaded withgenetic material isintroduced directly
into the body

3. Vector loaded with genetic
material introduced into cells

4. Cells containing
genetic material aregrown in a lab

insert genetic material into human cells, LVs take on therole of the final drug product. However, when used totransduce human cells ex vivo, such as in the generationof CAR T-cell therapies, LVs may be more appropriately
classified as drug substance. For CAR T-cell products andother ex vivo applications, separate quality target productprofile (QTPP) and critical quality attributes (CQAs)
should be developed for the vector material. Additionally,
vector manufacturing process residuals, impurities, andmultiplicity of infection (MOI) should be included in
the QTPP of the final drug product. This speaks to thecriticality of the vector in the eyes of regulatory bodies:
whereas the European Medicines Agency16 views LVs as
starting material, the U.S. Food and Drug Administration

Lentiviral Vector Manufacturing Process(FDA)17,18 simultaneously describes vector used for exvivo genetic modification of cells as bulk drug substance,
an active pharmaceutical ingredient, and a criticalcomponent of the final drug product. Regardless ofclassification, LVs must be appropriately characterized
and all assays used for quality control during its manufacture should be properly qualified and validated in aphase-appropriate manner. Within any investigational
new drug application (IND), a separate drug substancesection should be provided for vectors used for ex vivo
modification of cells, as outlined in Module 3 of theCommon Technical Document (CTD).19
The processes for development of the final drug product are often unique to the individual sponsor, however

A-CELL✼117most LVs in clinical and commercial use are supplied bycontract development and manufacturing organizations(CDMOs), where the development and manufacturing
process is already defined, often requiring only an adjustment of the transgene expression cassette within thegene transfer plasmid. The essential role of LV in the production of CAR T-cell therapies (and other cell and genetherapies) speaks to the importance of coordination andcommunication between the sponsor and the CDMO.
Helper plasmids are becoming increasingly available as
off-the-shelf products, the proprietary nature of which
may be protected under a drug master file (DMF).
Similarly, gene transfer plasmid templates and cell banksystems are becoming more widely available as “off-theshelf ” products. As such, the CDMO must procure theseessential components from qualified vendors that candemonstrate consistent quality of their products and solid control of risks associated with supply chain logistics.
While the sponsor is ultimately responsible for filings to
the health authority, obtaining all essential information
for the filings will require significant collaboration with
the CDMO.

Lentiviral Vector Manufacturing
Manufacturing of LV, like all biologics, can be divided
into upstream and downstream processes, with upstream
production steps aiming to produce high-titer vector
that is conducive to downstream purification. Upstream
processes vary depending on the cell line used for production and whether the cells are transiently transfected
or developed into stable producer cell lines. Adherent
cell cultures tend to accommodate the smaller-scaleneeds typical during developmental stages of productdevelopment, whereas suspension cultures are moresuited for large-scale production. Supplementation ofcell media (e.g., with serum or sodium butyrate) can
improve producer-cell viability and vector stability but
may present challenges to product safety and ease ofdownstream processing. Downstream processing steps
seek to maximize viable vector recovery while reducing
impurities that could compromise the efficacy or safety ofthe eventual drug product. Downstream steps follow thegeneral order of purification, enrichment/concentration,sterile filtration, and storage.

Lentiviral Vector Manufacturing Process

STARTING MATERIALS

According to the European Commission Directive
2001/83/EC,16 in the case of products consisting of viral
vectors, the starting materials shall be the components
from which the viral vector is obtained (i.e., the plasmids
used to transfect the packaging cells and the master cellbank [MCB] of the packaging cell line). Additionally,in the case of genetically modified cells (which would
include CAR T cells), the starting materials shall be thecomponents used to obtain the genetically modified
cells (i.e., the starting materials to produce the vector,
the vector, and the human or animal cells). The principles
of good manufacturing practices (GMP) shall apply fromthe bank system used to produce the vector onwards.18
The cell bank system used to produce LV traditionally consists of HEK 293 or HEK 293T packaging celllines. The HEK 293 cell line was developed in 1977 bytransfecting human embryonic kidney cells with sheared
adenovirus type 5 DNA.20 The gene encoding the SV40
T-antigen was then introduced into the 293 cell line tocreate the 293T cell line. Today, HEK 293T cells are generally preferred over HEK 293 cells due to their increased
cell growth and transfection efficiency, at least partiallyowing to the presence of the SV40 T-antigen.21,22
As discussed previously, the third-generation LVs
used most commonly utilize a 4-plasmid system: one
plasmid contains the gene(s) of interest and 3 refined“helper” plasmids contain viral sequences necessary
for production.6,10 The need to transiently transfect
4 different plasmids with each vector manufacturingbatch can be a source of variability in manufacturing
processes; thus, sponsors must ensure that plasmids areappropriately and consistently manufactured. Though
plasmids have historically been manufactured in custom batches, commercial production of “off-the-shelf ”
lentiviral helper plasmids is becoming increasingly
common. In addition, plasmid production companies
can often provide plasmids of varying grades, ranging
from research-grade plasmids for process development
purposes to those suitable for commercial production.15
Thoug h t he FDA’s Guidance for Industr y :Considerations for Plasmid DNA Vaccines for Infectious
Disease Indications23 does not specifically address plasmid
DNA products intended for noninfectious therapeutic
indications, information applicable to plasmids used in

A-CELL✼118

LV manufacturing can be drawn from its contents. As
such, all components used during LV production should
be sufficiently described in the manufacturing summary
included in an IND application. This includes detailed
descriptions of the plasmid construct generation andthe DNA sequence of the entire plasmid(s). In its 2020
Guidance for Industry: Chemistry, Manufacturing, andControl (CMC) Information for Human Gene Therapy
Investigational New Drug Applications (INDs),17 the FDA
describes bacterial MCBs as the starting material used
to generate plasmid DNA, which can then be used as amanufacturing intermediate to generate LVs. The FDA
recommends that the bacterial MCB be appropriately
qualified and that sufficient information on that qualification be submitted in the IND.17 Information submitted to support qualification should include certificatesof analysis for cell banks, executed batch records, andevidence of passed release tests that adequately establish
the safety, identity, purity, and stability of the microbial
cell preparation used in the bank. 17 Interestingly, aplasmid construct is used to generate the bacterial MCB(usually E. coli-derived). Often, this plasmid construct
is synthesized de novo under R&D manufacturingconditions. Within the IND, the sponsor should include
the full sequence of the plasmid used to construct the
MCB, and the sponsor should be aware of any safety andcross-contamination issues.
The genotype and source of the mammalian cell line
(e.g., HEK 293, HEK 293T) used to construct the vector
production or packaging MCBs and working cell banks(WCBs) must also be described in the IND. Additionally,both the MCBs and WCBs should be tested to ensurethat they are free from contamination with adventitious
bacterial and viral agents; all testing should be well
documented and controlled. Viruses are generally moredifficult to detect than bacterial contaminants and canbe of greater consequence should they be present in cell
culture. As such, sponsors should be careful to selectstarting and raw materials with a low risk of containing
adventitious virus. Process-specific raw materials should
be obtained from reliable sources that can demonstrateadequate control of their production. Raw materials can
include cell culture media and additives, reagents (e.g.,transfection reagents such as polyethylenimine [PEI]),
endonucleases, and consumables (e.g., cell culture

Lentiviral Vector Manufacturing Processunits, membranes, buffers, filters, resins, and cryovials).
Starting and raw materials should be subjected to aproper assessment of risk they could impose to vector
production; principles of risk assessments and criticality
determination are discussed in Chapter 4. Additional
guidance on viral evaluation of starting materials is foundin ICH Q5A (R1)24; guidance on viral testing throughout
LV production stages as well as specific testing methods
will be discussed later in the chapter.

Considerations for viral contaminationand clearance
The Consortium on Adventitious Agent Contaminationin Biomanufacturing (CAACB) recently publishedinsights for gene and cell therapy developers regarding
the most common viral contaminants in cell cultures,the sources of these contaminants, the cell lines affected,
and corrective actions that can be taken.25 Their findings
demonstrated that viral contamination in cell-based
manufacturing, though relatively rare due to existing
standards, can be extremely costly and detrimental to
manufacturing operations. Overall, human or primate
cell lines were more likely than rodent cell cultures tobe contaminated by viruses pathogenic to humans.
Only one case of HEK 293 cell-line contamination wasreported; human adenovirus type 1, which is pathogenic
to humans, was implicated in that case and the suspected source was the operator rather than the cell sourceor materials used in cell culture (e.g., serum or media
component).
Unexpected changes in cell culture behavior can be
an indication of viral contamination but many viral contaminants are not apparent. Virus-specific testing (e.g.,
polymerase chain reaction [PCR]) is often required for
detection, though even PCR may fail to detect low-level
viral contamination in raw materials prior to viral amplification. Because viral vector production generally starts
with fully characterized MCBs, they have a relatively low
risk of viral contamination from their starting cell sources. However, given the detrimental effects of potential
viral contamination, testing for adventitious virus should
be conducted at multiple timepoints in the LV manufacturing process (see Figure 7-7 in the Safety sectionof this chapter).25 Notably, testing for contaminants isrequired at the unclarified harvest stage, as they are most

A-CELL✼119

Figure 7-3: General upstream processing strategy for LV production via transient transfection of HEK 293T cells

likely to be detected prior to any downstream processing.
Again, guidance on viral testing throughout production
stages as well as specific testing methods are discussed ingreater detail in the Safety section of this chapter. Because
lentivirus is enveloped as well as temperature- andpH-sensitive, implementing known viral clearance methods (e.g., detergent/solvent, high heat, or extreme pH)
for LV processing is not possible.25,26 Therefore, the LV
manufacturing industry must rely heavily on prevention
and vector characterization methods to ensure patient
safety. Care must be taken to prevent viral contamination via thorough testing of raw materials and host cells
used in upstream processing.27 Downstream processing
methods (e.g., chromatography, filtration) can also act asclearance methods that will separate contaminating virus
from lentiviral particles without harming the activity
profile of the LV particles.25-27
UPSTREAM MANUFACTURING PROCESSES

Multiple factors must be considered in order to optimize
bulk production of LV during upstream processes.10 The
production mode (e.g., transient transfection vs stable
or inducible cell line, adherent vs suspension) must be
considered early in process development. Several considerations impact the scalability of cell expansion, where
viability of cells must be maintained while achieving

Lentiviral Vector Manufacturing Process

suitable cell density to meet yield demands. Such considerations include the type of cell line used (adherent
vs suspension), the equipment used for cell expansion(flasks/bottles for small batches vs bioreactors for large
batches), and the type of cell media and supplementation.
The general upstream processing strategy for LV is shownin Figure 7-3; individual steps will be discussed further
in subsequent paragraphs.

Transient transfection vs stable cell line
The predominant mode of LV production involves transient transfection of cells with optimized plasmid systems(Figure 7-1) that maintain critical elements for vector expression and packaging but eliminate unnecessary genesthat compromise safety and yield. Co-transfection of thegene transfer and “helper” plasmids can yield titers in
the 106 to 108 transducing units (TU)/mL range, thoughlarger transgenes upward of 7.5kb may affect yield.6,10,28
Titers are also impacted by cell density, incubation time,
temperature, pH, and plasmid concentration.10 In fact,
the optimal plasmid ratio can be so impactful on titerthat it is often considered proprietary information.
A wide variety of transfection reagents can be used for
LV production, each with unique implications in termsof scalability, cost, and efficiency. Calcium phosphateoffers an inexpensive option that is most suitable for

A-CELL✼120

Table 7-1: Examples of inducible and stable cell lines for LV production
Cell line

LV
generation

InductionsystemEnvelope

Reported titer

Adherent or
Suspension

Reference

GPRG-TL20IL2RG

3rd

Tet-off

VSV-G

5x107 TU/mL

Adherent

Throm 200935

293SF-PacLV

3rd

Tet-on,CumateVSV-G

3.4x107 TU/mL/
day

Suspension

Broussau200836293TsaGLOBE

3rd

Tet-onVSV-G

1.4x107 TU/mL

Suspension

Chen 202037

LentiPro26

3rd

NA

MLV
Amphotrophic
Envelope

106 TU/mL/day

Adherent

Tomas 201831

WinPac

3rd

NA

RD114-PR

1x106 TU/mL

Adherent

Sanber 201534

small-scale use. Calcium phosphate and plasmid DNA
form precipitates that attach to the cell surface and are
endocytosed. The need for constant mixing to ensure
consistent precipitate formation, the requirement tochange medium after transfection, and the sensitivityof the process to temperature, pH, and component concentrations makes this method difficult for large-scale
production.10 The use of lipofectamine, a cationic lipidthat entraps plasmid DNA within liposomes that fusewith the cell membrane, is limited by cytotoxicity, cost,
and the requirement for media exchanges post-application.10,29,30 Polyethylenimine, which forms polyplexes
with plasmid DNA that are endocytosed by cells, is lesssensitive to pH alterations and its lower toxicity precludes
the need for media exchanges following transfection. It
requires less plasmid DNA than calcium phosphate, isrelatively inexpensive, and can be used in both adherent
and suspension cultures, making it more suitable forlarge-scale LV production.10,29
Transient transfection offers fast and dependably consistent LV production but is suboptimal for large-scale
production due to the cost of plasmid and reagents, significant batch-to-batch variation in titers, and the needto clear both transfection reagents and residual plasmid
DNA.29 As such, a stable cell line that constitutively produces high-titer, high-quality, homogenous LV would be
ideal for large-scale use. Unfortunately, the generation of
stable cell lines for LV production has proven challenging, at least partly due to the VSV-G envelope featured
in most second- and third-generation plasmid systems.
Though VSV-G offers broad tropism and the potential

Lentiviral Vector Manufacturing Process

for high titers, its inherent cytotoxicity does not permit
constitutive expression and, therefore, its use is limited totransient transfection modalities.10,31 Expression of gag,pol, and certain transgenes can also introduce cytotoxicity.6 Through the addition or removal of tetracycline,
inducible systems such as Tet-on and Tet-off can limit
cytotoxicity by triggering gene transcription during only
key phases of LV production.6,10 Such inducible systems
have been used to produce LV used in studies of investigational therapies for Wiskott-Aldrich syndrome32 andX-linked severe combined immunodeficiency.33 However,the use of tetracycline requires removal downstream, andinducible systems carry the risk of “leaky” expression thatcan lead to uncontrolled LV production.10 Constitutive
producer cell lines successfully used for LV production
utilize an alternate envelope protein,31,34 offer lower titers
than those produced by transient transfection methods,
and have yet to be adopted into mainstream practice.
Though details of producer cell lines are often proprietary
information, examples of inducible and constitutive producer cells used for LV production are provided in Table
7-1. More complete listings and additional information
can be found in reviews by Perry et al.10 and Merten et al.6

Adherent vs suspension production
During early development stages, adherent cultures arecommonly used due to the innately adherent nature of
HEK 293T cells, the higher LV titers and cell densities
achieved, and the desire for flexible manipulation of cells
within small culture flasks and bottles.10 However, adherent cultures can be limiting during scale-up. Though

A-CELL✼121

multilayer flasks provide additional surface area for cellattachment and growth, they are bulky and require extensive open manipulation during upstream and downstream processing steps. The use of microcarriers, which
can be porous or solid, fails to achieve high LV titers due
to cell clumping, inconsistent delivery/removal of nutrients and oxygen, and compromised removal of toxic
metabolites.10 Fixed-bed bioreactors may be best-suited and most economical for large-scale expansion ofadherent cultures, allowing for cell expansion on a 3D
matrix that provides a balance of higher surface areawith less bulkiness.10,38 Though plagued somewhat bypoor cell distribution, optimized fixed-bed bioreactors
such as the iCELLis® and scale-X™ are able to achieve
LV titers close to 106 TU/mL.39 Examples of vessels
used for adherent cultures include HYPERFlask® and
HYPERStack® (multilayer, Corning); iCELLis® (fixed
bed, Pall); and scale-X™ (fixed bed, Univercells).
Suspension cultures are theoretically more ideal forlarge-scale LV production, as they can achieve greater
production volume without the need for attachment
surfaces.29 HEK 293T cells can be adapted to suspension
culture, though adaptation is generally accompanied bya loss of productivity, with titers around 106 TU/mL or
lower. Shear forces are higher for suspended cells andthey are inherently more difficult to transfect, as they
represent moving targets for transfection complexes.
Nevertheless, a gradual adaptation of HEK 293T cells
to suspension media has recently achieved LV titers of
108 TU/mL using 5-Liter shaker flasks.40
Suspension cultures minimize manual handling andenable both automation and in-line monitoring. As withall steps of CAR T-cell manufacturing, closed bioreactors
with weldable connections can minimize contamination
potential throughout much of the LV production process. The potential for shear damage to cells introducedby shaking or stirring can be difficult to control during
scaled-up production, particularly within large bioreactors, and media replacements required for transienttransfection modalities can be more challenging. As
such, it remains to be seen whether the high titers accomplished in 5-L shaker flasks can be achieved in larger-scale production. Stirred-tank bioreactors can scale
up to thousands of liters, whereas rocking bioreactors
offer a lower-shear environment at a lower scale (10-200

Lentiviral Vector Manufacturing ProcessL).10 According to Comisel et al., single-use stirred tankbioreactors may be the most cost-effective vessel type for
LV production in suspension-adapted cell lines, thoughcapacity limits are restrictive in high-dose, high-demand
scenarios.38 Examples of bioreactors used for suspension
cultures include Xcellerex™ XDR (stirred tank, Cytiva);
Biostat® (stirred tank, Sartorius); and WAVE (rocking
platform, Cytiva). Additional discussion of technologies
used for both adherent and suspension cultures is foundin Chapter 8 of this document and in the review by Perryet al.10

Serum vs serum-free production
Many LV production modalities utilize serum to
optimize both cell growth and vector stability. Major
detriments to serum addition include the need for removal of higher protein loads in downstream processes
and the risk of immunogenicity.10 Additionally, as the
majority of serum used for LV production is bovine-derived, the risk of bovine-related disease transmission isof utmost concern. The bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom during
the 1990s brought heightened attention to the dangers
of prion-mediated transmissible spongiform encephalopathies (TSEs) for human health. As no laboratory
tests are currently available to screen live cattle for theprions causing BSE infection, the sourcing of bovine
serum (country/herd/animal) is an important aspect ofoverall risk mitigation for any cell therapy product.41
In addition to concerns related to TSEs, the process oftreating fetal bovine serum (FBS) with gamma radiationto achieve viral inactivation is important for making
FBS GMP-compliant for use in LV manufacturing.17
To minimize risk of animal-related disease, there isa drive to remove serum from the production of LV.
Though serum is required to mitigate the cytotoxicity
introduced by calcium phosphate, PEI can be used in
the presence or absence of serum.10,29 Although adherent cells are difficult to grow in serum-free media,
HEK 293T cells can be adapted to suspension growthin serum-free media.6,40 Stable cell lines have also beensuccessfully adapted to serum-free media.29 The detailed
comparison between adherent and suspension cell
platforms has been described in an earlier section ofthis book chapter.

A-CELL✼122

Figure 7-4: General downstream processing strategy for LV intended for ex vivo therapies* Can be excluded if manufacturing process certified as fully aseptic.

DOWNSTREAM MANUFACTURING PROCESSES

Downstream processing (DSP) of LV seeks to maximize
vector recovery while minimizing impurities that could
negatively affect the efficacy or safety of the geneticallyengineered cell therapy drug product (i.e., the CAR T cell).
The degree of purity and concentration required depends
on the intended use of the product; purity is of utmost
importance during clinical trials where human safety and
CAR-T manufacturing performance could be jeopardized
by residual components. Typical process-related impurities
include process residuals such as PEI, plasmid DNA, hostcell nucleic acids, host cell proteins, serum, and leachables.10
Enveloped viruses such as lentiviruses are inherently
susceptible to physico-chemical stress such as changes
in temperature, pH, other culture conditions, and shear
stress.42 As such, steps used in downstream LV manufacturing must prioritize the stability of the viral particle.
Downstream purification techniques continue to advance;
yet, LV manufacturing typically results in overall DSP
yields of roughly 10-25%.43 Because of the short halflife of LV (~3-18 hours at 37°C38), multiple harvests arecommonly implemented in LV adherent processing, necessitating batch-style downstream efforts. A move toward
continuous processing and purification is evolving and is
yet to be broadly implemented in LV manufacturing.
Key uncertainties about continuous DSP remain, including whether upstream batches would be individually

Lentiviral Vector Manufacturing Process

tested, pooled, and then submitted to downstream processing, or whether harvests would be processed continually upon removal from the bioreactor and thenpooled at the end of downstream steps.43 Regardless ofapproach, consistent control of upstream processing
must be demonstrated and all processes used duringpooling should be properly validated. Sequential harvests
from the same bioreactor can be pooled as one sub-lot ofa batch, but defined release criteria (e.g., titer) and appropriate safety testing should be in place for each individual
sub-lot. ICH Q7 specifically states that out-of-specification batches of active pharmaceutical ingredient (e.g., LV,
according to the FDA definition) should not be blended
with other batches for the purpose of meeting specifications (e.g., titer). Additionally, blending processes
should be adequately controlled and documented, andthe batch record of the blending process should allow
for traceability back to the individual batches contained
within the blend.44
Continuous DSP also requires a solid understanding of LV purification principles. Steps such as depth
filtration, ultrafiltration, and chromatography can be
employed in a variety of sequences throughout the downstream process and often serve to clarify, concentrate,
and buffer exchange the LV harvests. The general DSP
strategy for LV is shown in Figure 7-4; individual steps
will be discussed further in subsequent paragraphs.

A-CELL✼123

Clarification/cell removal

Clarification occurs as the first step in DSP and typically involves microfiltration and/or centrifugation. The
complexity of clarification steps is highly influenced
by upstream manufacturing processes. Because newer
technologies (e.g., fixed-bed bioreactors) used foradherent culture perform a baseline level of cell removal (cells stick to the reactor material itself), simple
polymer-based filters are often sufficient for removing
leftover cell fragments.10 More complex clarification
strategies are required when using suspension culture
for LV production, as whole cells must be removed.
Cells can be sedimented by low-speed centrifugation, which also serves as a pre-filtration step toprevent clogging of the filter(s) in subsequent steps,
though centrifugation subjects LV to shear force andrequires open manipulation. Membrane filters feature
an inert polymer with pores of a specific diameter;
use of progressively finer filters can accomplish
significant clarification. Due the size of LV (80-120
nm in diameter), using filters with exceedingly small
pore diameter can result in product loss. For thisreason, the pore size of the clarification membranefilter is generally 0.45 μm.45 Depth filters retain cell
particles throughout sponge-like material composedof polymer, binder, and a filtration aid (typicallydiatomaceous earth [DE]). The use of DE can lower
LV titer; however, Valkama et al. reported functional
recovery close to 100% using a depth filter with DE
to clarify LV pseudotyped with VSV-G.45 Through design-of-experiment approaches, Labisch et al. recently
found that a DE concentration of 9 g/L achieved the
optimal balance between clarification and recovery.46
Recovery of >95% of titer via a depth filter without
DE has also been reported.47 The optimal filtration
technique for clarification clearly remains to be seen,
but in most cases, adsorption onto the filter or onto
a component excluded by the filter results in at least
some loss of LV titer. Notably, some LV can be recovered by flushing the filter with buffer.10,45
Two technologic advances are worthy of mention
when discussing clarification. Tangential flow filtration(TFF), though commonly employed during concentration steps, can be used with both membrane anddepth filters to separate suspension cells from LVs;

Lentiviral Vector Manufacturing Process

yields as high as 90% have been reported recently with
2-5 μm depth filters used in TFF.10,48 Another option
is low-shear centrifugation (i.e., kSep® by Sartorius),
accomplished through a careful balance of centrifugal
and fluid-flow forces. Capital cost is very high, but advantages include a closed system, scalability, and highproduct recoveries.49
An important step in DSP is nucleic acid reduction,
which is generally facilitated via the addition of nuclease(e.g., Benzonase®, Denarase®) during the clarification
stage or early purification stages. Nucleic acid impurities
can originate from either the plasmid DNA or producer
cell line DNA. Nuclease serves to reduce the size of any
residual DNA to no greater than ~200 base pairs toprevent the risk of any deleterious effects in recipient
cells.10 The amount of nuclease required will be higher
if added during clarification vs after an initial purification/concentration step. Nuclease will be removed
from the LV during purification and polishing steps. An
alternative to nuclease addition is the SecNuc™ platform
developed by Oxford Biomedica, which features either(1) co-transfection of producer cells with both vector
components and a plasmid that encodes a nuclease
enzyme, or (2) co-culture of vector-production cellswith nuclease-expressing helper cells.50

Chromatography
Chromatography, which is amenable to scalability andautomation, can be used after initial clarification tofurther separate viral particles from contaminants andimpurities by exploiting differences in their interactions
with stationary and mobile phases. These properties
can include size, charge, hydrophobicity, and binding
affinity. 10 Inherently, capture chromatography alsoachieves some degree of bulk product concentration,
as the virus is eluted in a smaller volume. Traditional
size exclusion chromatography (SEC) is somewhat
limited by the size of LV particles, as high molecularweight contaminants and residual plasmid/cell DNA
may not separate from viral particles. Additionally, SEC
is difficult to scale and may be more suitable for final“polishing” of the vector rather than bulk purification.6
Affinity chromatography (AC) requires the modification of LV envelope protein with a “tag” (e.g., histidine,
heparin, or biotin) that binds to an immobilized ligand.

A-CELL✼124

Segura et al. reported 53% recovery of VSV-G pseudotyped LV particles with removal of 94% of impurities
using heparin AC.51 Though having potential to yield
highly pure, concentrated product, AC is limited by
the need to modify the LV envelope, which could theoretically affect the viral biologic activity or introduce(in the case of heparin) a product of animal origin.10,42
Anion exchange chromatography (AEX), whereby
negatively charged vector particles bind to positively
charged chromatographic matrix, is a promising technology for large-scale LV purification. Early reports
by Scherr et al.52 and Kutner et al.53 highlighted LV
recovery of 68% and 76%, respectively, using AEX.
Separation is performed in bind-elute mode, utilizing
stepped concentrations of salt buffer (up to a final
concentration of ~1 M NaCl) for elution steps. As LV
is destabilized by high-salt solutions, dilution of the LV
bulk immediately after final elution must be performed
to prevent envelope degradation.10,42 Novel stationary
phases are being developed, including a cellulose nanofiber-based stationary phase that produces functional
vector yield of roughly 90%.54

Concentration
Some degree of LV concentration is accomplishedduring initial clarification and purification steps,
though additional concentration is generally required.
Concentration can be accomplished by sequential
rounds of centrifugation/resuspension or via SEC,
but tangential flow filtration (TFF), which allows
for reduced manual handling and high vector concentration, is particularly useful for large-scale virus
concentration.42 When run in ultrafiltration/diafiltration mode, TFF not only allows for volume reduction
and buffer exchange, but also removes low molecularweight impurities.10 TFF systems can utilize hollow
fiber membranes or cassettes comprised of various
membrane material (e.g., cellulose- or polyethylene
sulphone). All approaches offer high surface area tovolume ratios, though the use of flat cassettes can subject LV particles to higher shear forces. As discussed
previously, TFF can be used for multiple purposes(clarification, purification, and concentration), andis therefore employed at multiple steps of the overall
downstream manufacturing process.10

Lentiviral Vector Manufacturing Process

Final formulation

Formulation of LV can be performed via either chromatography (SEC) or TFF, with SEC being more limited
to small-scale operations. Using TFF, the final buffer
exchange step can be used to introduce a buffer systemthat provides maximal LV stability. Buffers commonly
employed include HEPES, HIS-HCl (both resistantto pH drift with temperature change), TRIS, or phosphate-buffered saline. Buffer excipients are often added,
such as recombinant human serum albumin (to promotestability) and sugars (as cryopreservatives and osmolarity
regulators).10,42 Alternatively, for ex vivo applications suchas CAR T-cell therapies, vector can be formulated into
the cell media used for culture of target cells.55 Thoughproteins and sugars found in traditional cell media may
help to stabilize the vector, formulating in cell media may
not allow for optimal stabilization and could result in LV
aggregation or adsorption onto container material. Thiscan be particularly problematic if the CAR T cell productrequires short-term freezing during transport.10,42

Sterile filtration or aseptic processing
Sterile filtration, commonly the final DSP step, utilizes a
0.2 or 0.22 μm filter to remove any aggregates or adventitious agents. Sterility assurance is a GMP regulatory
requirement, but terminal sterile filtration is generally
associated with LV loss of 30-50%. 10,45 Altering thetiming of sterile filtration within the overall DSP
sequence may improve LV recovery. For example, thedownstream processing protocol developed by Oxford
BioMedica has placed the sterile filtration step after
the first TFF-based concentration step and before thesecond (final) TFF step to reduce vector losses due toadsorption to membrane material at very high vector
concentrations.56 Alternatively, the sterile filtration
step can be skipped, provided that the manufacturing
process is certified as being fully aseptic. Validation of
aseptic processing (via media process tests) and useof a clean room (ISO 5) for all manipulation steps isrequired,57 and processes that forego sterile filtration
are likely to be examined closely by regulatory agencies. Incorporation of closed or semi-closed systems
increases the feasibility of aseptic processing,6,42,55 anda successful large-scale, semi-closed protocol thatskips the sterile filtration step has been reported.21 The

A-CELL✼125

Figure 7-5: Options for upstream and downstream processing of LVs
Upstream
Processing

Downstream Processing

LV production

Harvest andclarification

Purification

Transient transfection
vs stable cell line

Membrane microfiltration

Anion exchange
chromatography

Single/dual-layer
with progressively
finer pores

Traditional vs novel
stationary phases

Adherent vssuspension culture
Serum vsserum-freeVessel/bioreactor type

Normal flow or TFF
depth filtration

Sterile filtration(or certified aseptic
manufacturing)Affinity chromatography

Stabilizers/
cryopreservatives/
cell media

TFF

Vials/bags/syringes

TFF microfiltration
Depth filtration

Formulation/final
filtration/storage

Size exclusion
chromatography
Ultracentrifugation

Low-speed or low-shear
centrifugation

Nucleic acid reduction; cell debris,
particulate and protein removal
Concentration achievedalteration of DSP steps described by Oxford BioMedica
and the option to omit sterile filtration altogether
speak to the variability possible within DSP protocols.
Options for technology and procedures to employ atvarious steps of upstream and downstream processing
are shown in Figure 7-5.

Final vector product container closure
Equipment and materials used for final LV storage must
be compatible with the vector and any excipients used tostabilize the vector, contain low amounts of leachables/
extractables that could alter the quality of the vector, andbe amenable to freeze/thaw cycles (when cryopreservation
is likely). Physical adsorption to surfaces of syringes, bags,
and glass vials has been reported with LV, with the suggestion that temperature, protein concentration, and hydrophobic interactions between vector and material surfaces
can affect the rate and degree of adsorption.58 Notably,
careful choice of excipients can help to minimize adsorption to container surfaces, as demonstrated by Kumru etLentiviral Vector Manufacturing Process

al. with the use of glass vials.58 Rigid polymer vials andflexible bags are options for LV storage, and either may
be suitable when using vector material as drug substance,
as in the case of CAR T-cell production. Though tested
with expanded T-cell cultures rather than LV material
itself, ethylene vinyl acetate (EVA) bags and cyclic olefin
polymer (COP) vials with rubber stopper-aluminum seal
closures performed comparably in terms of container
cryopreservation performance.59
Importantly, vector filled into bags with sterile weldable connections (e.g., those reported by Schambach
et al. using Baxter Cryocyte™ Bags, Transfer Packs, andPlasma Transfer Sets) can enable closed processing andconformance with GMP standards during transduction ofhuman cells for CAR T-cell products.60,61 Bags that enable
closed processing may be particularly useful during largescale allogeneic CAR T-cell production. The choice of bags
vs vials could also be influenced by the required dosing
volume and whether vector is being used as drug productfor direct injection/infusion. Optimization of fill volume

A-CELL✼126

may help to reduce waste during transduction.38 Similarly,
larger bag sizes could have implications for the amount of
product sacrificed during required sterility testing.
PROCESS CHARACTERIZATION

LV process validation is composed of the aggregate
studies contributing to process knowledge (process
design, process characterization, process qualification,and ongoing commercial monitoring). LV and cellular
drug product process validation steps should be conducted at similar phases of clinical development forthe overall program. Proper characterization of the LV
manufacturing process is essential for minimizing risk
and ensuring consistent quality of the vector. Throughout
clinical development, process design is refined and critical process parameters (CPPs) are identified; the end
goal is a consistent process that minimizes potential
sources of process and material variability. Processcharacterization requirements may vary depending on
the intended application of the LV (as drug product ordrug substance), with formal process characterization
studies implemented once the final commercial process
is established. Vector used for ex vivo cell therapies suchas CAR T-cell products is more appropriately defined as
drug substance. As described in ICH Q8(R2)62:“At a minimum, those aspects of drug substances…
and manufacturing processes that are critical to productquality should be determined and control strategies
justified. Critical formulation attributes and process parameters are generally identified through an assessmentof the extent to which their variation can have impact on
the quality of the drug product.”
Process characterization is enabled by the applicationof design-of-experiment (DOE) approaches to determine
how factors affecting the process impact or interact withthe output of the process (with outputs generally representing the CQAs of the LV). Examples of where DOE
approaches can be applied to optimize LV manufacturing
steps include the following:• Raw material inputs• Temperature, pH, and time of manufacturing andhold steps• Clarification and/or sterilization steps (e.g., filter
material, pore size)

Lentiviral Vector Manufacturing Process• Chromatographic approaches to purification• TFF pressure and flow rates and/or centrifugation
time and speed• Choice and concentration of stabilizers and/or
cryopreservatives• Container closure systems
During process characterization, knowledge of
CPPs generated from DOE studies are used to define
the operating ranges for various process parametersthat ensure consistent performance of the process.
Analytical methods used in characterization studies
should be scientifically sound (e.g., specific, sensitive,
and accurate) and provide reliable results.63 Stabilityof manufacturing intermediates, which may include
material from collection or hold steps (e.g., temporarystorage of bulk harvest, purification intermediates, concentration steps) should also be assessed. This helps todetermine the maximum hold times and facilitates theestablishment of process limits.17
Analytical data obtained from comprehensive processcharacterization are used to develop in-process controls(IPCs), consolidation of which serves as the controlling
document for the manufacturing process. A well-characterized process, though time-consuming, not only lays thefoundation for process qualification and validation, but also
reduces the risk of lost batches (and wasted time and money) moving forward. A description of the DS manufacturing
process and process controls must be included in Module 3of the CTD; any changes or updates to the manufacturing
process/controls as product development proceeds should
be submitted as an amendment to the IND.17
PROCESS QUALIFICATION

Process qualification represents the stage at which theability to repeatedly produce in-specification LV at commercial scale is verified through process performance
qualification (PPQ). As defined in ICH Q7,44 performance qualification represents: “documented verificationthat the equipment and ancillary systems, as connected
together, can perform effectively and reproducibly basedon the approved process method and specifications.”
Process qualification represents an important stage ofoverall process validation, which is executed by a written
protocol that specifies the manufacturing conditions,

A-CELL✼127

controls, testing, and pre-defined success criteria. Prior
to performing PPQ runs, all utilities and equipmentused in production should be shown as suitable for theirintended use and able to perform properly. All raw materials used in PPQ must meet predefined specifications
and the personnel, equipment, facilities, and manufacturing processes used must reflect those expected duringcommercial production. Additionally, all methods used
to evaluate PPQ lots should be validated.
Process qualification and validation for LV manufacturing follows the general approach outlined in ICH
Q744 and the FDA guidance Process Validation: General
Principles and Practices.63 Most original IND submissions will not include PPQ data, but demonstrationof a protocol-driven PPQ with as many commerciallyrepresentative lots as possible is required prior to filinga BLA. Given that the intended applications of LV canvary, early discussions with regulatory agencies can help
to determine the level of validation required for a particular product and the degree of conformance to CGMP
standards required.

Comparability
Changes in process parameters for LV manufacturing
may require comparability studies to ensure that thepre- and post-change LVs are similar in terms of identity, purity, safety, and efficacy. In addition to showing
equivalency of manufactured LV, comparability exercises
may need to include a comparison of processes themselves. Depending on the clinical development phase of
the product and the risk imparted by the change in theprocess parameter, comparability may require evaluationof the cell therapy product attributes in a paired-arm
approach using the same cellular starting material transduced (e.g., splitting the leukapheresis starting material
from the same donor) with both pre- and post-change
LVs.18 This may be particularly relevant when a new
manufacturing site, scale, or strategy (e.g., automation)is introduced.18 Data from CPPs and results of in-processcontrols should be examined to better understand theimpact of introduced changes on the final LV preparation.64 Guidance on comparability studies for LV canbe found in ICH Q5E.65 Again, early discussion withregulatory agencies can help to determine expectations
for comparability studies.

Lentiviral Vector Manufacturing Process

Internal Versus External
Manufacturing and Testing
As thousands of sponsors develop new cell and genetherapies (CGTs), there will likely be more relianceon contract development and manufacturing organizations (CDMOs) for production of therapies orof key components such as plasmid DNA and viral
vectors. As such, CDMOs are key stakeholders in the
production of viral vectors, and sponsors and CDMOs
should create close partnerships.66 Depending on the
CGT sponsor’s internal manufacturing expertise andcapacity as well as the novelty of a therapy or component, a sponsor may choose to outsource production
at any early or late stage of development (i.e., fromprocess development, through process validation andultimately commercial production). Because CGT
sponsors generally seek to compress development
timelines, it is essential that the sponsor and CDMO
streamline the technology transfer process.67
Inherently, knowledge transfer between sponsor
and CDMO is more complex for a CAR T-cell therapy
than it is for an intermediate vector component. Many
manufacturing processes for CAR T-cell therapies are
unique to the sponsor, making the knowledge transfer
to a CDMO more detailed and complicated. Conversely,
the majority of CDMOs have well-defined vector production processes, and sponsors may request only a few
simple modifications to certain parts of the establishedprocess (e.g., plasmid constructs) to maximize output.
The VSV-G gene is subject to export control and associated licensure requirements, which is an important logistical consideration for transfer of plasmids between
the CDMO and sponsor.68 Exporting licenses may also
be required when certain other transgene components(e.g., porcine teschovirus-1 2A [P2A], foot-and-mouthdisease virus 2A [F2A] sequences) are included in theplasmid construct. Coordination between sponsor
and CDMO is critical for LV production, particularlyin the context of ensuring that all Bureau of Industry
and Security (BIS) Export Administration Regulations(EAR) requirements68 are met.

A-CELL✼128

TRANSFER OF MANUFACTURING PROCESS ANDANALYTICAL METHODSTechnology transfer between sponsor and CDMO
Successful technology transfer may be facilitated byalignment of expectations early in the sponsor/CDMO
relationship. BioPhorum recently published a set ofrecommendations, from a panel of sponsor and CDMO
representatives, for ownership of various production-related responsibilities.67 Their recommendations in five
key areas relevant to technology transfer and processvalidation are listed below and can be incorporated in
kickoff meeting documents or checklists:• Process design (e.g., identification of CQAs,
definition of the manufacturing process and control
strategy, design of process characterization studies)• Analytical methods (e.g., definition/qualification of
in-process and release testing, design, and ownershipof comparability studies)• Supply chain (e.g., critical material sourcing, testing,and supply strategy; raw material variability assessment and qualification)• Facility and equipment (e.g., process maps forequipment and personnel, assess CDMO facility
and procedures against sponsor process design andrequirements)• Regulatory and quality (e.g., final product releasestrategy and discussion with regulatory agencies,
regulatory filing preparation, definition of ongoingvalidation strategy)

Assay qualification and validation
Responsibility for identifying assays for product orcomponent release and characterization (e.g., potency),
in-process testing, and comparability studies appropriately lie with the sponsor, as they should define productrequirements. However, given the CDMO’s intricate
knowledge of their own facility design/operations andprevious experience with manufacturing similar products, they may be most qualified to recommend assays
for adventitious agents and/or residual plasmid/producer
cell DNA. The sponsor and CDMO often share responsibility for execution of assay qualification and validation.
Unique product-specific assays may be best qualified andLentiviral Vector Manufacturing Processvalidated by the sponsor since those methods may be
performed by the QC unit of the sponsor. Qualificationof standard, compendial assays could be the responsibility of the CDMO.67
PROPRIETARY PROCESSES

Either the sponsor or CDMO (or both) can protect proprietary LV manufacturing steps within a Drug Master
File (DMF). By definition, a DMF is a submission to the
FDA that “may be used to provide confidential detailed
information about facilities, processes, or articles used in
the manufacturing, processing, packaging, and storing of
one or more human drugs.”69 By design, the non-holding
party can refer to material contained in the DMF within
their IND. While the FDA will be able to reference material contained in the DMF, the non-holding party will
not have access to the material contained within. As such,
DMFs can represent a barrier to knowledge and technology transfer. Protection of proprietary information
may be challenging in the EU—most sponsors want to
conduct clinical trials and achieve commercialization in
the EU but filing of DMFs is not allowed/afforded there.
Additional regulatory guidance on DMFs can be foundin Chapter 2.

Product Testing and Characterization
Requirements for drug substance characterization to
be reported within Module 3 of the CTD are outlinedin the 2020 FDA Guidance: Chemistry, Manufacturing,and Control (CMC) Information for Human Gene
Therapy Investigational New Drug Applications (INDs).17
Requirements for characterization generally increaseas products move through different phases of development. Additional information can be found in the 2003
FDA Guidance: INDs for Phase 2 and Phase 3 Studies:
Chemistry, Manufacturing, and Controls Information.70
CRITICAL QUALITY ATTRIBUTES OF LV

As a critical component of the final product, viral vector
CQAs are important to consider early in the developmentof any CAR T-cell therapy. Information on identifying
and defining CQAs can be found in Chapter 4. Examples
of CQAs specific to LV are found in Figure 7-6.

A-CELL✼

129

Figure 7-6: Examples of CQAs for LV to be used in the development of CAR T-cell therapies
Infectious
titer
Biologicalpotency

Physical
titer

Potency

Sterility
CAR
transgene

Mycoplasma
Adventitious
virus

SafetyCQAs for
LV

Identity
Envelope
protein

RCL

PurityResidual
plasmid DNA

Producer-cell
DNA/proteins
Process-related(serum, reagents)

RELEASE TESTING

Testing is performed on every LV lot to confirm its
quality and alignment with specifications set for eachkey attribute. The understanding of appropriate testing,and corresponding testing limits, grows as LVs progress
through stages of clinical development and as the LV field
matures. During early clinical development, a set of assays
for LV quality should be performed after each process stepto help determine which steps are most critical and whichassays are best-suited to pick up on process changes that
impact the final LV product. As the LV product moves toward commercialization, assay selection, assay timing, andspecifications will be refined based on increased productknowledge. Examples of assays used for release testing andcommon references used to justify CQA specifications for
LVs are included in Table 7-2.
ASSAY QUALIFICATION AND VALIDATION

According to ICH Q2(R1), the objective of any assay used
to characterize a drug substance or drug product should

Lentiviral Vector Manufacturing Process

be clearly understood.94 Qualification and validation of
the assay should center on the following:
Accuracy
PrecisionSpecificity
Detection limit
Quantitation limit
Linearity
Range
The main objective of assay qualification (performed during development to enable clinical release
testing, typically with a reduced number of lots available that are fully representative of the clinical LV)
and validation (performed in preparing commercial
process validation with multiple lots of the final, fully
representative product) is to robustly demonstrate thatthe assay will serve its designated purpose.94 If changes
in LV production or analytical procedures occur at any
phase of development, revalidation of assays may berequired. The need for revalidation will depend on the

A-CELL✼130

Table 7-2: Release testing for key LV attributes.10,24,71,72
Attribute
class

Specification attribute(determined by CQA)
Transgene presence

Identity
Envelopein vivo and in vitro
assaysReplication-competent
LV

qPCR, PERT,
cytopathology

Mycoplasma
Sterility
Endotoxin
pH

Purity

PCR, ddPCR, NGS,
Sanger Sequencing
SDS-PAGE, MS,
immunoblotting,
ELISA

Adventitious virus(human, bovine, andporcine if animal-derived
materials used)

SafetyGeneralPotential assay(s)Osmolality
Appearance (color andclarity)

PCR, RT-PCR, cell
culture-based
assaysCulture-based
assaysLAL method: gelclot, chromogenic,
and turbidimetric
pH meter(potentiometry)
Osmometer
VisualResidual plasmid

qPCR

Residual host cell DNA,
total DNAqPCR, Picogreen,
DNA Threshold
assay

Host cell protein

ELISA, MS

Residual serum/
nuclease/transfection reagent/solvent

ELISA, MS,chromatography

Product-related
impurities: Interfering
particles, non-infectious
particles

ELISA, MS

Visible particulates

Visual inspection**

Common reference(s) for justification of CQA
specification*
21 CFR 610.1473
21 CFR 610.1473
ICH Q5A (R1)24
9 CFR 113.5374
9 CFR 113.4675
9 CFR 113.4776
Testing of Retroviral Vector-Based Human Gene
Therapy Products for Replication Competent Retrovirus
During Product Manufacture and Patient Follow-up.
FDA Guidance for Industry, January 202077
USP <63>78
USP <71>79
USP <85>80
USP <791>81
USP <785>82
USP <631>83
Guidelines on the quality, safety, and efficacy
of biotherapeutic protein products prepared byrecombinant DNA technology: Replacement of Annex
3 of WHO Technical Report Series, No. 814. (2013)84
Guidelines on the quality, safety, and efficacy
of biotherapeutic protein products prepared byrecombinant DNA technology: Replacement of Annex
3 of WHO Technical Report Series, No. 814. (2013)84
USP <509>85
USP <1132>86
Serum: 21 CFR 610.15(b)87
Nuclease: ICH M7(R1)88
Transfection reagents: ICH M7(R1)88
Solvent: USP <467>89
Chemistry, Manufacturing, andControl (CMC) Information for
Human Gene Therapy Investigational
New Drug Applications (INDs). FDA Guidance forIndustry, January 202017
USP <788>90
USP <790>91
USP <1790>92

continued on next page

Lentiviral Vector Manufacturing Process

A-CELL✼131

Table 7-2: Release testing for key LV attributes.10,24,71,72
continued from previous page
Attribute
class

Specification attribute(determined by CQA)

Physical/genomes titer

Strength/potencyPotential assay(s)Common reference(s) for justification of CQA
specification*

ELISA (p24), qPCR,
RT-PCR, HPLC

Chemistry, Manufacturing, andControl (CMC) Information for
Human Gene Therapy Investigational
New Drug Applications (INDs). FDA Guidance forIndustry, January 202017

Transduction ofcells followed byquantification of
Infectious/functional titer the pro-viral DNA
copy number
by qPCR or byimmunofluorescencewith flow cytometry

Chemistry, Manufacturing, andControl (CMC) Information for
Human Gene Therapy Investigational
New Drug Applications (INDs). FDA Guidance forIndustry, January 202017

Physical titer: infectious
titer ratio

Calculation

Chemistry, Manufacturing, andControl (CMC) Information for
Human Gene Therapy Investigational
New Drug Applications (INDs). FDA Guidance forIndustry, January 202017

Functional/biologicalpotency (transduced
primary cells)

Cell proliferation,
cytotoxicity,
cytokines

Potency Tests for Cellular and Gene Therapy
Products. FDA Guidance for Industry, January 201193* Harmonization based on filing jurisdictions** Visible inspection of all filled LV vials is a manufacturing criteria, captured on the batch record. However, some sponsors may include avisible particulate evaluation as part of the appearance method. Additional characterization methods for identifying inherent and intrinsic
particulates include light microscopy, DLS, SEC-MALS, TEM, AUC, and FFF-MALS
AUC, analytical ultracentrifugation; ddPCR, digital droplet polymerase chain reaction; DLS, dynamic light scattering; ELISA, enzyme-linked
immunosorbent assay; FFF, field flow fractionation; HPLC, high-performance liquid chromatography; LAL, Limulus amebocyte lysate; MALS,
multi-angle light scattering; MS, mass spectrometry; NGS, next-generation sequencing; PCR, polymerase chain reaction; PERT, productenhanced reverse transcriptase; qPCR, quantitative polymerase chain reaction; RT-PCR, real-time polymerase chain reaction; SDS-PAGE,
sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; TEM, transmission electron microscopy

extent of the change and its potential for impacting
the final LV product. All relevant data collected during
validation (and/or revalidation) should be submitted
within regulatory filings.
Examples of assays used to confirm various identity,
safety, purity, and potency attributes of the LV productare discussed in the sections that follow and listed inTable 7-2. Where applicable and established, Table 7-2
also provides common references used to justify attribute specifications; these may be of use when initiallydefining specifications and the QTPP in early-phasedevelopment. Chapter 9 of this document thoroughly
discusses analytical technologies.

Lentiviral Vector Manufacturing Process

IDENTITY TESTING

Testing of LV is performed to ensure the identity of thetransgene insert and to distinguish viral pseudotype(e.g., VSV-G, MLV envelope) if multiple vectors are
manufactured in the same facility. Assays can be nucleic
acid-centric and protein-centric. Examples of assays used
to confirm the vector genome and quantify viral protein
are listed in Table 7-2.
SAFETY TESTING

Testing LV preparations for adventitious virus,
Mycoplasma, bacteria, and endotoxin follows procedures common to any sterile biological product (Table
7-2).24,78,80 A safety concern specific to LV relates to

A-CELL✼132

Figure 7-7: LV production process timepoints for safety testing.
Process step

MCB

WCB

Expansion(vectorupstream)

Vectorharvest

Bulk

Finished
vector

EOPC

Testing• Retroviruses• Adventitious
viruses—in vitro—in vivo/
inapparent• Mycoplasma• Sterility• Endotoxin• Adventitious
viruses—in vitro• Mycoplasma• Sterility• Endotoxin• Bioburden• Endotoxin• Adventitious
viruses• Mycoplasma• Bioburden

the potential for replication-competent lentivirus(RCL). Though the plasmid systems used in traditional
third-generation LV vectors make recombinant virus
generation more of a theoretical than practical concern,
regulatory agencies require extensive testing for RCLs
in vector products.77,95 Additionally, risk assessmentand mitigation strategies to prevent the transmission of
SARS-CoV-2 via cell and gene therapy products is recommended by the FDA.96 Typical timepoints for safety
testing during LV production are outlined in Figure 7-7.

RCL testing
Theoretically, RCL could develop at any step during• Bioburden• Endotoxin• RCL• Sterility• Endotoxin• RCL• Adventitious
viruses—in vitro—in vivo/
inapparent

manufacturing of the LV product or during the expansionof ex vivo-transduced cells in culture due to recombinationof viral genes carried within the helper and gene transfer
plasmid. Testing for RCL at each step of the drug substance(i.e., LV) and drug product (i.e., CAR T cell) manufacturing
process would result in significant loss of material and time.
The latest FDA guidance on RCL testing reflects the accrued
evidence of safety associated with advances in LV design
and testing. As such, the FDA no longer recommends RCL
testing on WCBs and has revised recommendations regarding the amount of vector that should be tested.77 Current
recommendations for RCL testing are summarized in Table
7-3; techniques used for RCL testing are listed in Table 7-2.

Table 7-3: Material, frequency, and type of testing recommended to ensure RCL-free vector
and cell product.77
Material

Frequency of testing

Testing for RCL

MCB (stable cell line)*

One-time

Cells and supernatant

Vector harvest material-End-of-production cells-Vector supernatant

At product release

Cells and supernatant

Ex vivo transduced cells

At product release

Cells only*** Non-transduced packaging cell lines (MCB) are tested for retroviral activity of contaminating viruses**If accumulated manufacturing and clinical experience demonstrates that the transduced cell product is consistently RCL-negative, anagreement may be reached with the FDA to reduce or eliminate testing of ex vivo genetically modified cells.

Lentiviral Vector Manufacturing Process

A-CELL✼133

When cell testing is required, the FDA recommends testing 1% or 1x108 (whichever is less) pooled
vector-producing cells or ex vivo transduced cells by
co-culture with a permissive cell line. When supernatant testing is required, the FDA recommends testingat least 5% of the total supernatant by amplification ona permissive cell line. The FDA also considers current
manufacturing evidence, which indicates that <1 RCL/
dose equivalent is an achievable level for vector preparations intended for clinical use. As such, sufficient vector
supernatant should be tested to ensure a 95% probabilityof RCL detection if present at a concentration of 1 RCL/
dose equivalent (i.e., the volume of vector that would
be administered in a CAR-T dose based on LV volume
used at T-cell transduction). The sponsor should detailthe amount to be tested and provide a justification forthe proposed testing volume within the IND.77 Since
RCL would theoretically co-purify with the LV product,
the guidance could be interpreted to indicate that RCL
testing can be performed on the final LV, which would
confer additional material savings and ease of testing asmaller volume than the vector harvest.

SARS-CoV-2 testing
In its 2021 Guidance for Industry, the FDA outlined expectations for cell and gene therapy product manufacturers to perform a risk assessment that identifies, evaluates,
and mitigates factors that may allow for transmission of
SARS-CoV-2 via their products.96 The FDA added thatthe risk assessment and mitigation strategies should be
included in the appropriate regulatory submissions (e.g.,
IND, BLA, or DMF). The guidance notes that SARSCoV-2 has been shown to be capable of infecting andreplicating HEK 293 cells.96,97 Considerations specific to
LV production could include processes used to control
viral spread (e.g., minimizing open manipulation steps)
and contamination risk during manufacturing (e.g., employee screening practices). The FDA does not provide
specific testing recommendations for source material, cellbanks, in-process intermediates, or final drug products,
though such testing may be included in a risk mitigation
strategy based on the assessment of potential risk. The
risk mitigation strategy could potentially include screening EOPC cells for SARS-CoV-2 RNA.

Lentiviral Vector Manufacturing Process

PURITY TESTING

Purity tests seek to ensure that the LV product is free fromphysical, biological, and chemical contamination that
could affect the quality and safety of the final LV product.
Generating a good understanding of impurities, and the
toxicities they could confer, early in vector and processdevelopment will aid in developing acceptable tolerancesin the final LV product.66,71 Purity expectations set forth
by regulatory agencies must be satisfied, and will continue to evolve as the field of biologic therapies advances.72

Residuals
Sources of impurities include the host cell system used
to generate the LV product (e.g., residual host cell DNA
and proteins), residual plasmids used for transfection,
residual transfection reagents, non-functional vector(e.g., broken, immature, with insufficient envelope
protein, or those that have failed to package the vector
DNA98), residuals from culture medium (e.g., serum),and residuals from downstream purification processes(e.g., nuclease, chromatography resin components).
Host cell and plasmid impurities in the LV product may
interfere with the analytical methods used to test boththe LV and the CAR T-cell product. The ability of the
manufacturing process to reduce the amount, size, or
activity of residual host cell and plasmid DNA should bedemonstrated. In general, reduction of host-cell DNA to<10 ng per dose with fragments smaller than 200 bp isrecommended.84 Examples of technologies used to detect
impurities and common references used for justificationof allowable impurity levels are listed in Table 7-2.

Aggregates/Particulates
Biologically derived products, particularly those derived
from viruses, viral vectors, or proteins, may containinherent (those that are expected from the product itself) and intrinsic (generated within the manufacturing
process, potentially including silicone oil, rubber, glass,or stainless steel) particles or agglomerates. In such cases,
limits for visible particulates, which are generally considered a safety concern for patients, must be specified in
the approved regulatory application. Drug products forparenteral administration, including difficult-to-inspect
cell-based medicinal products, should be “essentially freeof visible particles.”91,92,99 A position paper by Mathonet et

A-CELL

✼134

al. reviews best practices for visual inspection processesof biotechnology-derived products, including associatedoperator training, quality control sampling, testing, andsetting of acceptance criteria that correspond to “essentially” or “practically” free of visible particles.100
Aggregates of DNA (host-cell or plasmid-derived)
can form during various stages of LV production72 anddownstream processing steps (and their order) can
influence the formation of aggregates. For example, if
nuclease is applied late in DSP, large-sized residual nucleic acid contaminants present after harvest and during
early DSP steps can capture vector particles, forming
aggregates that could result in vector loss.6 Particulates
should be characterized to confirm identity of inherent
and intrinsic nature.101 Methods and technologies used
for particle and aggregate detection, identity, and common references used for justification of allowable levels
are listed in Table 7-2. Testing for visible particles that
may arise from LV aggregates is also part of stability
testing (briefly discussed later in this chapter), as theirformation can be influenced by storage containers andconditions.
POTENCY TESTING

Potency is the quantitative measure of biological activity(generally expressed in units) resulting from a particularquality attribute. Potency or strength measures for LV
include physical titer and infectious titer. Physical titeris expressed as the number of viral particles per mL andreflects the total number of viral particles present (active
and inactive), whereas infectious titer measures how much
vector is available to transduce a cell and is expressed astransducing units per mL. Infectious titer is always lower
than physical titer, but provides a more accurate representation of the transducing ability of the vector.71
An emerging expectation from regulatory agencies
relates to biologically relevant potency.18 In the case of
CAR T-cell products, biological potency centers aroundhow the LV (e.g., titer, transduction) contributes to
CAR expression and cytotoxicity. Expectations fordemonstrating a solid understanding of LV contribution
to biological potency grow as the CAR T-cell productproceeds through product development stages. By thetime of pivotal clinical trials, characterization of thebiological potency conveyed by LV should demonstrate

Lentiviral Vector Manufacturing Processthat its contribution to the eventual CAR T-cell therapy iswell understood. Examples of technologies used to assess
potency of a LV product are listed in Table 7-2.

Multiplicity of infection

In ex vivo applications, Multiplicity of Infection (MOI)
is a parameter used to predict how LV titer (typicallymeasured with an in vitro assay, and/or in a non-primarycell line) correlates to infectivity of the primary target
cell. Since transducing titer may range between LV lots,
determination of the MOI allows for a variable volume
of LV to be used during CAR-T manufacturing based oncorresponding LV titer where:
MOI = LV titer (Transducing units/mL) × Transduction
volume (mL) / number of target cells

In theory, MOI should have a linear relationship between titer and infectivity of primary cells.102 In reality,
determination of MOI is complicated by factors such as
target cell health, LV stability, transduction reagent, incubation time, media exhaustion, and batch contamination.
Because of the inherent variability of the behavior
between LV and donor T cells, special care should betaken to determine MOI during LV and CAR-T processdevelopment. Like other LV and CAR-T parameters, establishing an accurate MOI depends on product knowledge and process consistency. The ultimate goal is to set
an MOI in the CAR-T batch record that will produce
consistent CAR-T product quality attributes. Examplesof MOI application for CAR-T manufacturing purposes
are provided in Table 7-4.
MOI as a CAR-T manufacturing process parameteris determined during CAR-T process development by
testing a titrated range of LV on primary cells103 in ascale-down model and confirming the candidate MOI in
full-scale development runs.18 Due to LV batch-to-batch
variation, MOI often needs to be determined experimentally for each lot of LV by transduction with serial
dilutions.18 Titration data from multiple LV lots can be
combined to determine the final MOI, a manufacturing
unit operation, for the CAR-T process. In CAR-T, characterization studies to titrate LV transduction efficiency
to determine response curves show that increased MOI
correlates to increased vector copy number and potencyof the primary (i.e., CAR-T) cell, with an eventual plateau.104 The approaches listed here reflect the industry

A-CELL✼135

Table 7-4: Examples of MOI application for CAR-T manufacturing purposes.
MOI (set parameter)

10

Viral titer (variable)

1x108 TU/mL

1x108 TU/mL

1x107 TU/mL

Number of target cells(may be variable due to donor cell growth)

100x106 cells

75x106 cells

100x106 cells

Transduction volume(input to manufacturing process)

10 mL

7.5 mL

100 mLstandard to date. However, the Considerations for theDevelopment of Chimeric Antigen Receptor (CAR) T
Cell Products Draft Guidance for Industry18 provides
additional clarity on MOI determination, such that a
lot release test (i.e., qualified and listed on vector specification) to determine the vector concentration thatcan be used to normalize the amount of vector used
for CAR-T transduction is recommended.
The ideal MOI will allow maximal transductionof target cells, with minimal toxicity105 and little LV
waste. Employing a characterization study to titrate
MOI during development is beneficial in the longterm, as an MOI set higher than necessary for GMP
production would result in excessive use of expensive
vector product.
STERILITY TESTING

Principles of sterility testing for LV products generally
follow those outline in USP <71>.79 However, LV used
as a drug substance (e.g., destined for use in an ex vivo
CAR T-cell product) may have some latitude withsterility testing because it’s not a direct injectable.
USP <71> states that, for liquid products with afinal volume of >100 mL per container, a minimum of
10% of the contents of the container (but not less than
20 mL) must be used for sterility testing on at least 2
different media. USP <71> also states that, for batches
containing more than 100 but less than 500 containers,
a minimum of 10 containers must be tested. Adheringto these requirements, particularly for LV packaged inbags (to enable closed processing during transductionof human T-cells), could result in substantial loss of
product and could substantially delay production of
the final CAR T-cell product. Importantly, the minimum quantity and number of containers to be tested

Lentiviral Vector Manufacturing Process

are qualified in USP <71> as being subject to otherjustification and authorization, suggesting that somelatitude in sterility testing may be afforded. The sponsor’s sterility testing schema should be outlined in the
LV Justification of Specification and aseptic control
strategy (if aseptic processing is implemented in lieu
of sterile filtration).

Stability
Stability testing is conducted throughout the lifecycle of
LV development and serves to provide key information
on how the vector is influenced by temperature andstorage container. According to ICH Q1A (R2), “stability studies should include testing of those attributesof the drug substance that are susceptible to change
during storage and are likely to influence quality, safety,
and/or efficacy. The testing should cover, as appropriate,
the physical, chemical, biological, and microbiologicalattributes.”106 As such, stability assays focus on assessing
key CQAs of the LV product, including those related
to identity, purity, and potency.107 Results of stability
studies help to establish the appropriate shelf-life of thevector and optimal storage conditions.17
As with drug substance characterization, regulatory
requirements for drug substance stability studies (andthe level of result detail) generally increase as drug
products move through advanced phases of development. A summary of available stability data, and an
outline of ongoing study design for the DS, should be
included in each DS-related section of Module 3 of the
CTD. Results from ongoing stability studies should be
updated in the IND on a regular basis. Information onthese requirements can be gleaned from FDA guidance(Table 7-5). The nature of the particular drug substance

A-CELL

✼136

Table 7-5: FDA guidance on stability program requirements for drug substance
Guidance document

Applicable guidance

Chemistry, Manufacturing,and Control (CMC)
Information for Human Gene
Therapy Investigational New
Drug Applications (INDs).
January, 202017

The types of stability studies (either conducted or planned) that will be used
to demonstrate that the DS is within acceptable limits are recommended to be
described in the original IND submission.
Stability studies may evolve with product development. As product development
progresses, consideration of stability studies required to determine an expiration
date is warranted and these items should be discussed at late-phase IND
meetings.
Information on qualification of analytical procedures used to generate stability
data should be included in the original IND.

INDs for Phase 2 and Phase
3 Studies: Chemistry,
Manufacturing, and Controls
Information. May, 200370

Any changes to the DS stability program from that described in earlier phases should
be provided in an information amendment.
Stability data from clinical trial materials used in Phase 2 and Phase 3 studies should be
provided in annual reports as data become available.
During Phase 3, a stability protocol to be used for formal stability studies should be
developed to ensure appropriate stability data are generated for filing at the NDA stage.

will determine which tests should be included in thestability program,70 and expectations for the DS stability program should be discussed during early meetings
with the FDA to ensure smooth transition throughdifferent phases of development.
A variety of analytical tools can be used to conduct stability studies. Thorough discussion of analytical methods
can be found in Chapter 9. The stability-indicating properties of methods can be assessed during assay development by testing with vector treated by forced degradation
conditions (such as multiple freeze-thaw cycles).17 Special
considerations for vector stability include maintenance of
infectious titer and functional potency. The potential forviral protein aggregates, which can create immunogenicity issues when the final drug product is administered
to a patient, must be assessed during long-term storage.
Aggregation detected in the stability studies, as evident
by visible particulates, indicates unstable formulation of
the final vector drug substance during long-term storage.
Acceptance criteria for real-time stability studies should
be derived from the LV release specification and available
stability data. As release specification acceptance criteria
are refined after accumulation of product knowledge,
stability study acceptance criteria can also be updated tomatch the release criteria. However, differences between
the acceptance criteria of the stability studies and theLentiviral Vector Manufacturing Process

release specification may be justified based on the observations from the real-time stability studies.106 For instance,increased degradation products (impurities) may be acceptable in stability studies with sufficient justification.108
VECTOR BULKS

For drug substance bulk material that is to be stored
after manufacture but prior to formulation and final
manufacturing, ICH Q5C107 states:“…stability data should be provided on at least
3 batches for which manufacture and storage
are representative of the manufacturing scale ofproduction. A minimum of 6 months stability dataat the time of submission should be submitted incases where storage periods greater than 6 months
are requested. For drug substances with storage periods of less than 6 months, the minimum amountof stability data in the initial submission should bedetermined on a case-by-case basis.”
The quality of DS batches used in the stability program should be representative of the quality of material
used in preclinical and clinical studies as well as that to
be made on a production scale.106,107 In essence, yourbulk stability protocols should provide data to cover

A-CELL✼137

Table 7-6: Examples of stability studies for bulk vector (IND to commercial).
Phase

Study type

Temperature

Quality

Lots

MethodStatusStudyduration

Preclinical(IND-FIH)106

Long-term

NominalGMP(Dev OK)

3

Qualified

12 months17,109

Phase 1106

Long-term

NominalGMP

1 to 3

Qualified

12 months17,109

Pivotal(registration lots)110

Long-term

NominalGMP

All PPQ

Validated

12 months17,109

Pivotal(registration lots)110

Stressed &accelerated

˚C -20, +5

GMP

3

Validated

12 months

Commercial

Long-term

NominalGMP

Up to 3

Validated

12 months109

the period between manufacturing of the bulk to theinitiation of fill finish (Table 7-6).
FINISHED VECTORICH Q5C states that “stability information should be
provided on at least 3 batches of final container productrepresentative of what will be used at manufacturing
scale.”107 Though this recommendation is specific to drug
product, even vector intended for final use as a drug
substance (as in the case of LV utilized in CAR T-celltherapies) should likely be held to the same standard.
Similarly, a minimum of 6 months stability data at thetime of submission should be submitted in cases wherestorage periods greater than 6 months are requested.
Retest periods and shelf life for stability should
be based on real-time data obtained at the long-term
storage condition. According to ICH Q1A(R2), fordrug substances intended for storage below -20°C (asis the case with cryopreserved LV, which is often stored
at a nominal condition of -80°C), the retest period forstability should be “treated on a case-by-case basis.”106
Stability protocols for finished vector should provide
data to cover the full development period (Table 7-7).
STRESSED AND ACCELERATED STABILITY STUDIESIn its Q5C and Q1A(R2) guidance, the ICH strongly
suggests that studies be conducted on the DS (in thiscase, finished vector) under stressed conditions.106,107
To perform accelerated (conditions that should cause
only a small amount of product degradation) andstressed (exaggerated storage conditions that introduce

Lentiviral Vector Manufacturing Process

significant degradation to the finished vector) studies,temperature increases are applied to the vector tointentionally degrade it. Results of these studies help
sponsors to understand how and to what extent thevector is degraded, what degradation products result,and can inform adjustments to vector formulation that
could potentially minimize degradation and provide adata set to evaluate product risks for storage or shippingand handling temperature excursions.
CONTAINER CLOSURE SYSTEM

Stability studies on vector bulk substance should be
conducted within containers used during production, whereas studies on finalized vector should be
performed on DS packaged in a container closuresystem that is the same as (or simulates) the packaging
proposed for storage and distribution.106 Smaller-scale
vessels may be used for vector bulk and finishedvector stability studies, but the containers should befully representative of the final container construction
materials and closure system. Small-scale stability samples should also be filled in a manner proportional tothat of the final container surface area and headspace.
The suitability of the container closure system must
be discussed in part 3.2.S.6 of Module 3 of the CTD.
Specifically, “the suitability should be discussed withrespect to, for example, choice of materials, protection
from moisture and light, compatibility of the materials with the drug substance, including sorption tocontainer and leaching, and/or safety of materials of

A-CELL✼138

Table 7-7: Examples of stability studies for finished vector (IND to commercial)
Material

Phase

Study Type

Temperature

Quality

Lots

MethodStatusStudy Duration

Finished vector

Preclinical(IND-FIH)106

Long-term

NominalGMP(Dev OK)

3

Qualified

3 to 5 years17,106,109

Finished vector

Phase 1106

Long-term

NominalGMP

1 to 3

Qualified

3 to 5 years17,106,109

Finished vector

Pivotal(registrationlots)107

Long-term

NominalGMP

All PPQ

Validated(PPQ)

3 to 5 years17,106,109

Finished vector

Pivotal(registrationlots)106,107

˚C -20, +5,
Stressed &room temp,
accelerated
+36

GMP

3

Validated

72 hr to months

Finished vector

Pivotal(registrationlots)106,107,111

CCIT

GMP

3

Validated

3 to 5 years

Finished vector

Pivotal(registrationlots)107

In-use conditions:
Stability of manufacturing
intermediates during
process characterization

GMP

3

Validated

In-useconditions andholds

Finished vector

Commercial

Long-term

GMP

Up to 3

Validated

3 to 5 years110

NominalNominalconstruction.” Additionally, container closure integrity
evaluations are required throughout the developmentof sterile products. During clinical stages, sterility testing of samples held at nominal conditions until the endof the study can be employed to confirm the continuing
sterility throughout the product’s shelf life or dating
period.111 In preparation for commercial filing, container closure integrity testing with validated methods
to assess container ingress (such as dye, microbial, andgas) can be implemented in the stability studies.111,112
EXTRACTABLES AND LEACHABLES

The assessment of extractables and leachables is important for any DS or DP that will be maintained in acontainer during production hold steps or final storage/
shipping. Depending on the exact means and extent of
DS integration into the final DP, and the time that the DS
spends in contact with container material, full extractable and leachable studies could be warranted, particularly if a novel container closure system is implemented(such as flexible bags, etc). That decision should be based

Lentiviral Vector Manufacturing Process

on a thorough assessment of the risk that any leachables
contained in the DS could pose to the final drug product.
Extractables are compounds that can be extracted fromthe container closure system in the presence of a solvent.
Extractables are tested for in the container material andrepresent a potential impact to the DS. Leachables arecompounds that leach into the final formulation fromthe container closure as a result of direct contact with theformulation. Leachables are tested for in the final formulation itself (contained within the closure system) andrepresent actual impact to the DS.113 Areas of concern
with extractables and leachables relate to vector CQAs
of efficacy and safety. At a minimum, discussion of the
DS container closure system in Module 3 of the CTD
should include suitability of the system with respect
to leachables, and every company should evaluate andprovide a profile of potential substances that could leachinto the bioprocess. In the case of CAR T-cell therapies,
it’s possible that leachables present within DS may besufficiently diluted during media-exchange and othersteps of the CAR-T manufacturing process.

A-CELL

✼139

References
1.

Kymriah. Prescribing information. Novartis; 2021. Accessed
June 18, 2021. https://www.novartis.us/sites/www.novartis.us/
files/kymriah.pdf.

2.

Breyanzi. Prescribing information. Bristol Myers Squibb; 2021.
Accessed August 6, 2021. https://packageinserts.bms.com/pi/
pi_breyanzi.pdf.

3.

Abecma. Prescribing information. Bristol Myers Squibb; 2021.
Accessed August 6, 2021. https://packageinserts.bms.com/pi/
pi_abecma.pdf.

4.

ClinicalTrials.gov. Accessed June 22, 2021. https://clinicaltrials.gov/ct2/results?term=lentiviral+vector%2C+CAR+T-cell+AND+CAR-T+cells.

5.

Southey F. Oxford BioMedica to capture 25-30% of lentiviral
vector market by 2026, predicts CTO. BioPharma-Reporter.
December 10, 2018. Accessed June 22, 2021. https://www.
biopharma-reporter.com/Article/2018/12/10.

6.

Merten OW, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016;3:16017.
doi:10.1038/mtm.2016.17.

7.

Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol.
1998;72(11):8463-8471. doi:10.1128/jvi.72.11.8463-8471.1998.

8.

Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM.
Development of a self-inactivating lentivirus vector. J Virol.
1998;72(10):8150-8157. doi:10.1128/jvi.72.10.8150-8157.1998.

9.

Zufferey R, Dull T, Mandel RJ, et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol.
1998;72(12):9873-9880. doi:10.1128/jvi.72.12.9873-9880.1998.

10. Perry C, Rayat A. Lentiviral vector bioprocessing. Viruses.
2021;13(2)doi:10.3390/v13020268.
11. Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein
M. LDL receptor and its family members serve as the cellular
receptors for vesicular stomatitis virus. Proc Natl Acad Sci U S
A. 2013;110(18):7306-7311. doi:10.1073/pnas.1214441110.
12. Amirache F, Lévy C, Costa C, et al. Mystery solved: VSVG-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the
LDL receptor. Blood. 2014;123(9):1422-1424. doi:10.1182/
blood-2013-11-540641.
13. Ghani K, Wang X, de Campos-Lima PO, et al. Efficienthuman hematopoietic cell transduction using RD114- and
GALV-pseudotyped retroviral vectors produced in suspension
and serum-free media. Hum Gene Ther. 2009;20(9):966-974.
doi:10.1089/hum.2009.001.
14. Müller S, Bexte T, Gebel V, et al. High cytotoxic efficiencyof lentivirally and alpharetrovirally engineered CD19-specific chimeric antigen receptor natural killer cells against
acute lymphoblastic leukemia. Front Immunol. 2019;10:3123.
doi:10.3389/fimmu.2019.03123.
15. Brown J. Supporting AAV and lentiviral vector development
and commercialization. Pharma’s Almanac. May 24, 2019.
Accessed June 23, 2021. https://www.pharmasalmanac.

Lentiviral Vector Manufacturing Process

com/articles/supporting-aav-and-lentiviral-vector-development-and-commercialization.
16. European Commission. European Commission Directive
2001/83/EC on the community code relating to medicinal
products for human use. November 2001. https://ec.europa.
eu/health/sites/default/files/files/eudralex/vol-1/dir_2001_83_
consol_2012/dir_2001_83_cons_2012_en.pdf.
17. US Food and Drug Administration. Guidance for Industry:
Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug
Applications (INDs). January 2020. https://www.fda.gov/media/113760/download.
18. US Food and Drug Administration. Draft Guidance for Industry: Considerations for the Development of Chimeric Antigen
Receptor (CAR) T Cell Products. March 2022. https://www.
fda.gov/media/156896/download.
19. US Food and Drug Administration. Guidance for Industry:
M4Q: CTD—Quality. August 2001. https://www.fda.gov/media/71581/download.
20. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics ofa human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36(1):59-74. doi:10.1099/00221317-36-1-59.
21. Ausubel LJ, Hall C, Sharma A, et al. Production of CGMP-grade lentiviral vectors. Bioprocess Int. 2012;10(2):32-43.
22. Gama-Norton L, Botezatu L, Herrmann S, et al. Lentivirus
production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. Hum Gene
Ther. 2011;22(10):1269-1279. doi:10.1089/hum.2010.143.
23. US Food and Drug Administration. Guidance for Industry:
Considerations for Plasmid DNA Vaccines for Infectious
Disease Indications. November 2007. https://www.fda.gov/
media/73667/download.
24. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q5A (R1): Viral safety evaluation of
biotechnology products derived from cell lines of human or
animal origin. September 1999. https://database.ich.org/sites/
default/files/Q5A%28R1%29%20Guideline_0.pdf
25. Barone PW, Wiebe ME, Leung JC, et al. Viral contamination in
biologic manufacture and implications for emerging therapies. Nat
Biotechnol. 2020;38(5):563-572. doi:10.1038/s41587-020-0507-2.
26. Cameron R, Smith K. Virus clearance methods applied inbioprocessing operations: an overview of selected inactivation
and removal methods. Pharm Bioprocess. 2014;2(1):75-83.
27. Scott C, Aranha H. Viral safety of viral vectors: special
concerns arise when the virus is the product. BioProcess Int.
2021;19(10):4-12.
28. al Yacoub N, Romanowska M, Haritonova N, Foerster J.
Optimized production and concentration of lentiviral vectors
containing large inserts. J Gene Med. 2007;9(7):579-584.
doi:10.1002/jgm.1052.
A-CELL

✼140

References, continued
29. McCarron A, Donnelley M, McIntyre C, Parsons D. Challenges of up-scaling lentivirus production and processing. J
Biotechnol. 2016;240:23-30. doi:10.1016/j.jbiotec.2016.10.016.
30. Wang T, Larcher LM, Ma L, Veedu RN. Systematic screeningof commonly used commercial transfection reagents towards
efficient transfection of single-stranded oligonucleotides.
Molecules. 2018;23(10):2564. doi:10.3390/molecules23102564.
31. Tomás HA, Rodrigues AF, Carrondo MJT, Coroadinha AS.
LentiPro26: novel stable cell lines for constitutive lentiviral
vector production. Sci Rep. 2018;8(1):5271. doi:10.1038/
s41598-018-23593-y.
32. Wielgosz MM, Kim YS, Carney GG, et al. Generation of alentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy. Mol Ther Methods Clin Dev.
2015;2:14063. doi:10.1038/mtm.2014.63.
33. Mamcarz E, Zhou S, Lockey T, et al. Lentiviral gene therapy
combined with low-dose busulfan in infants with SCID-X1.
N Engl J Med. 2019;380(16):1525-1534. doi:10.1056/NEJMoa1815408.
34. Sanber KS, Knight SB, Stephen SL, et al. Construction of stable
packaging cell lines for clinical lentiviral vector production.
Sci Rep. 2015;5:9021. doi:10.1038/srep09021.
35. Throm RE, Ouma AA, Zhou S, et al. Efficient constructionof producer cell lines for a SIN lentiviral vector for SCID-X1
gene therapy by concatemeric array transfection. Blood.
2009;113(21):5104-5110. doi:10.1182/blood-2008-11-191049.
36. Broussau S, Jabbour N, Lachapelle G, et al. Inducible packaging cells for large-scale production of lentiviral vectors inserum-free suspension culture. Mol Ther. 2008;16(3):500-507.
doi:10.1038/sj.mt.6300383.
37. Chen YH, Pallant C, Sampson CJ, et al. Rapid lentiviral vector
producer cell line generation using a single DNA construct.
Mol Ther Methods Clin Dev. 2020;19:47-57. doi:10.1016/j.
omtm.2020.08.011.
38. Comisel RM, Kara B, Fiesser FH, Farid SS. Lentiviral vector
bioprocess economics for cell and gene therapy commercialization. Biochem Eng J. 2021;167:107868. doi:10.1016/j.
bej.2020.107868.
39. Leinonen HM, Lepola S, Lipponen EM, et al. Benchmarking of Scale-X bioreactor system in lentiviral and adenoviral
vector production. Hum Gene Ther. 2020;31(5-6):376-384.
doi:10.1089/hum.2019.247.
40. Bauler M, Roberts JK, Wu CC, et al. Production of lentiviral
vectors using suspension cells grown in serum-free media.
Mol Ther Methods Clin Dev. 2020;17:58-68. doi:10.1016/j.
omtm.2019.11.011.
41. World Health Organization. WHO Guidelines on Transmissible Spongiform Encephalopathies in Relation to Biologicaland Pharmaceutical Products. 2003. https://apps.who.int/iris/handle/10665/68932.
42. Moreira AS, Cavaco DG, Faria TQ, Alves PM, Carrondo MJT,
Peixoto C. Advances in lentivirus purification. Biotechnol J.
2021;16(1):e2000019. doi:10.1002/biot.202000019.

Lentiviral Vector Manufacturing Process

43. Masri F, Cheeseman E, Ansorge S. Viral vector manufacturing: how to address current and future demands? Cell &Gene Therapy Insights. 2019;5(suppl 5):949-970. doi:10.18609/
cgti.2019.104.
44. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q7: Good Manufacturing Practice
Guide for Active Pharmaceutical Ingredients. November 2000.
https://database.ich.org/sites/default/files/Q7%20Guideline.
pdf.
45. Valkama AJ, Oruetxebarria I, Lipponen EM, et al. Development of large-scale downstream processing for lentiviral vectors. Mol Ther Methods Clin Dev. 2020;17:717-730.
doi:10.1016/j.omtm.2020.03.025.
46. Labisch JJ, Bollmann F, Wolff MW, Pflanz K. A new simplified
clarification approach for lentiviral vectors using diatomaceous earth improves throughput and safe handling. J Biotechnol. 2021;326:11-20. doi:10.1016/j.jbiotec.2020.12.004.
47. Marques J, Dillingham M, Beckett P, et al. Optimizing Viral
Vector Manufacturing for Gene Therapy. In: Pharmaceutical
Technology Biologics and Sterile Drug Manufacturing eBook;
Pharmaceutical Technology: Cranbury, NJ, USA, 2020.
48. Williams T, Goodyear O, Davies L, et al. Lentiviral vector
manufacturing process enhancement utilizing TFDF™ technology. Cell & Gene Therapy Insights. 2020;6:455-467.
49. Ksep® Systems. Sartorius. Accessed August 2, 2021. https://www.sartorius.com/en/products/process-filtration/cell-harvesting/ksep-systems.
50. Oxford Biomedica. SecNuc for AAV, Lentiviral Vectors andAdenovirus; Oxford Biomedica: Oxford, UK, 2019.
51. Segura MM, Garnier A, Durocher Y, Coelho H, Kamen
A. Production of lentiviral vectors by large-scale transienttransfection of suspension cultures and affinity chromatography purification. Biotechnol Bioeng. 2007;98(4):789-799.
doi:10.1002/bit.21467.
52. Scherr M, Battmer K, Eder M, et al. Efficient gene transfer into
the CNS by lentiviral vectors purified by anion exchange chromatography. Gene Ther. 2002;9(24):1708-1714. doi:10.1038/
sj.gt.3301848.
53. Kutner RH, Puthli S, Marino MP, Reiser J. Simplified production and concentration of HIV-1-based lentiviral vectors using
HYPERFlask vessels and anion exchange membrane chromatography. BMC Biotechnol. 2009;9:10. doi:10.1186/1472-67509-10.
54. Ruscic J, Perry C, Mukhopadhyay T, Takeuchi Y, Bracewell DG. Lentiviral vector purification using nanofiber
ion-exchange chromatography. Mol Ther Methods Clin Dev.
2019;15:52-62. doi:10.1016/j.omtm.2019.08.007.
55. Merten OW, Charrier S, Laroudie N, et al. Large-scale manufacture and characterization of a lentiviral vector produced
for clinical ex vivo gene therapy application. Hum Gene Ther.
2011;22(3):343-356. doi:10.1089/hum.2010.060.

A-CELL

✼141

References, continued
56. Truran R, Buckley R, Radcliffe P, Miskin J, Mitrophanous K.
Virus purification. World Intellectual Property Organization.
2009. International Publication Number WO 2009/153563 A1.
57. US Food and Drug Administration. Guidance for Industry:
Sterile Drug Products Produced by Aseptic Processing—Current Good Manufacturing Practice. September 2004. https://www.fda.gov/media/71026/download.
58. Kumru OS, Wang Y, Gombotz CWR, et al. Physical characterization and stabilization of a lentiviral vector against adsorption and freeze-thaw. J Pharm Sci. 2018;107(11):2764-2774.
doi:10.1016/j.xphs.2018.07.010.
59. Lyness AM, Kraft C, Hashimdeen S, Rafiq QA. Comparison of
rigid polymer vials and flexible bags for cryopreservation of T
cells. Cytotherapy. 2020;22(5):S148.
60. Iyer RK, Bowles PA, Kim H, Dulgar-Tulloch A. Industrializing Autologous Adoptive Immunotherapies: Manufacturing
Advances and Challenges. Front Med (Lausanne). 2018;5:150.
doi:10.3389/fmed.2018.00150.
61. Schambach A, Swaney WP, van der Loo JC. Design andproduction of retro- and lentiviral vectors for gene expression
in hematopoietic cells. Methods Mol Biol. 2009;506:191-205.
doi:10.1007/978-1-59745-409-4_14.
62. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q8(R2): Pharmaceutical Development.
August 2009. https://database.ich.org/sites/default/files/Q8_R2_
Guideline.pdf.
63. US Food and Drug Administration. Guidance for Industry:
Process Validation: General Principles and Practices. January
2011. https://www.fda.gov/media/71021/download.
64. European Medicines Agency. Questions and Answers: Comparability Considerations for Advanced Therapy Medicinal Products (ATMP). December 2019. https://www.ema.europa.eu/en/
documents/other/questions-answers-comparability-considerations-advanced-therapy-medicinal-products-atmp_en.pdf.
65. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.
Quality Guideline ICH Q5E: Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process. November 18, 2004. https://database.ich.org/
sites/default/files/Q5E%20Guideline.pdf.
66. White M, Whittaker R, Gándara C, Stoll EA. A Guide to Approaching Regulatory Considerations for Lentiviral-MediatedGene Therapies. Hum Gene Ther Methods. 2017;28(4):163-176.
doi:10.1089/hgtb.2017.096.
67. Cell and Gene Therapy Considerations When Assigning
Responsibilities During a Product Transfer Between a Sponsor
and a Contract Development Manufacturing Organization.
BioPhorum Operations Group Ltd; April 2021. https://www.
biophorum.com/download/cgt-considerations-when-assigning-responsibilities-during-a-product-transfer-between-a-sponsor-and-a-contract-development-manufacturing-organization/.
68. Federal Register. Commerce Control List: Clarifications to

Lentiviral Vector Manufacturing Process

the Scope of Export Control Classification Number 1C991 To
Reflect Decisions Adopted at the June 2019 Australia Group
Plenary Meeting. January 7, 2021. https://www.federalregister.
gov/documents/2021/01/07/2020-27754/commerce-control-list-clarifications-to-the-scope-of-export-control-classification-number-1c991-to.
69. US Food and Drug Administration. Drug Master Files: Guidelines. Updated November 16, 2017. Accessed August 25, 2021.
https://www.fda.gov/drugs/guidances-drugs/drug-master-filesguidelines.
70. US Food and Drug Administration. Guidance for Industry:
INDs for Phase 2 and Phase 3 Studies: Chemistry, Manufacturing, and Controls Information. May 2003. https://www.fda.gov/
media/70822/download.
71. King D, et al. Viral vector characterization: a look at analytical
tools. October 10, 2018. https://cellculturedish.com/viral-vector-characterization-analytical-tools/.
72. Moleirinho MG, Silva RJS, Alves PM, Carrondo MJT, Peixoto
C. Current challenges in biotherapeutic particles manufacturing. Expert Opin Biol Ther. 2020;20(5):451-465. doi:10.1080/147
12598.2020.1693541.
73. US Food and Drug Administration. Code of Federal Regulations. 21 CFR 610.14: Identity. 2020. https://www.accessdata.
fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm?fr=610.14.
74. US Food and Drug Administration. Code of Federal Regulations. 9 CFR 113.53: Requirements for ingredients of animal
origin used for production of biologics. 2021. https://www.
ecfr.gov/current/title-9/chapter-I/subchapter-E/part-113/subject-group-ECFRa4cbfb362190bc0/section-113.53.
75. US Food and Drug Administration. Code of Federal Regulations. 9 CFR 113.46: Detection of cytopathogenic and/or
hemadsorbing agents. 2021. https://www.ecfr.gov/current/
title-9/chapter-I/subchapter-E/part-113/subject-group-ECFRdf8f72db5f24969/section-113.46.
76. US Food and Drug Administration. Code of Federal Regulations. 9 CFR 113.47: Detection of extraneous viruses by thefluorescent antibody technique. 2021. https://www.ecfr.gov/current/title-9/chapter-I/subchapter-E/part-113/subject-group-ECFRdf8f72db5f24969/section-113.47.
77. US Food and Drug Administration. Guidance for Industry:
Testing of Retroviral Vector-Based Human Gene Therapy
Products for Replication Competent Retrovirus During Product
Manufacture and Patient Follow-up. January 2020. https://www.
fda.gov/media/113790/download.
78. United States Pharmacopeia. General Chapter <63>: Mycoplasma Tests. 2022. https://doi.usp.org/USPNF/USPNF_
M3687_01_01.html.
79. United States Pharmacopeia. General Chapter <71>. Sterility Tests. 2022. https://doi.usp.org/USPNF/USPNF_
M98810_01_01.html.
80. United States Pharmacopeia. General Chapter <85>. Bacterial
Endotoxins Test. 2022. https://doi.usp.org/USPNF/USPNF_
M98830_02_01.html.

A-CELL✼142

References, continued
81. United States Pharmacopeia. General Chapter <791>. pH.
2022. https://doi.usp.org/USPNF/USPNF_M99590_02_01.
html.
82. United States Pharmacopeia. General Chapter <785>. Osmolality and Osmolarity. 2022. https://doi.usp.org/USPNF/
USPNF_M99580_03_01.html.
83. United States Pharmacopeia. General Chapter <631>. Colorand Achromicity. 2022. https://doi.usp.org/USPNF/USPNF_
M99390_02_01.html.
84. World Health Organization. Guidelines on the quality, safety,
and efficacy of biotherapeutic protein products prepared byrecombinant DNA technology: Replacement of Annex 3 of
WHO Technical Report Series, No. 814. 2013. https://www.
who.int/publications/m/item/recombinant-dna-annex-4-trsno-987
85. United States Pharmacopeia. General Chapter <509>. Residual
DNA Testing. 2022. https://doi.usp.org/USPNF/USPNF_
M7146_02_01.html.
86. United States Pharmacopeia. General Chapter <1132>. Residual Host Cell Protein Measurement in Biopharmaceuticals.
2022. https://doi.usp.org/USPNF/USPNF_M8647_01_01.html.
87. US Food and Drug Administration. Code of Federal Regulations. 21 CFR 610.15(b): Constituent materials: Extraneous
protein; cell culture produced vaccines. 2021. https://www.
accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=610.15.
88. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Multidisciplinary Guideline ICH M7(R1): Assessment
and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. March 2017.
https://database.ich.org/sites/default/files/M7_R1_Guideline.
pdf.
89. United States Pharmacopeia. General Chapter <467>.
Residual Solvents. 2022. https://doi.usp.org/USPNF/USPNF_
M99226_07_01.html.
90. United States Pharmacopeia. General Chapter <788>. Particulate Matter in Injections. 2022. https://doi.usp.org/USPNF/
USPNF_M99586_02_01.html.
91. United States Pharmacopeia. General Chapter <790>. Visible
Particulates in Injections. 2022. https://doi.usp.org/USPNF/
USPNF_M7197_01_01.html.
92. United States Pharmacopeia. General Chapter <1790>. Visual
Inspection of Injections. 2022. https://doi.usp.org/USPNF/
USPNF_M7198_06_01.html.
93. US Food and Drug Administration. Guidance for Industry:
Potency Tests for Cellular and Gene Therapy Products. January 2011. https://www.fda.gov/media/79856/download.
94. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q2(R1): Validation of Analytical Procedures: Text and Methodology. November 2005. https://da-Lentiviral Vector Manufacturing Processtabase.ich.org/sites/default/files/Q2%28R1%29%20Guideline.
pdf.
95. European Medicines Agency Committee for Medicinal
Products for Human Use. CHMP/BWP/2458/03. Guideline
on development and manufacture of lentiviral vectors. May
26, 2005. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-development-manufacture-lentiviral-vectors_en.pdf.
96. US Food and Drug Administration. Guidance for Industry:
Manufacturing Considerations for Licensed and Investigational Cellular and Gene Therapy Products During COVID-19
Public Health Emergency. January 2021. https://www.fda.gov/
media/145301/download.
97. Zang R, Gomez Castro MF, McCune BT, et al. TMPRSS2 andTMPRSS4 promote SARS-CoV-2 infection of human small
intestinal enterocytes. Sci Immunol. 2020;5(47):eabc3582.
doi:10.1126/sciimmunol.abc3582.
98. Zhao Y, Keating K, Dolman C, Thorpe R. Characterizationof complete particles (VSV-G/SIN-GFP) and empty particles(VSV-G/EMPTY) in human immunodeficiency virus type1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety. Hum
Gene Ther. 2008;19(5):475-486. doi:10.1089/hum.2007.119.
99. Jere D, Sediq AS, Huwyler J, Vollrath I, Kardorff M,
Mahler HC. Challenges for cell-based medicinal products
from a pharmaceutical product perspective. J Pharm Sci.
2021;110(5):1900-1908. doi:10.1016/j.xphs.2020.11.040.
100. Mathonet S, Mahler HC, Esswein ST, et al. A Biopharmaceutical industry perspective on the control of visible particles
in biotechnology-derived injectable drug products. PDA J
Pharm Sci Technol. 2016;70(4):392-408. doi:10.5731/pdajpst.2015.006189.
101. Parenteral Drug Association. Industry Perspective on the
Medical Risk of Visible Particles in Injectable Drug Products.
2014. https://www.pda.org/docs/default-source/website-document-library/publications/industry-perspective-on-medical-risk-of-visible-particles-in-injectable-products.pdf?sfvrsn=2.
102. Zhang B, Metharom P, Jullie H, et al. The significance of controlled conditions in lentiviral vector titration and in the useof multiplicity of infection (MOI) for predicting gene transfer
events. Genet Vaccines Ther. 2004;2(1):6. doi:10.1186/14790556-2-6.
103. US Food and Drug Administration. FDA Briefing Document,
Oncologic Drugs Advisory Committee Meeting. BLA 125646:
Tisagenlecleucel, Novartis Pharmaceuticals Corporation.
https://www.fda.gov/media/106081/download.
104. Shahan S, Zhou S, Shorr R, Brieva T. Characterization of
CAR-T transduction parameters using a lentiviral vector. In:
Baksh D, Rietze R, Wall I, eds. Advancing Manufacture of Celland Gene Therapies VI. ECI Symposium Series; 2019. https://
dc.engconfintl.org/cell_gene_therapies_vi/31.

A-CELL✼143

References, continued
105. Pampusch MS, Haran KP, Hart GT, et al. Rapid transductionand expansion of transduced T cells with maintenance ofcentral memory populations. Mol Ther Methods Clin Dev.
2019;16:1-10. doi:10.1016/j.omtm.2019.09.007.
106. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q1A (R2): Stability Testing of New
Drug Substances and Products. February 2003. https://database.ich.org/sites/default/files/Q1A%28R2%29%20Guideline.
pdf.
107. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Multidisciplinary Guideline ICH Q5C: Quality of Biotechnological Products: Stability Testing of Biotechnological/
Biological Products. November, 1995. https://database.ich.org/
sites/default/files/Q5C%20Guideline.pdf.
108. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. ICH Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances. October 6, 1999. https://database.
ich.org/sites/default/files/Q6A%20Guideline.pdf.

www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.
cfm?fr=312.23
110. US Food and Drug Administration. Code of Federal Regulations. 21 CFR 211.165: Testing and Release for Distribution.
2021. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/
cfcfr/CFRSearch.cfm?fr=211.165.
111. US Food and Drug Administration. Guidance for Industry:
Container and Closure System Integrity Testing in Lieu of
Sterility Testing as a Component of the Stability Protocol for
Sterile Products. February 2008. https://www.fda.gov/media/76338/download.
112. United States Pharmacopeia. General Chapter <1207>: Package Integrity Evaluation—Sterile Products. 2022. https://doi.
usp.org/USPNF/USPNF_M99926_01_01.html.
113. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for
Human Use. Final Concept Paper: ICH Q3E: Guideline
for Extractables and Leachables (E&L). June 30, 2020.
https://database.ich.org/sites/default/files/ICH_Q3E_
ConceptPaper_2020_0710.pdf.

109. US Food and Drug Administration. Code of Federal Regulations. 21 CFR 312.23: IND Content and Format. 2021. https://

Lentiviral Vector Manufacturing Process

A-CELL✼144

CHAPTER 8

Manufacturing of
Cell-Based Therapies
CHAPTER LEAD

Bruce Thompson, Resilience
CHAPTER CONTRIBUTORS

Jasmin Kee, Kee Bio Ltd
Brent Rice, Precision BioSciences
Fabio Fachin, Takeda
Jim Beltzer, Latham BioPharm Group
Melissa DeFrancesco, Lyell Immunopharma
Michael Maguire, Avectas
Mark Sawicki, Cryoport
Ken Forman, IDBS
Marc Smith, IDBS
Rachit Jain, Merck
Qasim Rafiq, University College London

Chapter 8 | Contents
Introduction to Cell-Based Therapy
Manufacturing...................................................................146
Starting materials.......................................................... 147
Allogeneic vs. autologous considerations................ 147

Expansion....................................................................... 160
Process scale..............................................................161
Culture duration........................................................164

Aseptic Processing...........................................................148

Development and manufacturingconsiderations...........................................................164

Open vs. closed systems..............................................149

Expansion system summary.................................... 167

Physical connectivity....................................................150

Final Wash and Volume Reduction–Drug
Substance........................................................................ 167

Unit Operations for an Example CAR-T
Manufacturing Process.....................................................151
Thaw (for processes initiating from acryopreserved starting material).................................151
Cell washing (volume reduction and media
exchange)........................................................................ 152
Cell selection..................................................................156
Activation........................................................................158
Gene-editing/transduction..........................................158
Lentiviral transduction..............................................159
Gene editing..............................................................159
Electroporation......................................................... 160
Lipid nanoparticles................................................... 160
Mechanoporation..................................................... 160
Chemical transfection............................................. 160

Introduction to Cell-Based Therapy
Manufacturing
Cell-based therapy products can be categorized as being
either autologous or allogeneic products. Autologous
products are patient-specific and are derived from thepatient’s own cells. Allogeneic products are non-patient
specific, often sourced from cells of donors, and may
be genetically modified, for example by knocking out
the T-cell receptor (TCR) complex, to mitigate against
adverse immune responses. Allogeneic and autologous

Chapter 8: Manufacturing of Cell-Based Therapies

Formulation.....................................................................168
Fill and finish options.....................................................169
Cryopreservation............................................................171
Electronic Systems Considerations.............................. 172
Enterprise systems architecture overview................ 172
Enterprise systems in detail......................................... 173
Business systems....................................................... 173
Data systems.............................................................. 176
Operational systems................................................. 177
Enterprise systems recommendations...................... 178
IT considerations............................................................ 178
Conclusions........................................................................ 179
References........................................................................... 179

products each present their own unique challenges for
manufacturing products that meet the required standards
for safety, efficacy, and quality.
While a lot can be learned from small molecules andbiologics manufacturing (e.g., monoclonal antibodies),
the fundamental difference in the product and value
chain between cell-based therapies and traditional biopharmaceutical products is in the different manufacturing
paradigms related to the drug substance (DS) and drug
product (DP). In cell-based therapies, the DS contains theactive cell product (manipulated or nonmanipulated) that

A-CELL✼146

holds the therapeutic potential, and DP is the formulated
DS that is filled into final containers that are ready fordelivery to patients as therapy. For traditional biopharmaceuticals, separate manufacturing facilities, staff, andschedules for manufacture are typically employed for the
DS and DP. As such, storage and transport between the
DS and DP location can be critical. On the other hand,for cell-based therapy, while there is often a clear distinction between DS and DP, the same staff and facility
are often used for the full process. Fill and finish may
be performed separately in a contiguous portion of the
facility, where careful considerations should be made on
the hold times between the process portions to ensure
minimum impact on product quality. In addition, forautologous therapies in particular, other manufacturing
distinctions apply, such as a batch size of 1 and therefore
implications for release testing and exceptional release,variability and limited availability in starting materials,
and lack of terminal sterilization. Many of these concepts
are discussed in this chapter.
This chapter will cover the principles related to cellbased therapy manufacturing, in particular for a CAR-T
example, including the acquisition of starting materials,
activating and engineering the cells to produce the desired targeting moieties (e.g., chimeric antigen receptor[CAR] or TCR), expanding the cell population to achieve
the desired clinical dose, harvesting the cells, formulationof the DS, and finally, cryopreservation and storage of the
final product. Many additional considerations are highlighted, such as the source of the starting materials frompatients (e.g., autologous) or from healthy donors (e.g.,allogeneic), the connectivity between each unit operation, and the requirements for strict aseptic processing,as there is no final filtration or terminal sterilization stepin manufacturing cellular therapy products.
Additional considerations are discussed at the end of
the chapter regarding operational components of producing CAR-T products. These include the consideration ofelectronic systems for manufacturing execution (MES),
quality management (QMS), release testing and laboratory information management (LIMS), document management (DMS), and other systems designed to ensure
track and trace abilities, enhanced documentation, androbustness of the manufacturing and testing processes.

Chapter 8: Manufacturing of Cell-Based Therapies

STARTING MATERIALS

Quality therapeutic products start with quality raw
materials, whether those materials are a set of chemical
or biologic reagents, or the living cells that form thebasis of a CAR-T cell therapy. Whether the cellular
starting material originates from the patients themselves(autologous) or healthy donors (allogeneic), cells areusually collected through a process called apheresis,
where blood is separated into its components as it
passes through an instrument. The cell type of interest(i.e., white blood cells in the case of CAR-T therapies)
is collected into a bag, while the other blood components are returned to the patient. Other cell sources
and collection methods are available for other types ofcell-based therapies.
Technical and engineering challenges related tostarting materials arise as cell-based therapy products
progress towards commercialization. The increased
number of patients/donors and collection sites driven
by the increased commercial demand result in the increased variability in cellular starting material, which iscited as one of the major reasons cell therapy products
fail manufacturing runs,2,3 therefore driving standardization efforts on the apheresis process.4 Implementationof donor eligibility criteria as well as technical patientconsideration ensures safe administration to patientsand successful collection procedure, mitigating therisks in manufacturing the therapeutic product. Further
discussion on the apheresis process and its challenges,
technical considerations related to the instrument andprotocol optimization techniques, patient considerations, and donor screening and testing are covered in
Chapter 5.
ALLOGENEIC vs. AUTOLOGOUS CONSIDERATIONSAutologous and allogeneic cell-based therapy manufacturing process differences are reflected in the following
simplified descriptions:
An autologous cell therapy requires on-demand
manufacture, beginning with apheresis collection froma patient and ending with release of the product for
imminent use in that same patient.
An allogeneic cell therapy is manufactured by beginningwith apheresis collection from a donor and ending withrelease of the product for off-the-shelf use in many patients.

A-CELL

✼147

Figure 8-1: Autologous and allogeneic
generation of cell therapy products
Autologus

Allogeneic

Cell collection(leukapheresis)
In vitro genetic
modification
Expansion,
cryopreservation
CMC analytics,
batch release
Infusion

While both approaches share common challenges (e.g.,cell sourcing, raw material qualification, product testingand stability, cold chain shipping logistics), each approachpresents its own opportunities and challenges. The patient-specificity of the autologous therapies has the primaryadvantage that the eventual DP is native to the patient’s
body. The DP, therefore, should naturally avoid rejection
by the patient’s immune system. Additionally, it should not
actively reject the patient’s body (i.e., no risk of graft versus
host disease, GvHD). However, these potential efficacy andsafety advantages are met by the inconvenient reality thatpatients can vary significantly in age, health, weight, diseasestage and burden, prior treatment history, etc., all of which
can impact the quality or quantity of the starting material
for the manufacturing process (i.e., the patient’s own T cells).
Variability in the starting material naturally presents thepotential for significant variability in the cell therapy manufacturing process. But this inherent batch-to-batch process
variability is not necessarily a significant issue when the process only needs to produce a single dose for a specific patient.

Chapter 8: Manufacturing of Cell-Based Therapies

Allogeneic therapies, on the other hand, can in theory
provide the opportunity for greater economic viability
and impact a larger number of individuals by producing
batches capable of treating many patients. Accordingly,
the allogeneic process must provide more consistent production of these larger batches. In contrast with the autologous paradigm, the necessary optimization of process
and product performance within allogeneic cell therapy
production benefits from the ability to better control thedonor population for the starting leukapheresis material.
Donors may be selected by simple assessment of age andhealth status or by a more complex consideration of cellcharacteristics (e.g., phenotype) that are theorized toresult in the desired high batch yields and consistentproduct quality.
Process development and characterization efforts also
benefit from this improved control, since the starting material for these development activities will more naturally
resemble that for the subsequent clinical manufacturing
process. As a final note, the potential immunogenicity of
the allogeneic DP does present a greater potential safety
risk (i.e., acute rejection and via GvHD) for the patient.
Therefore, additional genetic modification and cell selection/depletion steps during production are necessary
to minimize this immunogenic risk, and further release
testing is necessary to demonstrate sufficient clearanceof this risk.
In summary, an autologous cell therapy process mustovercome greater variability in the starting material dueto the inherent variability among individual patients.
The allogeneic cell therapy process must overcome thepotential challenges associated with a more complex,
larger scale process, a more complex safety profile due tothe risk of GvHD, and the demand of generating consistently large batch yields. Specific disease prevalence andpatient population are factors to take into account when
deciding an autologous vs. allogeneic approach. For
diseases with a small patient population, an autologous
approach may still be favorable. On the other hand, theupside potential of developing an in-theory economicallyfavorable, off-the-shelf product that can positively impact
a much larger number of patients per batch encourages
the allogeneic pursuit.

A-CELL✼148

Aseptic Processing
Traditional biopharmaceutical manufacturing processes use terminal sterilization and/or filter sterilization
to destroy or remove contaminants and ensure final
product sterility. However, a specific challenge for celltherapy products is that terminal sterilization or filtration
cannot be used. This is because viable cells or cell-derived preparations, which are the critical elements offinal cell therapy products, cannot withstand terminal
sterilization procedures without being killed and are
too large to pass through sterilization-grade filter membranes. Additionally, due to the timing and logistics oftreatment delivery, product sterility test results are notalways available before administration of the product topatients. Thus, in cell therapy product manufacturing
settings, all process materials, equipment, and surfaces
are pre-sterilized using validated sterilization methods,
and aseptic processing must be used during production
to prevent the introduction of contaminants during all
steps of the manufacturing process.
Aseptic processing can be defined as the handlingof materials in an environment where the air supply,
materials, equipment, and personnel are regulated tocontrol microbial and particulate contamination. Aseptic
processing is required from the first process step where
sterility is considered necessary, to when the container
system with the final product is closed, through the product delivery to the patient. Successful aseptic processing
can be achieved by implementing the following measuresduring operations:• Provisioning personnel controls (gowning andaseptic technique training and protocols)• Environmental monitoring for quality and control• Validated cleaning and sterilization procedures ofcontainers, equipment, disposables, tools, and surfaces that come into contact with a DS or DP
A powerful tool for evaluating aseptic processrobustness is aseptic processing simulation (APS), in
which a process is run and analyzed under the worstcase contamination risk conditions. Unlike in traditional
biopharmaceuticals where the focus of APS is in the

Chapter 8: Manufacturing of Cell-Based Therapies

final fill process (due to the use of terminal sterilization
prior to the final fill), the burden of APS in cell therapy
manufacturing rests on a more complete representation of handling throughout the process. Cell therapy
product developers must also consider manufacturing
process boundary conditions unique to the context ofcell therapy. For example, developers must make surethat both initial cell/tissue harvesting and, if required,transport to the patient’s bedside are done under aseptic
conditions. By conducting operations using aseptic processing, developers can maximize their chances to avoid
the high cost of reprocessing or producing replacement
lots in cases of substandard product quality. Ensuring
a successful lot is critical as many cell-based therapies
are used to treat acute, malignant indications where thepatient often doesn’t have the luxury or health to wait
for a replacement batch. Aseptic processing also assurespatients and regulatory authorities that products are
manufactured with safety as a top priority.
OPEN vs. CLOSED SYSTEMSCell therapy product manufacturing must, at all times
and in all ways, account for the possibility of contamination introduced during steps in the manufacturing
process. This is because contaminants can undermine thesafety and efficacy of the product and introduce risks forpatients. While all biopharmaceutical processes are subject to such concerns, it is extremely important for celltherapy product manufacturing processes to minimize
contamination because they involve operations uponliving cells. As such, cell therapy product developers
must carefully consider whether to implement their manufacturing steps as open operations or closed operations.
In open operations, more common for traditional
biopharmaceutical manufacturing and earlier phases ofclinical trials, production occurs with process materials
exposed to the room environment. By contrast, closed
operations utilize facilities and equipment designed toensure that DS, intermediates, and DP are not exposedto the room environment at any point during production.
In recent years, due to cost and efficiency considerations,and to good manufacturing practice (GMP)-related
guidance from regulatory agencies, there has been an
industry-wide transition toward implementing closed

A-CELL

✼149

operations wherever possible. Cell therapy productdevelopers benefit greatly by learning from this trend
and supporting technologies that increase the adoptionof closed operations. Additionally, technology developers
are introducing new equipment to enable closed cellprocessing steps at an increasing rate.
Implementing closed operations, at any stage of
research or production, reduces contamination risk
by adventitious agents. This is because closed systems,
by design, use components that minimize the need forhuman contact with the operating system, and provide
physical barriers that serve both to reduce the risk ofcontamination by preventing contact with the room
environment and contain the product or intermediate ina location away from sources of possible contamination.
In addition, a closed operating system allows materials toenter or leave a system via predetermined control paths.
As an additional benefit, because closed operations areoften achieved by using specifically designed “plug andplay” equipment that can easily be set up and launchedby personnel, there is often a significant reduction intime and steps required for validation and operations.
Given the high costs for space in biopharmaceuticalmanufacturing settings, dedicating too many production
areas for separate cleanrooms (which are essential forminimizing contamination risks in open operations) andgowning areas can lead to unnecessarily excessive cost of
goods. Closed operations can allow for more efficient useof space, because closed-systems allow for multi-patientbatches to be produced in the same room, and fewerrooms needing to be designated for components orequipment (which are designed to have their own physical barriers) used in the system process. For more details
on facility design considerations for cell-based therapies,refer to Chapter 11.
In practice, closed systems are often more precisely
described as being functionally closed. This is because
process systems may be routinely or occasionally opened
to perform a maintenance procedure, add a reagent (e.g.,
media addition in a bag inside a BSC), or install a newcomponent, after which the system is returned to a closed
state through sterile welding or aseptic connections tothe subsequent unit operations, or a sterilization stepprior to use in the manufacturing process (subject to follow-up validation). Also, there may be situations where

Chapter 8: Manufacturing of Cell-Based Therapies

open processes are housed within isolators that comprisethe closed systems. These situations are also functionallyclosed and demonstrate the ongoing transition fromopen to closed operations as a general feature of evolving
industry practices. Process closure can be implemented
within the context of a larger operating framework thatinvolves multiple cleanroom suites that each constitutemodules of the overarching production process (with theaim of maximizing productivity of each module). Since
these modules are separated, the manufacturing process
as a whole is comprised of both open and closed aspects.
Developers must balance individual goals for productivity against the risk-reducing features of system closure
on a case-by-case basis. An example closure analysis ofa CAR-T process has been performed,5 which includes
assessment of contamination risks in each unit operation
as well as mitigation strategies.
Several factors can pose challenges for developers to
implement maximally closed operations, in large part
because the use of closed manufacturing processes acrossthe industry is still in its relatively early days. To begin
with, open operations have been an established part ofbiopharmaceutical manufacturing. A number of facilities
may require major redesigns to eliminate open operatingprocedures and will, therefore, require developers tocontend with potentially high upfront costs and logistical challenges. And, if developers require parts frommultiple suppliers, cases might arise where components
sourced from different suppliers are incompatible anddevelopers may need to establish custom guidelines
for use across suppliers or may need to arrive at othersolutions. Furthermore, since a lot of closed systemequipment is relatively new to the manufacturing space
and is designed with very particular functions in mind,
personnel will need proper training to handle unfamiliar or sensitive process elements such as tubing, valves,and connectors. Finally, with current GMP (CGMP)
guidelines put forth by regulatory authorities such as
the FDA and the EMA, developers that establish closed
systems must provide evidence that their systems are
fully closed to the extent that satisfies the definitions set
by the relevant guidelines. This will require developing
testing protocols and validation steps that adequately
demonstrate closure.

A-CELL✼150

PHYSICAL CONNECTIVITY

Single-use connection technologies exist wherever aconnection is made that allows materials to flow throughduring the manufacturing process or during final product delivery. Connectors are an essential part of any
technology to facilitate the flow of materials from one
unit operation to another without any leaks or breaks insterility, which could jeopardize product sterility or lossof product that will affect patients’ safety. Connectors
and proper connectivity are vital to ensure that the integrity of every point of the process is always maintained.
Connector type selection is also important. An aseptic,
fully closed connection can be made within a process
through aseptic connectors, such as AseptiQuik®, Opta®,
Kleenpak™, or sterile welding. Aseptic connection could
be a repetitive process; therefore, picking the right aseptic connector that is easy to use, genderless, and allows
for an effective, quick, accurate leak-free connection is
critical.6 A challenge that remains is that while sterile
connections are growing in availability, the disconnection process is not always sterile and still requires
controlled space to disconnect materials. Alternatively,
open style connections, such as luer and MPC, can beused, but developers must take into consideration thatthese connections have to be made in a highly classified
environment, such as a positive displacement laminar
hood, to avoid contamination during material transfer.
Developers must consider the pros and cons of eachconnector to ensure they use the best connector foreach process,7 so the process must be designed ahead oftime with consideration of process scaling for large scale
manufacturing to meet patient demand.
Testing of critical components such as connectors
must be performed both upstream, to protect the cellline, and downstream, especially in critical operations
after filtration, to ensure these components do not introduce particulates, bioburden, leachables, or extractables
into the final product, which could affect patient health.8
Two organizational bodies have created guidelines for
extractables and leachables on single-use assemblies:
US Pharmacopeia9 and BioPhorum.10 Smaller volume
samplings are performed during the cell therapy manufacturing process (for discussion on in-process sampling,refer to Chapter 9); therefore, connectors must be carefully selected when used for in-process sampling (e.g.,

Chapter 8: Manufacturing of Cell-Based Therapies

sampling manifolds) to maintain process sterility while
minimizing product waste. Connectors must always
maintain integrity and sterility; therefore, selecting theright connector is essential to safely and efficiently transfer therapy products to patients where every drop counts.

Unit Operations for an Example CAR-T
Manufacturing Process
The main manufacturing steps in CAR-T therapy production are:
• Cell isolation (leukapheresis)• Activation• Transduction and/or genetic editing• Expansion• Harvest and formulation• Cryopreservation• Reinfusion (post-chemo depletion)
The following figures (Figures 8-2 to 8-5) show an
overview of the manufacturing steps. Resting peripheral
blood mononuclear cells (PBMCs) are collected fromthe donor. This material may be cryopreserved within
24 hours and stored at -120°C. Afterwards, a controlled
thaw and wash step removes the cryopreservation material. Activation is achieved by ligating CD3 and CD28
with antibodies attached to magnetic beads or similar
reagents, followed by transduction with self-inactivating
lentivirus that codes for the gene of interest (e.g,. chimeric antigen receptor (CAR)). After transduction, residualmaterials are washed out. The cells are then expanded toachieve yields necessary for the final dose. The engineered
cells are de-beaded (as needed), washed, formulated, andplaced in bags or vials for infusion or cryopreservation.
Release testing is done following the manufacturing process to ensure product quality and safety. Sterility testing
is often done prior to cryopreservation while additional
critical quality attribute (CQA) testing is performed on the
final product (e.g, cryopreserved material). Considerations
for each of the unit operations are discussed in the following sections. Note that most of the discussions are focusedon autologous processes, with additional considerations
for allogeneic products.

A-CELL✼151

Figure 8-2: Autologous cell therapy manufacturing process (cell preparation to expansion)(Adapted from BioPhorum Cell therapy manufacturing maps.)11

Cell selection
media

Patient
cells
Cell wash
solution#1

To cell harvest(Figure 8-3)

Lentivirus
and reagents

Activation
media

Cell culture
media

Cell thaw(optional, if
frozen postcollection)

Cell
preparation(debulk /enrichment)

Cell selection

Cell activation

Gene editing(Viral delivery)

Cell expansion

Thaw cells
in bags /cryovials

Reducecontaminatingcell types(e.g. RBCs /platelets) fromapheresis orwhole blood

Select CD4+,
CD8+, CD25+
T-cells from
PBMCs

StimulateT-cellgrowth andenhanceuptake of viralmaterial

Deliver
genetic
material tonucleusExpand genemodifiedT-cells in staticor dynamic
culturesystems

Density
gradient
centrifugation

Affinity
purification
using
magnetic orother beads

Beads coatedwith activatorssuch asCD3/CD28

Lentivirus
or retrovirus
deliverysystemNon-bead
Technologies

Transductionusingspinoculation
or reagents

Water bathThawingdeviceInterleukins

THAW (FOR PROCESSES INITIATING FROMA CRYOPRESERVED STARTING MATERIAL)

Thawing is a critical processing step in cell therapy that, if
performed incorrectly, can lead to significant cell damage
via mechanisms such as intracellular recrystallization
or solute effects.12 Generally speaking, independent of
the container in which the starting material was frozen(e.g., cryobag, cryovial), thawing should be performed
in a controllable and rapid manner, so that extracellular
crystallization can dissolve rapidly to enable proper
cell rehydration. As such, within cell therapy GMP

Chapter 8: Manufacturing of Cell-Based Therapies

Wash out
excess virus

Single-use
bioreactors,
flasksCell culture
media,
cytokines,
growth factors
and/or serum

manufacturing, thawing is typically performed utilizing
a temperature-controlled warming apparatus such as awater bath or, more preferably, one of the many water-free(semi-)automated thawing systems now available in themarket (e.g., Plasmatherm by Barkey, ViaThaw by Cytiva).
These systems are most often composed of pre-heatedcomponents (typically pre-warmed at temperatures≥37°C) that are placed into contact with the frozen
container to rapidly increase the sample temperaturevia (mostly) conductive mechanisms. Depending on thethawing device of choice, thawing can be controlled by

A-CELL

✼152

Figure 8-3: Autologous cell therapy manufacturing process (cell harvest to fill and finish)(Adapted from BioPhorum Cell therapy manufacturing maps.)11

Finalcontainers &labelsCell wash
solution #2
From cell
expansion(Figure 8-2)

Debeadingreagent

FormulationbufferAdministration
to patient

Process step
Cell harvest

Debeading(optional)

Wash andconcentration

Formulation,
fill and finish

Cryopreservecellsuspension(optional)

Remove cellselection or
activation
media
from cellsuspension

Purify cellsuspension
andconcentrate
to the desired
volume foradministration

Place cellsuspension
in solution foradministration
or cryopreservation
Fill, inspect,
and label finalcontainer

Freezesuspension topreserve cells
duringshipment or
storage

Magnetic cell
separation

Tangential
flow filtration

Cryobags,
cryovials

Controlled rate
Freezer

Description
Pool cell
culturematerial andprepareto purify andformulate

Unit operation
Depth filtration
Tangential
flow filtration

Centrifugation

Centrifugation

either a qualified/validated thawing time (e.g., a fixed
thawing time approach) or via active sample temperature-monitoring probes capable of detecting an “end ofthaw” condition (e.g., a fixed end-of-thaw temperatureapproach), with the former approach currently being themost common within the industry. In either case, thethawing time (typically ranging between 1-10 minutes)
and “end-of-thaw” temperature are both container- andsample-specific, therefore requiring specific qualification/
validation for each type of starting material/container
combination, as well as consideration of the post-thaw
GMP operations that should be designed to minimize
the post-thaw exposure of cells to cryoprotectants suchas dimethyl sulfoxide (DMSO).

Chapter 8: Manufacturing of Cell-Based Therapies

CELL WASHING (VOLUME REDUCTION AND MEDIA
EXCHANGE)

Upon completion of thaw, the starting cellular materials
undergo a procedure—typically referred to as cell washing—aimed at removing the DMSO and other excipientsthat compose the cryomedia formulation that was utilized
to freeze the starting apheresis. Long-term cell exposure
to DMSO (>30-60 min) may result in negative cytotoxic
effects,13 thus driving the need to remove DMSO as rapidly
as possible.
From a mechanistic perspective, cell washing refers toprocesses in which the original (input) media containing thecellular samples is replaced, or exchanged, by a target media,
while targeting minimal nucleated cell loss or damage. A

A-CELL

✼

153

Figure 8-4: Allogeneic cell therapy manufacturing process (cell preparation to expansion)(Adapted from BioPhorum Cell therapy manufacturing maps.) 11

Activation
media

Healthydonor cells,
post-apheresis(Chapter 5)

Geneediting
reagents

Process
step
Cell thaw(optional,
if frozen
postcollection)

To cellselectionand harvest(Figure 8-5)

Lentivirus
andreagents

Expansion
media

Cell
isolation /enrichment

Cryopreserve(optional)

Thaw(optional)

Cell
activationGeneknockout(KO)

CAR genedeliveryCellexpansion

Isolatedesiredtarget cell
type fromapheresis
product

Preserve
and freeze
cells

Warm andthaw cells
prior tofurther
processing(option topool multiple
batches ofcells)

Stimulation
andproliferationof cells

To makethe cells
immunecompatible

To insert
gene ofinterest(GOI)

Expandgene
modifiedT-cells instatic or
dynamic
culturesystems

Affinity
purification
using
magnetic orother beads

Controlled
rate
Freezer

Water bath

Antibodycoatedbeads,
bags, flasks
Solubleactivation
reagents/
antibodies
Antigenpresenting
cells (APC)

Deliverysystems:
electroporation, nucleofectors, lipid
nano particles,
viral,
mechanicalsqueeze technology

AAV, lenti,
retrovirus

Single-use
bioreactors,
flasksDescription
Thaw cells
in bags /cryovials

Unit operation
Water bathThawingdeviceCell sorter(Fluorescence
activatedcell sorting FACS)

Bags orvials

Thawingdevicescell washing procedure in which there is no change in total
volume between the input (pre-wash) and target (postwash) samples is typically referred to as a media exchange
operation (see Protocol 1 in Figure 8-6). Conversely, acell washing procedure in which the post-wash volume issmaller than the pre-wash volume is referred to as a volume
reduction procedure (Protocol 2 in Figure 8-6).
Media exchange and volume reduction functions
are then often combined, as outlined in Protocol 3 in

Chapter 8: Manufacturing of Cell-Based Therapies

Cell culture
media,
cytokines,
growthfactors and/or serum

Technology:
ZFN, TALEN,CRISPR, RNAi

Figure 8-6, to provide a preparative workflow where thecells are not only transferred to a final target media but
also concentrated into a lower volume, and therefore
to a higher cell concentration. This combined media
exchange and volume reduction protocol is particularly
effective when handling large sample volumes, suchas those typically associated with bioreactor harvests
where sample volume might be up to several liters.
By first performing a volume reduction, this sequence

A-CELL

✼154

Figure 8-5: Allogeneic cell therapy manufacturing process (cell harvest to fill and finish)(Adapted from BioPhorum Cell therapy manufacturing maps.) 11

Finalcontainers &labelsFrom cell
expansion(Figure 8-4)

Formulationbuffer

Process step
CAR+ TCR- T
cell selection
and harvest

Debeading(optional)

Wash andconcentration

Formulation, fill
and finish

Cryopreserve
cell productRemove cellselection or
activation
media from cellsuspension

Purify cellsuspension andconcentrate
to the desired
volume foradministration

Final preparation ofcell product(Concentration,
Freeze medium,
excipients)
Fill final container
Perform final
inspection
Label finalcontainer

Freeze for longterm storage

Magnetic cell
separation

Tangential flow
filtration

Description
Obtain productpurityUnit operation
Affinity
purification
using magnetic
or other beads

Centrifugation

Cryobags,
cryovials

Controlled rate
FreezerFACS

significantly reduces the amount of washing volume
required to perform the subsequent media exchange.
Several cell washing methods/tools have been developed and are commercially available, each exploiting
different physics:14 these include centrifugation-based
approaches (CellSaver by Haemonetics, Sepax by Cytiva),
counterflow technologies (Rotea by Thermo Fisher, kSep
by Sartorius), acoustic-based devices (Ekko by Millipore
Sigma), and membrane-based technologies (Lovo by
Fresenius Kabi). Despite the difference in underlying
physics, selection of an appropriate washing tool istypically based on the following performance metrics:
Media Exchange Efficiency: The overall percentage
of media components (including base media, additives,

Chapter 8: Manufacturing of Cell-Based Therapies

cytokines) exchanged or depleted within one washingcycle. The higher the media depletion (or exchange)
percentage, the more efficient the washing procedure.
For example, given a target depletion level (e.g., 99.9%
or 2 log), it will take 2 cycles of a washing operation
exhibiting 90% media exchange efficiency to reach the 2log level, as opposed to 3 cycles for a system exhibiting
80% exchange efficiency.
Cell retention efficiency: The percentage of nucleatedcells (or specific cell subsets) retained during the washing procedure. The higher the nucleated cell retention,
the more efficient the technology. Conversely, washing
operations do not aim to retain enucleated cells. Platelets
whose size and morphology are significantly smaller than

A-CELL

✼155

Figure 8-6: Example Cell Washing Protocols
Cell retention

Media exchange

Cell retention

Volume reduction

Cell retention

Volume reduction

Protocol 1

Protocol 2

Media exchange

Protocol 3

that of nucleated cells, are therefore largely depleted
during a cell washing procedure. Red blood cells (RBC)
are another cell subset that can be partly depleted via
washing operations, although complete RBC depletion
requires dedicated cell selection steps as outlined in the
Cell Selection section below.
Throughput: The volumetric flow rate at which thesystem is capable of operating while providing the target
levels of cell retention efficiency. The higher the throughput, the more efficient the tool. Generally speaking, there
is a negative correlation between cell retention andthroughput, with higher cell retention being associatedto lower flow throughputs.
Minimal output volume: The smallest output volume
in which the system can deliver the final washed cellular sample. A low output volume is preferrable, as it
facilitates achieving a high cell concentration. This may
be an important consideration in closed cell washing

Chapter 8: Manufacturing of Cell-Based Therapies

technology depending on the required downstream
operations.
The performance metrics above should all be taken
into consideration when selecting the best washing tool
for a specific program or cell type. Additional aspects
such as cost of goods and data integration should also
be considered, particularly when mapping tool selection
towards GMP and commercial applications.
CELL SELECTION

After completion of cell washing (DMSO removal), thewashed apheresis material most often requires further
processing to yield a more consistent starting material
for the engineering and expansion steps. Variability ofthe starting material, particularly for patient-specificautologous therapies, increases the complexity of CAR-T
cell production, potentially resulting in a failure to meetthe dose requirements. As an example, it has been

A-CELL

✼156

demonstrated that specific cell populations can adversely
impact subsequent processing steps such as activation(e.g., the attachment of CD14+ monocytes to activating
Dynabeads®).15 Furthermore, there is evidence that RBCs
and monocytes interfere with the clinical efficacy of celltherapies.16-19 The evidence of the deleterious impact ofmonocytes is further reinforced by the strategy to deplete
these cells as a salvage operation when the patient starting material contains high levels of CD14+ cells and fails
to pass requirements for further processing. Therefore, inmost instances, pre-expansion cell processing is required
to select and enrich the T-lymphocyte cell population.
To address such challenges, cell therapy providers
have adopted different cell processing strategies, withsome opting to expand the entire nucleated cell fraction,
while others preferring to undertake cell enrichment
steps prior to activation, transduction/transfection, andexpansion. The exact nature of this preparative processing is dependent on the intended cellular fraction
required, and is often a trade-off between purity andyield. As outlined in the previous section, the initialprocessing steps typically involve the removal of platelets
and erythrocytes through a combination of washing andselection methods. Current approaches use legacy platforms adapted for lymphocyte enrichment, such as the
Cell Saver 5+ (Haemonetics Corp.) and the COBE 2991
Cell Processor (Terumo BCT). Newer systems include
the LOVO (Fresenius Kabi), Rotea (Thermo Fisher), andSepax C-Pro (Cytiva).
The aforementioned technologies support the washing and selection of specific cellular components resulting in the enrichment of the T-lymphocyte population.
Cell selection typically involves separation on the basis ofphysical characteristics, such as cell density or size, or on
the basis of immunophenotypic properties. Ficoll-Paqueis commonly used to deplete erythrocytes and granulocytes on the basis of density gradients (Ficoll-Paque hasa lower density than erythrocytes and granulocytes, but
a higher density than PBMC/MNC buffy coat). Typically,
Ficoll gradient separations require open processing,
however, adapted legacy platforms and newer systems
support GMP-compliant, closed, automated Ficollgradient separation.
While density gradient separation supports effective erythrocyte and granulocyte depletion, other

Chapter 8: Manufacturing of Cell-Based Therapies

cell processing approaches are required for monocyte
depletion and lymphocyte isolation. Two strategies are
employed to achieve this: (1) elutriation and (2) antibody
bead conjugates selection. Elutriation involves the useof counter-flow fluid and centrifugal force to separate
cellular fractions on the basis of both density and size.
The principal mechanism for elutriation involves introducing the heterogenous starting material into the system
where the material is separated in a spinning chamber via
centrifugal force. A buffer is introduced counter to thedirection of the centrifugal force, and it is this counter
flow buffer that separates the cells based on their sizeand density and supports cellular fractionations. These
cellular fractions can then be sequentially removed,
enabling the enrichment of the lymphocyte population.
Elutriation is an effective method for discriminating
between monocytes and lymphocytes. This technology
has been effectively employed for other biotechnologicalapplications, including dendritic cell vaccine production,20 and is becoming increasingly common in CAR-T
processes.21 Systems that support monocyte depletion
include the Elutra® (Terumo BCT), Rotea™ (Thermo
Fisher) and Sepax C-Pro (Cytiva).
An alternative approach to lymphocyte enrichment
is to use magnetic antibody bead conjugates, which canbe used to support either positive or negative selectionof target cells. This method has the additional advantage
of enabling the enrichment of specific T cell subsets
including CD4+, CD8+, CD25+, or CD62L+, and target
cell isolation can be based on one or more markers. The
antibody bead conjugates are introduced to a heterogeneous cell population where they attach to the target cell.
A magnetic field is then applied to the cellular material.
In the case of positive selection, the target cells are boundby the magnetic antibody bead conjugate and retainedby the magnetic field, with the non-target cells passing
through and separated as part of the waste stream. To
obtain the target cell population, there is a washing stepprior to removing the column from the magnetic field,
after which the enriched population can be eluted fromthe column. In the case of negative selection, the target
cell population is that which passes through the magnetic
field in the initial flow stream, not bound by the magnetic
antibody bead conjugates. GMP-grade, antibody-conjugated beads are readily available and are routinely

A-CELL✼157

used with systems like the CliniMACS Plus and Prodigy
platforms (Miltenyi Biotec). While this method provides
additional cellular subset selection, it requires additional
processing to remove the beads; however, the emergenceof biodegradable MicroBeads (Miltenyi Biotec) may obviate the need for debeading as the target cells are freefrom antibody fragments and magnetic labels.
Other emerging approaches for cell separation include more traditional cell sorting using fluorescence
activated cell sorting (FACS), buoyancy activated cellsorting (BACS), acoustic wave separation, and inertial
microfluidics. Recent advances in cell sorting haveincreased the capability to sort labelled cells to achieveenrichment of rare populations of cells. New instruments
by Miltenyi, Sony, Cellular Highways and others are able
to increase sorting speeds and reduce processing times,
making them more amenable to GMP processes. BACS
achieves cell separation through the use of buoyant
microbubbles that are composed of a gaseous core witha lipid, polymer, or protein shell. In the case of positive
selection, microbubbles are coated with antibodies tocapture the target cells. The microbubbles then float tothe surface of the sample where they can be collected.
Although primarily a research tool at present, commercially available, semi-automated platforms exist, including the X-BACS systems (Corning). Similarly, acoustic
wave separation technology has proven effective at the
research scale for cell separation. Although earlier interms of its technology readiness level compared withmore established approaches such as elutriation andantibody bead conjugates, acoustic cell processing is
a promising approach involving the use of ultrasonicwaves to enable a no-shear, solid-state approach to cellconcentration, washing, and label-free selection. The
Ekko system (MilliporeSigma) appears to be a promising
acoustic cell processing platform purporting to support
closed and automated, label-free cell processing. Inertial
microfluidics have also been proven to effectively separate white blood cells from red blood cells at the researchscale.22 These systems utilize a curved fluid channel thatcauses a higher flow rate on the outside of the channel
compared to the inside channel wall. Due to a variety of
fluid flow forces, this can ultimately allow for the separation of red and white blood cells between the outer

Chapter 8: Manufacturing of Cell-Based Therapies

and inner walls of the channel. Therefore, this process isable to achieve effective, label-free separation. However,this technology is still early in development. While afully-automated, inertial device has been demonstrated
at the research level, no commercial products are currently available.
ACTIVATION

Selected or isolated starting T cells undergo an activation
step to both enable the T cells to proliferate to facilitate
gene transfer (e.g., lentiviral and retroviral transduction
is more efficient in actively cycling cells) and expand
the starting number of cells to achieve sufficient cells
to meet patient dosing requirements. This activation isaccomplished by engaging the cluster of differentiation
3 (CD3) receptor on the T-cell surface with activating
monoclonal antibodies. The CD3 receptor provides theintracellular signaling capabilities, and is associated with
the TCR protein complex on the T-cell surface capableof recognizing specific molecular signals as part of theimmune response to pathogens, foreign particles, cells,
etc., and reacting to them by inducing a proliferative
response to deal with the immunological insult. An
accessory signal, known as “signal 2,” is required to provide co-stimulation and is thought to act as a confirming
signal during the activation process. Signaling throughthe CD3 receptor without co-stimulation results in T cell
dysfunction (anergy) or clonal deletion in an effort toavoid autoimmune events.23-26
This activation process is co-opted in CAR-T cellmanufacturing and used to expand T-cell populations
ex vivo to enable larger-scale cell production. One avenue
for activating T cells through the CD3 receptor involves
incubation with a monoclonal antibody, OKT3, to induce
activation. This method is often employed when using
bulk PBMCs in lieu of purified T cells. Interleukin-2(IL-2) often accompanies OKT3 use in the culture to
support T cell expansion, minimizing the need for external co-stimulation.27 Another method uses two separate
monoclonal antibodies targeting CD3 (signal 1) andCD28 (signal 2), co-stimulatory receptors mimicking
a more physiologically relevant activation event wherean antigen presenting cell (APC) would provide bothsignals to a T cell simultaneously. These two monoclonal

A-CELL

✼158

antibodies may be attached to a bead (e.g., Thermo CTS
Dynabeads), included as part of a nanoparticle matrix(e.g., Miltenyi Transact), or utilized in other solid substrate formulations (e.g., adsorbed to plates or coatedin bags). The prevailing outcome is similar in these
instances in that T cells get activated and become more
susceptible to transfer of the gene of interest via lentiviralor retroviral transduction. T cell activation with thesevarious methods requires downstream considerations.
CTS Dynabeads must be removed from the culture usinga magnetic device to both stop the activation event andremove potential process-related impurities prior toadministration to a patient. The Transact reagent can be
diluted to dissociate the nanoparticle matrix and stop theactivation event. Regardless of the activation mechanism,
sponsors should be aware of the residual impurities,
including murine antibodies, from that process step.
Several strategies for clearance rely on dilution and additional cell washing steps through downstream processing
operations, which will help with removal and minimize
the presence of these reagents in the final product.
GENE-EDITING/TRANSDUCTION

Following activation, T-cells are genetically engineered
to express the synthetic CAR. Significant advancements
have been made in the gene-editing technology/transduction strategies to be applied to CAR-T therapies, andare outlined below.

Lentiviral transduction
A therapeutic product dose should be comprised ofengineered cells that stably express synthetic antigens
or targeting receptors. In the example of CAR-T cells
expressing anti-CD19 targeting receptors, the transgene
may be introduced with a lentiviral vector (LV), which
has a safe integration character, to achieve persistentexpression in the patient’s cells. Lentivirus is an RNA
virus where the transgene of interest (e.g., CAR) is
integrated randomly into the genome of the host cell.
Currently, third generation LVs are most widely used,typically produced using a 4-plasmid transient transfection system comprised of 3 packaging plasmids28 and 1
self-inactivating gene transfer plasmid. Following transduction, the viral RNA is reverse transcribed to DNA,

Chapter 8: Manufacturing of Cell-Based Therapies

and, subsequently, the CD19 chimeric antigen receptor
is expressed and transported to the host T cell surface.
T cells are sourced from a selection process done on anapheresis product from the donor or patient. It should be
noted, given the clinical indication, such as pediatric acutelymphoblastic leukemia (ALL), adult B cell lymphoma, or
solid tumors, that the donor cells may be exhausted (e.g.,
exposure to significant immune activation events leaving
the cells with little proliferative capacity) or have been
pre-treated via multiple rounds of chemotherapy to theextent that the cells may be hard to transduce.
Activated T cells are incubated with the lentivirus as
the key component of the transduction process. Then,the virus is washed out before the expansion step. The
expansion continues until a sufficient dose is reached, withthe dividing cells continuing to express the transgene after
infusion into the patient. The challenge in manufacturing
autologous CAR-T therapies lies within the detail beyond
this simplistic overview. In each unit process step, there
are opportunities to improve and optimize each aspect ofthe process.
Much of the cost and complexity of CAR-T manufacturing and therapy can be attributed to the viral vector
component of the process. Manufacturing viral vectors
such as lentivirus is challenging in terms of standardization, stability, and yield. Improvements in the design ofthe lentivirus (including packaging efficiency), as well
as scale (e.g., 10L to 200L) are current areas of industry
focus. Increasing the transduction efficiency of the target
T cell is a crucial challenge, and control of the culture
conditions, including cell culture media, cell density,
vector titer, temperature, and bioreactor design will help
increase the process yield of engineered cells.
Additionally, there are ongoing efforts to find alternatives to viral transduction. One such effort is to achieve
an effective, transient messenger RNA (mRNA) engineered CAR-T cell. The use of mRNA delivered to thecytosol avoids the challenges of viral manufacture andoptimization. Such a therapy could be administered ina multi-dose regimen rather than a single-dose therapy.
Though, to date, equivalent efficacy has been shown in amurine model29 but not in the clinic, this route remains
an attractive prospect. Another potential route to achieving stable expression of anti-CD19 receptor on T cells is

A-CELL✼159

to edit-in the transgene at a site-specific location. Roth etal. demonstrated site specific knock-in in the literature,30
but have not yet translated it to the clinic.

Gene editing

Developers have several options to enhance the performance of a CAR. Some of these options involve thetransient or stable expression or inhibition of a factor. An
excellent example of one such therapeutic option is theinhibition of PD-1. Immunologists differ on whether thisinhibition should be transient or permanent. However,
it is a frequent target for knock-out using a gene-editing
tool. There are several clinically validated gene-editing
platforms available to developers, including Clustered
Regularly Interspaced Short Palindromic Repeats(CRISPR)/Cas9, Zinc finger nucleases, and megaTALs
among others. Of these, CRISPR/Cas9 is ubiquitous due
to its relative ease of use and flexibility.
The CRISPR/Cas9 system must be delivered without
an integrating viral vector or more commonly by electroporation (see below) using an mRNA guide and a Cas9RNP (ribonuclear protein). As the delivery is transient,
the editing tool will perform its edit and then dissipate.
However, the editing results will persist in the cell.
For a knock-in editing, this capability presents thepossibility of an entirely non-viral, site-specific integration of a persistent transgene. Such a system has thepotential to disrupt the current manufacturing process
for advanced therapies.

Electroporation
Electroporation (EP) is the current leading non-viral delivery platform for cell engineering. This technology has beenused in the production of clinical trial materials and relies
on passing pulses of electric current through a solution
containing cells. EP can be used to deliver a diverse range ofcargos, including plasmids, mRNA, proteins, transposons,and gene-editing tools. Commercially available EP units
include MaxCyte, Lonza Nucleofection, and Thermo Fisher
Neon. Other systems have been developed and qualified
for clinical use, including the Cellectis Pulse Agile. EP has
been used in clinical trials to introduce transgenes into
human primary cells for either transient or permanent
expression. In transient expression, the therapeutic must be
re-dosed. However, this also allows the clinician to monitor

Chapter 8: Manufacturing of Cell-Based Therapies

and escalate doses if needed. In the case of permanent expression, the EP system must deliver a transposon systemsuch as Sleeping Beauty, PiggyBac,31 or TcBuster™. The
transposon system has the advantage of conferring stable
expression; however, the insertion of the transgene is still
at a random location. Site-specific integration into a safe
harbor loci requires knowledge of the target sequences.
While EP is the most common non-viral delivery
system, it is widely reported that it can cause cell damage.32 Scalability and recovery challenges also exist. Theseissues notwithstanding, delivery efficiency and viabilityof functional cargos of commercial systems can be in theregion of 90% at smaller scales33 (for example at scales of
106-107 cells), but efficiency and viability generally trend
lower as scale increases. It should also be noted that mostcommercial EP systems are fundamentally semi-continuous processing systems.

Lipid nanoparticles
Lipid nanoparticles (LNPs) are biodegradable polymeric
structures that have been used in vivo and, more recently,
ex vivo to deliver mRNA and RNPs to cells. They are
currently being evaluated for the production of transient
CAR-T therapies,34 as well as for use in gene editing.
Billingsley et al. demonstrated that LNPs have equivalenttransfection efficiency to EP, but with lower cytotoxicity.35
LNPs have a clear advantage in terms of their scalability,
as there is no device component. Challenges include theneed to establish and optimize the LNP chemistry andto improve process reproducibility.36

Mechanoporation
SQZ Biotech has applied its cell squeezing technology toclinically relevant cell types. Material is delivered to thecytosol by passing a population of cells through a microfluidic chip that compresses them, causing membrane
disruptions that allow for the entry of macromoleculesfrom the solution. The process is continuous, fast, andvery controllable. However, scaling the technology requires significant parallelization in the context of T-celltherapy, where a target dose may be up to 109 cells. SQZ
Biotech has shown data highlighting minimal genetic
perturbation of the cells and excellent cell loading
efficiency.37,38 In addition, there are other emerging

A-CELL

✼160

technologies based on microfluidic chip formats that use
forms of mechanical perturbation to deliver cargo into
cells, such as those by CellFE and Kytopen.

Chemical transfection
Intracellular delivery by reversible permeabilization of
the membrane has been demonstrated as a fast, simple,
and scalable process.39 Avectas has synthesized mRNA
CD19-CAR cells at scale that are highly functional
and potent. This chemical transfection technology canalso deliver gene-editing tools with high-efficiency andviability and the technology can perform multiple, sequential edits. These qualities will be necessary for moresophisticated cell engineering processes.
EXPANSION

Expansion is a critical operation, epitomizing thestatement “the process is the product” as it determinesyield, has high impact on cost of goods (COGs), facility
design, batch success, pipeline product platforms, andthe ultimate quality and function of the final cell product.
Expansion is particularly challenging, as it is the longest
and, arguably, the most sensitive and unpredictable ofthe unit operations given the length of the operation andthe variability of the donor and patient material feeding
into it. Determination of the effective number of cells perdose from dose escalation studies will help identify thetarget process scale, the volume in which doses shouldbe packaged, and the minimal lot sizes to be produced.
Expansion is a constant battle between achieving desired
yield while ensuring the cells are not too differentiatedto be effective. The filling and cryopreservation unit
operations are dependent on the expansion yield, which
can be highly variable. Manufacturing operations scheduling can also be impacted as companies weigh accepting
variable yield on a predetermined harvest day versus
a variable harvest day based on the particular growth
profile of an individual patient’s cells.
The expansion unit operation offers a variety ofchoices regarding culture vessel and modality, culture
length, media delivery and feed strategy, and media
supplementation. It is also the unit operation that has
seen the greatest amount of technological development.
The selection of culture strategy and expansion unit

Chapter 8: Manufacturing of Cell-Based Therapies

operation are often based on the target cell number required for dosing, the target product profile (TPP), andmay have facility fit considerations. Having as much ofthis information as possible before deciding the detailsof the expansion platform will help ensure the processdelivers the correct cell product and can prevent being
burdened with a system incapable of delivering the desired product in the most efficient and effective manner.
For example, the TPP may elucidate dose requirementsthat dictate a particular expansion volume requirement.
Most processes start at the bench using a manual method
such as flasks or cell stacks. Moving from an open manual method to a closed and automated expansion method
is likely required to produce the required number of cells
and to meet regulatory requirements. Deciding how andwhen to automate a particular step or an entire process
can have consequences. Utilizing a particular single-use
disposable may increase costs or introduce particulates
or leachables into the process. However, reducing thechances of microbial contamination and the ability todecentralize a process to use a less skilled work force may
ameliorate such problems.
A major challenge for the expansion unit operation isthe variability associated with the starting material (healthy
donor material during research and development and patient material during clinical and commercial manufacturing). Because of this inherent variability, considerable caremust be taken during development to account for potential
performance differences between different healthy donors,
individual patients, and patient populations. A well-designed process will contain process control strategies forcritical process parameters, enabling effective responses tovariable performance and mitigating batch failures during
GMP production. The major expansion decision points
discussed here can be divided into three major categories:
process scale, culture duration, and development andmanufacturing considerations.
An important consideration when deciding on an
expansion platform and strategy is process scale. The
final required cell number is a major driver of equipment
and process decisions and varies greatly depending upontarget, indication, and type of cell product (i.e., CAR
vs. TCR, autologous vs. allogeneic). For example, anautologous product will likely be focused on scale-out

A-CELL

✼161

capability for manufacturing, while scale-up would be
more imperative for an allogeneic product. Culture
duration is another factor that needs to be considered
before deciding on an expansion platform. Shorter
cultures have more flexibility when it comes to culture
vessel and modality, while longer processes likely needto plan for nutrient delivery, waste removal over extended periods of time, and impact on facility design. For
T cells, shorter process retains the stem cell memory
phenotype and central memory phenotype, while in alonger ex vivo expansion process, an effector/terminal
effector and more short-lived in vivo population will
emerge.40 Specific choices regarding development andmanufacturing considerations are key to designing the
optimal process for a specific product. For example, aprocess that may seem simple to operate in a research
environment may encounter significant challenges
when the process is scaled-up (taken from benchtop andadapted to manufacturing scale) and run many times ina manufacturing facility. Knowing the specific needs, capabilities, and challenges of the intended manufacturing
facility is key to streamlining the transfer of the processfrom development into GMP production.

Process scale
The choice of process scale for cell products is highlyvariable depending on the type of product and is largely
driven by target cell numbers. Options for scaling include
either scaling-up or scaling-out, the choice of which canbe driven by autologous vs. allogeneic applications or bydelivery time and cost drivers. The first major decision
point when considering process scale is static vs. suspension culture (also referred to as 2-dimensional vs.
3-dimensional cultures). Each option offers particular
benefits and challenges and must be considered carefully
depending on the specific needs of each product.
Static culture options include traditional diffusion-driven gas permeable cell culture flasks (Corning),
convection-driven gas permeable cell culture flasks(Wilson Wolf G-Rex), and gas permeable culture bags
made from a variety of materials (Origen). These arebasic technologies, making them simple to incorporate,with limited training required for operators that areskilled in sterile culture technique. Most are similar to

Chapter 8: Manufacturing of Cell-Based Therapies

technologies used in a research and development setting
and are relatively inexpensive, making them frequent
choices for start-ups and early-stage development organizations. While simple to operate, flask and bag cultures
can be cumbersome when larger culture volumes are
required. Flasks are inherently limited in size by the surface area required for proper gas and nutrient exchange,usually capped at 1 L for immune cell culture, making thescale-up option the addition of more flasks. More flasks
also increase the number of open events and increase
the possibility of contaminating all or part of a batchor dose. Gas permeable bags are available in a variety
of sizes, but larger bags (1 L or greater) are challenging
to handle from a mechanical perspective as they lackstructure. Fitting large bags in incubators, moving themfrom an incubator to a BSC, and operations within the
BSC can present handling difficulties. The inability tomonitor the cells and culture conditions is a considerable
disadvantage to static platforms. To determine how theculture is growing, the static cell layer must be disturbed,
which may have negative effects on cell growth andculture environment. Manufacturers of such platforms
generally advise against resuspending the culture often
because of concerns related to cell growth. Recently,
more complex automated and semi-automated static
culture options, such as the Lonza Cocoon and Miltenyi
CliniMACS Prodigy, have been developed that provide
some of the process monitoring and control capabilities of suspension culture platforms (discussed below)
within a closed system designed to be more standalone.
However, these options come with constraints specificto static culture such as difficulty in cell counting andlimited scale-up ability.
Diverse options exist for suspension culture as several
technologies have been borrowed and adapted from traditional antibody cell culture. These options are generally
more amenable to scale-up than static culture platforms.
Foremost among these is the rocking motion bioreactor
platform, which has been widely adopted in cell therapy
manufacturing. Rocking motion cultures have been in
use for decades as part of seed trains or production cultures for antibody products. A significant benefit of thesesystems is the scale-up ability this platform provides.
Single-use bioreactor bags are available in a wide variety

A-CELL✼162

of sizes (from working volumes of 300 mL to greater than50 L). Allowing cells to grow in suspension removes thesurface area constraints of static platforms, while active
delivery of oxygen and other gasses creates an opportunity to achieve higher cell densities. In addition, the low
shear stress produced by the wave-like mixing strategy
allows the cells to remain fully in suspension, under
low mechanical stress, while oxygen-containing air isconstantly mixed into the culture.
Process analytical technology (PAT) is commonlyused to continuously characterize the expansion process. Manufacturers of these rocking motion bioreactor
systems have incorporated many process monitoring
and controls capabilities. Bioreactor bag options include
integrated process sensors for pH, dissolved oxygen, celldensity, and perfusion membranes of various pore sizes
for media exchange. Controls capabilities include controlof rocking angle and speed, ability to deliver a specificmix of gasses (air, O2, CO2, N2) via mass flow controllers
at desired rates, and automated temperature, pH, anddissolved oxygen control. Increased process monitoring
capabilities allow for intervention to respond to changing culture conditions. This is just the first step toward
a big data approach where many aspects of the culture
conditions are continuously measured.
Fed-batch feeding where a bolus of media is added
to a bag or vessel when a particular pH, cell density, or
metabolite concentration is reached is commonly usedin cell expansion. While this approach is expedient anda holdover from antibody manufacturing, it can result
in large swings in the concentrations of waste products
and feed streams. In some cell types, high concentrationsof lactate have been shown to inhibit growth. Most ofthese systems offer the ability to customize the expansion
protocol. Process steps can be written into programs thatan operator can manually execute or be automaticallytriggered by time interval or other input (i.e., pH, celldensity). Because cells are already in suspension, it is
not disruptive to the culture to count the cells as often
as needed. Bioreactor bags are equipped with sampling
ports to withdraw culture samples. It is important to ensure cells are in homogeneous suspension while a sample
is pulled to get an accurate cell density reading. Whilethese sampling ports are considered “closed,” care must

Chapter 8: Manufacturing of Cell-Based Therapies

be taken when pulling samples, as the ports will be exposed to the open environment for a short time. Rocking
motion bioreactor platforms can be easily scaled-out andscaled-up. Key factors for scale-out are the ability to run
the same program on multiple units, monitor cultures forconsistency, and employ single-use bags to enable simple
clean up and reactor turn over.
Another option with similar benefits to the rocking
motion bioreactor platform is the stirred-tank reactor.
This is the traditional workhorse of antibody production
processes but is just starting to be explored for immune
cell culture. The impeller-driven mixing mechanism has
the potential to produce significant shear stress to which
immune cells are likely quite sensitive. However, becauseof their various impeller designs that exist to promote
superior mixing and increase mass transfer under lower
shear stress, stirred tank reactors can achieve very highcell densities and perfusion mechanisms in these systems are not limited in the same way rocking motion
bag perfusion filters are, making them a potential option
for programs requiring particularly high cell numbers.
Another advantage of stirred-tank systems is the availability of high-throughput scale-down models capableof running larger design of experiment (DOE)-style andscreening experiments. One disadvantage of stir tankbioreactors is the high cost of validation while scaling aprocess to produce the cells needed for advanced clinical
trials and commercial manufacturing demands. Somenewer cell expansion platform technologies that use
bioreactor chambers that can be expanded during cell
culture are becoming available and may be an option forease of scale-up and reduced validation costs.41
Perfusion can be achieved by a number of mechanisms and varies with the expansion system. Rocking
motion bioreactors (up to a certain, limited working
volume of generally 10 L or less) can be equipped withperfusion membranes. These come in a variety of poresize options, depending on bag manufacturer and bag
size. Stirred tank reactors and other systems can employ
perfusion via hollow fiber tangential flow filtration (TFF)or alternating tangential flow filtration (ATF). Perfusion
strategies can be tested and determined via development
and “locked” via incorporation into an executable program for manufacturing. Some bioreactors such as the

A-CELL✼163

Xuri Cell Expansion System are unique in offering bothfed-batch and perfusion feeding capabilities.42,43
Hollow-fiber bioreactors adopted from kidney dialysis applications have been used to produce biologics formore than 20 years. Cells are cultured on one side of asemi-permeable membrane while metabolites, gas, andnutrients continuously perfuse through the membrane(Terumo, Quantum). The continuous addition of nutrients along with the constant removal of waste products
produces a much different cell microenvironment than
the fed-batch systems characteristic of most manual
methods such as flasks, stacks, and bags. Automated systems like rocking bags and stir tank reactors offer perfusion-feeding strategies especially when high cell densities
are required. A two-compartment culture system is advantageous in that it allows for an extra degree of control
not possible with other configurations. Without perfusion-feeding strategies, the accumulation of unwanted
and potentially inhibitory metabolites such as lactate andammonia can cause growth arrest. Perfusion limits may
become necessary to control the amount of media used
and therefore the cost of the process. The small harvest
volumes from hollow-fiber bioreactor cartridges areadvantageous for downstream processing. Hollow-fiberbioreactors are available in several configurations, froma pump and single-use cartridge system to automatedself-contained units. Different membranes with differing
molecular weight cutoffs are available to accommodate
a variety of applications. Scale is achieved by adding
multiple units (scale-out) making them a better fit for
autologous cell applications.
While single-use bioreactors offer many benefits,
operation and maintenance of this equipment requires
more training and investment in capital and processdevelopment efforts than most static culture options.
Specific needs for individual programs should be carefully
considered; pros and cons of various expansion systems
should be investigated,44 and costs and benefits properly
analyzed before deciding on the optimal platform.

Culture duration
Determining the length of the expansion unit operation
is variable, informed by the specific needs of a particular
program. These decisions should be driven by the target

Chapter 8: Manufacturing of Cell-Based Therapies

product profile (TPP) and the quality TPP (QTPP),
which define the required cell number and desired cell
phenotype. Shorter cell culture duration has the advantage of fewer population doublings, shorter manufacturing facility times, and will likely require less nutrients.
Longer cell cultures can produce more cells, but thosecells are likely to have more variable phenotypes, takeup more time in the manufacturing facility, and require
more complex nutrients and waste management strategies. Upon sufficient characterization of cell growth in agiven expansion platform, another approach is to havea standard culture duration regardless of cell number,which will improve operational efficiencies and makescaling operations easier.
Short expansion durations (a week or less) may besufficient for many autologous CAR cell therapy programs. These processes have the advantage of being lower maintenance from the perspective of nutrient delivery
and waste removal, and thus are likely more amenable
to static culture options than longer processes. Forprocesses requiring a week or more, nutrient delivery,
waste removal, and media component stability must be
carefully assessed and planned for. Perfusion is the mostcommon technology available for nutrient delivery andwaste removal, although this can be achieved in static
cultures as well. In static flask cultures, spent media canbe manually removed by careful pipetting, but adding
new media invariably results in cell layer disruption.
Alternatively, processes can be designed so that fresh
media or concentrated feed is added without removing
the spent media (fed-batch), reducing the number ofmanipulations and therefore simplifying the process.
For longer processes, it is necessary to also carefully determine the stability of media components. It isconceivable that for long processes, consideration may
need to be taken for producing multiple, staged lots ofmedia throughout production if particularly sensitive
media components are used. For perfusion processes,room temperature stability must be characterized andproper timing determined for any media changes (removal of unused fresh media at room temperature andreplacement with new fresh media from longer-term
storage temperature). Newer technologies for refrigerated storage of media and reagents that are integrated

A-CELL✼164

with the culture platform are on the horizon as more
automation allows for longer periods of unattended
processing.

Development and manufacturing considerations
A crucial aspect of expansion unit operation development
is the ability and efficiency of transferring the process fromthe development lab to a GMP manufacturing facility. The
two spaces are similar but there are distinct differences.
Knowing what these differences are and how to plan foraccommodating them is key to both smooth transfer andsuccessful GMP production, regardless of the expansion
platform chosen. There is, of course, greater flexibility
when planning to manufacture internally as coordination
between development and manufacturing should be morerobust and development is likely to influence the equipment, workflow, and design of the manufacturing space.
The early adoption of automated technologies is usually
met with skepticism because of high equipment, disposables, and validation costs, but should lead to savings in
fewer lost batches due to contamination, lower personnel
requirements, and increased ease of transfer to a manufacturing environment. Robust, scalable, automated processes
increase the facility throughput, therefore spreading the
upfront capital expenditures over more products, resulting
in reduction of COGs in the long run. If working withan outside vendor, such as a contract manufacturing organization (CMO), take care to align as early as possibleon technology, process details, workflow, quality systems,
etc. As in any professional relationship, clear and frequent
communication, and technical and knowledge transfer areoften key to success.
A “begin with the end in mind” approach to understand how the commercial and large-scale clinical trial
GMP manufacturing processes will operate and the necessary steps to transfer the process from the development
lab to the GMP manufacturing facility is highly advantageous for efficient and successful technology transfer.
Early collaboration between the process development,
manufacturing operations, and engineering functions
can aid the establishment of a process development strategy that is aligned with scientific, operational, and facility
needs. Development of the process for manufacturing isa key step for repeatable and reproducible production.

Chapter 8: Manufacturing of Cell-Based Therapies

Consultation with regulatory, Quality Assurance, andQuality Control functions is also beneficial at this timeto ensure that the larger scale clinical and commercial
processes can repeatably produce the same productswith the required quality as the smaller scale early phaseclinical products.
One important consideration for the expansion unit
operation is closed vs. open processing. As mentioned
earlier, traditional static culture flasks are simple from atechnology and operations perspective. These processes
are, however, considered to be open. Flasks must be
opened in a BSC to manipulate the cells. This poses a
contamination risk and is likely to prevent producing
more than one product at a time in a single suite during
GMP production. Static bags have the advantage of being
equipped with syringe ports so samples can be removed
and components added via a closed system. Bags can also
be connected via weldable tubing for closed transfer between bags. Challenges arise, however, when larger volumes are in use (i.e., wash steps, large media additions)
as it becomes more difficult to add and remove large
volumes requiring multiple welds or additions via syringe. This imposes a contamination risk either by many
repeated interactions with the port or by creating an
open step where liquids are either poured in or drainedout through an open connection (i.e., syringe withplunger removed, open drain port into waste vessel). The
automated systems discussed earlier (primarily, rocking
motion and stirred tank bioreactors, but also other systems such as the Lonza Cocoon, Terumo Quantum, andMiltenyi Prodigy) can largely be considered functionallyclosed. Most sampling in these systems is accomplished
via some version of a syringe port and the systems aredesigned to make external connections via weldable
tubing. The type and diameter of this weldable tubing canvary by manufacturer so it is important to plan carefully
to ensure that all components required for the expansion
unit operation (i.e., single-use reactors, media and wastebags, welders) are compatible with each other. Whether
open or closed, simpler processes with fewer steps andmanipulations have a lot of advantages, such as reduced
probability of cross contamination and reduced resource
requirements, leading to higher percentage batch successand lower COGs.

A-CELL✼165

Pre-filled media bags save time and remove tedium
from the process but can present connectivity challenges
due to the wide variety of weldable tubing sizes andmaterials. Needless to say, connections to other unit
operations should also be planned in advance to maintain closure, whenever possible, throughout the entire
production process. Sterile connections between unit
operations not only improve manufacturing efficiency
and safety in a clean room but tubing welders can produce sterile welds in a lower-class clean space. Movingsome or all of the unit operations out of the cleanroom
would result in lower manufacturing costs.
Another important consideration for the development and manufacture of cell therapy products is theability to monitor and control the process. Processmonitoring and control are tenants of quality by design(QbD) and serve as the mechanism by which operators
can react to process data in real time and use pre-determined “levers” to make planned process changes tokeep the expansion process on-track. At the very least
it is important to know what happened during the run.
This process monitoring is particularly important when
performing process changes during scale-up to ensure
process parameters are properly controlled and theresulting product quality is maintained. Data collection
through a laboratory information management system(LIMS) allows for electronic batch records, cell tracking throughout the process, lot-release test results, andsource material management. When a mix of automatedand manual processes are present, radio frequency identification (RFID) can be used to track a batch of cells.
Additional consideration for electronic systems, data
capture and documentation, and enterprise electronic
resources are discussed towards the end of this chapter.
When designing the expansion unit operation, there isoften a trade-off between operational simplicity andprocess monitoring ability. Simpler systems, such as
traditional cell culture flasks and static bags, often offer
little opportunity to monitor and control process conditions while more technologically advanced systems,
such as single-use bioreactors, offer a number of monitoring and control points. For example, a flask culture
can monitor temperature and be set at a specific CO2
percentage in an incubator while a bioreactor system

Chapter 8: Manufacturing of Cell-Based Therapies

offers monitoring and control of a number of parameters including both the setpoints and present values
for each parameter. Examples include temperature, pH,
gas flow, dissolved oxygen, pressure, rocking speed, androcking angle. Parameters in these systems can often bemonitored at user-specified intervals and recorded via
operating software.
Probe technology is evolving and many systems now
include single use or non-contact fiber optic probes
for pH and dissolved oxygen, eliminating the need forcalibrating, cleaning, and sterilization between runs.
Different probe technologies based on capacitance arebeing developed to monitor cell growth and viability,
which enable users to monitor cell growth without theneed to physically remove samples.
Many process parameters can be controlled andmonitored where the system will respond to changing
inputs to maintain specific setpoints or run specified
profiles. For example, gas mixture in these systems isoften controlled by mass flow controllers that alter flow
rates to maintain the specified percentages of certain
gasses (i.e., CO2, O2). Proportional integral derivative(PID) controllers are also included in many cases thatallow the system to maintain, for example, a specific pH
setpoint, while allowing the user to determine the speed
and intensity of the response by making adjustments to
the PID controller settings. In development, the ability
to monitor a number of parameters is imperative to
identifying and characterizing critical process parameters. In manufacturing, it creates the opportunity tooperate a more consistent process as more parameters
are monitored and controlled during operation. Having
this ability allows the cells in culture to experience a
more consistent environment, which should lead tomore predictable process outcomes.
A final consideration in the development of the
expansion unit operation is which process analytics toinclude during development and manufacturing. Usefultools for development can include cell counting andviability analysis, metabolite analysis, and offline pH/
gassing analysis via a blood gas analyzer. It is widely
accepted that cell counting and viability analysis isimperative to include in both development and manufacturing of cell therapies as this is the most direct read

A-CELL✼166

of cell health and expansion process success. Metabolite
analysis can be a very useful tool, particularly during development when it can help to inform process decisions(e.g., particularly around feeds and perfusion strategy),
and careful consideration should be made as to when
and where this analysis should be included. Real-time
metabolite monitoring will be the next step in automation, particularly in autologous cell processes wherestarting material variability requires precise process control. Several manufacturers provide different versions ofthese instruments and an array of assays is available. Useof these analyzers allows development scientists to understand more about the culture environment of the cells
and how process changes or donor cell variability impact
the culture environment. Blood gas analyzers provide
an offline reading of parameters such as pH, dissolved
oxygen (DO), and CO2 and serve as an important toolfor validating the accuracy of online measurements ofthese parameters. Online probes, while calibrated, often
experience drift, whereas offline measurements, which
employ multiple-point assessments during analysis, arewidely considered to be more accurate.
Many technologically advanced expansion systems
include the ability to correct online values to match thosemeasured offline. While very useful for development,these types of analyses may or may not be necessaryduring manufacturing. These instruments, especiallythose used for metabolite analysis, are often expensive
to purchase and costly to maintain from a reagent andoperations perspective. Keeping the instruments functioning properly requires trained users and, most often,
daily upkeep and maintenance. If an expansion process isrobustly designed during development, metabolite analysis in the manufacturing facility may not be necessary.
A blood gas analyzer, on the other hand, may still be
required to maintain the accuracy of online probe readings. Considerations should be made when including
such equipment in a manufacturing facility; DO changes
rapidly once exposed to air so the instrument should beplaced in relative proximity to the expansion system.
Movement of this material is often via syringe and thetransfer between expansion system and machine may beconsidered an open step (transfer to a closed tube is notadvised if an accurate DO reading is required).

Chapter 8: Manufacturing of Cell-Based Therapies

Expansion system summary
A wide variety of expansion system options exist for celltherapy products that vary greatly in degree of technology,
complexity, scale, and capital investment. There is no ideal
system that satisfies the goals and needs of every institution,so careful consideration should be made to the specificgoals, opportunities, and constraints of each individualorganization. The expansion system needs to support boththe process and product goals as the two are inextricably
linked (i.e., if a large number of cells are required for a
product, the expansion system is likely to require the ability
to perform media exchanges). This can be accomplishedmanually in a flask system or automatically via a numberof mechanisms (perfusion via absolute filtration, perfusion
via hollow fiber filtration, media reoxygenation via recirculation) available on more automated systems.
The major decision points around the expansion
unit operation include process scale, culture duration,and development and manufacturing considerations.
The determination of process scale weighs heavily on
whether to choose a static or suspension culture, while
culture duration informs decisions on nutrient delivery
and raises questions on how duration may affect TPPs
and QTPPs. Determination should be made on whether
scale-up or scale-out is more relevant to a given program(based on process needs and business drivers such as
time, cost and quality), as different expansion systems
offer benefits and pose challenges to each. Development
and manufacturing considerations will be specific tothe capabilities and constraints of each organization.
Will manufacturing be performed internally or externally? If internal, can systems be harmonized between
development and manufacturing? If external, how can
the process be designed in development while taking
into account inherent differences between the internal
development lab and the external manufacturing facility
where customers have little to no impact on instrument
and system choices? How much information should becollected during development and how much of that
information must be monitored during manufacturing? Regardless of program goals and process details,
all decisions should be guided by QbD principles so
the resulting process is as robust as possible from aphase-appropriate perspective.

A-CELL✼167

FINAL WASH AND VOLUME REDUCTIONDRUG SUBSTANCE

Upon completion of the cell culture phase, the expanded
cells are harvested from the bioreactor or culture vesselto undergo a final cell washing operation. This last cellwashing operation is performed to simultaneously fulfill
three main purposes:• Remove/reduce cellular impurities, additives, andnon-target media components below acceptablerelease levels.
• Reduce the overall sample volume to increase theoverall cell concentration to meet downstream
formulation requirements.
• Perform a media exchange to transition the cells
from the harvested culture pool to a basal formulation media.
Selection of an appropriate washing apparatus and protocol to fulfill the first requirement is particularly critical,
as further impurity reduction typically is not possible after
this step, with the exception of large particulates whose
removal might be possible via coarse post-formulation
filtration methods. In the “Cell washing” section, an overview of washing tools was presented, distinguishing thembased on metrics such as underlying physics, efficiency,
and throughput. In addition to such principles, the following factors should be considered towards appropriate
selection of washing tools for DS preparation:
Impurity removal considerations: The final washingprocedure should be designed to achieve sufficient depletion of contaminants and remaining media additives
from upstream processes (e.g., BSA, cytokines) to meetthe product-specific purity criteria necessary to satisfy
the release specifications. As such, one of the criticalsteps towards the selection of a final washing tool is thedefinition of “target depletion levels” for media components, additives, and other impurities. These target
depletion levels, and specifically the highest depletionlevel amongst all the components that need to be purified, will then determine the amount of volume washouts(or “volume equivalents,” e.g., how many times the total
original volume should be replaced with new media
volume) that a washing operation will need to ensure
the product is sufficiently purified.

Chapter 8: Manufacturing of Cell-Based Therapies

As an example, let’s take a harvest of a 1L cell culture
whose culture conditions involved the use of two different cytokines with concentrations of 100 IU/mL IL-2
and 1 IU/mL IL-6. Let’s also assume that the target wash
output volume is 1L (e.g., no volume reduction), and thatthe final post-wash acceptable cytokine concentrationlevels are 0.1 IU/mL for both IL-2 and IL-6. Based onthese parameters, the volume reduction must provide
a 3 log (1000x) reduction in IL-2 concentration, as IL-2
represents the media additives that require the highest
depletion level in this example (from 100 IU/mL to 0.1
IU/mL). Assuming the use of an automated washingsystem such as those discussed in the “Cell washing”
section, and assuming a 90% volume exchange at eachwashing cycle, a minimum of three washing cycles (fora total of 2.7 volume equivalents, or 2.7 L total washingvolume) will be required.
The optimal washing tool will provide the ability toperform the required amount of volume washout within
the shortest operational window, while providing thehighest cell retention.
Volume reduction considerations: Particularly
for large harvest volumes, where the required media
volume to perform the target volume washout might
be significant (e.g., in the example above, the total
washing volume would increase from 2.7 L to 27 L if
the culture volume went from 1 L to 10 L), the ideal
washing tool will provide the ability to concentrate thesample prior to performing the washing operation, as
this will significantly decrease the total required volume
to perform the required depletion of impurities. Several
off-the-shelf tools exist that provide combined wash andvolume reduction capabilities. The ideal final DS washing protocol will therefore be one in which the overall
sample volume is first significantly reduced, followed by
a washing procedure providing the required amount ofvolume washout based on the post-volume reduction
volume. Applying this improved volume reduction andwash protocol to the example above, and assuming a 10x
concentration step prior to washing, the overall culture
volume would first be reduced from 1 L to 0.1 L (while
the IL-2 cytokine concentration would still remain at 100
IU/mL) followed by a 2.7 volume equivalent wash, i.e.,
0.27 L (as opposed to 2.7 L without volume reduction),to deplete the IL-2 level down to 0.01 IU/mL.

A-CELL✼168

In addition to the media consumption considerations
listed above, an even more critical design parameter forthe volume reduction step is the “target DS concentration.” The volume reduction step will indeed need tobe capable of concentrating the cells to a concentration
higher than that required to perform the final downstream DP formulation steps. For example, assuming atarget 100 × 106 cells/mL DP concentration and a DP
formulation protocol in which the DS is diluted 1:1 withcryomedia, the final DS wash will need to concentrate
the cells to 200 × 106 cells/mL to enable the downstream
dilution step.
Media exchange considerations: A key function of
the final DS preparation wash is the ability to transfer
the cellular product from the media in which the cells
were cultured to a different buffer of choice that will
constitute the “basal formulation buffer” in preparation
for the successive formulation/DP manufacturing steps.
Typically, basal formulation buffers are DMSO-free, but
might contain excipients and other additives aimed atenhancing cellular function or recovery (e.g., dextrose).
As some of these additives could potentially be limited
in availability and high in price, it is sometimes favorable
to consider a final DS washing approach where the main
washing operation is performed using an excipient/additive-free buffer, followed by a final washing cycle ofone volume equivalent that will place the cells in the final
desired buffer composition.
Other considerations for selecting the appropriate
wash/volume reduction tools include cost of goods, data
integrity, and connectivity as described in the PhysicalConnectivity section.
FORMULATION

Formulation involves the mixing of the final cell productwith a cryoprotectant and a diluent or freezing media toarrive at the desired concentration of cells.
Optimal formulation is critical for the success of a
final cellular product that is stable, safe, efficacious, andmeets regulatory requirements. Formulation is the process of combining cells, buffers, proteins, ancillary materials, and cryoprotectants, and is carried out immediately
after the cells are harvested at the end of the manufacturing process. Formulation is a temperature-dependentand time-sensitive step since the harvested cells during

Chapter 8: Manufacturing of Cell-Based Therapies

this step are held in suboptimal environmental conditions and without nutrients. Appropriate formulation isneeded to stabilize the cells so they can withstand stressfactors such as temperature excursions, pH changes, andmechanical stress caused by handling, storage, shipment,
and bedside preparation.1
Formulation strategies involve selection of the appropriate cryoprotectants and other excipients. The selectionof excipients plays a key role in maintaining the CQAs of
the final product. In addition to commercially available
cryopreservation media, common formulation for mammalian cells historically includes 10-90% fetal bovine
serum (FBS), 5-10% dimethyl sulfoxide (DMSO), and1-20 million cells per milliliter that have been previously
resuspended in an isotonic electrolyte solution, such as
Ringer’s lactate or Plasmalyte 148. While the use of FBS
has been questioned, it is still permitted as an excipient.
Serum-free cryoprotective agents are commercially available and, although proprietary, developers can referencea Drug Master File (DMF) for the regulatory agencies.
Serum replacements include human serum albumin(HSA) and human platelet lysate (hPL). HSA is one of
the most popular excipients in cell therapy because it is
the most ubiquitous protein in blood and is known tocreate an optimal microenvironment for sustained cell
viability. It acts as a scavenger of toxins and other reactive
oxygen species, maintains pH, provides insulation, andmaintains cell viability during cryopreservation without
increasing osmolality.45 Additional components of the
final formulation can include dextran, which serves as
an osmotically neutral volume expander and as parenteral nutrition, sodium chloride as a normal saline
diluent, and stabilizers such as sodium caprylate andN-acetyltryptophanate that protect proteins such as HSA
from oxidative stress.1,46
The cryoprotectant (DMSO) addition rate and thetemperature at which this occurs must be carefully determined and rigorously controlled. If the cryoprotectant
is added too quickly the cells may lyse; if it is added too
slowly or at an elevated temperature toxicity becomesproblematic.
There is no standard recipe for the final formulationand each cell type may perform differently following
different manufacturing protocols. Additional factors
such as cell concentration, sample volume, and even

A-CELL✼169

sample container geometry may affect the final outcomes. Whatever system is implemented it is importantto perform both short- and long-term follow up testing
to ensure the viability of the product.
FILL AND FINISH OPTIONS

Fill-finish operations to load cell therapy products into
appropriate containers are the endpoint of a product’s
journey through a GMP-compliant manufacturing
process until the product is transported, thawed,
and infused into the patient. After the formulation
process is complete, the product is filled into the final
product container (typically bags or vials), labelled,
cryopreserved (if applicable), and stored in an appropriate environment (e.g., 2-8°C, -80°C freezers, or vapor
phase liquid nitrogen) while batch release testing andcertification is carried out. Following batch release,the product is packaged for distribution. The productis then transported to the clinical or commercial use
site while carefully controlling the cold chain. At site, if
supplied frozen, the product undergoes final thawing andpotential cell wash or dilution steps prior to dosing thepatient. The overall efficacy of the clinical product partly
depends upon the efficacy of the bio-preservation steps
within the process. A thorough understanding of the
impact of cryopreservation, thawing, and preparation of
the product for administration to patients is essential toensure its efficacy as the product is of most value at thispoint. These include formulation, aliquoting into bags
or vials, cryopreservation, short- or long-term banking,
maintaining the cold chain during transportation to theclinic, and the final thawing and potential cell wash or
dilution steps prior to dosing the patient.
In conventional practice, cell therapy products may
be presented in either vials or bags. Both of these options
have advantages and disadvantages, and developers mustchoose according to closure integrity (the container mustbe hermetically sealed and provide adequate physicalprotection to ensure specimen integrity is maintained
throughout processing, storage, and distribution), sample
stability (the container must ensure that the productremains stable over long periods of time), and ease ofaccess to the product. When deciding, developers should
consider all safety and quality standards and patientneeds. Selection of the final container should therefore

Chapter 8: Manufacturing of Cell-Based Therapies

take into consideration particulate levels, leachable andextractable profiles, and material integrity at a range oftemperatures achieved during cryopreservation, storage,
shipment, and the thawing process.
Traditionally, cells have been cryopreserved in vials.
Vials are especially valued for durability under extreme
temperature conditions and amenability to storage undera variety of conditions. Additionally, they can be moreeasily combined to meet a variety of doses requiredin early phase clinical dose escalation studies. As celltherapy processes are developed, washing cells, resuspending cells in freezing media, and finally aliquoting
into cryovials is a manual process mostly carried out ina BSC. Automated solutions for vial filling exist, having
been developed for pharmaceuticals and more recently
biologics. Vials are suitable for seed banks and large-scale
allogeneic cell therapies, but they are not as convenient
for autologous cell therapies due to the requirement to beprocessed in a BSC and thus unsuitable for administering
cells at a clinical site by the end user. Other drawbacks
include the size limitation (i.e., cell doses that may exceedbillions of cells may not fit in a vial) and the inability
to hermetically seal the system. Vial closure systems,
whether screw caps or rubber stoppers, do not allow
complete closure and therefore pose contamination risks
to the product. More recently, automated vial filling platforms have been developed that use more sophisticatedvials with filling ports and various retrieval options thatfacilitate sterile weldable connections.
Bags are commonly used as the final container forcell therapy products due to the existing abundance ofinfrastructure for processing, freezing, and storing bag
container systems from the long history of bag usage inblood banking systems. Cryopreservation of cells in bags
facilitates administering large numbers of cells at the clinical site with a sterile weldable connection, a luer lock, or a
spike connection. From the manufacturing perspective, theability to aseptically weld and hermetically seal cryobags isextremely beneficial and eliminates the need for a BSC or
similarly controlled environment. However, the fill process
can be uniquely challenging. While commonly used forlarger volumes, new bag formats are available in several
sizes between 5 and 10 milliliter volumes.
Even given the advantages bags offer, several drawbacks
do exist. There are cases where samples must be stored in

A-CELL

✼170

smaller volumes than is appropriate for a bag format; dueto the relatively brittle nature of the material out of whichmost bags are made, sample stability at typical storage temperature may be compromised, especially during transportand manipulation of the sample during thaw. Perhaps mostsignificantly, bags carry a risk of compromised container
integrity. A study by Khuu et al.47 investigated a series ofcatastrophic bag failures first noticed in 2001 that wereassociated with four specific bag lots made from poly
(polyethylene-co-vinyl acetate). While no serious adverse
patient effects occurred, extensive bag failures led to cell
product contamination, increased product preparation
time, increased antibiotic use, and increased costs to replace defective products. In another study48 investigatingcases from 2000 to 2006, integrity loss of 3.5% of bags wasobserved during bag thawing at 37°C.
Sealed or semi-closed vials, such as the AT-Closed Vial®
from Aseptic Technologies, offer both the scalability of traditional screw top cryovials and the closed system advantages of bags. The ability to fill and seal AT-vials without
removing the septum minimizes the contamination risk.
Both bags and vials are available with traceable bar coding
and RFID tags to identify and record batch information,
testing results, and other quality documentation. Manyof these containers are supported by the FDA DMF, withextensive data provided in their validation packages.
Many fill and finish protocols start out as manual
operations in a BSC. As lot sizes increase, because theoverall manufacturing process is scaled out/up, manual
operations quickly become a bottleneck. Several devices
have been launched into the marketplace that are capableof rapidly filling bags or vials with formulated cell products. While capable of quickly filling multiple bags or vials
and greatly reducing contact time in DMSO, some devices
lack the capability to cool formulated cell products andsome must be used in an isolator (Aseptic Technologies
M1 and L1, Sexton Biotech AF500). Other manufacturers
have automated the entire unit operation including mixing and cooling capabilities (Miltenyi Prodigy, Terumo
Finia, Sexton Biotech Signata) and the ability to fill bothbags and vials (in some cases). The current automatedofferings are somewhat limited when large-scale is needed
and may require multiple devices to accomplish the fill
and finish without increasing the DMSO exposure timeto levels that impact product quality.

Chapter 8: Manufacturing of Cell-Based Therapies

After products are filled, they must be appropriately
inspected and labelled. The type of label and printing ink
used needs to be suitable for adhesion to the primarycontainer, cryopreservation, and thawing of the productso that the label remains attached to the primary container
and legible. Incorporation of all the information requiredon the label can pose a challenge, especially when using
vials as the label area is often quite small. Labels that extend the printable area (e.g., “Flag” and “leaflet” labels) areavailable and development studies on thawing times andselection of appropriate thawing equipment should be
carried out with the chosen label to ensure establishmentof robust end-user protocols and product quality prior topatient administration. Labeling for licensed CAR T cells
must conform to the requirements in 21 CFR Part 201 and
21 CFR Part 610 Subpart G. Autologous CAR T products
must be labeled “FOR AUTOLOGOUS USE ONLY”, andthe label should also include at least two unique identifiers
to confirm patient identification prior to administration.49
For allogeneic products where batch sizes are much larger
than autologous product, the time taken to visually inspect
and label the product is much longer so it is important todesign the fill-finish process to ensure that all critical processparameters such as hold times and temperature are still met.
Ultimately, the choice of final container and label mustbe suitable to the developed manufacturing process, besufficiently scalable for commercial success, and must
accommodate the end-user. With the current available options that can offer simplicity, flexibility, and scalability, oneof these containers can likely suit cell therapy developers’
product needs.
CRYOPRESERVATION

For the clinical or commercial distribution of cell therapies,
the standard convention at this time is to perform cryopreservation prior to distribution either to the patient or for
temporary storage until required use, where the cells willbe thawed and administered. This is principally due to thefact that cryopreservation provides significant flexibility in
manufacturing and patient scheduling, facilitates the quality
control release process for a DP, and allows for transportation of the therapy to the patient. Working with fresh,non-cryopreserved cell therapy products increases the risk
to patients because all subsequent operational activities(labelling, packaging, batch record review, QP release, and

A-CELL

✼171

shipping) must be rushed so that infusion to the patient canoccur within the product stability time window. As such, thetime allotted for release testing and quality assurance review
is minimal, often resulting in patient infusions with limited
data available (e.g., interim sterility reads). Moreover, thelack of cryopreservation significantly increases the risk of acell therapy product shortage as only limited inventory canbe built, especially in the allogeneic space.
The current established best practice for T cell therapies is formulation in a DMSO-based cryoprotectant andprocessing into either blood bags (typical for autologous
therapies) or cryovials (common for allogeneic therapies),
followed by cell cryopreservation utilizing a highly reproducible, controlled-rate freezing system suitable for thetype of primary packaging (bags or vials) that can support
a cooling rate of −1°C min−1 or slower to preserve highlevels of cell viability. This is because the rate at which acell solution is brought from a physiological temperature toa cryogenic temperature has been shown to be a key factor
in cell survival. It has been demonstrated that rapid coolingrates (i.e., greater than −10°C min−1 have led to a notablereduction in viable cell count if the subsequent warming
process for administration is slower than 6.2°C min−1.50 The
loss in cell viability is believed to be due to the creation of
amorphous ice structures in the cells, which when subsequently thawed at slow rates lead to ice recrystallization
causing the mechanical disruption of cells. Implementationof a controlled cooling rate also decreases variability in theoutcome of the cryopreservation process.
There is no one-size-fits-all cryopreservation approachfor cell therapy processes. One needs to consider thetype of cell, cryoprotectants, cell concentration, and theprimary packaging in the optimization process for cell
cryopreservation. Each of these aspects could affect the
final product quality.
• Cryoprotectant. The cryoprotectant should be selected
such that the final product is stable, safe, efficacious,and meets regulatory requirements. Typically, to avoid
osmotic shock and temperature-related toxicity, cells
are cooled down to 4°C before the same temperature
DMSO-containing cryoprotectant is added.
Maximum contact time should also be assessed todetermine the operational time window before cell
viability and functionality drops.

Chapter 8: Manufacturing of Cell-Based Therapies• Cell concentration. The cryoprotectant’s power is bestleveraged within specific cell concentration ranges.
• Product container (bag or vials). The material andthe geometry of the final container will have an
impact on heat transfer and homogeneous cooling/warming rate of the product.
Recommendations would be to utilize QbD approaches to identify the CQAs that provide consistency
in meeting the TPP of the final product. This process
is particularly critical for cell therapies where, unlike
many other bioprocesses, the properties of the final
product (viability, recovery, and cell function) postthaw represent the cumulative effect of all of the processing steps and reagents used in the process, where
each step may influence the total number and viabilityof cells after completion of the manufacturing and cryopreservation processes. The current recommendation
for a minimum viability standard for cell therapies is atleast 70% (based on FDA guidance,51 and often higher)
post-thaw after both formulation and cryopreservation.
In addition, assessing post-thaw vitality or apoptosis aspredictive signals of cell viability drop, and post-thaw
functionality, ensures a more holistic readout of thequality of the cryopreservation process, therefore generating a stronger dataset to support decision-making
when locking down the process.
For cell therapies, best practices require that cells
be stored at temperatures below -150°C. Storage intemperature-controlled environments and systems toprevent temperature excursions are commonly used. The
stability of cells in storage is most strongly influenced
by two factors: transient warming and background ionizing radiation. Proper training and product handling
protocols can help reduce transient warming of samples
during retrieval of samples from a biostorage location.
In conclusion, the success of CAR-T manufacturing relies on a series of critical processes, from theprocessing of cellular starting material (apheresis) to
cryopreservation, to ensure product yield and quality.
Each step involved in the manufacturing process has
its own technical considerations and challenges, andcontinued work is being performed in the cell therapy
field to address these challenges and optimize eachprocess for various therapeutic applications. While

A-CELL✼172

this chapter covers the technical considerations of each
unit operation, manufacturers must also ensure the safedelivery of the cell therapy product to end users at clinical administration sites; therefore, cold chain storage,
distribution, and logistics must also be considered.

Electronic Systems Considerations
Enterprise control is the ability to combine control, intelligence, and process management to enable business
optimization that is inclusive of digital information (IT)and production operations (OT). It is the deliberate actof synchronizing business strategy with operational execution in real-time to enable closed-loop business control
across an enterprise. Additionally, it is the start of a datacollection capability that allows for data feedback loops
into process optimization. In the context of cell-based
therapy manufacturing, these entail considerations forelectronic systems for manufacturing execution (MES),
quality management (QMS), process information management (PIMS), release testing and laboratory information management (LIMS), document management(DMS), warehouse management (WMS), computerized
maintenance management systems (CMMS), and othersystems designed to ensure track and trace abilities,
enhanced documentation and robustness of the manufacturing and testing processes.
ENTERPRISE SYSTEMS ARCHITECTURE OVERVIEWEnterprise IT/OT Systems commonly follow the ISA
95 standard which is based upon the Purdue Enterprise
Reference Architecture (PERA). ISA 95 focuses on
segregation of the IT and OT domains with strictlevel-based control and dataflows intended to prevent
unauthorized access or changes to the OT systems. With
the introduction of Cloud and IIOT (Industrial Internetof Things) within the last 10 years, many institutions are
implementing a modified hybrid-based model to enable
Cloud/Edge technology at each level of the architecture.
While the hierarchy of ISA 95 details a logical separation of systems utilized for designing digital solutionarchitectures, the model can be simplified into Business
Systems, Data Systems, and Operational Systems.

Chapter 8: Manufacturing of Cell-Based Therapies

ENTERPRISE SYSTEMS IN DETAILBusiness systems

Business Systems are those traditionally used across theenterprise and encompasses commercial functions forsales, business operations for finance, accounting, andHR, and corporate business planning. One common
business system is the enterprise resource planning(ERP) system. Other examples include product life cycle
management (PLM), supply chain orchestration (SCO),
customer relationship management (CRM), and humanresource management (HRM). Reaching further intobusiness operations we find document management systems (DMS), warehouse management systems (WMS),
computerized maintenance management systems(CMMS), quality management systems (QMS), track
and trace (T&T) systems, learning management systems(LMS), and finite scheduling (FS) systems. These are typically of enterprise-scale, and often require a significant
investment in human resources, dollars, and time.
ERP

An enterprise resource planning (ERP) system is typicallya suite of integrated applications that an organization can
use to plan their main business processes. ERP systems
track the status of business commitments such as orders,purchase orders, payroll, warehouse management, financial operations, and planning. An ERP system facilitates
information flow between these business functions andmanages connections to outside stakeholders.
In cell-based therapy commercial manufacturing,
ERP systems are critical as the pharmaceutical manufacturer needs to keep track of all patient orders along withcritical must-have inventory. For manufacturers withallogeneic processes, ERP systems are also important as
one donor gives life to a number of recipients and the
ERP system keeps tracks of all this information.
The most common ERP systems used in the life sciences space are SAP, Oracle, and Microsoft Dynamics,
all of which are also available in the cloud.
SCO

Supply chain orchestration (SCO) platforms have recently evolved for advanced therapy medicinal products(ATMPs) or more commonly cell and gene therapy manufacturers. SCOs control the chain of identity (COI),

A-CELL✼173

which is a unique patient ID, and the chain of custody(COC), which ensures the name of anyone who has
touched the product and where the product has traveled
are recorded. To facilitate ATMP sector growth, thesupply chain infrastructure is critical in supporting theindustry from clinical trials to commercial distributionof life-saving treatments to more patients in a safer andmore secure manner. The Health Insurance Portability
and Accountability Act (HIPAA) in the United States andthe General Data Protection Regulation (GDPR) in theEuropean Union are the foremost regulations all SCO
platforms must adhere to. The patient journey, as some
vendors call it, is tracked within this platform. Similar toboth autologous and allogeneic processes, the first steps
include creating a unique identifier or COI for the donor /patient, drawing blood from the donor / patient (apheresis
process), centrifuging the product, securing the blood bagby printing barcoded labels, integrating with shipping andlogistic vendors to ship the blood bag to manufacturing
facility, preparing the final product for shipment back to
the hospital or clinic after manufacturing is complete, andthen infusing the final product into the patient.
Notable companies that use cloud-based solutions
to track needle-to-needle patient monitoring include
TrakCel, Vineti, Hypertrust, FarmaTrust, and Danaher(previously GE).
HUB Services

New to the industry and specifically valuable to the celland gene therapy industries is the entry of HUB Services.
HUB services provide connections between a patient
and their health care providers, pharmacies, distributors,
and insurance companies, while at the same time ensuring privacy as provided by HIPAA regulations.
HUB Service providers include large health providers
such as AssistRx, CareMetx (Walgreens), Omnicare
Specialty Care Group (CVS), OptumRx (United Health)and Sonexus Health (Cardinal).
FS

Finite scheduling (FS) systems in the cell and geneindustry are gaining prominence as more cell therapy
manufacturers reach clinical trials and patient population grows from a mere 100 at start to more than

Chapter 8: Manufacturing of Cell-Based Therapies

10,000 patients when the cell-based therapy productis fully commercialized. FS systems support resourcescheduling with finite capacity, production sequences
optimization, materials management, multi-scenery
analysis, dynamic constraints management, laboratory
room scheduling, and patient scheduling with direct
integration to SCO to allow nurses to book a slot forpatients. An FS system allows cell therapy manufacturers to do global and local production planning (patientorders) and dynamically allocate production orders.
The following benefits are obtained: improvement offinite capacity work plan and patient order tracking,production lead time reduction, inter-operational andline buffer reduction, prompt material calling from suppliers, inventory-level cost reduction, and improvementof transportation efficiency.
The most notable FSs are Körber Pharma PAS-X,
sedApta, and Bio-G (purchased by Emerson in 2019).
DMS

A document management system (DMS) is used to receive, track, manage, and store documents and eliminate
paper. They keep a record of the various versions createdand modified by different users (version tracking). It
is often viewed as a component of enterprise content
management (ECM) systems and related to digital asset
management, document imaging, workflow systems, andrecords management systems required by the company’s
QMS. Documents stored in a document management
system, such as procedures, work instructions, and job
aides, provide evidence of documents under control.
Failing to comply can cause fines, loss of business, or
damage to a business’s reputation. Important aspectsof document control include reviewing and approving
documents prior to release, reviews and approvals,
ensuring changes and revisions are clearly identified, ensuring that relevant versions of applicable documents areavailable at their points of use, ensuring that documents
remain legible and identifiable, ensuring that external
documents (such as customer-supplied documents or
supplier manuals) are identified and controlled, andpreventing unintended use of obsolete documents.
Paper documents have long been used in storing
information. However, paper can be costly and, if used

A-CELL✼174

excessively, wasteful. Document management software isnot simply a tool, but it helps manage access, and trackand edit stored information. A DMS is an electronic cabinet that can be used to organize all paper and digital files.
It helps businesses convert paper documents to digital
files and store them in a single hub after they are scannedand digital formats get imported. An important benefitof digital document management is the “fail-safe” environment it provides to safeguard documents and data.
The most common DMSs include Veeva’s Vault DMS,
Qumas, and Microsoft SharePoint.
CMMS

A computerized maintenance management system(CMMS) is a computer database of information about
an organization’s equipment maintenance operations.
This information is intended to help facility maintenance workers do their jobs more effectively by helping
to determine when equipment will require maintenanceand which equipment is predicted to fail. This helps
management make informed decisions to calculate thecost of equipment breakdown repair versus preventive
maintenance for each piece of equipment, possibly leading to better allocation of resources. Cell-based therapy
manufacturers spend a considerable sum of money on
equipment, as the equipment used in this space is very
intricate and complicated, and the CMMS helps ensure
the equipment runs as expected. CMMS data may also
be used to verify regulatory compliance. To properly
control the maintenance of a facility, information isrequired to analyze what is occurring. Manually, thisrequires a tremendous amount of effort and time. A
CMMS also allows for record keeping to track completed
and assigned work orders in a timely and cost-effective
manner. Cell-based therapy manufacturers have started
using CMMSs extensively to better control and organize
maintenance management.
The different components of a CMMS include, but are
not limited to, equipment data management, predictive
and preventive maintenance, labor, work order system,
scheduling and planning, vendor management, inventory
control, purchasing, budgeting, and asset tracking.
The most common CMMSs are Blue Mountain, SAP
PM, and IBM Maximo.

Chapter 8: Manufacturing of Cell-Based Therapies

QMS

A quality management system (QMS) is a collectionof business processes focused on consistently meetingcustomer requirements and enhancing their satisfaction. It aligns with an organization’s purpose andstrategic direction. It is expressed as the organizational
goals and aspirations, policies, processes, documented information, and resources needed to implement
and maintain it. The ISO 9000 family of standards isprobably the most widely implemented QMS worldwide–the ISO 19011 audit guidelines apply to both
pharmaceutical and medical devices—and deals withauditing management systems, including principles ofauditing, managing an audit program, and conducting
management system audits, as well as guidance on
evaluating the competence of the individuals involvedin the audit process.
The two primary guidelines for life science manufacturer QMSs and related services today are theISO 13485 standards and the US FDA 21 CFR 820
regulations.
Quality system requirements for the life science
industry are internationally recognized as a way to assure product safety and efficacy to customers. The FDA
constituted the rule in Code of Federal Regulations 21
CFR 820, which all medical device manufacturers must
follow. A typical quality system includes management
controls; design controls; production and processcontrols; corrective and preventative actions; material
controls; records, documents, and change controls; andfacilities and equipment controls. Pharmaceutical drug
manufacturers are regulated under a different sectionof the Code of Federal Regulations.
The QMS covers processes such as calibrations,internal audits, corrective actions, preventive actions,
identification, labeling, and control of non-conforming
products to prevent its inadvertent use, delivery or processing, and purchasing and related processes such as
supplier selection and monitoring.
Common IT systems to manage a QMS include
Veeva’s Vault QMS, TrackWise from Sparta (purchasedby Honeywell in 2020), SmartSolve from IQVIA, andMasterControl QMS.
LMS

A-CELL✼175

A learning management system (LMS) is used for theadministration, documentation, tracking, reporting,automation, and delivery of educational courses, training
programs, or learning and development programs.
Learning management systems are designed to
identify training and learning gaps, utilizing analytical data and reporting. LMSs are focused on online
learning delivery but support a range of uses, acting asa platform for online content, including courses, bothasynchronous- and synchronous-based. An LMS offers
classroom management for instructor-led training or aflipped classroom, used in higher education, but not in
the corporate space. Modern LMSs include intelligent
algorithms to make automated recommendations forcourses based on a user’s skill profile, and to extract
metadata from learning materials to make such recommendations more accurate.
The most notable LMS in this space is ComplianceWire.

Data systems
Data systems are key to the collection, storage, andsharing of manufacturing data. Newer data systems additionally add functions for the analysis of data as well as
sharing and collaboration of both data and insights garnered from it. For this simplified grouping, data systems
would include manufacturing execution systems (MES),
laboratory information management systems (LIMS),
process information management systems (PIMS), anddata historians.
MES/EBR

A Manufacturing Execution System (MES) is a computerized system used in manufacturing to instructand document the transformation of raw materials
to finished goods. An MES provides information thathelps manufacturing decision makers understand how
current conditions on the plant floor can be optimized
to improve production output. These systems work inreal-time to enable the control of multiple elements of the
production process (e.g., inputs, personnel, machines,
and support services). In cell-based therapies, theMES covers all steps in the manufacturing process andtypically it helps in reducing cycle time of the process
along with reviewing only critical deviations ensuring
compliance and data integrity to batch records. An MES

Chapter 8: Manufacturing of Cell-Based Therapies

includes electronic batch records (EBR), albeit MES ismuch more, and operates across multiple function areas
such as management of product definitions across the
product life cycle, resource scheduling, order execution and dispatch, production analysis and downtime
management for overall equipment effectiveness (OEE),
product quality, or materials track and trace. The MES
creates the EBR by capturing the data, processes, andoutcomes of the manufacturing process, and often can
result in elimination of paper batch records.
MES systems in the cloud can integrate with almost
any system and can be used as a source of truth for shop
floor manufacturing. MES or digital EBR systems in thecloud are advantageous for cell-based therapy manufacturers as they control the chain of identity (COI), chain ofcustody (COC), chain of condition, and time-to-delivery,
which are critical aspects for cell-based therapy manufacturers in the cell and gene space. MES applications
seamlessly integrate with supply chain orchestration(SCO) platforms in absence of a formal ERP system. An
MES system also keeps track of CQAs, critical processparameters (CPP), QTPP, and integrates with a PIMS forcross-product analysis of these parameters.
The most notable MESs in the life sciences space
include Körber Pharma (formerly Werum IT Solutions)
PAS-X, POMSnet Aquila from POMS, Emerson Syncade,and Rockwell FactoryTalk Pharma Suite.
LIMS

A laboratory information management system (LIMS)
is designed to improve laboratory productivity and efficiency by keeping track of data associated with samples,
experiments, laboratory workflows, and instruments. A
LIMS can feature specialized capabilities for researchand development laboratories, process development andmanufacturing laboratories, or bioanalytical laboratories.
The LIMS can also act as one source when using the foureyes principle, automating workflows and tracking all theimportant sample information, data, workflows, and QA/
QC results a lab generates each day.
The most common LIMSs in the life sciences space
include LabVantage, Thermo-Fisher LIMS, StarLIMS by
Abbott, and Labware.

A-CELL

✼

176

PIMS

Operational systems

A process information management system (PIMS) is
employed for process monitoring from the collectionof unique raw materials during apheresis through batch
production to clinical endpoint data. A PIMS supports
adherence to regulatory authority requirements like
continued process verification (CPV) by providing analytics for monitoring and reporting capabilities as wellas supporting investigations. These systems are unique
as one of the few systems designed to curate data frompaper, spreadsheets, and other data sources existing at
all levels of the organization. Common examples include
curation of data from paper batch records with data froma LIMS, ERP, Historian, and QMS.
One cell-based therapy usage of a PIMS system is for
management of V2V (vein-to-vein). The PIMS allows
linking blood characteristic information collected fromblood donations, the resulting manufactured materials
quality information, and non-patient identifying efficacy
data from hospital medical records to allow for visibility
into how the therapies are performing along with any
possible correlations of adverse effects due to manufacturing quality problems or raw materials.
Two popular PIMS providers include Skyland PIMS
and BIOVIA Discoverant.

Operational systems include automation and typicallyfollows ANSI S88 standards set by the industry. The
most common systems in this group are the supervisory control and data acquisition (SCADA) system,
distributed control system (DCS), data historian system,
environmental monitoring system (EMS), building
automation system (BAS), and equipment monitoring
systems. These systems use sensors placed across theentire plant to collect data and allow users to makeinformed decisions on the floor. Commonly used in
traditional pharmaceutical and biotech industries, thesesystems have gained prominence in the cell and genetherapy space as well. They are highly complex systems
that require an automation engineer and high capital tosupport the high operating cost.
For cell-based therapy manufacturers, althougheach of these is important, we cover the EMS and
BAS as these are prevalent at every cell-based therapy
manufacturer who has reached Phase 1 clinical trials.
DCS and SCADA systems are often needed for muchlarger scale processes such as large and small molecule
manufacturing.

Data historian

A data historian (also known as a process historian oroperational historian) records and retrieves production
and process data by time; it stores the information in atime-series database that can efficiently store data withminimal disk space and fast retrieval. Time-series information is often displayed in a trend or as tabular dataover a time range. As cell-based therapy sponsors accumulate large amounts of data from complex equipment,a data historian comes in handy where a cloud-based
system can integrate with any equipment and store thedata in real time (microseconds). Commercial off-theshelf data historians have several advantages over homegrown systems and off-the-shelf relational databases
such as off-the-shelf data acquisition interfaces with
control systems.
The most notable data historian systems include
OSIsoft PI (purchased by Aveva) and Rockwell
FactoryTalk Historian.

Chapter 8: Manufacturing of Cell-Based Therapies

EMS/BAS

Cell-based therapy manufacturers use an environmental monitoring system (EMS) for managing controlledenvironments as it is designed to evaluate, manage, andreduce the risks associated with everyday environmental
monitoring. An EMS controls the whole process, not justscheduling for individual sampling points; it also captures
all critical quality parameters for environmental controls.
These systems integrate with LIMS, MES, instrumentation,and mobile sample acquisition in a paperless environment.
Some key features include utilities monitoring, viable and non-viable sampling, comprehensive reporting
capabilities, automated investigation management androot cause analysis, facility map for visualization of
positive counts, integration to particle counters and air
samplers, and integration to process monitoring and QC
lab instrumentation.
Several notable EMSs include Novatek EM, ThermoFisher EM, and Siemens Life Science Laboratory.
Building automation systems (BAS) connect andautomate functions inside a building including lighting,

A-CELL✼177

HVAC, fire, and security systems. They can additionally monitor facility assets that may directly impact
environmental monitoring, such as doors that separate
environmentally sensitive spaces.
BAS vendors include Honeywell, Johnson Controls,
Schneider Electric, United Technologies, Emerson
Electric, and Siemens among other providers.
ENTERPRISE SYSTEMS RECOMMENDATIONS

For a commercial manufacturer annually handling
30,000+ patients, most of these enterprise systems are
critical as the volume of patients is high and it is crucial
that the entire business works in coherence to remove
silos and manage all data electronically in the cloud.
For a preclinical or clinical trials manufacturer,
the initial patient volume is small; therefore, only an
MES or EBR, PIMS, LIMS, DMS, and environmental
monitoring system are needed to minimize reliance on
paper and spreadsheets. From experience, these are theonly systems early-stage manufacturers use and havesignificantly progressed in their clinical trials while
capturing the relevant regulatory data at each stage.
IT CONSIDERATIONSRegardless of differences in specific technologies utilized
for the manufacturing process, a consistent and enduring challenge for every cell and gene therapy company is
the collection, storage, retrieval, analysis, and reportingof the data. When selecting technologies to employ,considerations into cost, resource investment, singleor multi-vendor, off-the-shelf (OTS) versus custom
solutions, schedules, and spreadsheets come into play.
Firms rarely go all in from the beginning with respect
to the IT environment. Instead, firms often purchasepiecemeal to solve “problems at hand,” incurring tremendous burdens on the organization as they take evolutionary steps forward. The best practice to minimize
costs and burdens is to envision the idealized final statefor data management and reporting, and then design
the steps necessary to reach that final state. Creating an“IT Maturity Model” is critical for planning and guiding
this IT investment roadmap.
A common problem to consider while developing thisroadmap is deciding to take the lowest cost, simplest, andseemingly easiest path forward for managing data (e.g.,

Chapter 8: Manufacturing of Cell-Based Therapies

Microsoft Excel). Putting 21 CFR Part 11 and validation
concerns aside, this decision also puts a burden on gathering, entering, contextualizing, tracking, and creating
the analytics for all that gathered data. Invariably, Tech
Ops leadership realizes that a large percentage of timeand resources are spent on managing spreadsheets.
At this point, it is time to consider new systems. An
obvious problem is the legacy data in the spreadsheetsthat will later need to be available for analysis in the
new tools. While Excel is often the easy decision at
start-up, as companies climb up their maturity model toearly-stage product development, they should consider
the critical timing for cutover to automated systems tokeep resources on revenue generating activities, rather
than spending months trying to unravel spreadsheets,
or worse—paper, with historical data.
As with every company, management of human capital (captive or outsourced) and human resources are IT
costs best optimized. It is recommended that companies
consider technology decisions that lower costs involving human capital to allow focusing funds on core IP
development and manufacturing. Due to advancements
in security architectures, cloud technologies are rapidly
being adopted in the life sciences arena, allowing firms
to redirect precious resources from large and expensive
IT data centers to more important IP challenges thatbring value to the business.
Whether a company should incorporate a single-vendor or multi-vendors with integration is important for the future of the business. Since the life
sciences industry tends to swing back-and-forth onwhich vendor approach is ideal, one should consider
the risks and benefits of both paths forward. If partnering with a single vendor, the therapeutic developer
should take into account whether the vendor can prioritize their needs given much of the control has beenplaced in the provider’s hands. Selecting a multi-vendor
approach mitigates this risk and provides flexibility to
the therapeutic developer, however integration among
vendors could be an issue and therefore needs to beevaluated thoroughly.
Deciding whether to use OTS or custom solutions
can be difficult. An OTS solution comes at a fractionof the cost of a custom solution but gives everyone thesame product, therefore capabilities rarely provide 100%

A-CELL✼178

of a therapeutic developer’s desired needs. However,OTS solutions can usually be installed and be ready to
run much faster, allowing a company to extract value
from these solutions sooner (often referred to as timeto-value). Additionally, the product vendor provides new
functionality and system maintenance. As a best practice,therapeutic developers should perform a gap analysis
of critical business requirements against current andplanned features. If gaps are determined to be critical,
they should assess whether there are other ways to fill thegaps at lower cost. If a custom solution is deemed the desired approach, they need to determine what budgetarysacrifices are needed to justify this development and therespective ongoing support.
Schedules must also be considered when selecting
technologies to employ, i.e., how early a good practice(GxP) system needs to be up and running for eacharea of the business, and whether validation is needed.
If validation is required, one needs to understand theimpacts of deciding to validate at initial deployment orlater by working with the QA organization. Additionally,risk-based approaches to validation that further accelerate the software’s time-to-value should be explored.

Conclusions
This chapter presents the principles of cell-based therapy
manufacturing, which involves numerous steps and unit
operations, each associated with its own complexity.
In particular, considerations for manufacturing of an
example CAR-T product are described. The complexityof this process warrants the implementation of diverse
technologies throughout its manufacture to ensure an
appropriate product yield is achieved to meet the clinicaldose requirements, while the product quality, safety, andefficacy is maintained. Continuous advancements in
production technologies are being made in the cell therapy field to allow for increased yield, while maintaining
product quality. In addition to the platform innovations(e.g., new gene editing techniques, new expansion hardware, automated fill and finish platforms), advancements
are also being made to support process improvements
through increased process control, incorporation ofdata analytics, and automation. These are facilitated byinnovations in hardware such as new non-invasive sensor
development, imaging capabilities, and software improvements to complement these innovative technologies.

References
1

2

3

4

5

6

Pike N, Dargitz C, Weist M. Freeze it and forget it: the value of
having a robust formulation and cryopreservation strategy forcell therapy manufacturing. Thermo Fisher Scientific. 2020
Clarke D, Aragon M. Optimizing the quality of cell therapy
starting materials. RegMedNet. October, 2018. https://www.
regmednet.com/optimizing-the-quality-of-cell-therapy-starting-materials/
Siegler SE. Autologous CAR-T Cell Manufacturing: Current
Standing and Future Directions. Genetic Engineering & Biotechnology News. September, 2017. https://www.genengnews.
com/insights/autologous-car-t-cell-manufacturing-current-standing-and-future-directions/
Cell Collection Standards for Cell and Gene Therapies. https://www.standardscoordinatingbody.org/project-cell-collection-standards-for-cell-therapies
Closure analysis of a mock autologous cell therapy process.
BioPhorum, December 2021. https://www.biophorum.com/download/closure-analysis-of-a-mock-autologous-cell-therapy-process/Andrews T. The standardization of single-use components
for bioprocessing. CPC White Paper 7003. 2017. https://www.

Chapter 8: Manufacturing of Cell-Based Therapies

cpcworldwide.com/Portals/0/Library/Resources/Literature/WhitePapers/Documents/CPC_WhitePaper_standardization_of_single-use_components.pdf
7Pendlebury, D. CELL & GENE THERAPIES: A GUIDE TO
SINGLE-USE CONNECTIONS - 10 Transferable Lessons From
The Bioprocessing Industry. February 20188
Mirasol F. Connector Integrity in Single-Use Biomanufacturing
Systems. BioPharm International 30 (11) 20179
USP <665> Plastic Materials, Components, and Systems Usedin the Manufacturing of Pharmaceutical Drug Products andBiopharmaceutical Drug Substances and Products
10 Best practices guide for evaluating leachables risk from polymeric single-use systems. BioPhorum, August 2021. https://www.
biophorum.com/resource/extractables-and-leachables
11 Cell Therapy Process Manufacturing Maps. BioPhorum, September 2020. https://www.biophorum.com/download/cell-therapy-process-map/
12

Lane N, Fuller BJ, Benson EE (Eds.). (2004). Life in the Frozen
State (1st ed.). CRC Press. https://doi.org/10.1201/9780203647073

A-CELL

✼179

References, continued
13

14

15

16

17

18

19

20

21

22

23

24

Fry LJ, Querol S, Gomez SG, McArdle S, Rees S, Madrigal JA.
Assessing the toxic effects of DMSO on cord blood to determine
exposure time limits and the optimum concentration for cryopreservation. April 2015. https://doi.org/10.1111/vox.12267
Anqi Li, Gina D. Kusuma, Dawn Driscoll, Nathan Smith, Dominic M. Wall, Bruce L. Levine, David James, Rebecca Lim, Advances in automated cell washing and concentration. Cytotherapy
23(9), 2021, 774-786. https://doi.org/10.1016/j.jcyt.2021.04.003.
Powell DJ Jr, Brennan AL, Zheng Z, Huynh H, Cotte J, Levine
BL. Efficient clinical-scale enrichment of lymphocytes for use
in adoptive immunotherapy using a modified counterflow centrifugal elutriation program. Cytotherapy. 2009;11(7):923-935.
doi:10.3109/14653240903188921
Assmus B, Tonn T, Seeger FH, et al. Red blood cell contamination of the final cell product impairs the efficacy of autologous
bone marrow mononuclear cell therapy. J Am Coll Cardiol.
2010;55(13):1385-1394. doi:10.1016/j.jacc.2009.10.059
Bryn T, Yaqub S, Mahic M, Henjum K, Aandahl EM, Taskén
K. LPS-activated monocytes suppress T-cell immune responses
and induce FOXP3+ T cells through a COX-2-PGE2-dependent
mechanism. Int Immunol. 2008;20(2):235-245. doi:10.1093/intimm/dxm134
Künkele A, Brown C, Beebe A, et al. Manufacture of Chimeric Antigen Receptor T Cells from Mobilized Cyropreserved
Peripheral Blood Stem Cell Units Depends on Monocyte
Depletion. Biol Blood Marrow Transplant. 2019;25(2):223-232.
doi:10.1016/j.bbmt.2018.10.004
Stroncek DF, Lee DW, Ren J, et al. Elutriated lymphocytes for
manufacturing chimeric antigen receptor T cells. J Transl Med.
2017;15(1):59. Published 2017 Mar 16. doi:10.1186/s12967-0171160-5
Berger TG, Strasser E, Smith R, et al. Efficient elutriation of
monocytes within a closed system (Elutra) for clinical-scale
generation of dendritic cells [published correction appears in J
Immunol Methods. 2005 Aug;303(1-2):152]. J Immunol Methods. 2005;298(1-2):61-72. doi:10.1016/j.jim.2005.01.005
Stroncek DF, Lee DW, Ren J, et al. Elutriated lymphocytes for
manufacturing chimeric antigen receptor T cells. J Transl Med.
2017;15(1):59. Published 2017 Mar 16. doi:10.1186/s12967-0171160-5
Jeon H, Jundi B, Choi K, et al. Fully-automated and field-deployable blood leukocyte separation platform using multi-dimensional double spiral (MDDS) inertial microfluidics. Lab Chip.
2020;20(19):3612-3624. doi:10.1039/D0LC00675K
Trickett A, Kwan YL. T cell stimulation and expansion using
anti-CD3/CD28 beads. Journal of Immunological Methods,
2003;275(1–2):251-255. ISSN 0022-1759. https://doi.org/10.1016/
S0022-1759(03)00010-3
Nagai S, Azuma M. The CD28–B7 Family of Co-signaling
Molecules. Co-signal Molecules in T Cell Activation. Advances
in Experimental Medicine and Biology, 2019;1189:25-51. https://doi.org/10.1007/978-981-32-9717-3_2

Chapter 8: Manufacturing of Cell-Based Therapies

25

26

27

28

29

30

31

32

33

34

35

36

37

Appleman LJ and Boussiotis VA. T cell anergy and costimulation. Immunological Reviews, 2003;192:161-180. https://doi.
org/10.1034/j.1600-065X.2003.00009.x
Smith-Garvin JE, Koretzky GA, Jordan MS. Annual Review of
Immunology 2009;27:1, 591-619. doi: 10.1146/annurev.immunol.021908.132706
Schwab R, Crow MK, Russo C, Weksler ME. Requirements
for T cell activation by OKT3 monoclonal antibody: role ofmodulation of T3 molecules and interleukin 1. J Immunol.
1985;135(3):1714-1718
Dull T, Zufferey R, Kelly M, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol.
1998;72(11):8463-8471. doi:10.1128/jvi.72.11.8463-8471.1998
David M. Barrett, Xiaojun Liu, Shuguang Jiang, Carl H. June,
Stephan A. Grupp, and Yangbing Zhao. Human Gene Therapy.
Aug 2013. 717-727. http://doi.org/10.1089/hum.2013.075
Roth, T.L., Puig-Saus, C., Yu, R. et al. Reprogramming human
T cell function and specificity with non-viral genome targeting.
Nature 559, 405–409 (2018). https://doi.org/10.1038/s41586-0180326-5
David C. Bishop, Leighton E. Clancy, Renee Simms, Jane
Burgess, Geetha Mathew, Leili Moezzi, Janine A. Street, Gaurav
Sutrave, Elissa Atkins, Helen M. McGuire, Brian S. Gloss, Koon
Lee, Wei Jiang, Karen Maddock, Georgia McCaughan, Selmir Avdic, Vicki Antonenas, Tracey A. O’Brien, Peter J. Shaw,David O. Irving, David J. Gottlieb, Emily Blyth, Kenneth P.
Micklethwaite; Development of CAR T-cell lymphoma in 2 of
10 patients effectively treated with piggyBac-modified CD19
CAR T cells. Blood 2021; 138 (16): 1504–1509. doi: 10.1182/
blood.2021010813
Martin P. Stewart, Robert Langer, and Klavs F. Jensen. Chem.
Rev. 2018, 118, 16, 7409–7531. Publication Date: July 27, 2018.
https://doi.org/10.1021/acs.chemrev.7b00678
Hilly-Foster J, Steger K, Carmen J, Shivakumar R, Brady J. Bolstering Therapeutic Efficacy and Speed to Clinic Using Non-viral
T- & NK-cell Engineering. MaxCyte, Gaithersburg, MD, USA
Rurik JG, Tombácz I, Yadegari A, Méndez Fernández PO,
Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK,
Mui BL, Albelda SM, Puré E, June CH, Aghajanian H, Weissman
D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat
cardiac injury. Science. 2022 Jan 7;375(6576):91-96. doi: 10.1126/
science.abm0594
Billingsley MM, Singh N, Ravikumar P, Zhang R, June CH,
Mitchell MJ. Ionizable Lipid Nanoparticle-Mediated mRNA
Delivery for Human CAR T Cell Engineering. Nano Lett.
2020;20(3):1578-1589. doi:10.1021/acs.nanolett.9b04246
Desai N. Challenges in development of nanoparticle-based
therapeutics. AAPS J. 2012;14(2):282-295. doi:10.1208/s12248012-9339-4
Tia DiTommaso et al., Cell engineering with microfluidic
squeezing preserves functionality of primary immune cells
in vivo. Proc. Natl Acad. Sci. USA. 2018; 115 (46). https://doi.
org/10.1073/pnas.1809671115

A-CELL

✼180

References, continued
38

39

40

41
42

43

44

Hanson J, McNally E, Foley K, et al. SQZ Microfluidic Platform
Enables Delivery of Gene Editing Materials and Transduction of
Difficult to Engineer Cell Types While Preserving Cell Phenotype and Function. Poster or Paper presented at: Keystone
Symposia: Engineering the Genome 2020
O’Dea S, Annibaldi V, Gallagher L, et al. Vector-free intracellular delivery by reversible permeabilization. PLoS One.
2017;12(3):e0174779. Published 2017 Mar 30. doi:10.1371/journal.pone.0174779
Saba Ghassemi, Selene Nunez-Cruz, Roddy S. O’Connor, Joseph
A. Fraietta, Prachi R. Patel, John Scholler, David M. Barrett,Stefan M. Lundh, Megan M. Davis, Felipe Bedoya, Changfeng Zhang, John Leferovich, Simon F. Lacey, Bruce L. Levine,
Stephan A. Grupp, Carl H. June, J. Joseph Melenhorst, Michael
C. Milone; Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells. Cancer Immunol Res 1 September 2018; 6 (9): 1100–1109. https://doi.
org/10.1158/2326-6066.CIR-17-0405
Ori Biotech. Insights. https://oribiotech.com/insights
Bowles P, Dalle Fusine I, Gratch YS, et al. A closed, integrated, and modular t-cell manufacturing strategy. Cytotherapy.
2018;20(5):S109. ISSN 1465-3249. https://doi.org/10.1016/j.
jcyt.2018.02.321
Briggs M. Cell expansion using Xuri W25. Presented at 8thTechnical Meeting of the Advanced Therapy Medicinal Product(ATMP) Manufacturing Community. February, 2014. http://
atmpmanufacture.org/wp-content/uploads/2016/10/GE_Mark_
Briggs.pdf
Nogueira DES, Cabral JMS, Rodrigues CAV. Single-Use Bioreactors for Human Pluripotent and Adult Stem Cells: Towards
Regenerative Medicine Applications. Bioengineering. 2021;
8(5):68. https://doi.org/10.3390/bioengineering8050068

Chapter 8: Manufacturing of Cell-Based Therapies

45

46

47

48

49

50

51

Mirabel C, Puente-Massaguer E, Del Mazo-Barbara A, et al.
Stability enhancement of clinical grade multipotent mesenchymal stromal cell-based products. J Transl Med. 2018;16(1):291.
Published 2018 Oct 24. doi:10.1186/s12967-018-1659-4
Hoogendoorn KH, Crommelin DJA, Jiskoot W. Formulationof cell-based medicinal products: a question of life or death?
Journal of Pharmaceutical Sciences. 2021;110(5):1885-1894.
doi:10.1016/j.xphs.2020.07.002
Khuu HM, Cowley H, David-Ocampo V, et al. Catastrophic
failures of freezing bags for cellular therapy products: description, cause, and consequences. Cytotherapy. 2002;4(6):539-549.
doi:10.1080/146532402761624700
Thyagarajan B, Berger M, Sumstad D, McKenna DH Jr. Loss of
integrity of umbilical cord blood unit freezing bags: description and consequences. Transfusion. 2008;48(6):1138-1142.
doi:10.1111/j.1537-2995.2008.01688.x
US Food and Drug Administration. Draft Guidance forIndustry: Considerations for the Development of Chimeric
Antigen Receptor (CAR) T Cell Products. March 2022. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/considerations-development-chimeric-antigen-receptor-car-t-cell-products
Baboo J, Kilbride P, Delahaye M, et al. The Impact of Varying
Cooling and Thawing Rates on the Quality of Cryopreserved
Human Peripheral Blood T Cells. Sci Rep 9, 3417 (2019). https://doi.org/10.1038/s41598-019-39957-x
U.S. Food and Drug Administration. Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic
Cell Therapy Investigational New Drug Applications (INDs).
Section IV,E,2. April 2008. https://www.fda.gov/media/73624/
download

A-CELL✼

181

Chapter 9

Analytical Technologies
for Cell Therapies
CHAPTER LEADS

Rupa Pike, Thermo Fisher Scientific
Taylor Zhang, Sana Biotechnology
CHAPTER CONTRIBUTORSMichael Sadick, Precision BioSciences
Joseph Laning, Astellas
Anja Fritsch, Confarma

Chapter 9 | Contents
Background......................................................................... 183

Replication competent lentivector............................190

Critical Quality Attributes (CQA) and Quality by Design(QbD).................................................................................... 184

Vector copy number....................................................191
Identity (610.14)................................................................191

Approaches to Analytical Assay Development.............. 184

Purity (21 CFR 610.13).......................................................191

Phase appropriate, fit-for-purpose assay development
approach.......................................................................... 184

Molecular Assays to Confirm Gene Editing.................... 192

Compendial assays......................................................... 185

Potency (610.10)...............................................................191
Genomic Characterization................................................ 193

Pathway for assay/method validation........................... 185

Impurities............................................................................ 193

General guidance on assay development,
qualification, and validation........................................... 186

Cell Line Identity................................................................. 193

Categories of Analytical Assays....................................... 186

In-use stability................................................................. 194

For information only (FIO) assays.................................. 186

Stability................................................................................ 194
Stability testing to support shelf life............................. 194

Analytical Assay for Cell Characterization..................... 188

Process Analytical Technologies (PAT)........................... 194

Safety (microbiological safety,
610.12, 610.13,610.40)........................................................188

Non-invasive and non-destructive in-process testing

Sterility........................................................................ 188

for cellular analysis......................................................... 196

Pyrogen testing........................................................... 189

Process analytical technologies for media nutrientsand metabolites................................................................197

Mycoplasma................................................................190

Conclusions..........................................................................197

Gram stain...................................................................190

References........................................................................197

Background
Cell-based therapies play a vital role in transforming
human health as they offer solutions for many unmet
medical needs. The commercial launch of CAR-T
products Kymriah™, Yescarta™, Tecartus™, Breyanzi™,
Abecma™, and most recently Carvykti™ generated
tremendous excitement and have resulted in moretherapies advancing through clinical trials towards
commercial approval. These advancements have fueled the need to develop robust and well-characterized
analytical methods that are critical for demonstrating
safety, identity, purity/impurity, quantity/strength, andpotency of the final products. Effective analytical technologies also play a vital role in choosing raw materials,
controlling the manufacturing process, determining
the quality of intermediates, and ensuring a safe andeffective final product.

Chapter 9: Analytical Technologies for Cell Therapies

Cell-based therapies are unique and are often described as “the process is the product” where the final
product is comprised of living cells. The complexities associated with cell therapy manufacturing require a broad
set of analytical tools. It is critical to develop and employ
analytical assays early in the process development stage
since they provide insights into the attributes of the cells
throughout the manufacturing process, and help enable
process changes and control of the final product.1
This chapter focuses on the approach to analyticalassay development, progress of analytical technologies,
and challenges related to process and product characterization for cell-based therapies. While this chapter
uses a CAR-T product as an example to illustrate thevarious analytical technologies used during manufacturing and final product testing, many of the principles
outlined here are applicable and beneficial to a broader
profile of cell therapy products.

A-CELL✼183

Critical Quality Attributes (CQA) andQuality by Design (QbD)
According to ICH Q8, critical quality attributes (CQAs)
can be defined as the physical, chemical, biological, or
microbiological properties of the cell product that should
be within an appropriate limit, range, or distribution toensure the desired product quality. These properties are
identified early in the product development process.
Their criticality is determined using quality by design(QbD) principles, as outlined in Chapter 4, and arecontinuously refined throughout the development bycharacterization studies. Successful characterizationof these attributes through implementation of analytical technologies depends on product understanding,including:• Interlink between CQAs of the product (including
potential CQAs from early product development)
and analytical methods• Performance attributes of the process• Performance attributes of the methods• Key characteristics of the various starting raw andin-process materials
In the case of cell therapies, it is vital to develop analytical assays that build upon the relationships between
donors’ or patients’ starting materials, raw material
variables (media, supplements, cytokines/growth factors, extracellular matrix substrates), product variables(expected mechanism of action, cell phenotype, marker
expression, proliferation profiles, metabolic profile,apoptosis, etc.), and process variables (medium perfusion
or exchange rate, feeding regime, pH, dissolved oxygen,etc.). This is to ensure that product quality is maximized
and CQAs are maintained within the expected ranges
throughout the product life cycle. Such strategies involve
designing several specific assays during the early process development stage and subsequently refining andstreamlining select assays for good manufacturing practice (GMP) as the product’s CQAs and characteristics,
and the manufacturing process are better defined. This
ensures standardization and reproducibility, reduces deviations, increases quality, and consistently meets releasecriteria for the final product. This also ensures a smooth

Chapter 9: Analytical Technologies for Cell Therapies

transition from clinical to commercial manufacturing.
Some unique challenges associated with cell therapies
include inherent variability in the starting material, lackof reference materials, limited quantity of manufactured
lots, multiple and diverse mechanism of actions, paucity
of cells available for quality control (QC) and stability
testing, and inability to sterilize the final product. Despite
several advancements, cellular therapeutics often experience challenges and delays in clinical trials due to a lackof suitable analytical methods that are reproducible andmeet the regulatory standards.

Approaches to Analytical Assay
Development
PHASE APPROPRIATE, FIT-FOR-PURPOSE ASSAY
DEVELOPMENT APPROACH

Fit-for-purpose is a commonly used phrase when discussing method validation. The concept maintains thatthe level of validation should be tailored to an assay’s
intended use. For example, qualification is acceptablefor early development, but full validation is required for
the Biologics License Application (BLA) or Marketing
Authorization Application (MAA) stage. The decision
to develop fit-for-purpose assays should be made in theearly stages of product development. Fit-for-purpose assays in cell therapy are designed for their usability by the
QC and development labs. Figure 9-1 shows an example
of an assay development pathway. It is important to notethat the exact activities for each phase can vary based on
the therapeutic program, for example Phase II might beperformed separately prior to the pivotal Phase III.
Bioassays are a good example to consider in a discussion of fit-for-purpose assays as they have inherent
variability, require extended culture periods, use multiple(variable) cell types, and generate more than one readout.
While at times it may be necessary to validate a complex
bioassay, it is worth considering in early product development whether there are simpler alternative assays or
surrogate assays that are easier to develop in a shorter
time. Using several assays during early development
may help to identify orthogonal methods that may be
more straightforward to validate.3 This approach could
also provide several options for an assay that adequately
controls potency, instead of “starting from scratch” after

A-CELL

✼184

Figure 9-1: Traditional assay life cycle development roadmap2
Pre-clinical &non-GLP Tox

GLP Tox/
IND Enabling

Phase I/IIa

Initial/Immediate Assay

Next-level Assay

Early Phase Qualification• Enable early work

• Method selection,
development, andoptimization• Limited repetition (‘n’)• Limited parameters• Wider acceptance criteria

More reflective of MOA;
high potential for goodprecision, accuracy androbustness.

Data trending, with eye
towards criteria to be usedin late phase validation.

• Candidate selection
Minimal development
desired; possibly notprecise or robust.

an assay is deemed inadequate late in development. These
could then be bridged to or complemented by moreelaborate potency methods, with subsequent validation
prior to Phase III.
Assay or method qualification typically happens during
early clinical phases. During qualification, understanding an assay’s key performance parameters (specificity,
sensitivity, accuracy, precision, etc.) is vital, however,there is no pre-set acceptance criteria and robustness is
not studied in detail. Complete validation is performed
after the manufacturing process is “locked down” andthe product is in its final matrix because the validation
process is matrix dependent. This typically happens prior
to initiation of clinical Phase III studies or before the BLA/
MAA stage. Analytical technologies demand a strategic
approach for assay qualification and validation due tointra- and inter-assay variabilities and intrinsic differences
between distinct cell types. Performing a risk assessment
exercise early in the development helps identify the CQAs
(this topic is addressed in Chapter 4), and ultimately helps
inform the timing of qualification and validation for thecorresponding release assays. For “for information only”(FIO) assays (discussed in later sections), such risk assessments can inform whether validation is necessary.
COMPENDIAL ASSAYS

Only limited qualification is required for compendial
assays as they are driven by strict pharmacopeial (USP/
EP) requirements. For assays such as appearance, pH,
and osmolarity, only a single qualification run with theappropriate reference material is required before conducting a GMP sample analysis.

Chapter 9: Analytical Technologies for Cell Therapies

Phase III
& BLA

Late Phase “Phase­appropriate validation”• Full number repetition (‘n’)• All parameters• More stringent
acceptance criteria (based
upon generated data)• Robustness testing

Post-licensure

Data trending andmethod performance
evaluations• Method evaluations
and comparability,with revalidation asnecessary

PATHWAY FOR ASSAY/METHOD VALIDATION

The pathway to assay validation is illustrated in
Figure 9-1, and is typically achieved through the following steps:• Protocol-driven exercise with predefined acceptance criteria• Non-compendial method validation• Validation according to ICH Q2 guidelines
For validation, the following parameters are generally
evaluated:• Specificity• Linearity• Accuracy• Precision (repeatability, reproducibility, and intermediate precision)• Range• Limit of quantitation (LoQ)• Limit of detection (LoD)• Compendial method validation (verification of
product with the method)
It should be noted that, depending on the purpose of
the assay, not all parameters are required for validation.
For example, limit of detection is often not required forpotency and purity.
It is often challenging to follow the roadmap presented in Figure 9-1 for analytical assay developmentin cell therapies. Some of the challenges include starting material (e.g., patient sample) variability, limited

A-CELL

✼185

numbers of lots and small lot sizes for autologous products, inherent variability of the production process,variability in potency, and product stability concerns.
This is especially true for autologous therapies as the
final product is administered in small lot sizes to one
patient. Typically, only a small amount of in-housereference material is available for conducting multiple
tests and the distinctive nature of cell therapy makes itchallenging to develop commercial reference standards
for long-term use.
GENERAL GUIDANCE ON ASSAY DEVELOPMENT,QUALIFICATION, AND VALIDATION

After an analytical method is selected for product testing,it evolves through development and optimization to
become qualified for use in clinical trials.4
1. Qualify and validate high-risk assays that measure safetyparameters (e.g., mycoplasma and endotoxin tests) for
use in humans.
2. During process development, qualified assays should becontinuously subjected to re-optimization or requalification if procedural adjustments are necessary tomaintain suitable performance.
3. Before method validation, performance history
should be established to predetermine acceptancecriteria for indicated validation parameters.
4. A complete GMP validation of assays used forrelease, stability, and in-process testing is required forsubmitting a BLA or MAA. Ideally, assays should bevalidated prior to initiation of pivotal clinical studies.
When changing an assay, a risk assessment should be performed to determine how the assay change impacts evaluation of the cell product. The requalification strategy of assays
upon change is dependent on the degree of the change.5

Categories of Analytical Assays
Analytical assays can be grouped into distinct categories:n Use of assays at specific stages during the manufac-

turing process. These include analytical assays for:• Starting material (e.g., apheresis product, cord blood,bone marrow, cell lines)

Chapter 9: Analytical Technologies for Cell Therapies• Intermediate material (in-process analytics)• Final product (identity, purity, impurities, potency,
and safety)—these tests are also used to determine
product stability
n Types of assays needed to characterize various proper-

ties of the cells as a final product. These include:• Cellular properties (cell number, cell size, cellmorphology, phenotype, genomic stability)• Biochemical properties (cell viability, metabolism,
gene and protein expression)• Cellular responses and functions (cell activation,
cell proliferation/expansion, cell differentiation,
cell function such as cytokine secretion, antigen
recognition, and cytotoxicity)

For CAR-T manufacturing, the FDA recommends
appropriate in-process testing to be conducted at relevant process steps to achieve and maintain control ofthe manufacturing process.5 Results from these tests
can be used to guide manufacturing decisions at criticalsteps, such as when to change media or harvest the cells.
In-process testing regimens for CAR T cells typicallyassess multiple parameters (e.g., viability, cell number,cell phenotype, CAR expression). Figures 9.2-9.3 depict
an example testing paradigm for an autologous CAR-T
product throughout the manufacturing process. Details
on each analytical assay are described in the subsequent
section.
It is important to note that the exact testing strategy
for every product is different depending on the indication of interest and modality, therefore, risk assessment
is critical during process development to determine
which tests are necessary at which time points. If certain
impurities at a certain process step are considered highrisk, assays to identify or quantify them need to be doneat that specific point during the process.
FOR INFORMATION ONLY (FIO) ASSAYS

Oftentimes a sponsor or manufacturer may decide to use
assays or tests that are under development to evaluate product characteristics or method performance capabilities.
These tests are designated as “for information only” (FIO).
FIO assays, while not used for final product lot release,are vital for understanding additional quality attributes of

A-CELL

✼186

Figure 9-2: Autologous cell therapy testing paradigm (cell preparation to expansion)
Process Step
Cell thaw(optional, if
frozen postcollection)

Testing

Cell
preparation(debulk/
enrichment)• Cell counts• Cell viability• Safety
Endotoxin
Gram Stain
Sterility
Mycoplasma(optional)

Cell
Selection• Cell counts• Cell viability• Purity
RBC, platelet
depletion, %WBC
• Identity
T cell markers
to establish abaseline prior
to selection.

Cell
activation• Cell counts• Cell viability• Identity
CD4+,CD8+

the cellular product and the mechanism of action (MOA).
They are also helpful in gathering more information about
the key steps of the manufacturing process or final product. FIO assays are indispensable as backup assays if there
are problems validating existing release assays (e.g., theassay is too inherently variable, supply shortage, or discontinuation of critical materials). FIO assays can be tracked
throughout process changes to reinforce comparability orhelp identify noncrucial characteristics (e.g., determining
time-points in culture when cells stop producing a specificcytokine even if that does not affect its target function inpatients). After sufficient information is gathered, specification and release criteria can be redefined and qualified

Geneediting(viraldelivery)• Potency(optional)
CytokinereleaseCytotoxicity

Cellexpansion• Cell counts• Cell viability• Identity%CAR+• Cell counts• Cell viability• Purity
Process/productrelated impurities• Identity%CAR+CD3+• Potency
Cytokine release
Cytotoxicity

in later stages of clinical trials.
A phase-appropriate strategy may be considered for
the FIO assays. The purpose of Phase I is primarily toevaluate drug safety. If the FIO assay attributes could potentially impact patient safety, they should be well-characterized to ensure no adverse impacts on patient safety.
Health authorities may require the qualification of FIO
assays for such safety related attributes and implementation of the FIO safety assays on the certificate of analysis.
As part of the development, FIO assays could enable a
deeper understanding of MOA and contribute to theimplementation of MOA as they may be reflective ofpotency before the pivotal trials.

Figure 9-3: Autologous cell therapy testing paradigm (cell harvest to fill & finish)
Process Step
CellharvestDebeading(optional)

Wash andconcentration

Testing

Chapter 9: Analytical Technologies for Cell Therapies

Formulation,
fill and finish

Cryopreservation(optional)

Cell counts
Cell viability

Cell counts
Cell viability
Safety:Sterility
Mycoplasma
Endotoxin
Gram Stain
RCL
Vector copy number

PurityResidual
plasmid DNA
Residual beads
Contaminant
cell types
Identity%CAR+CD3+
Potency
Cytokine release
Cytotoxicity

A-CELL✼187

A cautionary approach in FIO assay development anddisclosure may prove beneficial because if the FIO assays
are deemed to be strong from a safety and potency perspective, the regulatory agencies may require the sponsor
to convert such FIO assays into final lot release assays.
If sponsors are not prepared for such a demand, it will
delay the regulatory submission timeline.

at every step of the manufacturing process and can beassured by testing of raw materials, testing of intermediate samples, minimizing contamination risks during
manufacturing, and testing the final product prior torelease. Safety testing is further categorized into sterility,
pyrogen testing, mycoplasma, gram stain, replicationcompetent lentivirus, and vector copy number.

Analytical Assay for Cell
Characterization

Sterility

The Code of Federal Regulations (CFR) is the codification of the general and permanent rules published in theFederal Register by the departments and agencies of theFederal Government. Title 21 of the CFR (CFR 21) is reserved for rules of the Food and Drug Administration. 21
CFR Part 610 is reserved for General Biological ProductsStandards that also applies to cell therapy products.
The analytical assay-related regulations in 21 CFRPart 610 can be subdivided into the following categories:n Safety (610.12, 610.13, 610.40)• Sterility• Pyrogen testing• Mycoplasma• Opportunistic viruses (adventitious agents)• Vector copy numbers• Replication-competent lentivirus (RCL)n Identity (610.14)• Specific test to distinguish it from other products
manufactured/tested at the same siten Purity (610.13)• Free of extraneous materialsn Potency (610.10)• Measurement of biological function/activity
n Constituent Materials (610.15)• Ingredients, Preservatives, Diluents, Adjuvants,
Excipients, Ancillary materials
SAFETY (MICROBIOLOGICAL SAFETY, 610.12,
610.13, 610.40)

Safety testing is conducted to confirm that the productis not contaminated with bacteria, mycoplasma, fungi,
adventitious viruses, endotoxins, or replication-competent viruses derived from viral vectors. Safety is critical

Chapter 9: Analytical Technologies for Cell Therapies

Sterility testing is required to demonstrate the absence of
viable micro-organisms. The sterility test is a conventional
test that is based on the growth of microorganisms (bacteria, yeast, and fungi) in two culture media incubated at
two temperatures with a final visual reading after 14 days.
The methodology of this test is harmonized between the
different pharmacopoeias (USP, Ph. Eur, and JP) since2010. As stated in Ph. Eur 5.1.1, sterility testing cannot
guarantee that the product is sterile but it does help to ensure that the validated and controlled production processprovides assurance of the sterility of the finished product.
This chapter presents some differences to consider
between cell therapy products to that of typical pharmaceutical drug products. Cell therapy products have one ormore of the following characteristics:• Short life span (hours to days)• Aseptic manufacturing with several steps, often
manual, in different locations (hospital, clinic,
therapy center)• Limited or very limited quantity of product• Each batch is unique (one patient, one batch) for
autologous products• Used in the context of treatment for life-threatened
patients (cancer, rare genetic diseases)• Very high manufacturing cost
• High or very high cell concentration• Thermosensitive• May not contain preservative agents or protection
against micro-organisms• The manufacturing process (temperature, media,
raw material, presence of oxygen) is very favorable
to microbial contamination
Most of these characteristics make the applicationof the traditional sterility test technically or economically impossible.

A-CELL✼188

Since 2010 and the evolution of alternative microbiology methods, several rapid sterility methods havebeen validated for the release of pharmaceuticals. Thesemethods are still based on growth but the result could
be accelerated by changing the reading method. The best
known are the Milliflex® rapid technique based on ATP
detection or the BACT/ALERT® or BD BACTEC™ thatis based on CO2 emitted during growth. With the emergence of these rapid microbiology methods, several guidance documents were published on the implementationof such an approach, e.g., the European Pharmacopoeia
Chapter 5.1.6, the Parenteral Drug Association (PDA)
Technical Report No. 33, and USP <1071>.
The ATP-based methods can interact with the cell
products and the CO2-based methods take more than
one week to produce a result. However, these methods have paved the way for the development of new
methods with guidelines on validation methodologies.
It is important to point out that validating alternative
methods remains complex and time consuming, andrequires expertise in several areas including statistics.
Alternative microbiology methods can solve the timeissue but do not address the challenges presented by theavailability of a limited number of samples. The European
Pharmacopoeia has started to address this issue of sample
quantity with a dedicated chapter, 2.6.27: MicrobiologicalControl of Cellular Products. In 2012, the FDA removed
the notion of minimum quantities to be tested from 21
CFR 610.12 and replaced it by a justification of the quantity to be tested. This point relating to quantities is still
unclear and is a crucial issue for the industry.
Based on experience and in accordance with the evolution of regulations, the sterility test must be based on thegrowth of microorganisms since non-growth techniquessuch as qPCR are not able to demonstrate the viabilityof a microorganism and its ability to grow. The second
important feature of the ideal method is the ability to investigate quickly in case of a positive test. Investigation is
key to understanding the source of the contamination andto take appropriate action if the product has already beendelivered to the patient. The third important feature of theideal method is automation of a data analysis process thatis specific to the microorganisms.
In this context, developing a hybrid method withprior enrichment and measurement of the evolution ofChapter 9: Analytical Technologies for Cell Therapies

the quantity of microorganisms by sensitive and specificmethods could be one solution, increasing sensitivity, reducing time-to-result, and limiting the amount of sample
needed for the analysis.

Pyrogen testing
For cell therapy products, the FDA Guidance for Industry“Chemistry, Manufacturing, and Control (CMC)
Information for Human Gene Therapy Investigational
New Drug Applications (INDs),” issued in January 2020,
requires testing for endotoxin to verify the absence ofpyrogenic contaminations. The EMA guidelines havea similar requirement. The endotoxin assay is basedon an enzymatic reaction of limulus amebocyte lysate(LAL) to endotoxin, which can be detected by clotting or
quantified using kinetic methods based on chromogenic
substrates. The composition of the products generally
allows testing according to USP <85>, EP 2.6.14, and JP
4.01 after simple dilution of the cell suspension to remove
interference with the assay. Control of in-process intermediates can be achieved with cartridge-based systems,
allowing generation of test results within minutes, while
testing for batch release is generally performed using
validated chromogen kinetic methods.
Testing for endotoxin as described in USP <85>, EP
2.6.14, and JP 4.01 is the primary assay used for measuring the presence of pyrogens, but there are other assay
formats emerging that provide relevant information on
the pyrogen content of a sample. The endotoxin test is
specific for endotoxins but pyrogens are a diverse groupof contaminants whose common denominator is theirability to induce a fever reaction after contact with humanmonocytes/macrophages, so testing for endotoxin alone
might not be sufficient to verify the absence of pyrogens.
Among those, microbial contaminations by gram-positivebacteria cause pyrogenicity that is not detectable with theendotoxin assay. EP 5.1.10 requires verifying the absenceof non-endotoxin pyrogens during the production process and in the final product as a prerequisite for using theendotoxin assay for batch release, while 21 CFR 610.13
requires demonstrating the equivalence of the chosen
test method to the rabbit pyrogen test, which effectively
also means that the absence of non-endotoxin pyrogens
must be demonstrated. For this demonstration, the rabbit
pyrogen test is universally accepted, but it is tedious and

A-CELL✼189

prone to misinterpretation due to individual differencesin the biological reaction of the test animals. A full in
vitro alternative is available with the monocyte activation test, where pyrogenicity is measured based on thecytokine expression of human monocytes/macrophages
after contact with the product. This method is also usefulfor evaluating the potential impact of bioburden on the
pyrogenicity of the cellular product.

Mycoplasma
Mycoplasma testing is performed to ensure that the
product is free from mycoplasma contamination. The
compendial methods for mycoplasma detection, as described in USP <63> and EP 2.6.7, are increasingly being
replaced by nucleic acid-based techniques (NAT) as they
provide faster results and, depending on the mycoplasma
strain, higher assay sensitivity.
Validation design guidelines for NAT are described in
EP 2.6.7, JP XVII “Mycoplasma Testing for Cell Substrates
used for the Production of Biotechnological/Biological
Products,” and general chapters EP 2.6.21 “Nucleic Acid
Amplification Techniques,” and USP <1223>.
Real-time polymerase chain reaction (PCR), also
called quantitative PCR (qPCR), is the preferred method
for cellular samples because this technique allows detecting almost all Mollicutes species that can contaminate cell
cultures, even fastidious strains that cannot be detectedwith the compendial culture method. Moreover, fast results (few hours to 1 day) can be obtained in comparison
to the culture method (28 days).
Despite its effectiveness for cell-based therapies products, some analytical points must be considered:• Kit or PCR reagents must detect a broad range of
mycoplasma (Mollicutes) species. More than 100
species is recommended.
• As mycoplasma can adhere to the cell surface, cells
must be tested. Testing of supernatant only mightlead to false-negative results.
• Due to the possible co-purification of cellular
DNA and potential mycoplasma DNA, PCR
chemistry must be carefully considered. It ishighly recommended to use hydrolysis probes
such as TaqMan® probes. Use of unspecific DNA
stains should be avoided to prevent unspecific

Chapter 9: Analytical Technologies for Cell Therapies

amplification and false-positive results.
• Cell concentration must be known for each test
and properly controlled. Depending on the chemistry and the primers/probe design, concentration
above 1-5 million cells per input volume maylead to PCR inhibition and sometimes to false(low)-positive results. A matrix-specific validation
by testing a defined cell concentration or a “worstcase” (highest) concentration is a good analytical
approach to prevent unexpected results.
• As qPCR does not guarantee a 100% results
reliability rate, using a back-up detection kit or
identification by DNA sequencing of the qPCR
amplicon are appropriate options.
• To detect as low as 10 CFU/mL as recommended
by Pharmacopeia, input volume tested should be
at least 1 mL (using mycoplasma standards with
GC/CFU ratios <10). If this volume is not available
for testing, use a “hybrid” mycoplasma method,combining enrichment steps by culture withsubsequent PCR detection.

Gram stain
Gram stain is a fast method to confirm the absence ofbacteria, either gram-positive or gram-negative bacteria.
The staining procedure reveals peptidoglycans present
in the bacterial cell walls and a positive stain indicatesmicrobiological contamination.

Replication competent lentivirus
Replication-competent lentivirus (RCL) could theoretically occur, but has not been observed with third-generation replication-incompetent self-inactivating (SIN)
vectors. Detection of the vesicular stomatitis virus G(VSV-G) gene in a drug product (DP) can be used as the
RCL surrogate. RCL can also be tested by the co-culture
method as part of the vector release specification.
The design and optimization of any PCR methods involves the selection of the primers, probes, PCR reaction
parameters, and PCR reaction reagents. Due to the natureof the qPCR reaction, it could be subject to potential PCR
inhibition from the matrix of the sample or preanalyticalprocesses. Alternatively, droplet digital PCR can be usedinstead of qPCR to partially alleviate the effects of matrix
interference and remove the need for standard calibration.

A-CELL

✼190

Vector copy number
Vector copy number (VCN) is a surrogate measure of
lentiviral transduction, which detects vector transgene
integration into the host cell genome. It is intrinsicallyrequired for the CAR protein expression and efficacy.
Lentiviral transduction presents a theoretical safety riskof insertional oncogenesis, thus VCN is also considered asafety assay.6 In the VCN method, genomic DNA (gDNA)
extracted from the DP is used as a template in a multiplexed, qPCR reaction to monitor vector integration.
IDENTITY (610.14)

The identity of the final product is established using
unique distinguishing characteristics to confirm that the
product contains the intended cellular and noncellularcomponents. The manufacturing process/workflow incell therapies can range from days to weeks with various
cell manipulations including but not limited to selection, activation, expansion, and cryopreservation. Cells
are subjected to various media, cytokines, and culture
conditions (static, dynamic, hypoxia, normoxia, etc.).
Such complex workflows can introduce both intended
and unintended changes. Identity testing is essential toaddress the concern that the unintended changes do notchange the biological nature and function of the cells or
lead to phenotypic drift in the final cell product.
Identity testing of cell therapy products includes theuse of flow cytometry as it is well established in the research community and has translated well to GMPs. Flowcytometry entails the staining of live (or fixed) cells withfluorescently labeled antibodies that recognize cell surface
or, in some cases, intracellular expressed proteins.1 For
example, all hematopoietic stem cells express CD34 andall T-cells CD3, Mesenchymal stem cells (MSCs), a very
common cell type in clinical trials, must be positive for
CD73, CD105, and CD90 and negative for CD45 andCD34. Other markers may also be tested for absence (e.g.,
CD14/CD11b, CD79alpha/CD19, and HLA-DR).
PURITY (610.13)

Purity testing is carried out to evaluate and confirm thatthe final product contains minimal undesired components, such as contaminating cell types, dead cells or
debris from dead cells, and residual process reagents that
may affect the purity of the cells.

Chapter 9: Analytical Technologies for Cell Therapies

In cell-based therapies, it is impossible to ensurethat the final cell population will be 100% homogenous,
since the starting material is human material and the
manufacturing process involves multiple steps that affectcells differently. A cut-off for minimum purity must be
decided based on the mode of action of the cell productand potential impacts to safety. Likewise, there should bea cut-off for cell viability, post-production, or post-thawing, and for all other cellular components.
Flow cytometry is often used in identity and purity
methods with potentially different instrument platforms.
The phenotypes of the CAR-T cells are important since
the cells are the therapeutic agents, which could impact
the efficacy, PK, and safety. Phenotypes currently considered critical for product release are viability, %CD3+,
and %CAR+. The staining and gating of the different
cells could be method dependent. The fluorescenceminus one (FMO) gating, the selection of dye and antibody conjugation, and the expression levels of surface
markers are considered to ensure the balanced responses
and accuracy of the method. The representative samples
are important as it is not unusual to observe potential
impurities inferences.
POTENCY (610.10)

Potency is defined as the quantitative measure of biological activity and is necessary to confirm that the productpossesses the inherent or induced biological functions
relevant for the treatment of the intended clinical indication. Ideally, potency assays measure all aspects of thetherapeutic and biological ability of a product.
For CAR-T cells, important aspects include transferof the CAR gene into T cells and ability of the transferred
genetic material to elicit the desired biological effect in impacted T cells. If the product includes more than one active
ingredient (cell type or cell line), the potency assays shouldbe able to measure the potency of all active ingredients.
Potency assays should be quantitative and can include animal-based bioassays, cell-based bioassays, or
analytical assays (which are designed to measure theimmunochemical, molecular, or biochemical characteristics of the product). In general, health authorities
prefer a cell-based bioassay, but are open to inclusionof analytical assays to enable early clinical development. In addition, animal-based bioassays are often

A-CELL

✼191

discouraged to limit animal use where possible. Useof an analytical assay for a marketed product typicallyrequires data to demonstrate that the assay provides
acceptable control over relevant product attributes andthat a cell-based bioassay is not needed. Some of thechallenges encountered in establishing potency tests
for cell-based therapies include the inherent variabilityof starting material, availability of limited material for
testing, heterogenous product composition, sub-optimal assay precision, lack of established reference standards, incomplete understanding of MOA, multiple
active ingredients, limited stability, and the potentiallycomplex fate of a product in vivo.
Potency tests, along with a number of other tests,
are performed as part of product conformance testing,7
comparability studies,2 and stability testing.3 These tests
are used to measure the product attributes associatedwith product quality and manufacturing controls, andassure the identity, purity, strength (potency), andstability of products. Similarly, potency measurements
are used to demonstrate that only product lots thatmeet defined specifications or acceptance criteria areadministered during clinical investigation and following
market approval.8
During early-stage development, extensive characterization should be performed to generate knowledge on
MOA and support the appropriate potency. For CAR-T
products, potency characteristics commonly tested
include the number of transduced T cells and the functionality of antigen-specific T cells such as cell killing andcytokine release (e.g., interferon-gamma [IFN-γ] releasein response to CD19-expressing target cells).9,10 Potency
characteristics of other cell therapy products have to be
defined based on the putative MOA. Often a combinationof several (often cell-based) assays is required to generatea more holistic picture of Drug Product potency.
While there are functional assays in place for certain
cell therapies like CAR-T products, the complex natureof the MOA of most cell therapies presents a limitation
to potency assay development. In the case of CAR-T,
the current IFN-γ production and cytotoxicity assays
still cannot accurately predict the product’s clinicaloutcome. Different technology platforms to monitor
the cytokines include AlphaLISA, Meso Scale Discovery(MSD), and Luminex.

Chapter 9: Analytical Technologies for Cell Therapies

It is therefore necessary to monitor clinical outcomes
and use statistical methods and correlative analysis todetermine relationships between the product CQAs andthe clinical outcomes. This exercise allows for a better understanding of the manufacturing variables that impact
product quality and clinical outcomes, which ultimately
dictate the product control strategy (topic covered in
Chapter 10).
Potency assays reflecting the MOA might require
combinations of extended cell cultures combined withmethods to determine target gene expression, morphological cell characterization, or determination of cellularfunction. As projects mature through different clinicalphases, a thorough evaluation should be performed to
implement the potency on the most reliable and QC
friendly platform.
Ideally, potency assays should be executed using
full-dilution curve analyses, allowing constrained fouror five-parameter fit logistics to be used. To date, thishas been difficult to achieve when assessing cell-based
therapeutics (autologous or allogeneic CAR-T). As
a result, there may be a tendency to use minimal (oreven single) dilution of therapeutic (i.e., a single E:T
ratio) and determine an absolute amount of cytokine
generated at that E:T ratio in the case of CAR-T cells.
This may be, unfortunately, misleading as different
CAR-T lots/donors may provide dose/response curves(e.g., holding target cells constant, and titration of the
CAR-T cells) that are not equivalent, resulting in different rank-order of cytokine production by the different
lots/donors depending upon the specific E:T ratio used.11
Thus, using the current approach of one fixed E:T ratio,
it is possible to derive a misleading interpretation of DP
potency. This could be especially important with the useof allogeneic CAR-T cells, where understanding the “offthe-shelf ” potency of the DP might be used to determine
consistency.

Molecular Assays to Confirm
Gene Editing

With the increasing understanding of cell biology andgenetic editing capability, gene knock-in and knockout are being introduced in new cell therapies to bothconfer the antigen specificity and modulate the immune

A-CELL

✼192

interactions such as PD1 checkpoint pathways. For
example, CRISPR technology has been used to produce
the T cell receptor (TCR) cell therapy that has both PD1
knock-out and transgene knock-in into the TRAC loci.
To monitor the genetic modification, qPCR and droplet
digital PCR (ddPCR) were developed.
Additionally, as a nuclease (e.g., CRISPR) is used to prepare the T cell genome to receive and insert the CAR gene,
the potential for an improper insertion (off-target) mustalso be assessed, generally via droplet digital PCR (ddPCR).
Additional need for molecular control arises fromcell therapies generated from pluripotent stem cells.
Clone selection and generation of Master and Working
Cell Banks carry the risk of introducing point mutationand chromosomal aberrations into the DP, which need
to be tightly controlled to prevent interference with or
potentiation of oncogenic properties.

Genomic Characterization
Allogeneic therapies may require genomic characterization as long-term culture of cells can alter the genomic
composition and lead to chromosomal abnormalities.
Such abnormalities may further affect protein expression,
cell function, safety, and purity, therefore the genomic
stability/normality of the material should be assessed at
multiple timepoints.12
The timepoints where genomic characterization ismost important are:
1. The source cells during establishment of master andworking cell banks.
2. Manufacturing processes that including genetic
modification of a cellular product.
3. Manufacturing processes that rely on extended
culture and cell expansion to meet the final dose.
Currently, several methods are used to assess thesechanges, including g-banded karyotype, chromosomal
microarray, fluorescence in situ hybridization (FISH),or sequencing.13 It is important to note that all methods
have limitations or trade-offs in resolution and sensitivity. Therefore, genomic characterization may need to be
tailored to the product and processes being utilized in
manufacturing.

Chapter 9: Analytical Technologies for Cell Therapies

Impurities
Impurities can be categorized as product- or process-related impurities. In CAR-T therapies, product-related impurities can originate from leukapheresis products and arecomprised of non-T cells, residual tumor cells, non-viable
T cells, and non-transduced T cells, all of which could bemonitored by the flow cytometry methods.
In other certain cell therapy products, product-related impurities can refer to its pluripotent origin, whereresidual pluripotent stem cells need to be tightly controlled. The lower limit of quantification (LLOQ) in flow
cytometry methods is typically not sufficient to support
the acceptable impurity levels (e.g., <0.01% residual pluripotent cells in the DP). DNA-based methods such as
qPCR and ddPCR are currently being evaluated for theirpotential to be integrated in respective DP release panels.
Process-related impurities include materials employedor added during product manufacturing such as residual
beads, antibiotic, or cytokines. As we introduce newreagents such as activation and gene editing reagents,
we also need to demonstrate process control and develop
new analytical detection methods. A stage-appropriate
approach should be taken for control of process-related
impurities. The approach should be based on a risk
assessment for each impurity, and strategies to control
each impurity should be prioritized according to risk.
Full process and method validation for clearance ofprocess-related impurities is typically not required untillate-stage or before BLA filing. If activation beads areused during manufacturing, a key impurity assay in therelease specification could be a residual beads assay (e.g.,
imaging technology). Overall, the detection and processvalidation of impurities clearance must be demonstrated
or the high-risk impurities could be monitored as part of
the release specification.

Cell Line Identity
Confirmation of cell line identity (distinct from cell
type identity) is strongly encouraged for cell-based
therapies that utilize cell lines as starting materials.
A profile of cell lines used as starting/source material
should be established at the earliest possible timepoint
followed by regular testing at specific pre-determined

A-CELL

✼193

time points to ensure confidence throughout the manufacturing process.
Cell line identity testing can be accomplished via
short tandem repeat (STR) testing or a similar method.
STR can discriminate cell-based products from one
another as well as identify contaminating cells.

Stability
In addition to release testings, some of the assays discussed so far are used to determine the stability of the celltherapy products. The FDA recommends that productstability studies for CAR-T cells should include testing
to determine product shelf life, as well as in-use stability,5which covers a much shorter time frame (e.g., hours todays) to establish the duration cells can be held under
specific conditions.
IN-USE STABILITY

The purpose of in-use stability testing is to establish aperiod of time during which a product can be used while
retaining quality within an accepted specification oncethe container is opened. In-use stability is critical fordelivering reproducible results for cell products (freshly
applied or frozen), significantly influencing productquality. Example studies that can be done to determine
in-use stability include determination of maximum holdtime between product thaw and administration. For CAR
T cells that are administered fresh, stability information
for the intended hold time between final formulation andadministration should be provided.5 According to the
EMA Guidance on In-use Stability Testing of Human
Medicinal Products (CPMP/QWP/2934/99), the test
should be designed to simulate the use of the product inpractice as closely as possible, taking into consideration
the fill volume of the container and any dilution/reconstitution before use. Throughout the duration of the in-use
stability test period, the product should be stored underconditions as recommended in the product literature.
The appropriate attributes of the product susceptible
to change during storage should be determined overthe period of the proposed in-use shelf life. These can
include, but might not require, all analytical assays forcell characterization listed in the earlier section.

Chapter 9: Analytical Technologies for Cell Therapies

STABILITY TESTING TO SUPPORT SHELF LIFE

Product stability testing is required to ensure that thecell therapy product (DP) is, at a minimum, stable forthe duration of the clinical trial.14 To determine theproduct shelf life, stability indicating assays should be
identified through stressed stability samples studies.
The stability-indicating assays should be related to thefunctional, phenotypic, and physicochemical propertiesof the DP. Accelerated stability should be evaluated tosupport the product handling and short excursion ofstorage conditions. According to the existing productinserts for marketed therapies, the formulation andstorage conditions are often based on dimethylsulfoxide (DMSO) and cryopreservation. A detailed study ofcryopreservation of autologous CAR-T products has
been conducted that revealed changes in mitochondrial
dysfunction, apoptosis signaling, and cell cycle damage
pathways.15 The study also confirmed that the function
and phenotypes in the release panel are not impacted in
the target storage condition. At such low temperaturesand using an adequate formulation, the stability of the
CAR-T drugs was demonstrated in the study.
An illustrative example of a stability testing plan isprovided in Table 9-1. Note that not all attributes need
to be tested at all time points. The frequency of testing
could vary based on the product-specific applications.
The shelf life and storage conditions could be justified by
the stability data with the representative cellular productsin the right container. Under optimized cryopreserved
storage conditions, the DP can be stable to support theclinical and commercial applications.

Process Analytical Technologies (PAT)
In-process testing or process analytical technology (PAT)
is defined as a system for analyzing and controlling the
manufacturing process through timely (in-process)measurements of:• CQAs• Overall performance attributes of raw materials
and (cellular) byproducts• Overall final product quality• Real-time evaluation of the manufacturing process

A-CELL✼194

Table 9-1: Example stability testing plan for an autologous CAR-T product to determine shelf life.
Attribute category

Test

Time points following lot release (months)
0

3

6

9

12

18

24

36

Viable cell countCell counting

✓

✓

✓

✓

✓

✓

✓

✓

Cell viability

Cell counting

✓

✓

✓

✓

✓

✓

✓

✓

SafetySterility (USP <71> / EUR Ph 2.6.27)

✓

✓

✓

Identity% CAR+ CD3+

✓

✓

✓

✓

✓

✓

✓

✓

Potency

Cytokine release (ELISA)

✓

✓

✓

✓

✓

✓

✓

✓

Due to the inherent variability of starting cellular material and changes in the characteristics of cells as they gothrough the manufacturing process, a significant level ofin-process testing is required to ensure product quality andconsistency. Table 9-2 lists the most common PAT technologies that could be used for cell therapy development.
Many challenges exist for offline analysis used incell therapy. The analysis often occurs outside the GMP
manufacturing facility in a QC lab or a contract testing✓organization. In some cases, the results are available at alater time, after the process is complete. Sample handling isanother challenge as the samples need to be drawn several
times, which may contribute to contamination risk. The information cannot be used for real-time process modulating.
Online PAT provides an opportunity to gather the
information in a timely manner, and potentially provide
this information to improve the process in a timely andefficient manner.

Table 9-2: Example in-line and at-line PAT technologies for cell therapy process monitoring16
Technology

Measurement

NIR spectroscopy

Cell culture metabolites (glucose, glutamine, lactate, ammonia), viable andtotal cell density, osmolality

Raman spectroscopy

Cell culture metabolites (glucose, glutamine, lactate, ammonia), viable andtotal cell density, osmolality

Fluorescent sensors

pH and dissolved oxygen

Refractive index

Compositional changes

Multi-wavelength fluorimetry

Amino acids

Holographic imaging

Cell shape/size, cell viability

Impedance

Biomass/cell viability

Turbidimetry

Biomass

HPLCMedia components (amino acids, sugars, proteins, metabolites)

LC-MS

Media components (amino acids, sugars, proteins, metabolites)

Coulter counter

Biomass/cell viability

Imaging

Cell size/shape, cell viability

Photometric analyzers

Cell culture metabolites (glucose, glutamine, lactate, ammonia)

Chapter 9: Analytical Technologies for Cell Therapies

A-CELL✼195

NON-INVASIVE AND NON-DESTRUCTIVE INPROCESS TESTING FOR CELLULAR ANALYSIS

Real-time inline monitoring of cells as they undergo avariety of manipulations in a closed system during GMP
manufacturing is important to evaluate the process parameters and maintain the CQAs of the final product.
Such sample monitoring efforts at multiple time points
within the workflow, while essential, also pose significant
risks. Sampling of cells is invasive and can introduce
contaminants into the production line. It can also terminally remove cells that constitute the final product andtherefore is destructive.
Non-invasive and non-destructive inline monitoring
solutions that directly interface with closed automatedsystems for cell analysis offer real-time assessment in astandardized and reproducible manner.17 They enabledirect assessment of phenotype, morphology, viability,
proliferation, and functionality of cells throughout theworkflow. Such technologies can also monitor cell health,pH, oxygen levels, and metabolites and help in determination of optimal process parameters.
Currently there are a few technologies that offer such
options. One example is the Ovizio inline, automatedmicroscope that generates reliable viable cell counts
based on their patented double differential digital holographic microscopy (D3HM) technology. Sampling ofcell populations can be remotely carried out in real-time,
without any labelling or cell removal. Its versatility makes
it compatible with off-the-shelf stirred tanks, rocking
motion bags, or any static and dynamic culture vessels.
Other examples are PreSens (Precision Sensing)
Optical sensors, Sonotec ultrasonic sensors, and BugLabsensors that can measure some cell attributes without
direct contact. Single use sensors can play a vital role ifthey can be redesigned as non-invasive or no-contact
sensors.
Cell viability can be measured based on radiofrequency impedance technology. This can be achieved bysingle-use probes integrated into rocking bags or bags
placed in incubators (dynamic/static culturing of cells)
that allow for continuous monitoring of cell expansion
in bags vs. daily monitoring or monitoring on specificdays/time points. This technology is particularly helpful
as sample availability and access to the culture system isa limitation in cell-based therapies.

Chapter 9: Analytical Technologies for Cell Therapies

The technologies for monitoring the phenotypes andfunctions of cell therapy are largely offline, as in the QC
methods. Online technologies are challenging to develop
because of cell complexities. However, with the increasing development of cell therapies, further advances foronline or inline analysis are expected.
PROCESS ANALYTICAL TECHNOLOGIES FOR
MEDIA NUTRIENTS AND METABOLITESFor CAR-T cell therapy, the media is designed specificallyto promote T cell expansion. The media contains aminoacids, growth factors, cytokines, other nutrients, andmetabolites, where PATs are available for each of theseanalytes. Various analytical tools have been utilized tomonitor the media, metabolites, and secreted proteins in
the cellular therapeutic process. For example, chip-based
capillary electrophoresis-mass spectrometry (CE-MS)
has been utilized to provide all the essential amino acids
concentration information in less than 10 minutes.18
Cytokines are important components of the T cell
culture and have been monitored by immune assays
with platforms such as MSD or AlphaLISA. The immunoassays provide the specificity and sensitivity forthese analytes. Other technology could potentially be
developed to monitor the cytokines. For example, an
online device based on monolith was designed to enrich low concentration of proteins and coupled with MS
detection. The integrated setup can analyze the protein
concentration down to pM range with cytochrome C as
the model protein. Such sample enrichment may enable
the analysis of low concentration proteins by liquid
chromatography–mass spectrometry (LC-MS).19
Non-invasive methods (Raman or NIR detectors)
have been developed for monitoring cell attributes using
sensors where the sensors are not in physical contact withthe cells or media. For example, optical sensors such asnear infrared and Raman spectroscopy are non-invasive
and can provide continual in-line monitoring of parameters associated with cell metabolism such as glucose andglutamine consumption and production of lactate andammonia. Raman Spectroscopy can be used for cell cycle
state and chemometric modelling to monitor nutrientsand metabolites in real-time without the need for sampling the cultures, thus reducing the chances of contamination and human errors. However, analytical data from

A-CELL✼196

online optical systems is typically not amenable to directinterpretation. Further advancement in process analyticaltechnologies will enable process development scientists tomodulate the process based on the real time inputs.

Conclusions
Diverse analytical technologies have been implemented to characterize cell therapy products to ensure thequality, safety, and efficacy of this promising new modality. Continuous advancements in analytical tools arebeing made in the cell therapy field to allow for timely
process monitoring and for proper implementation ofcontrols. These include new molecular assays to shorten
the release time, multi-parametric platforms integrated
throughout the process, multivariate data analysis, smart
analytics, and data integration. They are facilitated byinnovations in hardware such as new non-invasive sensor

development, increased resolution in imaging capabilities, and software improvements to complement these
innovative technologies.
Advancements in cell-based therapies are criticallytied to enhancements of analytical assays. The applicationof artificial intelligence (AI) techniques and machinelearning is beginning to be recognized in the advanced
therapy field to build predictive models for personalized
treatments with engineered stem cells, immune cells,
and regenerated tissues. AI could enable data learningand prediction of clinical outcomes, thus optimizing
cell therapy clinical trials through precise planning,
simplified patient recruitment and retention, resulting in
reduced complexity and costs.20 Complementing current
analytical tools with machine intelligence could have an
exponentially high impact on the cell therapy field todramatically reduce product release time and acceleratecommercialization.

References
1

Carmen J, Burger SR, McCaman M, Rowley JA. Developing
assays to address identity, potency, purity and safety: cellcharacterization in cell therapy process development.
Regenerative Medicine. 2012;7(1):85-100. doi:10.2217/
rme.11.105

2

Rios M, Moore AR. Analytical Testing Strategies for CAR
T-Cell Products. BioProcess International 17(11-12)sNovember-December 20193

Wiwi C. Analytical considerations for cellular therapy
manufacturing. Cell Gene Therapy Insights 2016; 2(6), 651661. doi:10.18609/cgti.2016.071

4

Ritter N, Russell R, Schofield T, et al. Bridging Analytical
Methods for Release and Stability Testing: Technical, Quality
and Regulatory Considerations. BioProcess International
14(2) February 2016

5

Considerations for the Development of Chimeric
Antigen Receptor (CAR) T Cell Products. US FDA Draft
Guidance for Industry. March 2022. https://www.fda.gov/
regulatory-information/search-fda-guidance-documents/
considerations-development-chimeric-antigen-receptor-cart-cell-products

6

7

Wang X, Rivière I. Clinical manufacturing of CAR T cells:
foundation of a promising therapy. Mol Ther Oncolytics.
2016;3:16015. Published 2016 Jun 15. doi:10.1038/
mto.2016.15
Potency Tests for Cellular and Gene Therapy Products. US

Chapter 9: Analytical Technologies for Cell Therapies

FDA Guidance for Industry. January 2011. https://www.
fda.gov/regulatory-information/search-fda-guidancedocuments/potency-tests-cellular-and-gene-therapyproducts
8International Conference on Harmonization: Final
Guidelines on Stability Testing of Biotechnological/
Biological Products (ICH Q5C). 61 FR 36466. July
10, 1996. http://www.fda.gov/downloads/Drugs/
GuidanceComplianceRegulatoryInformation/Guidances/
ucm073466.pdf

9Tyagarajan S, Spencer T, Smith J. Optimizing CAR-T Cell
Manufacturing Processes during Pivotal Clinical Trials.
Molecular Therapy - Methods & Clinical Development.
2020;(16):136-144. ISSN 2329-0501. https://doi.
org/10.1016/j.omtm.2019.11.018

10

Levine BL, Miskin J, Wonnacott K, Keir C. Global
Manufacturing of CAR T Cell Therapy. Molecular Therapy- Methods & Clinical Development. 2017(4):92-101. ISSN
2329-0501. https://doi.org/10.1016/j.omtm.2016.12.006

11

Kiesgen S, Messinger JC, Chintala NK, et al. Comparative
analysis of assays to measure CAR T-cell-mediatedcytotoxicity. Nat Protoc 16, 1331–1342 (2021). https://doi.
org/10.1038/s41596-020-00467-0

12

U.S. Food and Drug Administration. 21 CFR 610.18.
Cultures. 2021

13

McIntire E, Taapken S, Leonhard K, Larson AL. Genomic

A-CELL

✼197

References, continued
Stability Testing of Pluripotent Stem Cells. Current Protocols
Stem Cell Biology. 2020;52(1):e107. doi:10.1002/cpsc.107
14

U.S. Food and Drug Administration. Guidance for FDA
Reviewers and Sponsors. Content and Review of Chemistry,
Manufacturing, and Control (CMC) Information for Human
Somatic Cell Therapy Investigational New Drug Applications(INDs). US Department of Health and Human Services,
Rockville, MD, USA, 1, 1–39 (2008)15

Panch SR, Srivastava SK, Elavia N, et al. Effect of
Cryopreservation on Autologous Chimeric Antigen Receptor T
Cell Characteristics. Molecular Therapy. 2019(27-7):1275-1285.
ISSN 1525-0016. https://doi.org/10.1016/j.ymthe.2019.05.015

16

Marshall D, Ward S, Baradez MO. Requirement for smart inprocess control systems to deliver cell therapy processes fit forthe 21st century. Cell Gene Therapy Insights 2016; 2(6), 683-689.
ISSN: 2059-7800. doi:10.18609/cgti.2016.074

Chapter 9: Analytical Technologies for Cell Therapies

17

DePalma A. Sensors Coming with More Options. Genetic
Engineering & Biotechnology News, August 2017. https://www.
genengnews.com/magazine/299/sensors-coming-with-moreoptions/18

Gilliland WM Jr, Ramsey JM. Development of a Microchip
CE-HPMS Platform for Cell Growth Monitoring. Anal Chem.
2018;90(21):13000-13006. doi:10.1021/acs.analchem.8b03708

19

Naldi, M., Černigoj, U., Štrancar, A., & Bartolini, M. (2017).
Towards automation in protein digestion: Development of amonolithic trypsin immobilized reactor for highly efficient online digestion and analysis. Talanta, 167, 143-157.

20

Sniecinski I, Seghatchian J. Artificial intelligence: A joint
narrative on potential use in pediatric stem and immune
cell therapies and regenerative medicine. Transfus Apher Sci.
2018;57(3):422-424. doi:10.1016/j.transci.2018.05.004

A-CELL✼198

CHAPTER 10

Control Strategy
CHAPTER LEAD

Nolan Polson, GlaxoSmithKline
CHAPTER CONTRIBUTORSMax Tejada, Kite Pharma
Neil Leatherbury, Precision BioSciences
David Geoghegan, Spark Therapeutics
Quentin Vicard, Ori Biotech

Chapter 10 | Contents
Introduction......................................................................200

Control Strategy as a Function of Clinical Relevance...217

Risk-based approach to control strategy...................201

Correlative analysis to connect drug product
CQAs to clinical outcomes........................................... 217

Control strategy and regulatory expectations.......... 202
Chapter aim.................................................................. 202
Process Parameter Controls......................................... 202
Process characterization.............................................204
Process validation.........................................................210
Process comparability...................................................211
Material Attribute Controls............................................. 211
Raw and starting materials...........................................211
Ancillary materials........................................................ 212
Procedural Controls....................................................... 212
Aseptic processing....................................................... 213
Personnel....................................................................... 213
Environmental monitoring and control offacilities and equipment...........................................214

Impact of CQA levels on direct patient outcomes..... 217
Divergence between autologous and allogeneic
approaches....................................................................219
Improvements in CQA understanding throughout
the product life cycle....................................................219
Evaluation of Total Integrated Control Strategy.......... 220
Tools to develop an integrated control strategy........ 221
Control strategy evolution from clinical tocommercial development............................................ 221
Establishing Meaningful Specifications.......................223
Early-stage development............................................ 223
Clinical-to-commercial development........................ 224
References......................................................................225

Testing Controls.............................................................. 214
Routine testing..............................................................214
Periodic testing............................................................. 215
Due to similarities in control strategy development across various types of cell therapy products, some of the overall content and flow of this chapter was
based on, but adapted from when applicable, Technical Report No. 81: Cell-Based Therapy Control Strategy, a document published by the Parenteral Drug
Association (PDA). The authors of this A-Cell chapter acknowledge the work of the authors and editors in constructing the PDA document.
For further details on the PDA document, please refer to:
Parenteral Drug Association. Technical Report No. 81: Cell-Based Therapy Control Strategy. https://www.pda.org/bookstore/product-detail/4638-tr-81-controlstrategy-for-cell-therapy. February 2019.

Introduction
Manufacturing control strategy represents a comprehensive, risk-based approach to ensuring that a generated drug
product is of the highest quality. It seeks to minimize variability in all inputs and processes that impact the ultimate
quality and clinical performance of the product. According
to the International Council for Harmonisation (ICH)
Quality Guideline Q10 definition,1 control strategy is:
A planned set of controls, derived from current productand process understanding that ensures process performance and product quality. The controls can include parameters and attributes related to drug substance and drug

Chapter 10: Control Strategy

product materials and components, facility and equipment
operating conditions, in-process controls, finished productspecifications, and the associated methods and frequency
of monitoring and control.

Underlying the definition of control strategy is therecognition that both product and process understanding
evolve throughout the life cycle of drug product development. The robustness of the initial control strategy
depends on the extent of scientific knowledge available
at idea inception. Development should start with a basic understanding of the quality target product profile(QTPP, Chapter 3), built on a general idea of what the

A-CELL✼200

Figure 10-1: Overall control strategy development.

Product Understanding
DefineQuality
TargetProduct
Profile(QTPP)

Identify
Potential
CriticalQuality
Attributes(CQAs)

Process Understanding
Process
Characterization
Analytical
MethodDevelopment
Sources of
Variability
Product History

Establish theControl Strategy
Process
Parameter
Controls

Material
Attribute
Controls

Procedural
Controls

Testing
Controls

Clinical
OutputRisk Assessment

Adapted from PDA Technical Report No. 81 ; Cell-Based Therapy Control Strategy, with permission of PDA, Inc.

product must eventually accomplish. Early on, criticalquality attributes (CQAs, Chapter 4) described in the
QTPP may be informed by preclinical data, processing
runs, and rudimentary understanding of the various
sources of risk to the product or the patient. As process
and analytical methods are more fully characterized, anincreased understanding of how critical reagents, criticalprocess parameters (CPPs), and in-process and release
controls shape the control strategy begins to inform
how the manufacturing process can be manipulated to
optimize favorable profiles of CQAs. Valuable information
comes in later-phase development, as clinical efficacy andsafety data begin to emerge, and in post-approval settings
where real-world patient outcomes are reported. As such,the control strategy must evolve over time as more islearned about the product and about how various process
inputs affect its quality or performance.
A holistic integrated control strategy is created fromsmaller-scale control strategies that address all essential
parts of the manufacturing system: process parameter
controls, material attribute controls, procedural controls,
and testing controls (Figure 10-1). Clinical data encircles
and influences further characterization and refinement of
all elements of the control strategy as new data become
available, long-term safety profiles are established, andas increased understanding of patient heterogeneity andChapter 10: Control Strategy

variability are obtained. Patient-centric data not only
confirms the true criticality of various quality attributes,
but it may also help to identify aspects of the manufacturing process where control of variability is less essential.
Thus, the control strategy not only addresses the life cycleof the cell therapy product, but also the life cycle of its
own, characterized by continuous improvements as new
knowledge is gained. In its refined state, it incorporates athorough understanding of all risks to the product and,most importantly, to the patient.
As new product knowledge is gained, sharing and managing that knowledge across all involved parties (e.g., celltherapy developer, vector and cellular drug product production facility, contract development and manufacturingorganizations (CDMOs), and clinical trial management
organizations) helps to assure the ongoing effectivenessof the control strategy. Breakdown of knowledge sharing
at any point could compromise the integrated control
strategy and threaten quality and commercialization of
the cell therapy product.
RISK-BASED APPROACH TO CONTROL STRATEGY

Quality-by-design (QbD) principles of criticality analysis and risk management underlie the formation ofa control strategy that can guide and grow with a celltherapy product through various stages of development

A-CELL✼201

(Chapter 4). This risk-based approach is supported by
the European Medicine Agency’s (EMA’s) Committee
for Advanced Therapies and is discussed in two recently
published guidelines.2,3 Additionally, the U.S. Food andDrug Administration (FDA) has published several guidance documents that outline a risk- and science-based
approach to ensure product quality and patient safety.4-8
Risk analysis should be employed early in development
and be continued through all validation steps and commercial scale-up. Knowledge of various quality attributes can beused to score each attribute according to the severity of harm
it poses to the patient, the likelihood of its occurrence, andthe ability of analytical methods to detect its occurrence.9 As
the goal is to minimize overall risk to patients, stringency ofcontrols can often be increased for CQAs that pose medium-to-high risk to patients. Though the initial risk assessment is often based on limited product knowledge, as moreis learned about CQAs and CPPs, the risks that various inputs
pose to overall product quality become clearer. Enhanced
understanding of these risks helps to define appropriate
control points and testing stringency and is essential for thedevelopment of a robust integrated control strategy.
CONTROL STRATEGY AND REGULATORYEXPECTATIONS

A validated control strategy that allows consistent production of a safe, pure, and potent drug product with definedstability is central to regulatory approval of any cell therapy
product. Demonstration of the control of all processes(e.g., laboratory controls of analytical methods, control ofincoming raw materials) through their initial conception,
improvement, and optimization is essential. Expectations
for demonstrating control of cell therapy processes have
evolved over the last decade, both within the UnitedStates and European Union (EU) regulatory communities.
Chapter 2 addresses regulatory expectations as productdevelopment moves through key timepoints.
Touchpoints with regulatory agencies occur at allstages of drug development, from the pre-Investigational
New Drug (IND) meeting through all phases of clinicalstudy. The integrated control strategy, along with datafrom process characterization and validation studies, ispresented to regulatory agencies within filing documents;
any changes to product specifications or alterations of thecontrol strategy post-approval must be submitted andChapter 10: Control Strategy

approved by regulatory bodies. If the manufacturing
process is incompletely characterized or insufficiently
validated, the manufacturing process may lack robustness to support commercialization and the strategy may
not be accepted, further delaying the commercializationof the cell therapy drug product. Thus, taking the timeto establish, monitor, and improve the holistic control
strategy is the most efficient approach for any company
producing a cell-based therapy. Regulatory responses
to filings regarding all aspects of the integrated control
strategy such as raw material controls, procedural controls, CPPs, in-process controls (IPCs), and intermediate/
release specifications will also help inform overall strategy across programs. In addition to facilitating regulatory
approval, a solid holistic control strategy framework willallow the cell therapy developer to remain competitivein this rapidly expanding field as key clinical questions
surrounding cell therapy begin to be answered.
CHAPTER AIM

This chapter seeks to guide the reader through thedevelopment of a robust integrated control strategy
for a hypothetical chimeric antigen receptor (CAR)-T
cell therapy product in the setting of current regulatory
framework and guidelines. The content will illustrate
steps in the risk-based approach to control strategy generation. Differences between allogeneic and autologous
approaches to CAR-T cell therapy production will behighlighted where applicable.

Process Parameter Controls
After establishing a list of CQAs (e.g., cell concentration,
vector copy number, % CAR+ CD 3+) or potential CQAs
(e.g., viability, residual plasmid DNA) essential for the
CAR-T cell product efficacy and safety, the manufacturing
process must be developed and rigorously evaluated toensure consistent delivery of a quality product. Initially,a broad map of the production process should be createdutilizing tools such as a simple flow diagram, deployment
flow chart, SIPOC (supplier, input, process, output,
customer) diagram, or IPO (input, process, and output)
diagram. The product obtained after each step of the
production process should be described in detail and thespecific parameters that govern the overall process must be

A-CELL✼202

Table 10-1: Example IPO diagram for CAR-T expansion unit operation (assuming a 3-D suspension
culture in a rocking motion/stirred tank bioreactor)
Controllable parameters• Number of single-use bioreactor bags

• Culture duration• Rocker/agitation speed• Cell seeding density• Incubation temperature• Feed volume• Media volume for each bag• %CO2• Feed timing• Dissolved oxygen
Unit Operation Inputs

Cell expansion unit operation steps

Unit Operation Outputs

Starting materials

1. Seed cells and media into bioreactor bag

Performance attributes• Cells from previous step
(activation / gene editing)• Cell counts• Cell viability

Raw materials

2. Incubate for 5 days with a set agitation speed / profile• Culture medium• Serum• Culture feed
• Antibiotics

3. Add media feed

Consumables

4. Incubate for 5 additional days

• Purity: dead cells,
contaminating B cells,
process-related impurities(e.g., remaining beads)

5. Obtain cell count / viability• Identity: % CAR+ CD3+

• Single-use bioreactor bags
• Transfer bottles / bags
• Serological pipettes• Pipette tips
• Filters• Sterile centrifuge tubes• Welder blades

Quality attributes• Safety: sterility, endotoxin,
mycoplasma

6. Harvest; transfer cells to another bag for further
processing• Potency: cytotoxicity,
cytokine release

Equipment• Incubator• Biological Safety Cabinet (BSC)• Tissue culture pipettor• Rocking motion bioreactor/
stirred tank bioreactor• Aseptic welder• Grade B / Grade C room
Adapted from PDA Technical Report No. 81: Cell-Based Therapy Control Strategy, with permission of PDA, Inc.

identified. An example of a flow diagram for an autologous
CAR-T manufacturing process, including the process stepand associated unit operations, is presented in Chapter 8.
An example IPO diagram for the expansion step of CAR-T
manufacturing is shown in Table 10-1. Such process
mapping exercises should be performed for all steps/unit
operations involved in the overall manufacturing process.
Risk to the quality of the product can be introduced
during any step of the manufacturing process making
selection of appropriate process parameters essential.
The criticality of various process parameters is ultimately
determined by evaluating their risk to CQAs, which are

Chapter 10: Control Strategy

monitored throughout the manufacturing process toensure that the QTPP is met. Not all process parameters
will be deemed to contribute risk to the overall quality ofthe product; those that are truly critical go on to become
essential elements of the manufacturing control strategy.
Due to the inherent nature of cell therapy, the influenceof patient heterogeneity on process parameter controls
should be well-understood. This may require performing additional characterization studies using numerous
healthy donor materials so that a clear understandingof patient contribution versus process contribution to
overall process variability can be determined. That being

A-CELL✼203

said, patient-derived starting material may have intrinsic properties that affect CAR T manufacturing due todisease state and prior treatment; therefore, when using
patient-derived starting material, additional manufacturing process development may be recommended forautologous CAR T cells.10 A variance component analysis approach, which estimates the contribution of eachrandom effect to the overall variability within a process,may be used to decouple various sources of variability(e.g., patient, process, analytical) to help inform the mostappropriate process control strategy during development
and commercialization.
PROCESS CHARACTERIZATION

To be completed efficiently and allow manufacturers to movequickly from late-stage clinical trials to global commercial
filings, process characterization should take in a comprehensive, stepwise, organized approach. Oversimplificationor shortcuts in process characterization studies to achieve
quicker development timelines could lead to less-than-robust
processes that may result in delayed regulatory approvals or
difficulties with commercial manufacturing if processes are
insufficiently understood. Process characterization is typically
performed within a well-developed and qualified scale-down
model, allowing the large number of experiments required
to characterize a process and to select the best-performing
ancillary material (e.g., media, viral vector, transfection
reagent) to be executed on a small scale. Attempting to fully
characterize a process at a manufacturing scale may be bothcost- and resource-prohibitive.
During initial process characterization, one-factorat-a-time (OFAT) studies may serve as a starting point tobetter understand how single inputs affect process outcomes.
However, design of experiments (DoE) approaches are preferred over OFAT studies because they provide information
on how multiple inputs interact with each other. Knowledgeof CPPs generated from DoE studies are used to define theoperating ranges for various process parameters that ensure
consistent performance of the process. One such range is
the Proven Acceptable Range (PAR). As defined by ICH
Q8(R2),11 a PAR is:
A characterized range of a process parameter for which
operation within this range, while keeping other parameters constant, will result in producing a material
meeting relevant quality criteria.

Chapter 10: Control Strategy

In 2017, the EMA issued a Question and Answer document12 to facilitate a better understanding of parameter
ranges and how they should be presented in regulatory filings.
The document further defines a PAR as allowing deliberatechange in one parameter without changing the other parameters outside their normal operating range (NOR) or target.
The PAR should be adequately justified for all process parameters, regardless of whether they are critical or noncritical.
The document goes on to define a NOR as a region aroundthe target operating conditions that contains common operational variability (variability that cannot always be controlled
to a precise value). The NOR does not represent deliberate
adaptation of the process and does not cover a parameter
range that affects the quality of the process output. If a NOR
were to affect the quality of the process output, then a PAR
for that parameter should be established. The disclosure of
NORs within regulatory filings is intended to better quantify
the actual uncontrollable operational variability of processparameters and should be presented as what is practicallyachievable by available equipment.
Data obtained from comprehensive process characterization studies are used to confirm CPPs and to develop
meaningful IPCs, which together serve as the controlling
documentation for the actual manufacturing process. The
IPCs establish guidelines and define limits for process parameters and operations, deviation from which could result in an
adverse impact to product quality. IPCs should be performed
at key intermediate stages of the manufacturing process andmay cover a variety of quality aspects:13• Molecular (genomic integrity, identity; vector copy
number [VCN]; transduction efficiency; on- and off-target modifications)• Cellular (target cell identity/purity; cell growth kinetics;
cell viability; hold-time stability)• Process-related (temperature, pH, media consumption,
dissolved oxygen and/or carbon dioxide, metabolite
concentration, agitation, acid/base addition, perfusion rate)• Microbiological (presence of infectious particles, absenceof adventitious contaminants, sterility, mycoplasma)
To have a comprehensive and effective process characterization program, it is beneficial to have the most representative
process conditions and equipment to facilitate meaningfulsmall-scale, high-throughput process screening and development. The entire system must be considered, including

A-CELL✼204

the equipment and software required to generate, evaluate, and analyze all process-related data. In the case ofa CAR-T process characterization, this complete systemshould include flow cytometry for the cell phenotype
characterization during selection/activation, the automated small-scale bioreactor for expansion, metabolite
analyzer, representative process analytical technologies
for in-process testing and characterization (including
visualization of cell killing assays, as required), as well as
the software to generate DoE study results based on thedata generated. The higher the degree of automation ateach of these steps, the faster the process characterization
will be. It is important to note that analytics and software
capabilities need to be synergistic to avoid a bottleneck
where more data is generated than the software can analyze. Figure 10-2 shows a potential solution for a CAR-T
manufacturing process that can be used to efficiently
generate process-related data (potential CPP), evaluate
the CQA for each condition tested in your DoE plan, andeventually analyze all these data to confirm the CPP. This
example is not meant to serve as a recommendation forany particular equipment.

Failure Mode and Effects Analysis (FMEA)
As a part of the risk-based approach to establishing ameaningful integrated control strategy, risk assessment
tools such as the Failure Mode and Effects Analysis(FMEA) can be employed. Other risk assessment tools
to determine criticality of a product’s quality attributes are
discussed in Chapter 4. The FMEA provides a method toevaluate potential failure modes for processes and theirlikely effect on outcomes and/or product performance,15
and is calculated as Severity (the impact of failure) ×
Occurrence (the probability of failure) × Detection (thedetectability of failure). Each of the three factors are assigned a score, and when multiplied will result in the Risk
Priority Number (RPN), which ranks the failure mode,prioritizes risks, and evaluates risk mitigation.16 Since it

Figure 10-2: Potential solution for characterization of a CAR-T manufacturing process.
Synergy between manufacturing unit operations and data analytics ensures a comprehensive process characterization.14
Donor material
sampleprep

T-cellselection &activation

Transduction

Expansion

Harvest,Formulation& FillCharacterization

Administration

pH, DO, temperature,stirring speed, metabolites, media

Live cell imaging(Incucyte� SX5)

Flow cytometry(iQue� 3)

High throughput Cell Culture(Ambr� 15)
Transduction
efficiency, T-cell
activation

CAR-T cellpotency
CAR-T cell
memory,exhaustion,
killingLive cell imaging(Incucyte� SX5)

Microsart� ATMP
Flow cytometry(iQue� 3)

Baseline
phenotypeand function

Flow cytometry(iQue� 3)

DoE and MVDA software(SIMCA� / MODDE�)DoE setup and multivariate data analysis to determine CPP according to defined CQA

Rapid sterility
testing

Source: Adapted with permission from Sartorius webinar “Accelerating CAR-T process development: The power of process insight.”
https://www.sartorius.com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/accelerating-car-t-process-development-process-insight-webinar.

Chapter 10: Control Strategy

A-CELL✼205

Table 10-2: Example FMEA for CAR-T expansion process.
Potential
Cause(s)In what ways canthe Process Step or
Input fail?

What is the impact
on the Key Output
Variables once it fails?

What causes the
Key Input to go
wrong?

Insufficient cell
growthOperator
error

RPN
(Risk
Priority
Number)Current
Controls

SxOxD

What are theexisting controls
and proceduresthat prevent the
Cause or the
Failure Mode?

Improvement
Actions
Recommended

Detection

Potential Failure
Effect(s)Occurrence

Potential FailureMode(s)Severity

process stepor input

Failure Mode and Effects Analysis for process steps and inputs

1

1

3

3

Training, SOP /batch record

Verify operator
training

5

1

3

15

Training, SOP /batch record

Verify operator
training; prepare extra
media and bioreactor
disposables

Cell counter
failure;operator error

4

1

3

12

Training, SOP /batch record

Equipment
requalification /calibration; verify
operator training

What are the actions
for reducing theoccurrence of thecause, or improving
detection?

Media preparation
Incorrect
concentration ofserum / growth
factors added tomedia

Improper transfer Contaminationof cell culture
media into
bioreactor bag

Operatorerror, defectin single-use
disposables

Cell seeding
Incorrect number Insufficient cellof cells seeded
growth to fulfill
dose requirements
Cell expansion
Incorrect
incubation
temperature

Cell death;insufficient cell
growth to fulfill
dose requirements

Incubator
failure

4

1

1

4

Extra
incubators
available

Equipment
requalification/ calibration;
additional incubatortemperature monitor

Incorrect pH
control

Cell death;insufficient cell
growth to fulfill
dose requirements

pH controller
failure;operator error

2

1

3

6

Training, SOP /batch record

Equipment
requalification /calibration; verify
operator training

Incorrect DO
control

Cell death;insufficient cell
growth to fulfill
dose requirements

DO controller
failure;operator error

4

1

3

12

Training, SOP /batch record

Equipment
requalification /calibration; verify
operator training

Incorrectbioreactor
agitation speed

Cell death;insufficient cell
growth to fulfill
dose requirements

Bioreactor
failure;operator error

3

1

2

6

Training, SOP /batch record

Equipment
requalification /calibration; verify
operator training

Incorrect feed
concentration /feed timing

Insufficient cell
growth to fulfill
dose requirements

Operator
error

2

1

1

2

Training, SOP /batch record

Equipment
requalification /calibration; verify
operator training

Reduced cell yield

Incomplete
transfer due
to operator
error

4

2

1

8

Training, SOP /batch record

Verify operator
training

5

1

3

15

Training, SOP /batch record

Verify operator
training

HarvestInsufficient cellrecovery fromharvestImproperContamination
connection for celltransfer out of thebioreactor bag

Chapter 10: Control Strategy

Defective
connector,
weld failure,
operator error

A-CELL✼206

requires scoring for occurrence and detectability, FMEA
relies on product and process understanding and is typically performed in later stages of development (e.g., during
the control strategy development stage), where sufficient
data is available to predict occurrence and detectability.
FMEA principles can be applied to all aspects of the manufacturing process, including equipment and facilities, andmight be used to analyze a manufacturing unit operationand its effect on CQAs. It identifies elements/operations
within the system that render it vulnerable. The output/
results of FMEA can be used as a basis for identification of
improvement actions in the control strategy, particularly
for process inputs associated with a high RPN.
An example of FMEA applied to a CAR-T expansion
process is presented in Table 10-2. In this case, each factor
is assigned a severity, occurrence, and detection score

between 1 and 5; the highest overall risks are conferredby process inputs that present the greatest impact offailure (severity) from possible contamination or reduced cell yield that could result in insufficient dose forthe patient. Further characterization of these high-riskprocess inputs (i.e., how these parameters affect the final
product CQAs) should be performed, which will theninform the improvement actions for future operations.

Design of Experiments approaches
A key tool used during process development and characterization is DoE, which utilizes statistical and analyticalmethods to determine the impact of multiple inputs (e.g.,
process parameters) on product CQAs.17 The DoE approach can help to identify process parameters that havethe greatest impact on CQAs (the CPPs) and to identify

Figure 10-3: Example DoE including multiple donors, highlighting inherent patient heterogeneity.
Material from 7 donors are each processed 5 times and results are analyzed in triplicate, revealing that donor variance
Figure 10-3
comprises 82.4% of the total variance in the data.18

Variable 1

Average=0.87991993

Process
Donor

Variance components

Donor variability
Process variability
Analytical variability
Total variability

Donorvariance makes
Donorvariance
upup82.4%
makes
82.4%of the totalof thevariancetotalvariance
Variability as astandard deviation foreach of the variancecomponents

σσTotal2 = σDonor2 + σProcess2 + σAnalytical2 = 0.00439+0.00650+0.000291=0.00533
Source: Ashton R, et al. Presentation at: CASSS Cell and Gene Therapy Conference. June 10, 2019. Reprinted with permission.

Chapter 10: Control Strategy

A-CELL

✼207

Figure 10-4: Example coefficient plot for T-cell
expansion study in small-scale bioreactors.
The size and direction of the relationship between eachfactor and fold expansion was investigated while otherfactors were held constant.19
0.3

Effect on fold expansion

secondary relationships that may influence CPPs orthogonally. These CPPs proceed to more in-depth characterization, whereas parameters that impart the lowest risk
to CQAs may be excluded from further characterization.
Thus, DoE approaches increase the understanding of
truly critical elements of the overall process as well as
identify secondary relationships of materials or processparameters that may exert influence on the critical parameters, while also improving the efficiency of processcharacterization. DoE helps to minimize the number ofexperiments required to obtain the necessary process information without compromising the output, by ensuring
a minimum interaction between all experiments. A casestudy of a DoE involving one donor is provided in the
text that follows. Importantly, due to potential patient
heterogeneity, use of multiple donors in the DoE may benecessary (Figure 10-3).

0.2
0.1
0
-0.1
-0.2

Case Study

IL-2

Seedingdensity

pH

Media
A

Media
B

An example of DoE used to determine the optimal culture
conditions for primary T cells in a small-scale stirred tankbioreactor (Ambr® 15) is presented below (Table 10-3). The
goal of this DoE is to screen different parameters (factors)
and evaluate the effect of these factors on T-cell expansion,
as measured by two responses: cell growth (fold expansion)and cell viability. Other parameters, such as temperature(37°C) and culture volume (10 mL), are kept constant. A
linear, reduced combinatorial design DoE approach wastaken, with replicates of 4 experiments.
The result of the DoE is analyzed and presented in acoefficient plot (Figure 10-4), which represents the size anddirection of the relationship between each factor in the modeland the response variable (fold expansion) while other terms
are kept constant. Based on these results, higher fold expansion can be achieved through higher IL-2 concentration,
lower seeding density, higher pH, and the use of Media A.
With the knowledge gained from this initial DoE, asecond round of experiments was performed with a reduced number of factors: pH, IL-2 concentration, and dissolved oxygen (DO) (Table 10-4), while keeping seeding
density at 5×105 cells/mL and using Media A. The results

Table 10-3: Factors screened in DoE for optimizationof T-cell expansion in small-scale bioreactors.14

Table 10-4: Factors screened in second DoE for
optimization of T-cell expansion.14

Factors

Optimization Range

Factors

Optimization Range

IL-2 concentration (U/mL)

50; 125; 200

IL-2 concentration (U/mL)

50; 125; 200

Seeding density (cells/mL)

5×10 ; 1×10

pH

7.0; 7.3; 7.5

pH

7.2; 7.4

MediumMedia A, Media B

DO (%)

50; 70; 90

5Source: Hupfeld J, et al. Optimization of T-Cell Expansion Using the Sartorius T-Cell
Exploration and Characterization Solution. Sartorius application note: https://www.sartorius.com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/optimization-of-t-cell-expansion-app-note. Reprinted with permission.

6

Source: Adapted with permission from Sartorius webinar “Accelerating CAR-T process development: The power of process insight.” https://www.sartorius.
com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/accelerating-car-t-process-development-process-insight-webinar.

Chapter 10: Control Strategy

A-CELL

✼208

Figure 10-5: Coefficient plots for T-cell expansion and viability from the second optimization study.
Second optimization for fold expansion and viability identifies IL-2 concentration as a CPP with strong positive impact.19
4
0.15

0.1

2
Effect on viability

Effect on fold expansion

3

0.05

0

0
-1

-0.05

-0.1

1

IL-2

pH

DO

-2

IL-2

pH

DO

Source: Hupfeld J, et al. Optimization of T-Cell Expansion Using the Sartorius T-Cell Exploration and Characterization Solution. Sartorius
application note: https://www.sartorius.com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/optimization-of-t-cellexpansion-app-note. Reprinted with permission.

show IL-2 concentration as the most important factor forboth fold expansion and viability; IL-2 concentration istherefore identified as a CPP. This DoE also indicates thathigher DO might have a negative effect, and pH is shownto have a small effect on the responses (Figure 10-5).
To better visualize the outcome of this study, a sweet
spot plot can be generated from the DoE results (Figure10-6), which shows the optimal process range that can beapplied in future scale-up. In this case, to achieve the predetermined CQA of ≥8-fold expansion and ≥90% viability,
the T-cell expansion process should be operated with IL-2
concentration above 160 U/mL and DO below 85%.
Further characterization should then be performed
to confirm that the optimal process conditions obtained
through the DoE fulfill the CQA requirements. In thecase of CAR-T manufacturing, characterization of potency can be performed following expansion and harvestby quantifying CAR-T killing of target tumor cells. In theexample below, CD19+ CAR T cells (effector cells) were
co-cultured with Ramos cells (antigen-positive target
cells) at different ratios, and selective cell killing wasmonitored over time using live cell imaging (Figure 107). Effective killing of antigen-positive cells was observed.
More extensive potency characterization can be done invivo or in humanized mouse models.

Chapter 10: Control Strategy

Figure 10-6: Sweet spot plot to inform optimal
ranges of process parameters to achieve certain
CQAs.
Optimal process range was identified to achieve aminimum fold expansion of 8 and viability of 90% while
pH, stirring speed, seeding density, and medium typewere kept constant.19

Source: Hupfeld J, et al. Optimization of T-Cell Expansion Using the Sartorius T-Cell
Exploration and Characterization Solution. Sartorius application note: https://www.
sartorius.com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/
optimization-of-t-cell-expansion-app-note. Reprinted with permission.on.

A-CELL✼209

Figure 10-7: Characterization of CAR-T potency through quantification of cell killing assay.
CD19+ CAR T cells were co-cultured with antigen-positive target cells (A) or antigen-negative target cells (B) at different
target: effector (T:E) ratios. Target cells were labeled in green and images were taken at 72 hours following co-culture.14(A)

Antigentargetcells (Ramos cells)(A) Antigen positive
targetpositive
cells (Ramos
cells)

Target cell quantification(GCU × μm2 / image)+ control non-transduced cells

Ramos
T:E 1:0.25
T:E 1:0.5
T:E 1:1
T:E 1:2
T:E 1:3

3x1006

Increasing CAR-T cells+ CD19 CAR-T cells (1:2)

2x10061x1006

0
0

24

48

72

96

120

96

120

Time (h)Antigentargetcells (Jurkat cells)(B) Antigen negative
targetnegative
cells (Jurkat
cells)

Target cell quantification(GCU × μm2 / image)+ control non-transduced cells

JurkatT:E 1:0.25
T:E 1:0.5
T:E 1:1
T:E 1:2
T:E 1:3

3x1006

Increasing CAR-T cells+ CD19 CAR-T cells (1:2)

2x10061x1006

0
0

24

48

72

Time (h)Source: Adapted with permission from Sartorius webinar “Accelerating CAR-T process development: The power of process insight.”
https://www.sartorius.com/en/applications/cell-and-gene-therapy/cell-therapy/cell-therapy-guide/accelerating-car-t-process-developmentprocess-itnsight-webinar.

PROCESS VALIDATION

Following development of the IPCs, process performance
qualification (PPQ) runs are executed at commercial manufacturing scale to validate the process and confirm that itprovides adequate control of product quality. The number
of PPQ runs required will depend on adequacy of starting
materials, level of risk assessed, and complexity of both themolecule and the process.
Guidance regarding the overall approach to processvalidation has been provided by the FDA.20 This guidance

Chapter 10: Control Strategyaligns process validation activities with the product life
cycle concept, incorporating three key stages of processvalidation:• Process design, during which the commercial manufacturing process is defined based on knowledge gained
during clinical development and scale-up activities;• Process qualification, which seeks to evaluate whether the process is capable of reproducible commercial
manufacturing (via the execution of PPQ runs); andA-CELL✼210

• Continued process verification, which provides
ongoing assurance that the process remains in a stateof control during routine production.
PROCESS COMPARABILITY

Changes to the manufacturing process (vector or drug
product) are inevitable throughout the development,commercialization, and post-approval life cycle management of a cell therapy product. These changes may
occur when new knowledge is gained about CQAs and
CPPs, when processes are scaled-up and/or transferred
between manufacturing sites, or when process improvements or technological advances such as automation
occur. Manufacturers must demonstrate that resulting
vector or drug product are comparable before and after
the change. According to ICH Q5E,21 the goal of comparability exercises is to ascertain that pre- and postchange drug product is comparable in terms of quality,
safety, and efficacy. As such, manufacturers must provide
relevant technical information (based on a combinationof analytical testing, biological assays, and clinical/
nonclinical data in some cases) showing that processchanges will not have an adverse impact on the quality,
safety, and efficacy of the drug product. Similarity ofquality profiles must be demonstrated across validationbatches and the methods used to detect differences must
be appropriately selected and justified. In many cases, apaired-run approach for both vector and drug productmanufacturing changes is utilized to ensure that the truecomparison of process changes can be evaluated while
minimizing the potential impact of donor variability
in the cell therapy space that may confound a moreconventional comparability strategy used for biologics.
More details on comparability expectations throughout
the product life cycle are provided in Chapter 2.

Material Attribute Controls
Materials used during manufacturing processes can besources of significant variation across all types of drug
products.53 Materials of poor quality can introduce contaminants and impurities that impact the viability, purity,
potency, and safety of the final product. According toEU guidelines on good manufacturing practices (GMP)
for biologically active substances and medicinal products

Chapter 10: Control Strategy

for human use, control of material quality is especially
important during the manufacturing of cell therapy
products, where final sterilization is generally not possible and the ability to eliminate microbial by-products
is limited.22 Material controls can be established once
critical material attributes (CMAs) and potential risks
generated from materials are identified. Control strategies must be defined for each CMA so that variations canbe detected, managed, and minimized.
RAW AND STARTING MATERIALSThe selection of high-quality raw and starting materials
is paramount to ensuring the quality of a final CAR-T
cell product (Chapter 5, Chapter 7). Risks associatedwith raw materials can range from viral contaminationof processing reagents to heavy-metal impurities present
within excipients used for final product formulation. Raw
material quality can also vary between lots and suppliersand can be impacted by changes to supply-chain logistics.
For example, purified enzymes obtained from different
sources may vary in their specific activity, whereas hydrolysates may vary in their composition. Materials of biologic origin are often poorly characterized, but avoidanceof their use is rarely feasible in the manufacturing of celltherapy products. Where biological raw materials must
be used, a key to controlling overall risk to the final drug
product lies in demonstrating the ability to eliminate risks
from raw material variability.53
Starting materials for CAR-T cell products include
primary cells of human origin (Chapter 5) and vectors(Chapter 7). Several safety risks are inherent to cells
sourced from humans:53• Materials can be contaminated with adventitious
agents (e.g., bacteria, viruses, fungi, parasites)• Materials can have tumorigenic potential• Materials can elicit an immunogenic reaction
For autologous CAR-T cell products, primary cells
are extracted from individual patients with cancer andone batch represents one patient. Hence, variation andunpredictability between patients is the major cause ofbatch-to-batch variability in drug product production
processes. Conversely, for allogeneic CAR-T cell products,
a lot or batch derived from a single healthy donor’s cells

A-CELL

✼211

can be used to treat multiple patients (or an individual
patient with multiple doses). With either approach,viable cell count estimates can be misled by cellular
impurities within apheresis materials, such as myeloid
cells or circulating tumor cells. Complicating the processfurther, primary cells begin to change soon after removalfrom the body and cell viability can quickly be compromised in the absence of adequate controls. Changes tomorphology, cell growth potential, and cell-surface
markers may impact the ultimate performance of thecell construct. As such, developing strategies to ensure
the uniformity and consistency of apheresis processes isof utmost importance. Strategies to control the quality ofcollected cells include auditing of the apheresis center tobetter understand and standardize collection protocols,equipment selection to standardize processing conditions,equipment maintenance, personnel training, storage ofthe apheresis product, and shipping conditions prior to
drug product manufacturing.53 Additionally, a thoroughunderstanding of donor screening and testing requirements implemented by apheresis providers is warranted,
including their collection of donor history (e.g., previous
treatment exposure, prior illness, complete blood countwith differential, and absolute lymphocyte count), which
could impact the quality of incoming material that may
impact the probability of manufacturing success during
downstream processing. All components of the transportation process must be well-characterized, including theimpact of transport time and temperature on cell viability
and growth potential.
Inherently, the cell sourcing strategy for allogeneic
CAR-T cell products diverges from that of an autologous
product. The aim of an allogeneic CAR-T cell productis to be off-the-shelf ready for infusion into a recipient.
Allogeneic CAR-T cell s are derived from healthy donor
cells that have been gene-edited to allow administration
to patients unmatched for human leukocyte antigen(HLA). Donor screening criteria for allogeneic products
are extremely robust, seeking to eliminate the introduction of any adventitious agents that could cause diseaseor any immunogenic material that could precipitate graftversus-host disease (GVHD). In addition, phenotypicmarkers (e.g., PD1, CD27, and CD8) on donor cells canbe important predictors of CAR-T cell expansion andresponse in patients. As such, screening and selecting

Chapter 10: Control Strategy

healthy donors whose T-cells express a high frequency ofthese markers may improve response to allogeneic CAR-T
cell products.23
Vectors are effectively treated as starting materials
when used to manufacture genetically modified cells forcell therapy products, even when that modification occurs
ex vivo, as with CAR-T cell products. According to the
EMA, “principles of GMP shall apply from the banksystem used to produce the vector onwards.”13 Whereas
EU regulatory agencies very clearly view vector (and allcomponents used to make the vector) as starting material,13 language from the FDA is less clear, referring tovector as a “critical material.” 24 Nevertheless, expectations
regarding the vector are similar across regulatory bodies,
as vector attributes influence the final drug product. For
example, vector attributes have a direct impact on thecell therapy product VCN post-transduction. Further
discussion on control strategy, as it relates to vector, is
included in Chapter 7 of Project A-Gene.25
ANCILLARY MATERIALS

Primary cells, as well as the intermediate and final CAR-T
cell product, come into direct contact with multiple
ancillary materials during production. These materials
can vary widely in quality depending on the source andvendor, and therefore should be characterized to better
understand the risk they impart to the CAR-T cell manufacturing process, final product, and patient. The FDA
recommends that human- or animal-derived components
are not sourced from geographical areas of concern forpotential viral and/or transmissible spongiform encephalopathy agent contamination and that components be
tested appropriately for adventitious agents.10 In addition,sponsors should qualify ancillary materials for quality,
safety, and potency through vendor qualification programs and incoming material qualification programs, as
appropriate. Principles of ancillary materials risk management, qualification program, and reagents consideration
for cell-based therapies are covered in Chapter 6.

Procedural Controls
Having procedural controls in support of current GMP
manufacturing operations allows both the vector andthe CAR-T cell therapy manufacturer to mitigate risks

A-CELL✼212

imposed by personnel, facility design, equipment, andenvironmental conditions. Procedural controls should
be clear, concise, and leave no room for interpretation
by manufacturing staff. This drives consistency into processes, making them more robust and reducing overallvariability as much as possible. Manufacturers should
continually assess whether improvements should bemade based on periodic reviews for trend deviations/investigations and as a routine part of their internal quality review or annual product quality reviews as requiredby health authorities.
ASEPTIC PROCESSING

Manual processing of CAR-T cell products is becoming less common due to advances in automation andclosed-system manufacturing processes. Where manual
processing continues to be used, minimizing introduction of contaminants and impurities relies heavily on theindividual operator’s understanding of microbiology andaseptic technique.53 In semi-automated and closed-loopsystems, manipulation by individual personnel is more
limited, making overall product quality less dependent
on the individual operator. In such systems, aseptic
processing relies on ensuring that equipment and its
associated materials (e.g., tubing, vials) are properly
sterilized.
The need to describe aseptic controls in the overall
control strategy is discussed in both EMA and FDA
documents.2,26 Sufficient control must be demonstrated
for individual operator training, qualification of manufacturing procedures, aseptic manufacturing, andmicrobiology. Process simulations can be used to validate
manual and semi-automated/closed-loop aseptic processes, as described by both the FDA26 and the Parenteral
Drug Association (PDA).27,28
PERSONNEL

A complex, experienced workforce poised to adapt to a
rapidly evolving field is required to develop and manufacture CAR-T cell products. Safe, effective, and consistent manufacturing of CAR-T cell products requires
establishment of a quality culture at the organizational
level as well as a staff with in-depth product knowledge
and thorough training in both aseptic manufacturing andproduct-specific processes. Significant emphasis must be

Chapter 10: Control Strategy

Figure 10-8: Foundation for the environmental
monitoring program.

Environmental
monitoring
program

Cleaning/disinfectant
studies, sample site selection/
qualification,and data management

Facility design and qualification

placed on education, practical experience, and demonstrated competency of the personnel.53 The European
Commission recommends that manufacturing personnel“possess the appropriate qualifications, adequate practical experience relevant to intended operations, and [havea] clear understanding of their responsibilities, including
knowledge of the product appropriate to the assigned
task.”2 It also recommends that, prior to participatingin routine manufacturing operations, personnel should
participate in successful aseptic process simulation tests.2
Similarly, the World Health Organization provides policies for appropriate personnel hygiene levels and healthconditions, as well as procedures for preventing the transmission of communicable diseases from raw and starting
biological materials.29,53 Personnel must be trained, andretrained as necessary, to perform their assigned responsibilities adequately.30 This extends to personnel involvedin the cleaning and maintenance of areas used for aseptic
processing.31 In addition, manufacturing facilities musthave appropriate redundancy in both personnel andorganizational structure to ensure thorough review of
all manufacturing and testing activities.

A-CELL✼213

ENVIRONMENTAL MONITORING AND CONTROLOF FACILITIES AND EQUIPMENT

Environmental monitoring is an important control
element when manufacturing CAR-T cell products.
Facility design (Chapter 11) provides the foundation ofa successful environmental monitoring program (Figure10-8); design and qualification staff must consider
elements such as surface finishes and cleanability; airfiltration, flow, and pressurization; flow of waste frompersonnel, material, and equipment; gowning procedures
and certification; and the number of personnel to beaccommodated by the facility.53 For autologous CAR-T
cell products, the facility may need to accommodate theprocessing of multiple individual patient batches in parallel. Design considerations are different for allogeneic
CAR-T cell products, where one large batch destinedfor multiple patients is produced at a time. Regardlessof approach, the environmental monitoring program
should be supported by cleaning and disinfectant studies32 as well as sample site selection/qualification and data
management. Several resources exist for the developmentof a successful environmental monitoring program.2,26,33
Facility controls should consider the segregation ofmaterials, product, and personnel such that mix-ups
and cross-contamination are minimized. The type of
product being manufactured must also be considered(i.e., whether autologous or allogeneic processing will
be performed). Equipment must be “fit for the purpose”
of CAR-T cell manufacturing and controls should beestablished for cleaning and sanitizing all equipment.34

Testing Controls
Quality control tests for CAR-T cell drug products center on demonstrating a favorable CQA profile by direct ororthogonal controls using validated methods throughout
the entire production process. At a minimum, testing
strategies must show that all drug product release specifications are met prior to release and infusion into patients.
According to the ICH Q6B, a specification “establishes
the set of criteria to which a drug substance, drug product, or materials at other stages of its manufacture should
conform to be considered acceptable for its intended
use.”35 As such, quality control tests must consider themechanism of action of the final drug product in the

Chapter 10: Control Strategy

overall control strategy. Specification limits and theprecision required by the analytical methodologies used
for testing must consider all product-related knowledge
and should reflect accumulating consensus and advances
within the rapidly evolving CAR-T cell therapy field.
Specification limits may also be tied to clinical outcomesthrough correlative analysis techniques to evaluate thepotential impact of CQA profiles on clinical endpoints
such as safety, efficacy, and pharmacokinetic profiles.
Testing can be divided into routine testing (i.e., release testing, as required by regulatory authorities anddone on every lot prior to disposition) and periodic
testing (i.e., testing performed on sufficient batches todemonstrate that a process is in a continuous state ofcontrol via validation, comparability, stability, or periodic monitoring studies). Quality tests are a key part ofany manufacturing control strategy. Phase-appropriate
testing controls can be employed to support early development where limited knowledge about the process
and product is available. The analytical methods used tosupport characterization and release should be constantly
evaluated during development, and improvements madebased on increased product and process understandingor where technological improvements are available toimprove reliability, robustness, and reduce variability.
As such, the tests themselves must be developed andimproved throughout clinical development, with final
commercial validation performed to support commercial
licensure. The final control strategy should define when
testing should be performed, which assays will be used,
and the acceptable range for each CQA that ensures a
product of acceptable quality. Additional information on
analytical method strategies is found in Chapter 9.
ROUTINE TESTING

In-process testing monitors the quality of cells, clearanceof process-related impurities, or safety attributes by
testing CQAs at critical production steps or intermediate product stages. The understanding of appropriate
in-process testing for any CAR-T cell product grows
throughout the development life cycle of the product.
Early on, assays that monitor product quality and patient
safety should be performed after most process steps
to determine which steps are most critical and whichassays are best able to pick up on process parameters

A-CELL✼214

that impact quality attributes of the intermediate or final
drug product. As the product moves into later phases ofdevelopment, an evolution of the control strategy continues as analytical methods are optimized, new methods
are potentially added, and appropriate acceptance criteria
for in-process and release testing are established based onprocess characterization and clinical development studies. Final specifications of a commercial drug productshould also encompass relevant real-world experience obtained through clinical studies, where correlative analysis
can be performed to better inform the practical relevanceof the observed range of CQAs and the potential impact
on clinical efficacy, safety, or pharmacokinetic parameters.
The benefit of correlating to clinical studies lies in theability to justify potentially wider specification acceptancecriteria that have been demonstrated safe and efficacious
across the broad range of clinical experience. Due to thegreater inherent variability of patient material within thecell therapy space, the ability to correlate CQA profiles toclinical outcomes to justify wider specification acceptancecriteria will allow greater flexibility for patient dosing
across the broader range of manufacturing experience.
Routine release testing is performed on every lot to
confirm alignment with established specifications. When
combined with in-process testing, control of the manufacturing process and final drug product from batch-tobatch can be demonstrated.36 Release tests center aroundcharacterizing products according to their identity,
potency, purity, safety, and dose/viable cell number (seeTable 10-5 and Chapter 9).
PERIODIC TESTING

Full characterization of manufacturing processes requires
more than in-process and release testing of intermediate
and/or final drug product. Even extensive release testing
only confirms that a particular process was successful;
however, it does not fully demonstrate control of theprocess itself. PPQ runs and other periodic testing (e.g.,comparability, stability, periodic monitoring) are required
to ensure that the process remains in a state of control.
According to the FDA’s Guidance for Industry on
Process Validation, the PPQ is part of process qualification and seeks to “confirm the process design anddemonstrate that the commercial manufacturing
process performs as expected.”20 In general, PPQ runs

Chapter 10: Control Strategy

Table 10-5: Examples of release/characterization testing for CAR-T cell products.

Goal/target of assay

Attributebeing
testedViable cell number

Dose

Sterility
Mycoplasma
Replication-competent lentivirus

SafetyVector copy number
CAR+ cells
T-cell markers (e.g., CD3+, CD4:CD8 ratio)
Count/concentration of transgeneexpressing cells

Identity

Markers of cell viability
Residual LV particles or plasmid DNA
Undesired cell types (host tumor cells,
WBCs, B cells)

PurityT-cell activation reagents/beads
Cytotoxic potential
Interferon-γ secretion

Potency

CAR+: chimeric antigen receptor-positive; LV: lentiviral vector;
WBCs: white blood cells

will feature a higher level of sampling and scrutiny ofprocess performance than what is common during actualcommercial production. In some instances, PPQ runs
may be performed at the extremes of normal operating
ranges (NORs) to confirm that process control remains
intact at these extremes.
The PPQ protocol must specify the manufacturingconditions, controls, testing, sampling plan, analyticalmethods, acceptance criteria, and expected outcomes.
PPQ lots should be manufactured using the intended
commercial process by the personnel who will routinely
be in charge of each production step, and should meet thestandards described within the specification. Once a sufficient number of PPQ runs have been performed, a full PPQ
summary report documenting results, deviations (alongwith product impact statements), and an assessment ofadherence to the written PPQ protocol should be prepared.

A-CELL✼215

Incorporation of stability testing into the control
strategy provides a high degree of confidence that the
drug product is stable within its expected shelf lifeand under all transport, storage, and conditions of use
during patient administration. According to the UnitedStates Pharmacopeia (USP) chapter 1049, “protocols toestablish stability of a patient-specific therapy should use
materials from multiple donors and at least three lots.”37
Stability during process holds at the manufacturer orat the medical center(s) should be assessed, as should
stability at key in-process points if drug substance isstored before final processing and filling (as drug product). Assays used in stability testing must be capable ofdetecting changes in the relevant CQAs being measured.
Robust stability-indicating assays are required to test
for CQAs that may be expected to degrade over time instorage; these assays must verify that storage conditions
maintain the purity and potency of the drug productwithin predetermined specification ranges and within acertain time frame, which will then dictate the productshelf life. Notably, aberrant potency assay results can beindicators of changes in stability. With CAR-T cell therapies, cryopreservation is the key to a stable platform, as itenables transportation of both the leukapheresis material
and the final drug product back-and-forth between the
manufacturing site and medical center. Cryopreservation
principles are discussed in detail in Chapter 8.
Significant guidance on stability data requirements
can be found in ICH Q5C and ICH Q1A(R2).38,39 In general, for either drug substance or drug product, data fromat least three batches representative of the manufacturing
scale of production should be provided, with “representative” applying to manufacturing process, storage
conditions, and containers. If claimed shelf-life is greaterthan 6 months, a minimum of 6 months of data should
be provided at the time of submission to regulatory
agencies (for shelf lives of <6 months, data requirements
should be considered on a case-by-case basis). Where
material volume is sufficient as an output of the drug
product manufacturing process during pre-approval andpre-licensing, the recommended intervals for stability
testing frequency are as follows:• ≤1 year shelf-life: stability studies monthly for the first 3
months, then at 3-month intervals thereafter

Chapter 10: Control Strategy• >1 year shelf-life: stability studies every 3 months
during the first year of storage, every 6 months during
the second year, and annually thereafter (note: this may
be applicable to allogeneic CAR-T cell approaches)
Though stability testing ideally follows ICH guidance,cell therapies are often constrained by small lot size andlimited starting materials. As such, acceptable creative
approaches to stability testing exist, including bracketingand matrix designs where multiple lots are utilized toholistically build an “ICH-like” picture of the stability
profile.40 In these cases, more than three batches may
be required to build the holistic stability framework.
Additionally, reduced stability testing may be appropriate
after approval or licensure where data are available that
demonstrate adequate stability.39
An additional stability testing consideration relates tothe need to maintain potency during the in-use (postthaw) phase, prior to recipient infusion, which also may
be extended in allogeneic approaches. Regulatory guidance on in-use testing is sparse; the EMA41 advises that:“The appropriate physical, chemical, and microbial
properties of the product susceptible to change duringstorage should be determined over the period of the
proposed in-use shelf life. If possible, testing should be
performed at intermediate time points and at the endof the proposed in-use shelf life on the final remaining
amount of the product in the container.”
Data obtained from in-use stability testing helps
support information contained in the label regarding the
preparation, storage conditions, and period during which
the drug product can be used. Typical in-use studies aremeasured in hours, so care should be taken to ensurestudies carried out can be tested immediately to verify
potential impact of storage post-thaw prior to patient
administration. For further discussion on product andin-use stability, including an example stability matrix fora CAR-T product, refer to Chapter 9.
Whenever a change is made to manufacturing materials or processes, a risk of altering one or more criticalquality attributes of the drug product exists. As such,comparability studies on pre- and post-change drug
products must be conducted to show that the resultant
products are similar in terms of efficacy and safety. Muchof the existing guidance on comparability studies stems

A-CELL✼216

from ICH Q5E,21 with insights on comparability incorporated into recent guidance from both the FDA24 andthe EMA.42 Comparability claims should not rely solely
on routine release specifications; rather, a prospective
comparability study design should accompany all filings and may include characterization studies, forced
degradation studies, and long-term stability studies.
Additionally, retrospective analyses comparing efficacy,
safety, and pharmacokinetics of pre- and post-change
product can be utilized to show comparability.43 Due tothe inherent variability of patient starting material, splitrun approaches should be utilized to ensure that withina specific donor, the process change can be assessed prechange vs post-change. Having sufficient paired runs willallow statistical analysis of the process differences to bewell understood beyond the inherent material variability
impact on such studies. Including a comparability testing
protocol within the control strategy to support any futurechanges could demonstrate a commitment to upholding
the utmost efficacy and safety of a CAR-T cell product.

Table 10-6: Clinical factors examined incorrelative analyses.44,45

Control Strategy as a Function ofClinical Relevancepatient-specific characteristics can be examined for theirrelationship to clinical outcomes of interest (Table 10-6).44,45
The type and depth (i.e., univariate, multivariate) of correlative analysis performed will depend on the relationshipbetween the CQA and clinical outcome being examined:45

CORRELATIVE ANALYSIS TO CONNECT DRUG
PRODUCT CQAS TO CLINICAL OUTCOMES

Correlative analysis employs statistical methods, subject-matter review, and advanced analytics infrastructures
to explore potential associations between drug productquality attributes (particularly those related to purity,
strength, and potency) and clinical outcomes (i.e., efficacy,
safety, and pharmacokinetic profiles).44 These analyses caninform the true criticality of quality attributes as well as thetolerance levels for their specifications. Correlative analyses also facilitate a better understanding of how variation
within quality attributes impact both product performanceand patient outcomes. The end result may enable streamlining of the manufacturing process by pointing out where
flexibility lies in the process or the product.
Specific to CAR-T cell products, numerous genotypic, phenotypic, and functional CQAs measured during
release and characterization can impact efficacy, safety,
and pharmacokinetic profiles at the patient level (Table
10-5). Through correlative analysis, these CQAs andChapter 10: Control Strategy

FactorPotential correlative
relationshipPatient
characteristics

Age
Prior treatment exposure
Tumor characteristics
T-cell attributes

Clinical efficacy

Overall response rate
Complete response rate
Best overall response
Duration of response
Progression-free survival

Clinical safety

Cytokine release syndrome
Cytopenia
Neurotoxicity
Oncogenesis (or lack thereof)

Pharmacokinetics/
pharmacodynamics

Area under the curve
Maximum concentration
Time to maximum concentration• Categorical (e.g., responder/non-responder; cytokine
release syndrome [yes/no]; neurotoxicity [yes/no])• Time-to-event (e.g., progression-free survival; durationof response)• Continuous (e.g., area under the curve, maximum
concentration)
IMPACT OF CQA LEVELS ON DIRECT PATIENT OUTCOMES

The statistical effect size (i.e., magnitude) of the correlative relationship between a CQA and patient outcome andany resultant shift of the risk/benefit profile of the drug
product are used to judge the impact and clinical importance of variability within the CQA.45 Variability can be
introduced via patient starting material, manufacturing
processes, or analytical methods. Variation in manufacturing processes that seek to clear viral contamination
or remove process reagents (e.g., serum) have obvious
ties to patient safety, as do analytical methods to detect

A-CELL

✼217

Figure 10-9: Example of correlative analysis for a CQA (a) with two different scenarios illustrated (b).45
A
Number of lots

lower
middleupper
middle

lower

upper
CQA distribution

B

Scenario 1: Durable efficacy
is the same across range

Proportion in response

100

CQA
quartiles

80

Scenario 2: Trend in reduced durable
efficacy at lower end of the range

upper
upper middle
lower middle
lower

60

100
80
60

40

40

20

20

0
0

3

6

9

Time

12

15

18

0
0

3

6

9

Time

12

15

18

Source: Larson RP. Cell & Gene Therapy Products (CGTP) Virtual Symposium. June 8-10, 2020. Reprinted with permission.

contaminants such as mycoplasma. However, the impact
of variation in patient starting materials, which generally
represents the largest source of variation in autologous
CAR-T cell therapies, is oftentimes less clear.44 Given that
this variability is unique and inherent to the patient, it
may not result in an adverse impact on patient safety or
product efficacy, even if it results in a related CQA fallingoutside of the accepted specification range establishedduring preclinical drug development. As such, it may bejustifiable to push specification limits for certain CQAs
to the edges of their clinically relevant distribution when
it can be shown that no difference in the risk/benefit profile is imparted by attribute levels at the upper or lower
extremes of the distribution range.44,45 An example of thisis found in Figure 10-9, which groups CQA distribution
by upper and lower quartiles to illustratively determine
the overall clinical benefit across the range of clinicalexperience. Scenario 1 of Section B of the figure demonstrates an example where a specification may be justified
by the entire range of clinical experience due to similar
clinical benefit. Conversely, Scenario 2 of Section B may

Chapter 10: Control Strategy

represent a situation where a tighter specification may
be required to maximize patient benefit based on clinicalbenefit distributions. By expanding the specifications tothe broader ranges of clinical experience, delay of productdelivery caused by unnecessarily stringent specifications
can be prevented.
Several examples illustrate the value of performing
correlative analysis and considering wider acceptancecriteria for certain CQAs. Out-of-specification cell viability did not compromise the clinical safety or efficacy oftisagenlecleucel in patients with acute lymphoblastic leukemia, non-Hodgkin lymphoma,46 or diffuse large B-cell
lymphoma.47 Similarly, no significant difference in cell
viability was seen among responders and nonresponders
to axi-cel therapy among patients with relapsed/refractory large B-cell lymphoma.48 Broader specification limits
for VCN, which is often kept below 5 due to concern
for random integration and insertional oncogenesis,49,50
may be appropriate when a robust understanding of thevector’s insertion sites is present and when correlative
data show that clinical safety is not compromised.44,51

A-CELL✼218

DIVERGENCE BETWEEN AUTOLOGOUS AND
ALLOGENEIC APPROACHESIMPROVEMENTS IN CQA UNDERSTANDING
THROUGHOUT THE PRODUCT LIFE CYCLE

Correlative analytical strategies for autologous and allogeneic CAR-T cell products take somewhat different
approaches, owing largely to the unique challenges toefficacy and safety that each pose. With allogeneic
CAR-T cell products, optimization of attributes that
impact clinical efficacy focus largely on those related
to potency, particularly given the extended shelf life ofallogeneic products. Additionally, pharmacodynamic
considerations such as how quickly the allogeneic CAR-T
cell product is cleared by the recipient’s immune system
can significantly impact efficacy. Following lymphodepletion, infusion of allogeneic CAR-T cell products may
be followed by host rejection and a corresponding lossof clinical effect. As such, the degree of lymphodepletion present in recipients and the rapidity of immune
reconstitution can significantly impact efficacy. From asafety standpoint, correlative analyses focus on allogeneic
product quality attributes that could potentiate GvHD or
infectious disease transmission.

Initial CQA specifications for a drug product are based onlimited manufacturing and preclinical data. As experiencegrows with the product through clinical production andprocess and product characterization studies, and as clinical data begins surfacing in pre-pivotal trials, enhanced
understanding of CQAs and their appropriate specification levels will follow. As development proceeds towardcommercialization and product knowledge is gained,
regulatory agencies generally expect to see tightening of
acceptance criteria as part of the refined control strategy,44
though this may be less applicable and feasible with autologous cell-based products. As such, proposals for broader
acceptance criteria should be accompanied by extensive
correlative data that justifies the request. In these cases,
it may be advantageous to engage in direct regulatoryinteractions to have a robust scientific discussion about
proposed specifications, risk/benefit profiles for patients,
and potential impact on timely infusion to patients withpoor disease prognosis.

Figure 10-10: Components of a total integrated control strategy.52

Characterization
Studies

Manufacturing
Controls (Process
Controls &Setpoints, IPCs)

Process
Validation

Patient Variability(Apheresis)

CPV

Procedural
Controls

Raw Materials
Controls

Comparability

INTEGRATEDCONTROLSTRATEGYGMP Processing

Specifications

Stability
Specification

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission.

Chapter 10: Control Strategy

A-CELL

✼219

Figure 10-11: The integrated control elements matrix is used to score occurrence and detection of each attribute.

Attribute 1

x

x

x

x

Attribute 2

x

x

x

x

Attribute 3

x

x

x

x

x
x

x

x

x

Stability

Process
validation

IPC action limit/
acceptancecriteria

x

x

x

x

x

x

x

Attribute 4

Extendedcharacterization

The Proposed Control Strategy’s
ability to detect and control of theattribute are considered

DP release testing

Raw material
testing

Quality attribute

Process
knowledge

Experience withcommercial
process

Process knowledge determines the likelihood (occurrence) that the
attribute will be present in sufficient quantity in the drug product tocause the effect considered when assigning the severity score.

xxx

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission.

Evaluation of Total Integrated
Control StrategyT﻿he culmination of product development integrates anentire control system that supports a comprehensive control strategy to ensure product quality and drug productreliability to serve the needs of patients. Presenting a
total integrated control strategy (ICS) to the health authorities demonstrates a cumulative control of the entire
manufacturing process, including GMP procedural andfacility controls, raw material controls, robust processcharacterization to drive meaningful in-process controls,
periodic testing controls (e.g., PPQ, continued processverification, comparability, stability), as well as routine
testing during drug product release. All these controls,in addition to the target patient population, disease indication, potential impact of variability from the starting
patient material, and correlation to clinical outcomes canbe used to demonstrate a holistic control of CQAs wellbeyond just the release specification (Figure 10-10). The

Figure 10-12: The product quality risk assessment determines the final residual risk to patients from each attribute.
The Severity score and the Occurrence score are
multiplied to arrive at a Preliminary Hazard level.
This reflects the risk to patient without considerationof the testing program.

Quality attribute

Severity score

The Preliminary Hazard Level is multiplied by the detection
score to determine the final residual risk to patients fromeach attribute.

Preliminary
Occurrence score hazard level

Detection

Final residual risk

Attribute 1

3

5

15

1

15

Attribute 2

2

7

14

3

42

Attribute 3

1

1

1

2

2

Attribute 4

6

7

42

1

42

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission.

Chapter 10: Control Strategy

A-CELL

✼220

Figure 10-13: Utilizing quality by design principles to develop an integrated control strategy.

Manufacturingexperience

FIH• Hypothesis-driven
QTPP based on
platform CQAs

• Preliminary Control Strategy• Develop productcharacterization
strategy

Pivotal• Control Phase appropriate CQAs
and pCQAs through risk-based
approach• Execute product / processcharacterization studies• Perform preliminary Correlative
Analysis to confirm CQAs basedon clinical outcomes (safety,
efficacy)• Initial ICEM / PQRA to assess
process capability, initial control
strategy, and preliminary risk topatients

Commercial• Develop commercial enabling
Integrated Control Strategy

Commercial filing

Preclinical• Perform final Correlative
Analysis to confirm correlationof CQAs to clinical outcomes• Confirm total Integrated
Control Strategy (post PPQ) to
support commercial product• Final ICEM / PQRA to assess
process capability, commercial
control strategy, and final risk
to patients

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission.

total ICS should reflect a risk-based approach to establishing and assessing the overall risk posed by various
attributes to patient safety and product quality. As such,
the ICS helps to identify attributes that need to be evaluated to demonstrate process control and consistency.
TOOLS TO DEVELOP AN INTEGRATED CONTROLSTRATEGY

An integrated control elements matrix (ICEM) captures
the impact of process elements on product quality attributes, compiles control elements and defines where they
are applied in the process, and ultimately serves to define the control strategy. In combination with a productquality risk assessment (PQRA), the ICEM applies theprinciples of ICH Q9: Quality Risk Management to the
ICS. The ICS contained within the ICEM is integrated
in the sense that it accounts for attribute criticality,
process understanding, and testing controls.52
Following a similar approach to FMEA (discussed
earlier in the chapter), the combined ICEM/PQRA

Chapter 10: Control Strategy

approach evaluates the ICS for residual risk to patients
posed by various attributes. First, the ICEM is used toscore occurrence and detection of the attribute basedon process understanding and analytical method capabilities (Figure 10-11). The PQRA assigns a severity
score based on potential impact to patient safety andproduct efficacy to the attribute and then determines
the overall residual risk to the patient by considering
severity, occurrence, and detection (Figure 10-12).52
CONTROL STRATEGY EVOLUTION FROM
CLINICAL TO COMMERCIAL DEVELOPMENT

According to ICH Q9,15 consideration should be given
to improving the control strategy over the life cycle ofproduct development. The unique aspects of CAR-T
cell therapies requires that the industry leverage tools
such as ICEM and PQRA to develop phase-appropriate control strategies that adapt over time (Figure
10-13). Early-phase control strategies are reflective of
historical/initial knowledge of both the manufacturing

A-CELL

✼221

Figure 10-14: Pathway from early-phase to commercial control strategy.
Early Phase Control Strategy Development

Quality Target
Product Profile(QTTP)

Initial Critical
Quality
Attribute (CQA)
Assessment

Pre-Pivotal
Process
Parameter Risk
Assessment

Clinical Process
Control
StrategyForms the basisof design for thedevelopment ofdrug product

Identification ofpotential criticalquality attributes
based on process
knowledge and
QTPP

Identificationof and initialrisk assessment
for processparameters

Process Control
Strategy for clinicalphase production

3. Correlative Analysis(CQA
Clinical Data)
PIVOTAL
CLINICAL
EXPERIENCE

Critical Quality
Attribute (CQA)
Assessment
Identification of
Critical Quality
Attributes based on
pivotal experience

1. Process Characterization• PC studies
• PPQ
• CPV

Process
Characterization
Master Plan

Process
Characterization
Reports

Documentationof planned
characterization
studiesDocumentation ofindividual completedcharacterization
studies

Process
Characterization
Summary Report

Process Parameter
Risk Assessment
and Parameter/
Attribute
Classification
Documentation of
Parameter / Attribute
Risk Assessment andClassification

Summary ofcompletedcharacterization
studies

2. ProductCharacterization• CQA studies• Impurity studies

Range
Justification
ReportDocumentationand justificationof proposed
Commercial
Control Ranges

Commercial
Process
Control
Strategy
Proposed Process
Control Strategy
for commercial
production

Commercial Control Strategy Development
Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission.

process and the drug product characteristics. The commercial control strategy is refined based on an evolving
knowledge gleaned from product/process characterization, characterization of patient variability, andcorrelative analyses that tie product quality attributes
directly to clinical outcomes in the pivotal clinical trials. Establishment and utilization of early-phase control
strategies can be used to guide and provide structure tointernal product development stages, whereas a refinedcommercial control strategy is an essential component

Chapter 10: Control Strategy

of any biologics license application filing. An overview
of the pathway followed to develop phase-appropriate
control strategies is contained in Figure 10-14.
Importantly, different control strategies could beapplied at different sites during both early-phase andcommercial development.15 Differences could be due
to equipment, facilities, systems, and CDMO-related
confidentiality considerations. Where CDMOs are
involved, seamless knowledge transfer between the
CDMO and sponsor (in both directions) is essential.

A-CELL✼222

Establishing Meaningful
Specifications
CAR-T cell products represent a relatively new therapeutic modality. As such, literature on specification
development strategies for CAR-T cell therapies islacking. Guidance can be gleaned from ICH Q6B, 35
which discusses general approaches to specification setting for biological products. A specification, as definedby ICH Q6B, is a list of tests, references to analyticalprocedures, and appropriate acceptance criteria that
describe the numerical limits, ranges, or other criteria
for the tests described. Specifications are critical quality
standards proposed and justified by the manufacturer

and considered as conditions of approval by regulatory
bodies. As such, specifications are a key part of a total
control strategy that seeks to ensure drug product quality and consistency. The establishment of specifications
should consider manufacturing processes, stability of the
drug substance and drug product, data from preclinicaland clinical studies, and the capabilities of analyticalprocedures.35
EARLY-STAGE DEVELOPMENT

Like all elements of the control strategy, specification development takes on a life cycle approach. Early specifications are based on limited manufacturing experience and/or historical data from experiences with similar products.

Table 10-7: Examples of early-phase autologous CAR-T cell product specifications.52
Quality
Attribute
Appearance
Identity

PurityStrength

Parameter

Methodology

Specification

Color

Compendial

Description of color

ClarityCompendial

Description of turbidity

Confirmation of identity

Flow cytometry

Anti-XXX CAR+ cells detected (identity
confirmed)

Cell viability

Fluorescent
microscopy and image
analysis

T-cell purity/
immunophenotype

Flow cytometry

Product-related
impurities

Flow cytometry

Process-related
impurities

ELISA or other suitable
methodologies

Initial specifications based on historical
process understanding, initial impurity
risk assessment or tox assessment

CAR + viable T cells

Flow cytometry> XX CAR+ cells/mL (strength may be usedin lieu of potency)

Transduction controls

qPCR

Initial specifications based on platform
knowledge (product, process), patient
population, health authority guidance,and risk-based approach. Strength
alternative orthogonal control, develop inlater phase of development

Endotoxin

Compendial

XX EU/mL

Mycoplasma

Compendial

Not detected

Sterility

Compendial

No growth

SafetyInitial specifications based on platform
knowledge (product, process), patient
population, health authority guidance,and risk-based approach

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission

Chapter 10: Control Strategy

A-CELL✼223

Table 10-8: Examples of commercial-phase autologous CAR-T cell specifications.52
Quality Attribute

Parameter

Methodology

Specification

Color

Compendial

Description of color

ClarityCompendial

Description of turbidity

Confirmation of identity

Flow cytometry

Anti-XXX CAR+ cells detected(identity confirmed)

Cell viability

Fluorescent
microscopy and image
analysis

T-cell purity/
immunophenotype

Flow cytometry

Product-related
impurities

Flow cytometry

Process-related
impurities

ELISA or other suitable
methodologies

Meaningful specification establishedper process characterization/
impurity risk assessment

Strength

CAR + viable T cells

Flow cytometry> XX CAR+ cells/mL

Potency

Antigen-specificfunction

BioassayTransduction controls

qPCR

Product-specific acceptance criteria
established per clinical correlative
analysis (potency, transduction
controls)

Endotoxin

Compendial

XX EU/mL

Mycoplasma

Compendial

Not detected

Sterility

Compendial

No growthAppearance
Identity

PuritySafetyMeaningful specification establishedper clinical correlative analysis

Source: Polson N. Presentation at: CASSS Cell and Gene Therapy Conference. June 9, 2021. Reprinted with permission

They may also take into consideration knowledge ofthe patient population that the product is designed totreat, guidance from the health authorities, and initialassessments of risk posed by product- or process-related
impurities. Specifications for CAR-T cell products generally center around the quality attributes of appearance,
identity, purity, strength, and safety (Table 10-7).44,52

acceptance criteria employed at the commercial stage
should strongly consider data gleaned from correlative
analyses that bridge clinical outcomes to the specifications themselves, with the ultimate goal of setting
patient-centric specifications (Table 10-8).44,52

CLINICAL-TO-COMMERCIAL DEVELOPMENTAs development programs progress through clinical
trials and toward commercialization, regulatory bodies
generally expect specification acceptance criteria to betightened. This is often accomplished via a toleranceinterval-based approach that is founded on an understanding of process capability. However, process capability should not be the sole determinant of specification
acceptance criteria. As discussed earlier in the chapter,

Chapter 10: Control Strategy

A-CELL

✼224

References
1.

International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q10: Pharmaceutical Quality System.
June 4, 2008. https://database.ich.org/sites/default/files/
Q10%20Guideline.pdf

11. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q8(R2): Pharmaceutical Development.
August 2009. https://database.ich.org/sites/default/files/Q8_
R2_Guideline.pdf

2.

European Commission. EudraLex—The Rules Governing
Medicinal Products in the European Union. Volume 4:
Guidelines on Good Manufacturing Practice specific to
Advanced Therapy Medicinal Products. November 22, 2017.
https://ec.europa.eu/health/sites/default/files/files/eudralex/
vol-4/2017_11_22_guidelines_gmp_for_atmps.pdf

3.

European Medicines Agency. Draft Guideline on the Riskbased Approach According to Annex I, Part IV of Directive
2001/83/EC applied to Advanced Therapy Medicinal Products.
January 2012. https://www.ema.europa.eu/en/documents/
scientific-guideline/draft-guideline-risk-based-approachaccording-annex-i-part-iv-directive-2001/83/ec-appliedadvanced-therapy-medicinal-products_en.pdf

12. European Medicines Agency. EMA/CHMP/CVMP/
QWP/354895/2017. Questions and Answers: Improving theUnderstanding of NORs, PARs, DSp and Normal Variabilityof Process Parameters. June 6, 2017. https://www.ema.europa.
eu/en/documents/scientific-guideline/questions-answersimproving-understanding-normal-operating-range-norproven-acceptable-range-par_en.pdf

4.

US Food and Drug Administration. Guidance for Industry
and Food and Drug Administration Staff: Regulatory
Considerations for Human Cells, Tissues, and Cellular
and Tissue-based Products: Minimal Manipulationand Homologous Use. July 2020. https://www.fda.gov/
media/109176/download

5.

US Food and Drug Administration. Guidance for industry:
Evaluation of Devices Used With Regenerative Medicine
Advanced Therapies. February 2019. https://www.fda.gov/
media/120266/download

6.

US Food and Drug Administration. Guidance for industry:
Expedited Programs for Regenerative Medicine Therapies
For Serious Conditions. February 2019. https://www.fda.gov/
media/120267/download

7.

US Food and Drug Administration. Guidance for industry:
Manufacturing Considerations for Licensed and Investigational
Cellular and Gene Therapy Products During COVID-19
Public Health Emergency. January 2021. https://www.fda.gov/
media/145301/download

8.

US Food and Drug Administration. Guidance for industry:Same Surgical Procedure Exception Under 21 CFR 1271.15(b):Questions and Answers Regarding the Scope of the Exception.
November 2017. https://www.fda.gov/media/89920/download

9.

Tang Y; US Food and Drug Administration. Quality bydesign approaches to analytical methods -- FDA perspective.
Presented at: 2011 American Association of Pharmaceutical
Scientists Annual Meeting and Exposition; October 25, 2011;
Washington DC. Accessed June 11, 2011. https://docplayer.
net/11954697-Quality-by-design-approaches-to-analyticalmethods-fda-perspective-yubing-tang-ph-d-fda-cder-ondqaaaps-washington-dc-october-25-2011.html

10. US Food and Drug Administration. Draft Guidance forIndustry: Considerations for the Development of Chimeric
Antigen Receptor (CAR) T Cell Products. March 2022. https://www.fda.gov/media/156896/download

Chapter 10: Control Strategy

13. European Medicines Agency. EMA/CAT/GTWP/671639/2008.
Guideline on Quality, Non-clinical and Clinical Aspectsof Medicinal Products Containing Genetically Modified
Cells. November 12, 2020. https://www.ema.europa.eu/en/
documents/scientific-guideline/guideline-quality-non-clinicalclinical-aspects-medicinal-products-containing-geneticallymodified_en-0.pdf
14. Kellner K. Accelerating CAR-T process development: the powerof process insight. Sartorius webinar: https://www.sartorius.
com/en/applications/cell-and-gene-therapy/cell-therapy/
cell-therapy-guide/accelerating-car-t-process-developmentprocess-insight-webinar
15. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q9: Quality Risk Management.
November 2005. https://database.ich.org/sites/default/files/
Q9%20Guideline.pdf
16. Castillo FC, Cooney B, Levine HL. Biopharmaceuticalmanufacturing process validation and quality risk
management. International Society for PharmaceuticalEngineering. 2016. https://ispe.org/pharmaceuticalengineering/may-june-2016/biopharmaceuticalmanufacturing-process-validation-and
17. Politis SN, Colombo P, Colombo G, M Rekkas D. Design ofexperiments (DoE) in pharmaceutical development. Drug Dev Ind
Pharm. 2017;43(6):889-901. doi:10.1080/03639045.2017.1291672
18. Ashton R, Dave K, Stoops J. Variance components analysis
for autologous therapy process development. Presentation at:
CASSS Cell and Gene Therapy Conference. June 10, 2019.
19. Hupfeld J, Wercberger R, Albersdoerfer, M, et al. Optimizationof T-Cell Expansion Using the Sartorius T-Cell Exploration andCharacterization Solution. Sartorius application note: https://www.sartorius.com/en/applications/cell-and-gene-therapy/celltherapy/cell-therapy-guide/optimization-of-t-cell-expansion-appnote
20. US Food and Drug Administration. Guidance for Industry:
Process Validation: General Principles and Practices. January
2011. https://www.fda.gov/media/71021/download
21. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for
Human Use. Quality Guideline Q5E: Comparability of
A-CELL✼

225

References, continued
Biotechnological/Biological Products Subject to Changes in
Their Manufacturing Process. November 18, 2004. https://www.gmp-compliance.org/files/guidemgr/Q5E.pdf

33. Parenteral Drug Association. Technical Report No. 13 (Revised2014): Fundamentals of an Environmental Monitoring Program.
2014. https://store.pda.org/TableOfContents/TR13_TOC.pdf

22. European Commission. EudraLex—The Rules Governing
Medicinal Products in the European Union. Volume 4: Good
Manufacturing Practice for Medicinal Products for Humanand Veterinary Use; Annex 2: Manufacture of Biological Active
Substances and Medicinal Products for Human Use. June 2012.
https://health.ec.europa.eu/system/files/2019-02/2018_annex2_
en_0.pdf

34. European Medicines Agency, Committee for Proprietary
Medicinal Products. CPMP/QWP/054/98. Decision Trees for the
Selection of Sterilization Methods: Annex to Note for Guidance
On Development Pharmaceutics. April 5, 2020. https://www.
ema.europa.eu/en/documents/scientific-guideline/supersededannex-note-guidance-development-pharmaceutics-decisiontrees-selection-sterilisation_en.pdf

23. Graham C, Jozwik A, Pepper A, Benjamin R. Allogeneic
CAR-T cells: more than ease of access?. Cells. 2018;7(10):155.
doi:10.3390/cells7100155

35. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q6B: Specifications: Test Procedures andAcceptance Criteria for Biotechnological/Biological Products.
March 1999. https://database.ich.org/sites/default/files/Q6B%20
Guideline.pdf

24. US Food and Drug Administration. Guidance for industry:
Chemistry, Manufacturing, and Control (CMC) Informationfor Human Gene Therapy Investigational New Drug
Applications (INDs). January 2020. https://www.fda.gov/
media/113760/download
25. Alliance for Regenerative Medicine and National Institute forInnovation in Manufacturing Biopharmaceuticals. Project
A-Gene: A case study-based approach to integrating QbD
principles in Gene Therapy CMC programs. June 24, 2021.
https://alliancerm.org/manufacturing/a-gene-2021
26. US Food and Drug Administration. Guidance for Industry:
Sterile Drug Products Produced By Aseptic Processing—
Current Good Manufacturing Practice. September 2004.
https://www.fda.gov/media/71026/download
27. Parenteral Drug Association. Technical Report No. 22 (revised2011): Process Simulation for Aseptically Filled Products. 2011.
https://store.pda.org/TableOfContents/TR2211_TOC.pdf
28. Parenteral Drug Association. Technical Report No. 62:
Recommended Practices for Manual Aseptic Processes. 2013.
https://store.pda.org/ TableOfContents/TR6213_TOC.pdf
29. World Health Organization. WHO Good Manufacturing
Practices for Sterile Pharmaceutical Products. 2011. https://www.who.int/medicines/areas/quality_safety/quality_assurance/
GMPSterilePharmaceuticalProductsTRS961Annex6.pdf
30. US Food and Drug Administration. Code of Federal
Regulations Title 21 Part 1271: Human Cells, Tissues, andCellular and Tissue-Based Products. April 1, 2020. Accessed
June 14, 2021. https://www.accessdata.fda.gov/scripts/cdrh/
cfdocs/cfcfr/CFRSearch.cfm?fr=1271.170
31. European Commission. EudraLex—The Rules Governing
Medicinal Products in the European Union. Volume 4: Good
Manufacturing Practice for Medicinal Products for Humanand Veterinary Use; Annex 1: Manufacture of Sterile Medicinal
Products. November 25, 2008. https://www.gmp-compliance.
org/files/guidemgr/annex%2001[2008].pdf
32. Parenteral Drug Association. Technical Report No. 70:
Fundamentals of Cleaning and Disinfection Programs for
Aseptic Manufacturing Facilities. 2015. https://store.pda.org/
TableOfContents/TR70_TOC.pdf

Chapter 10: Control Strategy

36. Li Y, Huo Y, Yu L, Wang J. Quality control and nonclinicalresearch on CAR-T cell products: general principles and key
issues. Engineering. 2019;5:122-131.
37. United States Pharmacopeia. General Chapter <1049>.
Quality of Biotechnological Products: Stability Testing Of
Biotechnological/Biological Products. 1996.
38. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q1A (R2): Stability Testing of New Drug
Substances and Products. February 2003. https://database.ich.org/
sites/default/files/Q1A%28R2%29%20Guideline.pdf
39. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for
Human Use. Quality Guideline Q5C: Stability Testing of
Biotechnological/Biological Products. November 1995. https://
database.ich.org/sites/default/files/Q5C%20Guideline.pdf
40. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for Human
Use. Quality Guideline Q1D: Bracketing and Matrixing Designs
For Stability Testing of New Drug Substances and Products.
February 2002. https://database.ich.org/sites/default/files/
Q1D%20Guideline.pdf
41. European Medicines Agency. Note for Guidance on In-Use
Stability Testing of Human Medicinal Products. March 2001.
https://www.ema.europa.eu/en/documents/scientific-guideline/
note-guidance-use-stability-testing-human-medicinalproducts_en.pdf
42. European Medicines Agency. Guideline on Quality, Non-Clinicaland Clinical Requirements for Investigational Advanced Therapy
Medicinal Products in Clinical Trials. Draft. January 2019.
https://www.ema.europa.eu/en/documents/scientific-guideline/
draft-guideline-quality-non-clinical-clinical-requirementsinvestigational-advanced-therapy_en.pdf
43. Cockroft A, Wilson A. Comparability: what we can learn fromthe review of advanced therapy medicinal products. Regen Med.
2021;16(7):655-667. doi:10.2217/rme-2021-0026

A-CELL

✼226

References, continued
44. Zhang T, Larson R, Dave K, Polson N, Zhang H. Developing
patient-centric specifications for autologous chimericantigen receptor T cell therapies. Curr Opin Biomed Eng.
2021;20:100328. doi:10.1016/j.cobme.2021.100328
45. Larson RP. Leveraging product quality attribute correlative
analysis with clinical outcomes to establish control strategies
for gene-engineered T cell therapies. Cell & Gene Therapy
Products (CGTP) Virtual Symposium. June 8-10, 2020.
46. Pasquini MC, Hu ZH, Curran K, et al. Real-world evidence oftisagenlecleucel for pediatric acute lymphoblastic leukemia andnon-Hodgkin lymphoma. Blood Adv. 2020;4(21):5414-5424.
doi:10.1182/bloodadvances.2020003092
47. Chong EA, Levine BL, Grupp SA, et al. CAR T cell viability
release testing and clinical outcomes: is there a lower limit?.
Blood. 2019;134(21):1873-1875. doi:10.1182/blood.2019002258
48. Locke FL, Rossi JM, Neelapu SS, et al. Tumor burden,
inflammation, and product attributes determine outcomes ofaxicabtagene ciloleucel in large B-cell lymphoma. Blood Adv.
2020;4(19):4898-4911. doi:10.1182/bloodadvances.2020002394

50. Lu XV. Regulatory considerations for manufacturing and testingof investigational chimeric antigen receptor (CAR) T-cell
products. U.S. Food and Drug Administration. Presentation
at: Measurement Challenges for CAR-T Biomanufacturing.
January 29, 2016. Gaithersburg, MD. https://www.ibbr.umd.
edu/sites/default/files/public_page/Xiaobin%20Victor%20
Lu_FDA.pdf
51. Montini E, Cesana D, Schmidt M, et al. The genotoxic potentialof retroviral vectors is strongly modulated by vector design
and integration site selection in a mouse model of HSC genetherapy. J Clin Invest. 2009;119(4):964-975. doi:10.1172/
JCI37630
52. Polson N. Autologous cell therapy phase appropriate control
strategies from early clinical development to commercialization.
Presentation at: CASSS Cell and Gene Therapy Conference.
June 9, 2021.
53. Parenteral Drug Association. Technical Report No. 81: Cell-Based
Therapy Control Strategy. 2019. https://www.pda.org/bookstore/
product-detail/4638-tr-81-control-strategy-for-cell-therapy

49. Zhao Y, Stepto H, Schneider CK. Development of the FirstWorld Health Organization Lentiviral Vector Standard: Toward
the Production Control and Standardization of LentivirusBased Gene Therapy Products. Hum Gene Ther Methods.
2017;28(4):205-214. doi:10.1089/hgtb.2017.078

Chapter 10: Control Strategy

A-CELL✼227

CHAPTER 11

Manufacturing Strategy,
Facility Design,
and Manufacturing
Operations
CHAPTER LEADS

Jasmin Kee, Kee Bio Ltd
Peter Walters, CRB
CHAPTER CONTRIBUTOR

Kanti Thirumoorthy, Neogene Therapeutics

Chapter 11 | Contents
Introduction................................................................... 229

Cleanrooms and classification............................................ 244

Facility Design Process Overview...............................230

Cleanroom classification for open and closed
processes.............................................................................. 245

Definition................................................................................230
Design...................................................................................... 231

Cleanroom environmental monitoring........................... 246
Heating, ventilation, and air conditioning (HVAC).............247

Design reviews and contamination control
risk assessment................................................................... 232

Operational design considerations......................................... 248

Build, commissioning, qualification, and validation........ 233

Gowning operations............................................................. 249

Manufacturing start-up to manufacturing operation...... 233

Chain of identity and chain of custody.............................. 249

Construction Methods and Design Layout........................... 233

Segregation.......................................................................... 250

Construction methods.......................................................... 233

Multi-patient and multi-product facilities........................... 250

Processing room layout options......................................... 235

Manufacturing Start-Up and Operation................................. 251

Dedicated production space.......................................... 236

Personnel training and gowning.......................................... 251

Ballroom design................................................................237

Process simulation and aseptic process validation.......... 252

Dance-floor design (segregated unit operations)........... 238

Line clearance.......................................................................252

Support rooms..................................................................... 239

Cleaning and disinfection.....................................................252

Facility personnel and material flows.................................. 241

Cleaning validation.............................................................. 253

Unidirectional flows and facility design............................ 241

Environmental monitoring program................................... 253

Equipment selection and workstation layout.................... 242

References................................................................................. 254

Design practices for workstations...................................... 243

Appendix: List of Acronyms.................................................... 254

Specific considerations for commonly usedequipment............................................................................. 244
Biological safety cabinets.................................................. 244
Incubators.......................................................................... 244

Introduction
The growth of the cell and gene therapy (CGT) sector
has led to an increasing demand for facilities suitable
for manufacturing these products at a clinical andcommercial scale. The options for cell-based therapy
developers are to manufacture in-house through use ofexisting facilities or building new facilities; outsource
manufacturing to a contract development and manufacturing organization (CDMO); or rent current good
manufacturing practice (CGMP) space in a hybrid model
where the cell therapy manufacturer utilizes their own
employees in a facility overseen by the facility owner.
In contrast to more traditional biologic or vaccine

manufacturing facilities, which are typically designed
to accommodate large-scale processes using equipment
such as bioreactors, stainless steel vessels, and chromatography systems, cell-based therapy manufacturing
processes are traditionally more akin to research laboratory scale with equipment such as biosafety cabinets,
centrifuges, microscopes, and single-use plasticware to
execute operations, especially for autologous products.
Allogeneic products produced on a larger scale are on
the horizon and utilize bioreactor-based processes withpredominantly single-use systems. Many cell-based therapy product developers are in the process of establishingin-house manufacturing facilities to contend with theincreasing demand for cell-based therapy manufacturing

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼229

capacity, the relative lag and lack of suitable capacity
availability from CDMOs and rentable spaces, and toprovide flexibility of supply and retention of intellectual
property.
Designing and establishing new facilities for cellbased therapy manufacturing that meet required regulatory standards is a challenging task. It is often one of thelargest capital expenditures related to cell-based therapy
product development due to factors such as the need forhighly specialized equipment, the need for facility flexibility to accommodate current and future manufacturing
processes, and evolving process scales in anticipation ofincreased market demand. A key feature of cell-based
therapy product manufacturing is the requirement
for full aseptic processing rather than sterile filtrationor final product terminal sterilization as in the case of
traditional biologics. Therefore, the facility design must
minimize the risk of product contamination throughout the process from incoming raw materials to final
product storage to ensure product quality and patient
safety. Autologous processes pose design challenges toconcurrent manufacturing of multiple, small-scale, patient-specific batches, where mid to large-scale allogeneic
processes require the design to accommodate processes
at the boundary of manual processing capability andfuture introduction of automated technologies that are
still in development.
The goal of all biopharmaceutical manufacturing
facilities is to produce consistent, compliant productsthat meet regulatory requirements at a sustainable cost.
The facility design process translates process, operational,
engineering, and regulatory requirements into a tangible
facility and requires dedication of time and resourcesfrom all functions of the cell therapy organization
coupled with specialist input from architectural, engineering, and construction organizations. The design andconstruction of a manufacturing facility is integral to thereliable and reproducible execution of the manufacturing
process operation and should be defined and controlled
in line with qualification and validation principles set
out in regulatory guidelines for production of asepticallyprocessed products and CGMP.
This chapter describes an overview of the facility
design process, key regulatory requirements, design layout, operational design considerations, and qualification,

validation, and manufacturing start-up processes to
evolve initial facility requirements into a fit-for-purpose
operational facility for cell-based therapies. While someof the concepts presented here (e.g., equipment androom arrangement considerations) are geared towards
autologous therapies, discussions relating to risk assessment and management are applicable to other types ofcell-based therapies.

Facility Design Process Overview
Designing CAR-T and cellular therapy facilities to be
fit-for-purpose requires an integrated approach throughwhich the facility is designed to meet the manufacturing
process, operational, regulatory, and business requirements. The facility design ultimately contributes to theconsistency of delivering conforming products throughfacilitation of the manufacturing operation and activities as well as driving the capital and operating costs.
Typically, architectural and engineering biopharmaceutical design specialists are engaged by cell-based therapy
developers to lead the design process. This begins withthe definition of requirements, includes translation ofoperational and regulatory needs, and ends with the final
agreed design. Successful facility design requires expert
input and dedication of time from multiple functions
within the cell-based therapy developers including
manufacturing operations, quality, regulatory, engineering, project management, and business management
functions. The resourcing and management of a facility
design process should therefore be carefully plannedand considered. The evolution from facility design tomanufacturing of regulated products can be broadly described in the following stages—definition; design; build,commissioning, qualification and validation (CQV);
manufacturing start-up and manufacturing operation—
as described below and summarized in Figure 11-1.
DEFINITION

All facility design processes should begin with a User
Requirement Brief (URB) that contains a clear definitionof the business objectives and purpose of the facility.
This includes the activities and functions within thefacility such as manufacturing, R&D, administration, andquality control as well as details on location, capacity,

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼230

throughput, product lines (single or multiple), and future
requirements. Information on the processes and analytical testing requirements for the products intended tobe manufactured in the facility, and utility, automation,
safety, and maintenance requirements should also be included. In addition, a detailed process definition describing the manufacturing processes should be developed.
These documents set the foundation for the design effort.
At this stage, cross-functional engagement and internalalignment from the cell-based therapy developer is important to minimize changes and impacts on time, cost,and quality.
A feasibility study may be used to assess and refine therequirements and can provide high-level facility design
options and associated estimated delivery costs. This may
include assessment of different site locations and types
such as greenfield, brownfield, and retrofits of existingbuildings.
For many cell and gene therapy organizations, themanufacturing processes are not always completely
defined when entering into the facility design process.
In this case, particular focus on areas of the process that
impact the design in terms of cost, compliance, andease of scaled operations should be considered in linewith proactive discussion with Process Development
functions.
It has been common to design facilities with manually operated open processes and benchtop-oriented
equipment which can be adequate for early- to mid-stage
clinical development programs. However, later-stage
manufacturing demands should be considered at thedefinition stage to ensure that the facility can accommodate future scaled-up and scaled-out manufacturing
processes and meet regulatory expectations, particularly
with regard to compliance with CGMP standards. A
challenge for cell-based therapy developers is to balance
the needs of the business at the time of the definition
stage and future needs if products prove to be successful
through clinical trials. Designing for future expansion
without impacting ongoing manufacturing should
be considered to balance initial capital outlay withpotential future disruption to operations. In addition,
best practice and regulatory trends are moving towards
adoption of closed systems wherever possible; increased
utilization of single-use equipment, process aids andautomation to reduce variability and contamination
risks associated with manual operation steps; and monitoring specifically designed to ensure the maintenanceof aseptic processing. The URB should also consider
the manufacturing process evolution in line with future
regulatory expectations.
An additional consideration at the facility definition
stage is whether different/multiple products are to be
manufactured in the facility. In this case, facilities must
be designed with appropriate segregation, containment
level, and cleaning/disinfection procedures to mitigate
opportunities for possible cross-contamination. Further
consideration for appropriate containment and biosafety
requirements should be taken into account particularly
when viral vectors are used in the manufacturing process
such as in CAR-T therapies.
DESIGNThe facility design process evolves the URB and process
definition into a blueprint used to build the facility.
Several design phases can be adopted to translate therequirements into a functional design meeting operational, regulatory, and business needs. Biopharmaceuticalspecialists including architects and process, mechanical,
civil, electrical, and automation engineers work alongside
the cell-based therapy developer specialists during thedesign stage. Typical design phases are:
• Preliminary/Concept Design: This phase of designtranslates the URB and process definition document
into a practical facility layout that satisfies the needsof all end-users including manufacturing, quality
control, engineering, facility, quality assurance,
regulatory, and senior management functions. An
important first step at this stage is to develop the
URB and process definition into an agreed scope of
requirements and agreed process definitions for thefacility that forms the basis of the design. The process
definition is often accompanied by process flow
diagrams and block flow diagrams that outline theprocesses to be manufactured in the facility and aid
the development of the facility layout and operations.
Different design options are discussed at this stage to
produce a conceptual facility layout with associateddrawings showing the flow of personnel, materials,

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼231

and waste as well as cleanroom classifications andpressurization cascades. For facilities that handle
biological agents with a potential hazard to humanhealth (e.g., viral vectors), biosafety requirements
are also assessed and included at this design stage
along with general Environmental, Health and Safety(EHS) requirements. Cost estimates and projectschedules are also produced at this stage. Facility
operational philosophy documents are developed
including segregation and cross-contamination control, facility flows, gowning, biosafety, and supportive
heating, ventilation, and air conditioning (HVAC)
and automation philosophies, which feed the facility
layout development to a conceptual design. From theprocess definition, equipment lists and utility andelectrical requirements are produced. Identificationof back-up and emergency power requirements (e.g.,
uninterrupted power supplies) for the building andcritical equipment are also considered at this stage.
Typically, system impact assessment and component
criticality assessments are also undertaken to identify
equipment and systems that have a direct, non-direct, or no impact on the quality of the product.
These assessments form the foundation for thecommissioning, qualification, and validation stage.
• Detailed Design: This phase of design translates theconcept design package into an architectural andengineering package for construction. User requirements specifications (URSs) and functional design
specifications for the facility, equipment, and utilities
are produced that specify in detail what is requiredand how the systems should work. The URSs may
also be produced at the end of the concept designphase depending on the needs of the cell-based
therapy developer. These documents and the concept
design package form the basis for development ofdetailed engineering drawings such as piping andinstrumentations diagrams (P&IDs), electricalwiring, and HVAC design, as well as enabling theequipment and materials procurement process.
Some equipment and materials with long lead times
may be procured in the concept phase to enable theinstallation schedules to be met. It is typically during
this phase that the local jurisdiction is engaged anddrawing plans are submitted for construction permit
approval. Final cost estimates and project timelines
are also provided at this stage for executive management review.

Design reviews and contamination control
risk assessment
At each design phase, design reviews should be conducted to ensure that the proposed design meets theoperational, safety, and regulatory requirements. This includes assessment for compliance with CGMP regulatory
guidelines, manufacturing operability, good engineering
practice, and safety and maintenance operability reviews.
Given that product safety is of primary importance withrespect to the eventual delivery of cell-based therapy
products to patients, the design review should also be
supported by a contamination and cross-contamination
control risk assessment.
The contamination control strategy should take into
account the aspects of the process design that are meant toprotect the product from contamination. Contamination
control measures should be designed into each part ofthe production process, featuring control procedures
such as cleaning, decontamination, sterilization, andtransfer methods for primary packaging materials, consumables, product intermediates, and waste. The process
design should also consider the implementation of closed
processing systems or isolators to drive containment to
the equipment level and put less stress on maintaininga high classification of the background manufacturing
environment.
Manufacturers should comprehensively assess risks
associated with possible sources of contamination andshould, subsequently, implement measures to prevent
contamination commensurate with the risks. This may
include revising the design to reduce the risk or prospectively developing acceptable operational measuresin conjunction with the operations and quality assurance
teams to mitigate the risk.
It should be noted that the regulatory, operability, andsafety reviews are live documents and should be reviewed
and updated on a periodic basis through the process andfacility lifecycle.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼232

BUILD, COMMISSIONING, QUALIFICATION,
AND VALIDATION

Facility construction is conducted by qualified specialist
contractors who should have experience with the specificrequirements for construction of biopharmaceutical facilities. The construction begins with the preparation of theselected site, building the superstructure, making the facility watertight, installing utilities, and then installing the
HVAC, cleanrooms, equipment, and internal components
with appropriate waste containment strategies to achievea fully functioning facility. An “as-built” drawing for theconstructed facility is provided in a handover package.
Following the construction phase, the facility iscommissioned, qualified, and validated (CQV) to
demonstrate that the facility with equipment is fit for
the intended purpose. The CQV process systematicallydemonstrates that a facility being installed will offer ahigh degree of quality assurance such that manufactured products will consistently meet quality requirements through Installation Qualification, Operational
Qualification, and Performance Qualification (IQ/
OQ/PQ). Installation Qualification (IQ) verifies thatthe facility is installed in accordance with the detailed
design; Operational Qualification (OQ) ensures thatthe facility operates in accordance with the functional
design specification; and Performance Qualification (PQ)
ensures the facility performs in accordance with the user
requirements specification. The CQV process is conducted through documented test procedures and the level oftesting and documentation is determined by the system
impact assessments, which identify the impact of thesystem on the process. Associated Standard Operating
Procedures (SOPs) are also developed for operation of
the facility, utilities, and equipment to support the CQVprocess and typically forms the start of the personnel
training process.
MANUFACTURING START-UP TO MANUFACTURING
OPERATION

Following the handover of the qualified facility to theend-user, operational start-up activities, process qualifications, and validations need to be performed before
entering manufacturing operations that can provide data
packages to support regulatory licensure and approval toproduce clinical or commercial products. This includes

completion of critical elements of a quality management system (QMS) that include SOPs, establishing anenvironmental monitoring baseline, training of manufacturing and facility maintenance operators, processsimulations, and cleaning validations.

Construction Methods andDesign Layout
There are a number of construction methods and design
layout options that can be used to meet the requirements ofthe facility set out in the definition phase. These influence
the design, cost, construction time, and operability of the final facility. Key factors include the method of construction
for the building and cleanrooms; segregation philosophiesand flows of people, materials, and equipment through thefacility; gowning requirements, cleanroom classifications,and HVAC design and systems to ensure chain-of identity(COI) and chain-of-custody (COC) of the product during
processing. There are many design solutions that canmeet the requirements and periodic design reviews areundertaken alongside risk assessments governed under thequality risk management (QRM) processes to ensure thatthe design meets CGMP, aseptic processing, operational,
and business requirements. These topics are discussed in
more detail in the following sections.
CONSTRUCTION METHODS

Pharmaceutical facilities have traditionally been “stickbuilt” where they are constructed at the selected site using individual parts (e.g., wooden or steel studs, drywall,
brick, and mortar). Selection of building construction
material must take into account risks of material decay(rot, mold), which could pose safety concerns to the
product. The advantages of a stick-built construction are
the flexibility to amend the design after it is built; it is not
restricted to pre-determined dimensions, and often has
the lowest cost per square foot.
However, for cell-based therapy companies, speed andflexibility for a facility to grow with the evolution of thecompany are also key drivers in addition to cost. This has
led to the more recent adoption of modular construction
methods, which employ networks of offsite-constructed,
pre-fabricated cleanroom components. This method ofdelivery especially lends itself to greenfield facilities

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼233

Figure 11-1: Typical facility design stages
Design stage
Definition

Preliminary/concept design

Facility build andCQV

Detailed design

Manufacturing
start-up to
operation

PurposeExplores facility design
options to translate
the URB and process
definition into anagreed concept design

Develops theconcept design
into an architecturaland engineering
package forconstruction

Facility construction.
Cleanroom, equipment,
and utility installationand qualification
to hand over ademonstrated fit-for-­purpose facility

Operational setup and training fortransition into routine
operation

Cell therapy
organizationdeliverables:

Architecture& Engineering
deliverables:

Architecture& Engineering
deliverables:

Architecture& Engineering
deliverables:

Cell therapy
organizationdeliverables:• User Requirements
Brief (URB)• Process definitionby the cell therapy
organisation• Scope of Requirements• Process Definition(final)• Operational Philosophies
including segregation,
gowning, biosafety,
automation, HVAC
• Equipment & Utility List
• Concept Design­Layout,
personnel and material
flows, clean roomclassifications andpressure cascades• Estimated cost andschedule• GMP, safety, andoperability review• User Requirements
Specifications (URS)
including facility,
equipment, utilities• P&IDs• Electrical wiring
diagrams• HVAC diagrams• Fabrication andconstruction
documents• GMP, safety, andoperability review• As-built drawings• Facility handover
documents• Finalized SOPs
• Personnel training• Environmental
monitoring program• Process simulations• Cleaning validations

Define businessobjective, purpose
and scope of the
facility

Outputor large, open warehouse retrofits. Using the modular
facility approach, cleanrooms can be manufactured using
either modular wall panels (which are assembled together onsite to create the entire manufacturing space within
the greater facility structure) or fully modular solutions(the entire processing space is manufactured offsite as awhole, and then shipped to the site for installation inside
the greater facility structure).
There are several advantages to the modular approachover stick-built. The production site is constructedto facilitate the use of modular units, and the actual
cleanrooms are produced simultaneously in anotherlocation. Units can be assembled quickly and efficiently
at the given facility construction site (which allows formore rapid deployment or removal) and can save several

Cell therapy
organizationdeliverables:• IQ/OQ/PQ
procedures andreports• Training documents• SOPs­—facility,
utilities, equipment,
and training

months in overall construction time compared to more
traditional methods of biopharmaceutical manufacturing facility construction. Typically, given the parallel
nature in which modular delivery and site construction
operations can be carried out, the more modular you
drive your facility, the more you can compress yourconstruction schedule. Thus, fully modular solutions
are generally the fastest way to realize a facility. Also, as
the majority of the modular units are constructed offsite,
it lends itself to the requirements of a “clean build” tominimize dust and debris and reduce facility cleaning
time before operational hand over. It is possible to add
or remove modular processing rooms from the facility
with minimal interruption to ongoing operations, helping align the facility with manufacturing capacity needs

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼234

and market demand. Modules can either be installed Table 11-1: Construction Method Comparison
in an open shell space or assembled as configurable
units to become a biopharmaceutical factory when all
Stick-Built
ModularFullyof them are put together. In the context of cell-based
Panel
Modulartherapy product manufacturing, which frequently
Systemmakes use of viral vector technologies and patient
ScheduleHigh
MediumLowcells, modular units are typically tighter in terms of
Cost
Low
MediumHigh
construction and airflow control, potentially further
Flexibility
HighMediumLowenhancing segregation and containment particularly
On-siteHigh
MediumLow
for processes utilizing biological agents requiring theConstruction
implementation of higher biosafety levels (e.g., BSL3).
Risk
When considering modular construction methods,
consider dimensional constraints that are associatedwith standardized modules and if this is suitable for thedesign as “optimized templates” that can be consistently
overall facility design. During the construction phase,
produced and deployed as facility demands increase
there may be a slightly higher onsite construction risk
and production space scale-out is required. This bein terms of component fit, as the modular components
comes especially useful in cases where consistency is
may be difficult to adapt to unforeseen site constraints.
of paramount importance, and leveraging a known
In this case, the duration of shutdown for repairs anddesign for production expansion brings with it strategic
degree of invasive procedures for repair (e.g., particlealignment for manufacturing operations. This can be an
generation from on-site adjustment) should be further
advantage within sites, between sites, and to help trainconsidered. However, this risk is usually offset in a
ing, operations, and technology transfer of processes.
modular approach with a reduction in onsite safety
Processing room layout options
risks (due to the volume of offsite construction) and areduction in onsite startup risks (modular components
There are various options for the layout of the procan be pre-tested offsite prior to shipment). Modular
cessing rooms for cell-based therapy manufacturing
approaches are also typically more costly than a stickprocesses. When designing the layout, operational,
built approach, so the balance of speed, cost, andquality, and regulatory requirements, in particular
contamination potential must be considered.
CGMPs, must be considered to enable repeatable deWhile a modular approach can, in theory, be aplivery of compliant products. As cell therapies cannot
plied at any conceivable scale, in practice, the number
be sterile filtered or terminally sterilized, the layoutof modular systems and transportation difficultiesof the processing and support rooms must consider
associated with larger scale facilities (e.g., high floor
the needs of aseptic processing. The chosen layout
to ceiling heights, overly large ballrooms) renders theshould also mitigate the risk of contamination andfully modular approach to be most suitable for procross-contamination through appropriate segregation
cesses with smaller footprint requirements. Autologous
of processes and flows of people, products, materials,
processes and facilities that are designed to manufacand waste.
ture individual patient batches in dedicated processing
Ideally, the facility design should be based aroundrooms, for example, tend to require smaller operating
the rigorous definition of the processes and the operspaces, and thus lend themselves especially well to fully
ations to be performed, with associated QRM-based
modular solutions.
policies. Often processes are not finalized at the time
The use of modular designs, in particular fullyof facility design, however, developers should be able
modular processing rooms, lends itself to standardizato reference well-defined operational criteria that cantion. A modular approach can offer manufacturers aserve as the basis for the definitions of the processes toframework to establish the production spaces in their
be performed, while maintaining capacity for process

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼235

improvements and equipment replacement as part of aquality by design strategy. Details that can directly contribute to facility design considerations and requirements
include the identity and scale of the unit operations tobe performed; the duration and proximity requirementsof each unit operation; the materials to be used in theprocess; whether operations are open, fully closed, orfunctionally closed; personnel and required waste flows;
and the required proximity of support operations suchas testing in-process control samples, filling, and cryopreservation. A risk assessment (discussed in Chapter4) should be performed with regard to the operational
details of manufacturing to document and justify thefacility design requirements.
Facility designs must meet CGMPs as describedin the applicable country regulations and regulatory
guidelines. From a regulatory perspective, facility
designs that promote process movement throughout
the facility that mitigate the risk of mix-ups andcross-contamination are preferred. To this end, the following factors should be taken into account: whether
the process is to be performed in a single room forthe process duration; whether the production room isdedicated to a single patient for autologous products;
and, if the process is to be performed in a larger space,whether there are dedicated workstations for the duration of the process, or whether the process moves
through the space as it is performed. Risk assessments
and QRM-based policies should inform all aspects of
the approach to facility design.
Additionally, the planned utilization of space andequipment should be carefully considered. In the caseof autologous therapy production, operations cannot be
scaled up beyond the scope of a single patient. Instead,
operating spaces must be scaled out to accommodate
larger patient populations and achieve greater facility
throughput capacities. Inefficiencies resulting from
suboptimal floorplan and equipment-related design
could, therefore, be correspondingly scaled out, resulting in larger and more expensive spaces than otherwise
required. Thus, it is important to properly define efficientworking designs that can be copied and scaled out tomeet product capacity needs. In the case of allogeneic
therapies which are typically campaign manufactured
at larger scale, it is important to define efficient working

designs that can be scaled up and maximize the utilization of the space in the facility.
Some common facility design approaches are discussed below. It should be noted that a facility designneed not commit entirely to one approach or another.
The facility design should be born out of an evaluation of
the intended process or processes to be performed, therisks associated with each, and the risk-based management approach for the site. In many cases, a combinationof the following approaches may be used to create aneffective and efficient manufacturing facility design.
Dedicated production space

In light of the processing demands for autologous cellbased therapies where segregation of individual patientbatches is paramount, processing rooms dedicated to the
production of a single patient lot have become a common
facility approach for early clinical-stage production.
Since each room is separated from every other room,
this design approach maximizes process segregation
by eliminating (as much as possible) the potential forcross-contamination between patient lots or mix-up of
operational materials (Figure 11-2).
For autologous therapies, because the production
space is dedicated for the duration of the lot and theequipment and footprint cannot be reused for other
patient lots during processing hold steps or downtime,
dedicated production spaces are inefficient in termsof space and equipment utilization in the facility. In
addition, dedicated process room designs are typicallyused for end-to-end production of autologous therapies that may include both open and closed processes.
In this case, open processes are often carried out in
Biological Safety Cabinets (BSCs) and require a higher
background cleanroom classification compared to fully
closed processes as they incur higher operational costs.
If isolators are used to contain the open processes andallow for a lower background cleanroom classification,
the capital and maintenance cost of the isolator as wellas ease of operation need to be evaluated, especially
if a scaled-out production model is being considered.
While the dedicated production space is most utilized forautologous approaches, this design may also be suitable
for allogeneic approaches unless the scale is too great for
the room size restrictions.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼236

Figure 11-2: Example of Dedicated Production Space design
Each processing room can accommodate an end-to-end autologous process.
Processing Room 1

Processing Room 2

Ballroom design

The ballroom concept refers to a large, open manufacturing area that has the flexibility to accommodate
different types of processes and equipment with minimal
structural segregation (Figure 11-3). Due to the larger
footprint and tendency toward flexibility, the ballroom
design lends itself to processes and products that arelikely to change over time.
For autologous processes, multiple workstations, eachused for an individual patient lot, are required to enable
the most efficient use of the ballroom design, and canbe more space and cost efficient compared to utilizing
dedicated production rooms. However, the operation ofthis design is more prone to mix-ups and cross-contamination as there are likely multiple operations being staged
or performed within the same room at the same time.
For this reason, in support of robust QRM, ballrooms areespecially conducive for the adoption, implementation,and enhancement of process closure where possible andthe use of isolators where process closure is not possible. Because closed systems are characterized by total
separation from the surrounding environment, closed
equipment may be co-located in the ballroom space, allowing for more production per square foot of processing
room. Process steps that are prone to, or pose higher
contamination risks that may impact adjacent operations
should be segregated, either within specifically designed
containment equipment or with performance conducted

Processing Room 3

Processing Room 4

outside the ballroom in a specifically designed separate
space (see Dedicated production space). When designing
the workstation layout and detailed operations in a ballroom design, rigorous attention should be focused on
COI and COC to ensure segregation of materials andpatient batches. Particular attention should be focusedon any common equipment such as in-process testingstations, incubators, and cold storage that are used tohouse materials for multiple patient batches and should
be governed by the QRM.
For allogeneic products that are likely to utilize acampaign manufacturing approach, the ballroom has theadvantage of flexibility with the large open manufacturing space and ability to house different equipment andprocesses. Suitable, validated cleaning activities between
campaigns and consideration of equipment validation
processes in the master validation plan should also be
investigated if equipment is to be changed between
campaigns. If the process has open processing steps,
consideration should be given to the use of isolators toenable a lower cleanroom background classification. If
BSCs are utilized with the required higher cleanroom
classifications, the larger footprint ballroom may incur
significantly increased operational costs, and the risks
surrounding cross-talk between BSCs should be carefully
identified and managed.
Because of the inherent features of ballroom facility
design, less facility airlocks are needed (compared to

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼237

Figure 11-3: Example of ballroom design
For autologous processes each workbench is typically used for a single patient batch with appropriate
segregation. For allogeneic processes each workbench may house different unit operations. (PAL:
Personnel Air Lock)
PAL-in
PAL-out

other design approaches) and all equipment can beplaced and operated in one large, flexible space. Thus,
ballroom facilities allow for a measure of creativity indeveloping a variety of production scenarios. As a result, the ballroom design lends itself well to scale up ofallogeneic processes as well as scaling-out of autologous
processes as organizations progress through clinical trials
to commercial scale. Ballroom spaces that feature a highdegree of process closure and automation can facilitate
reduction of environmental classification, reduce operator requirements, and allow for greater facility throughput, improved quality, and consistency in production.
Additionally, from the standpoint of sustainability,
facilities utilizing ballroom spaces have the potential togenerate smaller overall footprints.
Dance-floor design (segregated unit operations)

The dance-floor approach is a hybrid between the utilization of dedicated rooms to segregate different batches in ascale-out operating model and the ballroom design wheresegregation is achieved through closed processes and operational procedures to provide a flexible manufacturing
space that lends itself to a scale-up operating model. In
the dance-floor design approach, the production unit operations are divided across a number of adjacent smaller
spaces. The individual rooms can be connected throughthe walls or passthroughs to maintain specific process

segregation or by common corridors where appropriate
operational procedures driven by the QRM would need
to be in place to ensure segregation. The dance-floor
design allows unit operations to be segregated, and canmaximize equipment and space utilization by decontaminating and turning over the space for the next patient orprocess (Figure 11-4). This configuration allows different
levels of cleanroom grades to be applied to each room
depending on the process requirements (opened/closed)
and allows for appropriate containment of process steps,
such as when handling viral vectors.
Due to the increased level of subdivision for the
production space, the dance-floor approach does notoffer as much flexibility as the ballroom approach andwill likely incur increased airlock requirements over a
single open ballroom space due to the required transitions to and from each individual room. It may also
require greater operational planning and coordination
on decontamination between patient lots. Therefore,
dance floors are most appropriate when processes use
the same facility, equipment, and platform technologies
in different locations or when only performing a single
process. While the dance-floor approach has segregation
advantages over the ballroom design, it does not provide
the segregation of the fully dedicated processing roomdesign to maximally implement COI and COC.
The dance-floor design could be suitable for

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼238

Figure 11-4: Example of: (A) Dance floor facility layout, and (B) schematic of the flow of multiple
product lots using a dance floor philosophy.
Suites are arranged based on unit operations, where each suite is dedicated for specific process steps and is connectedto adjacent suite by passthroughs. Product lot flows are processed in each suite after appropriate cleaning and line
clearance. Note that not all manufacturing unit operations are presented in this diagram. (BSC: Biological Safety Cabinet;
INC: incubator; TFF: Tangential Flow Filtration; CRF: Controlled Rate Freezer; LN2: liquid nitrogen).

A
Suite 1
Selection & activation

Suite 2
Expansion & harvest

Suite 3Purification,
formulation & fill,cryopreservation

Suite 4Storage

Pass throughPass throughPass throughLN2 storage

B
DAY 1 → DAY 2 → DAY 3 → DAY 4 → DAY 5 → DAY 6 → DAY 7 → DAY 8 → DAY 9 → DAY 10 → DAY 11 → DAY 12 → DAY 13 → DAY 14 → DAY 15
PRODUCT 1(LOT A)

Suite 1
BSC +incubator

Suite 2
BSC + incubator

PRODUCT 1

Suite 4
Suite 3
SharedTFF + filler
freezer+ CRF
storage

Suite 1
BSC +incubator(LOT B)

Suite 2
BSC + incubator

PRODUCT 2••

Suite 4
Suite 3
SharedTFF + filler
freezer+ CRF
storage

Suite 1
BSC +incubator•

Suite 2
BSC + incubator

Suite 4
Suite 3
SharedTFF + filler
freezer+ CRF
storage

UNIT OPERATIONS
Suite 1: Selection & Activation
Suite 2: Expansion & Harvest
Suite 3: Purification, Formulation & Fill, Cryopreservation
Suite 4: Storage•

allogeneic processes if the individual rooms are sized
appropriately for the unit operations. The initial isolation and expansion process steps for both allogeneic andautologous processes are often a similar scale with thedifferences in scale and space required occurring at thelater expansion, formulation, and filling process steps.
Therefore, the dance-floor design can be space efficientand operationally efficient for cell-based therapy companies planning to manufacture allogeneic and autologous
products.

Support rooms
Supporting areas for the processing rooms include warehousing, gowning, cold and frozen storage, personnel

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼239

Table 11-2: Comparison of Facility Designs
Segregation

Process
ClosureFootprint
Utilization

CapitalCost

Operational
Cost

Flexibility

Dedicated

Full segregation

Open (mostsuitable) or
closedLeastefficient

High

High

Within each room.
Suitable for scaleout. Autologous andallogeneic (spacepermitting)Ballroom

Segregation byprocess closure
and operational
procedure

Closed (mostsuitable)or openprocesses
contained byequipment

Most
efficient

Low

Low/Middle

Within large room.
Suitable for scale-up.
Autologous andallogeneic

Dance Floor

Unit operations
segregatedOpen orclosed –adapted byroom

Moderate
efficiency

MiddleMiddleCleanroom grades
can be adapted byroom. Autologous
and allogeneic

and material airlocks, and cleaning storage. These must
be designed in conjunction with the processing rooms
to ensure appropriate segregation, mitigate the risk ofcontamination and cross-contamination, and facilitate
operationally efficient flows for people, materials, andproduct through the facility.
The warehouse should be appropriately sized for theneeds of the facility and in line with the supply chain
and logistics strategy. It should have appropriate systems
for tracking materials and quarantine status and haveprocedures that minimize opportunities for mix ups,
especially if the facility supports multiple products. The
most common strategies are either to receive, store, test,
and release materials in an area connected to the main
production facility or to carry out these activities offsite
and have a smaller short-term warehousing area within
the production facility. For both cases, the warehousing space design should consider areas for inspection,
sampling, and testing of incoming materials; quarantine,
released, and rejected material and consumables storageat each required temperature; final product storage; andtransportation packaging areas.
If liquid nitrogen (LN2) is being utilized for product storage or transportation, correct ventilation andoperations health and safety requirements need to be
implemented. Spaces for cryostorage should be O 2
monitored and have sufficient warning and mitigation

systems. Selection of flooring material is important
where LN2 is used as some flooring materials (e.g., epoxy resin flooring) can crack on contact with LN2 spills
compared to other flooring materials (e.g., polishedconcrete) that are more resistant to cracking.
Gowning specific activities should have their own
dedicated spaces within the facility, such as locker rooms
and personnel air locks (PALs). Design of these spaces
should consider the quality of environment appropriate
for the gowning being performed, and the number of
personnel required to be able to perform gowningconcurrently. These spaces should consider operational
aspects such as shift changes, total personnel the facility
is designed to accommodate, and the number of gowningcomponents and steps performed. The gowning processitself is inherently associated with high numbers of particulate shedding from operators and non-processing
room clothing; HVAC for gowning spaces should anticipate the number of personnel concurrently gowning toensure gowning-generated particulates can be effectively
flushed from the space to maintain the desired environment classification. If improperly sized, gowning spaces
can impede the gowning procedures and ultimately bottleneck facility operations. Within the gowning rooms,
storage of the gowning wear (overgowns, head covers,
hair nets, shoe covers, gloves, etc.) and waste disposal of
packaging and sanitizing materials should be considered

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼240

for optimal operation. People are often the most common
cause of contamination, so adequate space to allow forcorrect gowning before handling the product is critical.
Personnel and material airlocks are utilized in facilities to transition between different classifications ofcleanrooms and typically require some level of roompressure rebalancing. Transitioning through airlocks
can require significant time and therefore spaces
should be sized so that the movement of people andmaterials to the end destination meets the needs of the
manufacturing program and any operational working
hour constraints. Modeling software can be employed
to determine the optimum balance of airlock size andpersonnel flow. When exiting process spaces where
open operations are performed, a best practice is to havede-gowning activities occur in a separate airlock fromgown-in activities. This mitigates any process materials
on the operators’ gowns from cross-contaminating otheroperators coming into the processing space. For material
airlocks, sufficient space should be allowed for laydown
areas to clean materials, the decontamination materials
themselves, and waste disposal for spent cleaning materials and discarded packaging.
Cleaning and decontamination storage areas can beoverlooked in initial facility designs but are critical to enabling the required cleaning processes. The room should
be designed for adequate storage and preparation ofcleaning agents and cleaning equipment as well as waste
disposal. Cleaning storage rooms should be located in
the facility to enable unidirectional cleaning from higher
classifications to lower classifications if possible. If there
are steps in the production process that are specificallysegregated due to higher risk of cross-contamination,cleaning of these spaces should be evaluated and may
require additional procedures or flows. The location
should also consider any needs for sinks, drains, andsupply of potable and compendial water, which can be asource of contamination.
Storage rooms for environmental monitoring equipment
such as air samplers are also required and the locations
need to be considered as part of the design.
Media preparation rooms generally require BSCs
for manual open additions to media (such as cytokines
and serum) and an appropriate supportive backgroundenvironment. Depending on the utilization of the space,
it is often used for other open-process support activities such as sterile container change and sterile tubing
preparation.

Facility personnel and material flows
The process rooms and support rooms must be designed
so that personnel and materials can flow through a facility to minimize the risk of contamination. With each
evolution of design layout considered through the facility
design phase, flow diagrams are produced to communicate the operational intent for the manufacturing facility.
Personnel flow diagrams show personnel entry into thedesignated controlled manufacturing area through gowning and de-gowning stages to exit from the controlled
area. This includes personnel entering the facility to operate the process and to clean the facility. Material flows
show entry of raw materials and consumables into thefacility, through the manufacturing areas and exit routeof materials as process waste, cleaning waste, or as partof the product, including flows through labelling, storage,
and final packaging prior to shipping out of the facility.
The flow of any intermediates and quality control (QC)
samples for in-process and outsourced testing are also
produced. Manufacturers of cell-based therapy products
are also required to specifically show the movement of all
patient cellular materials. In this way, the robustness ofthe design can be thoroughly interrogated and whether
risks for contamination or cross-contamination havebeen properly identified, understood, and suitably
mitigated. An example facility flow diagram is shown in
Figure 11-5 below.
Unidirectional flows and facility design

To minimize the risk of contamination in cell-based therapy manufacturing facilities, the use of unidirectionalflows is often employed. A unidirectional flow of personnel, raw materials, intermediates, products, equipment,
and waste can minimize the risk of confusion between
different products or product components, avoid the riskof cross-contamination, and minimize the potential foromission or wrong application of manufacturing or control steps. In general, in a strictly unidirectional workflow
scenario, after personnel change out of street clothes andChapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼241

Figure 11-5: Example facility flow diagram showing flow of personnel, materials, product, and waste.

Personnel
PAL-OUT

Materials
Product
Samples

Production suite

MAL

Waste

PAL-INCRF room

don facility uniforms or scrubs in a changing room, theflow should start with a PAL for personnel to gown andprepare for entering the production space. Within the
production space, appropriate restrictions should be inplace to control movement between different manufacturing rooms, and entry or reentry into classified areas.
Parallel processing rooms should be separated by ingoingand outgoing PALs and clean corridors should be clearly
distinguished from dirty ones based on the facility flow
QRM. There should be separated areas designated forreceipt and storage of materials and reagents, quality
testing, and manufacturing processes. The exit shouldbe through a de-gowning room to prevent personnel
from passing back into the clean gowning area after
exiting. It should be noted that access to and from themain gowning room depends on many factors, but it iscommonly through a single vestibule.
However, it should be noted that unidirectional flowat all times and across all aspects of the facility is spaceintensive, not always buildable, and not strictly required
from a regulatory standpoint. Manufacturing spaces
should be assessed for factors like what processes are tobe performed within, the level of segregation required,the degree of process closure, and the potential implication to the operator and their external gowning throughprocess performance. For areas where less stringent
requirements may be allowed, alternative facility designs can create more efficient space usage and flows.
For example, unidirectional flow may be establishedspecifically for critical process rooms, with bi-directional
flow and less stringent controls for less critical support
spaces. Dedicated gown-in and gown-out PALs thatconnect to an open common corridor may be employed
where operators are not deemed to be a source of crosscontamination. If unidirectional flow is not feasible in
the facility, segregation in time for movement of starting
materials, product, and waste should be implemented inconjunction with appropriate risk assessment.

Equipment selection and workstation layout
The layout of equipment in each cleanroom space should
be designed to enable consistent operations, mitigate therisk of cross-contamination, and optimize cleanroom
space. As cell-based therapy manufacturing processes
can be in early stages of development and scale when
designing facilities, there should be particular focus onareas of the process that are not conducive to meetingthese needs. For autologous processes that are replicatedthroughout facilities, exacerbation of operational inefficiencies and oversized facilities should be avoided.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼

242

Workstations are defined here as specific sets of equipment chosen and arranged to facilitate an intended manufacturing operation. This may be manual or automatedprocesses including single or multiple unit operations. For
autologous processes it is more common for workstations
to include multiple unit operations for a single patientbatch. For allogeneic processes, it is more common forworkstations to be segregated by unit operation due to thelarger scale. Depending on the complexity of the workstation, equipment can include any number of components
such as biosafety cabinets (BCSs), incubators, centrifuges,
microscopes, electroporators, cell separators, environmental isolators, and automated cell processors.
Equipment selection should consider compatibility
with cleaning and sanitization agents, auditable datacollection systems to meet data integrity guidelines,
and connectivity with other equipment. Heat loads andparticulate generation from equipment should also beassessed to ensure that cleanroom environments can be
maintained within specification when all equipment inan area is fully operational.
Design practices for workstations

The layout and arrangement of equipment within the
production space can be critically important to theoverall success of the manufacturing operation and reproducible production of the process. The workflow ofactivities in cell-based therapy manufacturing processing
rooms must be organized in a purposeful and methodicalmanner. The flows of personnel, materials, samples, andwaste should be identified and designed to ensure thatconditions that promote opportunities for cross-contamination or material mix-ups are removed.
Personnel circulation, not just at the equipment but
from process step to process step as well as general circulation through the room, must be accounted for in theoverall space design. As manufacturing operations havereasonably predictable durations and typically are conducted in a methodical series of process steps, temporalsegregation can be leveraged to aid circulation. The design
should account for areas for preparation of incoming
materials and consumables, laydown areas, in-processsample transfer, and waste removal to reduce overlapping flows during operations. Waste handling and flows

should be segregated from the handling and transportof any incoming raw or in-process materials to mitigate
cross-contamination opportunities. Particular attention
should be paid to waste liquid removal as there are often
limitations on sinks or drains in the area where there areunit operations that require water (e.g., incubators).
Equipment should be arranged to provide adequate
room to conduct planned operations and should accountfor maintenance access and cleanability. Equipment on
wheels or mounted on a cleanroom cart that can be
moved to provide accessibility, and connectivity to adjacent equipment, walls, or panels (process tubing, power
cabling, utility connections, etc.) should be considered.
Equipment located in classified cleanrooms will requireroutine exterior decontamination and cleaning, the levelof which will depend on the environmental classificationand the site procedures. Sufficient space should be given around the systems (for example, from an adjacentwall) to allow proper cleaning and decontamination
procedures.
Placement of equipment within a cleanroom space cansignificantly impact the air flows within the cleanroom.
In processing spaces where open aseptic operations are
conducted (for example, within a BSC), control and protection of the airflow in the critical zone is paramountto mitigating contamination of the process material. It is
critical for these spaces that the arrangement of equipment works to maintain both the critical zone airflow
pattern and also the intended airflow within the supporting processing room environment. Equipment mustnot be in positions where they will disrupt air velocities
across the face of the critical zone (for example, the sashof a BSC) or where they will impede airflow into HVAC
low wall return ducts and disrupt the overall room airflow patterns. Considerations should be taken to ensure
processing spaces with high loads of large equipment areable to maintain environmental conditions and airflow
patterns appropriate to the required classification. Smokestudies may be used to confirm that airflow is not being
impeded. Production spaces with mobile equipment or
cleanroom carts should have designated locations forstowing both while in and out of operation to mitigate
chances of them being in front of a low wall return anddisrupting room conditions.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼243

Specific considerations for commonly usedequipment
Biological safety cabinets and incubators are commonlyused critical equipment in cell-based therapy manufacturing. Specific considerations and best practices aredescribed below for these items of equipment.
Biological safety cabinets

A biological safety cabinet (BSC) provides a cost-effectivemeans to deliver the highest classified environment toprotect open processes used in cell-based therapy manufacturing from contamination. Due to the high risk ofcontamination posed by open processes, the ease of use
and placement of a BSC in a cleanroom is paramount.
The effectiveness of a BSC is highly dependent on its
positioning within its surrounding environment as disruptions to the sash airflow can impact the critical zone
environment internal to the BSC. Careful evaluation must
be taken when locating a BSC within a processing space.
Adjacent equipment and workspaces should be evaluatedon their likeliness to disrupt and impact room air flow
and quality, and therefore their potential to impact theenvironment internal to the BSC. Ideally, BSCs should
be positioned with the sash facing away from doors,
personnel circulation zones, or other equipment or
spaces expected to create irregular airflow conditions (i.e.,
rocking or rotating equipment, incubators, other BSCs). If
such an arrangement is unavoidable, an appropriate distance should be given between the sash and the opposite
disturbance to allow for room driven HEPA airflow toreturn to a regular airflow pattern at the face of the BSC.
Incubators

Incubators maintain an optimal cell growth environment
by controlling temperature, humidity, and carbon dioxide concentration. Incubators typically maintain the atmosphere to contain 5% CO2. CO2 can be supplied fromlocal, stand-alone cylinders near to the incubators orfrom a centralized distribution system with piping runs
to supply the CO2 to the incubators across the facility. In
general, it is best to have the CO2 sources located outside
the sterile area to mitigate environmental contamination
risks when performing change-overs or refills. If cylinders are used to supply gas, they should be securely anchored, clearly labelled, and have the correct regulating

valves attached. The tubing should be appropriate for thepressure of the gas used and securely fastened to avoid
any leakage of carbon dioxide. In all cases, a 0.22-µm porosity, non-wettable filter should be used on the input gaslines. Though major deviations in CO2 levels may occur
due to door opening activities or a change in pH of themedium, CO2 levels should be checked monthly using acalibrated CO2 meter. To manage risk for contamination,
the incubator should be emptied, dried, and cleaned withappropriate, non-corrosive disinfectant at a frequency
determined by risk assessment. There are several incubator designs and consideration should be given to thosethat have multiple small inner doors to reduce the change
in conditions in the incubator when the door is open,
individual trays where material containers can be easily
moved in and out of the incubator, and enclosed water
reservoirs used for humidification. Risk of contaminationfrom the humidifying water can be further controlled
by adding antibacterial or antifungal agents or planned
routine disinfection, though these should be assessed toensure that the agents are validated to be nontoxic withrespect to the cell-based therapy product material.

Cleanrooms and classification
Cell-based therapy products cannot be sterile filtered or
terminally sterilized, and therefore must be manufactured in cleanrooms using aseptic processes. Cleanrooms
are designed to mitigate the risk of contamination of
products through control of the concentration of airborne particles and viable organisms that pose risks to
product quality and sterility. The appropriate selectionof the cleanroom environment to mitigate the risk ofcontamination is critical for successful manufacturing.
Regulatory authorities such as the FDA (U.S.), MHRA
(U.K.), and EMA (EU) specify the standards that characterize the different tiers of cleanroom environments
and are an integral part of CGMP guidelines. The CGMP
standards are based on the allowable number of various
sized particles, defined in size category stages from 0.1
microns to 5 microns, in a given volume of airspace withina cleanroom. They also provide guidance of the cleanroom
air quality expected for different types of processes.
Different regulatory authorities have different naming
terminology for the cleanroom environment tiers andthere are some slight differences between the cleanroom

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼244

specification and monitoring requirements, which aresummarized in Table 11-3 below. The ISO-14644-1
classification system (as defined by the International
Organization for Standardization) provides a set ofstandards for cleanroom particle levels that may be referenced by manufacturers worldwide, thereby allowing
facilities to calibrate cleanrooms using established benchmarks. For manufacturers in the U.S. market, facilities are
currently designed in accordance with the ISO standard,
which in 2001 replaced the previously utilized cleanroom
Class standard denoted by Federal Standard 209E.
It should be noted that ISO-14644-1 uses metric units
and references a standard particle diameter starting at
0.1 microns, while Federal Standard 209E, the previous
cleanroom standard, used imperial units and referenced
a standard particle diameter starting of 0.5 microns. The
two sets of standards are often easily confused (evenin manufacturer spec sheets), and even though legallycancelled, Federal Standard 209E is still widely, thougherroneously, used. If Federal Standard 209E is encountered, developers should keep in mind that a Class 10,000(Federal Standard 209E) cleanroom corresponds to theparticle threshold equivalent of an ISO Class 7 (ISO14644-1) cleanroom. Nevertheless, the ISO standard
should be the reference for all new facility spaces being
designed to accommodate U.S. markets.
For manufacturers operating in the EU market, cleanroom standards are defined in terms of Grades under
EU GMP standards as outlined in EudraLex Volume 4,

Annex 1. Cleanrooms are certified as being Grade A, B,
C, or D with Grade A cleanrooms having the strictest
environment (in terms of air cleanliness), and Grade D
cleanrooms having the least strict environment.
Where cell-based therapy companies are seeking
product licensing from different countries, it is advised todesign and operate the facilities to the strictest applicableguidelines.

Cleanroom classification for open and closed
processes
Open manufacturing processing steps must be performed within the highest cleanroom ISO 5/Grade A in
operation environment for adequate aseptic processing
conditions. If the ISO 5 critical working zone is maintained within a BSC or a restricted access barrier system(RABS), the supporting background environment must
be classified ISO 7/Grade B.
A difference between the EU (EMA) and U.S. (FDA)
requirements is that if the ISO 5 critical working zone
is maintained within a closed system isolator, the cleanroom background environment may have a supporting
background environment of Grade D with appropriate
risk assessment and validation. In contrast, the FDA
requires ISO 8 depending on the operation.
Regardless of process closure, the regulatory expectation is that aseptic processing steps take place within acleanroom environment. Therefore, processes contained
within either closed systems or isolators are expected to be

Table 11-3: Comparison of EU vs. U.S. GMP Cleanroom Standards
Cleanroom Standard
U.S. (FDA)

Max number of particles/m3
U.K. (MHRA) / EU (EMA)

At rest≥ 0.5 μm

At rest≥ 5 μmIn operation≥ 0.5 μm

In operation≥ 5 μmClass

ISO

EU GMPAt restEU GMP
In operation

100

ISO Class 5

A/B

A

3 520

20

3 520

20

1 000

ISO Class 6

10 000ISO Class 7

C

B

3 520

29

3 520

2 900

100 000

ISO Class 8

DC

352 000

2 900

3 520 000

29 000D

3 520 000

29 000Not definedNot Defined

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼245

Table 11-4: EU vs. U.S. GMP Requirements for Microbial Contamination in Operation
ISO Class/Annex 1 Grade

FDA

EU GMP Annex 1

Active air sampling,
CFU/m3

Settle plates(diameter 90 mm/
4 hours)

Active air sampling,
CFU/m3

Settle plates(diameter 90 mm/
4 hours)

5/A

1

1

<1

<1

6

7

3

-

-

7/B

10

5

10

5

8/C

100

50

100

50

-/D

-

-

200

100

1

Annex 1 Grade B cleanroom dynamics are roughly comparable to ISO Class 7 cleanrooms for microbial
contamination in operation. At rest, Annex 1 Grade B are cleaner than ISO Class 7 cleanrooms and are morecomparable to ISO Class 5 cleanrooms.
1

supported by a minimum of an ISO 8 environment. These
include processes that use closed system equipment andsterile connectors, tube welders, and sealers to connect
within and between unit operations.
Cleanroom environmental monitoring

Periodic monitoring of cleanrooms is required to demonstrate control over both viable and non-viable particles
found in critical areas of the operation. The cleanroom
air, critical cleanroom surfaces, and personnel working in
the cleanrooms should be monitored as necessary. Viable
and non-viable particulates are monitored using a combination of settle plates, contact plates, and air sampling.
Settle plates are considered the standard passive air
sampling method and make use of petri dishes that contain sterile growth media (Tryptic Soy Agar) and measure viable particulates in the cleanroom air. The plates
are placed in designated locations in the cleanrooms fora designated time before processing in quality control
laboratories and assessment for viable particulate growth,measured as colony forming units/m3 (CFU/m3).

Active air samplers draw in a defined volume of airthat passes over a media plate. This plate is incubatedand will reveal viable organisms and indicate the number of viable particles per cubic feet or liter. Non-viable
particulates are measured using particulate counters thatdraw in a defined volume of air, which is analyzed in realtime using laser-diode technology to count the numberof non-viable particulates.
Contact plates also contain sterile growth media ina petri dish and are designed for dabbing onto criticalcleanroom surfaces or areas of the operators’ gowningthat are near critical processes (e.g., fingers and wrists).
The locations and frequency of the sampling andmonitoring are determined through risk-based analysis
and through the results obtained during the cleanroom
qualification. It should be noted that the requirementsof the FDA and EMA differ slightly in the acceptable
limits for microbial contamination for ISO 5/grade A
environments (Table 11-4 and Table 11-5). The stricter
requirements should be utilized if companies are licensing products in both jurisdictions.

Table 11-5: EU GMP Recommended Limits for Microbiological Monitoring of Clean Areas During Operation
Grade

Air sample, CFU/m3

Settle plates (diameter
90 mm), CFU/4 hours

Contact plates (diameter
55 mm), CFU/plate

Glove print(5 fingers), CFU/glove

A

<1

<1

<1

<1

B

10

5

5

5

C

100

50

25

-

D

200

100

50

-

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼246

ISO-14698 Biocontamination Control describes
standards for systems to control, monitor, and evaluatebiocontamination in cleanrooms. It should be noted that
the ISO-14644-1 and ISO-14698 classification systems
do not provide limits on viable particles, leaving action
and target values for the user to set as appropriate. For
manufacturers in the U.K. and EU markets, ISO-14698
has been superseded by EN 17141, which is considered tocontain more detail on the areas covered by ISO-14698.
EN 17141 has an emphasis on microbiological control
rather than biocontamination control and describes
potential sources of microbial contamination, the use of
the risk assessment approach, and developing an effective
environmental monitoring plan. There is also greater
emphasis on data recording, trending, alert and action
limits, and data integrity.

Heating, ventilation, and air conditioning (HVAC)
Cell-based therapy facilities must be designed, constructed, and used to minimize the possibility for the introduction and retention of particles inside rooms whereaseptic processing is to occur to mitigate the potential
for contamination. HVAC systems control airflow supply
rates and airflow patterns in the cleanrooms to eliminate
sub-micron airborne contamination originating frompeople, processes, and equipment and maintain the requirements of the cleanroom classification. The HVAC
system works to control the number of particulates,
microbial load, temperature, humidity, and pressure.
HVAC systems utilize air handling units (AHUs)
to draw in air (outside or recirculated) that is filtered,cooled, or heated and processed to remove any excess
humidity. The air is then supplied to the cleanrooms
through high efficiency particulate air (HEPA) filtersthat are designed to retain a very high degree (typically
99.995%) of particulates. The air in the cleanroom is thenreturned to the HVAC system through in-room exhaust
ducts. There are several HVAC design options and an
initial HVAC philosophy should be developed at theconcept design stage to consider factors such as the air
recirculation rate, AHU zoning, and HVAC maintenance.
The air exhausted from the cleanroom area can either
be recirculated and mixed with the outside air before
returning to the cleanroom, or it can be a single pass system where the air is completely exhausted. Single pass air

removes the risk of any process related contaminants inthe cleanroom being transferred back into the cleanroom
or to other cleanrooms in the same AHU zone of the
facility, but is more energy intensive and therefore morecostly to operate than a recirculated system as outside air
must be continuously treated for temperature and humidity. Recirculated air is commonly utilized in aseptic
facilities, but manufacturers must consider the risk oftransferring process contaminants from a cleanroom that
may not be removed by HEPA filters. Single pass air isgenerally considered for rooms that are processing highrisk materials (for example, viral vectors), for biologicalsafety level (BSL) containment, or as a zoning segregation
strategy (may be less expensive to single pass a small
room than carry another dedicated AHU). The use of
single pass air should be evaluated as part of the total
cleanroom HVAC design strategy.
The location of exhaust and intake for AHUs should
be considered. AHUs can be dedicated to each cleanroom in a facility or zoned where one AHU services
multiple rooms. During the facility design phase, HVAC
diagrams that indicate AHU zoning, room pressurization, and room classifications are developed. The designof the AHUs to serve dedicated cleanrooms or multiple
cleanrooms should primarily be based on mitigating therisk of cross-contamination in the facility. The risk levelvaries across different facilities and depends on factors
such as the total volume of air to be processed by the
AHU, the number of concurrent products planned to be
manufactured, the effectiveness of the gowning protocol,
the specific manufacturing processes, and the equipment
employed. It should be noted that when developing acomprehensive facility HVAC scheme, manufacturing
cleanrooms and facility areas outside the cleanroom
should never be tied in and served by the same AHU.
Dedicated AHUs are usually smaller and have (at anindividual level) reduced capital cost per unit compared
to larger AHUs that serve multiple areas of a facility.
However, more dedicated units are required, and operational costs can be greater as there are more units to test
and maintain compared to a zoned design.
The HVAC system also controls the pressurization
between cleanrooms and the pressurization strategy
acts as another measure to maintain the cleanroom environment and reduce the risk of cross-contamination.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼247

Pressure cascades are designed on the principle to facilitate the flow of particulates away from areas where theproduct and process are most prone to contamination toprotect the product and maintain the required cleanroom
classifications. In practice, pressurization schemes facilitate flow away from the highest classified cleanrooms
and areas of high particulate generation (e.g., gowning
rooms) to lower classified cleanrooms. Where there arepass-throughs between cleanrooms HEPA filtration
should also be used to create “active” pass-throughs formaterial transfer into or between critical areas, especially
if there is a change in classification between the rooms
and pressure cascades applied. The CGMP regulations
provide guidance for pressure differentials between different grade cleanrooms that are adjacent to each other.
Pressure differentials are also utilized for health andsafety purposes to facilitate the requirements of rooms
with BSL designations where infectious materials ororganisms may be handled.
Finally, to maximize the effectiveness of the HVAC inhigher classified spaces, air flows within the cleanroom
should be designed to be directional where possible,
where the air flows down through the ceiling HEPAs
and out of the room through low-level exhaust ducts to
sweep down and out any particulates and contaminants.
The positioning of equipment in the cleanrooms should
be considered in combination with the locations of
the low-level exhaust ducting to enable unobstructed
air flow. In general, it is good practice to locate large
equipment away from the exhaust ducts and not directly
underneath supply ducts. Equipment that is known togenerate particulates, such as tube welders, should belocated in the airflow path if possible. Smoke studies are
required to demonstrate adequate air flow through thecleanrooms to mitigate the risk of contamination.
In case of AHU failure, measures such as the use of
redundant AHUs, fail-safe isolation valves at criticalduct branching points, and a standby power supply
should be in place to re-establish containment. HVACs
supplying airflow to critical operating spaces, such as
ISO 5 or Grade A spaces for open aseptic operations,should be powered by uninterruptible power to reduce
the risk of potential process contamination in the eventof site power loss.
Testing and maintenance of the HVAC system, andin particular the HEPA filters, is required on a periodic
basis to ensure the continued control of cleanroom airand classification. During the design phase, manufacturers must consider the access to the HVAC system andthe type of HEPA filter installation, which can be either
accessed for testing and maintenance from inside thecleanroom or via walkable ceilings outside of the cleanroom. Walkable ceilings reduce the need for maintenancepersonnel and tools to be taken into the cleanroom area
but are more costly to install. The selection of the type of
HEPA filter installation should consider the disruption
and risk to the operation.
OPERATIONAL DESIGN CONSIDERATIONS

In parallel with the physical layout and engineering design factors, operational aspects that enable the practicaland efficient operation of a facility should be consideredat the design stage. Cell-based therapy product manufacturing for autologous processes involves many separate
unit operations that require manufacture of multiple
patient lots from the same product line potentiallywithin the same production space. Allogeneic products,
although not patient-specific, also have multiple batches
manufactured within the same production space and anincreasing number of cell-based therapy developers haverequirements for manufacture of both autologous andallogeneic product lines in the same facility. Regardlessof the type of product, the design of the facility must
always maintain conditions to ensure the safety, efficacy,and quality of products and minimization of the risk ofcross contamination. Tracking and segregation strategies
should be implemented with respect to factors including(but not limited to) adventitious agent contamination,
viral vector contamination control, cell material COI and
COC, raw materials, consumables, and specific productand process considerations. For manufacturers utilizing
viral vectors in transfection or transduction steps, the degree of process closure being implemented should also be
factored into the overall evaluation of the necessary levelof segregation required. Key operational design considerations for segregation in multi-patient and multi-productfacilities, COI and COC are discussed here. Operational
gowning considerations are also discussed as personnel
are integral to the manufacturing operation but present
the largest risk of contamination to the product.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼248

Gowning operations
To minimize the risk of contamination introduced byhumans, personnel must adhere to well-defined gowning
protocols commensurate with the requirements of thecleanroom classification. For higher classifications, suchas Grade B/ISO 7, aseptic gowning is required. Aseptic
gowning practice is fundamentally the process of “wrapping” personnel to isolate the body from the cleanroom
environment and keep individuals from shedding particulate contaminants into the cleanroom environment (tothe extent that is possible). This wrapping consists of layersof gloves, masks, and covers that are specially designed tominimize the amount of particulate that enters the cleanroom environment. Thus, specialized cleanroom garments
help to ensure that the particles emitted by the bodies of
personnel do not diminish the sterility of the environment.
Access to cleanrooms of higher classifications should
be strictly limited to trained personnel wearing proper gowning in accordance with the facility protocol.
Gowning apparel should not be worn outside of thecleanroom. The exact garment system should be selected based on specifications for the particular cleanroom
applications. Often, personnel must not wear gowningapparel over street clothes, but must instead remove theirstreet clothes and don a polyester under-suit. Gloves,
facemasks, and goggles are put on after the under-suitand outer gown have been donned. A further aspect of
the gowning system is the maximum length of time thata gown can be worn. In ISO 7/Grade B areas, gowns
are typically worn only for the length of the operator
shift, either to be discarded if single-use or submitted
for laundering if reusable. In lower grade cleanrooms,
the same gowning apparel might be worn by an operator
for several sessions throughout the day. Generally, as
individuals perspire and stress the gowning materials
through manual operations, the integrity of the gown
weakens.
Gowning apparel can be either reusable or single-use/
disposable. Reusable gowns are usually manufactured
using 100% continuous filament polyester or continuous
filament polyester/carbon combination yarns and must
be re-laundered as specified by a protocol. Disposable
garments are non-woven and are constructed of polyolefin. All cleanroom gowning garments are designed
to allow air to pass through, but retain particulates andthus, functionally act as filters. Efficacy is comparable between disposable and non-disposable gowns; the choice
to use either depends largely on economic and practicalconsiderations. Important factors to keep in mind whenchoosing appropriate gowning materials include thickness, weight, flexibility, filtering properties, durability,
and comfort. The nature of the tasks involved in the
production process, regulatory requirements, and any
specific customer requirements will also impact choices.
In addition to gowning procedures in the gowningarea of the facility, it must be noted that gowning procedures truly begin at home, with adequate bathing or
showering, shaving, brushing of teeth, and maintenanceof hair. If needed, non-silicone skin moisturizers should
be applied to reduce skin flakes. Cleanroom environments are prohibitive for the use of make-up, hair gels,hair sprays, perfumes, aromatic after-shave lotions, orbody lotions. While working, all employees must wash
their hands with soap and water before entering thecleanroom environment. Personnel must repeat gowning procedures if re-entering after exiting (such as after
eating or using the toilet).
For general reference, IEST-RP-CC003.3 contains
guidelines for gowning configurations and suggested
frequency for change of garments based on the requirements of the ISO 14644 classifications.

Chain of identity and chain of custody
Chain of identity (COI) and chain of custody (COC)
are key records that form part of a manufacturing lot
genealogy required for all pharmaceutical products
to ensure patient safety and product quality as set out
in regulatory guidance. COI and COC is of particularimportance for cell therapies as it is critical that patientsare treated with the product intended due to the presenceof living cells donated from the patient themselves, in thecase of autologous therapies, or from donors, in the caseof allogeneic therapies. COI is a record associating a donor’s unique identifiers connected with the sample being
processed from order through collection, manufacturing,administration, and post-treatment monitoring. For allogeneic therapies manufactured from cell banks, the linkto the donor is through the lot number for the cell bankused for manufacturing. COC is a record of data points
from collection through product administration of all

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼249

actions performed, all staff involved, and the associatedlocations, dates, and times.
In facilities that process patient material, as operations move through the manufacturing process fromworkstation component to workstation component orfrom workstation to workstation, it is critical that all raw
and starting materials, intermediates, QC samples, final
products, and product- or batch-specific components andequipment are tracked accurately to prevent mix-ups orcross-contamination. This tracking should, therefore, berobust enough to establish a COI and COC that, in both
dedicated and multi-product facilities, should support
segregation and ease of identification between different
lots. Special attention must be paid when cryogenic
storage is critical to the process and timing of productdelivery to patients. This includes the flow of product outof the processing area into the storage area and throughshipment, as well as stored product that requires further
processing in the production areas (e.g., master cellbanks used in the production of working cell banks).
Tracking can be paper-based or through validated electronic traceability systems (or a combination of both).
For example, a validated electronic traceability program
based on barcoding could be used, in which each printed
label contains multiple unique identifiers (e.g., patientID, lot #, or process #) and is reconciled as part of line
clearance. Meanwhile, a COC documentation system canbe used to track and document the movement, handling,
and temperature of materials through every step in the
manufacturing workflow and logistics chain.

Segregation
Processing areas should be segregated with respect toincoming and outgoing streams of raw materials, consumables, intermediates, final product, and QC samples.
Separate areas should be in place for unloading, storage,
and movement between cleanrooms. The most importantsegregation should, arguably, be between the route forincoming process materials and the route for outgoingprocess waste. There should be designated personnel
for waste collection and transfer, dedicated secondary
containers for material/product/waste transport throughshared spaces, and a dedicated waste disposal area basedon waste type and hazard level. Ultimately the manufacturing plan in terms of physical segregations, flows of

personnel and materials, and any temporal segregations
should be supported by the site QRM policy and justified
by associated risk assessments.
All raw materials, consumables, and cellular productmaterial should be tracked and checked before entering or exiting a controlled area. For processes where
incoming cellular material is processed to isolate therelevant cells for further processing, a dedicated room
for starting material isolation should be considered. If
this is a required manufacturing step for an allogeneic
process or for allogeneic processes that utilize cellbanks, the cells may then be placed in freezers arranged
or installed with appropriate controls (such as with anautomated system) to ensure separation between thestarting cells and any final frozen products. When readyfor use in production, cellular starting material shouldbe transferred in temperature-controlled containers into
the intended processing rooms. Regardless of processclosure, operations should be carefully evaluated for potential risks for contamination and cross-contamination,and the operational space should be designed to mitigate
those risks. Regulatory guidance and QRM should also
be referenced to determine which processes can be
performed side-by-side in the same room and which
processes must be conducted in segregated areas of the
facility. After processing, the final cell product should betransferred out of the processing room (for example, viaa pass-through) and transported to an inspection area.
The status of the material should be tracked through allstages of the production process using the conceptual
frameworks of COI and COC.

Multi-patient and multi-product facilities
In multi-patient facilities, stringent spatial segregation ofprocessing areas must be implemented to minimize thepotential for product mix-up or cross contamination. If
any amount of parallel processing is being considered,risks for cross-contamination should be evaluated. For
example, open transduction of one product generally
should not be performed within the same room as open
processing of another product line (it should be noted,
however, that there exist ways of potentially mitigating
risks, such as the use of closed processing). Overall,criteria for segregation may be based on factors such as
product types, origin of starting material, BSLs, required

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL

✼250

storage conditions, and processing materials (e.g., useof viral vectors for transduction). Procedures such as
fumigation of manufacturing rooms, enhanced physicalsegregation within the manufacturing area, single-passfiltered air, and HEPA filtration of exhaust air may berequired, especially if products involve the use of potentially infectious viral vectors. Additionally, emphasisshould be placed on using, wherever possible, single-use
consumables and equipment, product-dedicated equipment, and automated closed systems. Cleaning of the
manufacturing area and equipment must be validated todemonstrate adequate removal of any active viral vector
particles and any byproducts of patient-individualized
production processes.
If the facility is intended to support manufacturingof multiple products (e.g., viral vectors and the final cell
product in the case of CAR-T manufacturing), a carefulassessment of cross-contamination risks and biosafety
requirements should be performed, with mitigation measures integrated into the facility design. A major design
factor is typically whether the facility is intending to support multiple products through temporally segregatedproduction campaigns, or concurrent production withfacility elements in place to ensure process segregation.
If planning for temporal segregation in production, arobust sanitization and product turnover procedure willneed to be developed to provide segregation between different products. If planning for concurrent manufacturing, a robust QRM strategy will need to be developed anddocumented to ensure appropriate mitigation measures
are in place to protect against cross contamination andmix-up events between product materials.

Manufacturing Start-Up and Operation
Following handover of the facility after construction
and facility validation, several manufacturing start-up
activities are required before production of qualificationbatches to produce data for regulatory licensure canproceed. These include training of personnel, running
process simulations, validating aseptic and cleaning processes, establishing line clearance processes to segregatebatches, and establishing environmental monitoringbaselines and programs. All these activities form partof the overarching contamination control strategy andcontribute towards the repeatable production of compliant products.

Personnel training and monitoring
Personnel conducting manual operations present a significant risk of contamination in all production processes. Therefore, personnel training and behavior are key tothe implementation of an effective contamination control
strategy. Personnel training should be frequently updated as required and should consist of a curriculum thatcovers theoretical, practical, and CGMP aspects of basic
microbiology, personal hygiene, and aseptic techniques.
Records of personnel training should be maintained in acontrolled document management system. Further, giventhat appropriate and effective gowning is essential in all
facilities where aseptic techniques are required, personnel must be trained on the methods and importance ofgood gowning techniques. Gowning techniques must be
monitored and assessed regularly through practices suchas visual observation and contact plate sampling and is arequirement for Grade B/ISO 7 areas.
As specified in regulatory guidance for aseptic filling,
personnel monitoring is required in facilities engaging in
aseptic processing. The purpose of personnel monitoringis to ensure minimal introduction of particulate contamination into the cleanroom and to demonstrate andconfirm that personnel comply with aseptic techniqueprocedures. This monitoring is particularly importantbecause most cleanroom contamination can be traced tohumans (personnel shed large numbers of skin cells asskin flakes); monitoring is especially critical for situations
where maintenance of aseptic conditions can be especiallytricky, such as during planned open operations ( thatfrequently occur during cell-based therapy product manufacturing). Ultimately, the most important outcome of
personnel monitoring is to minimize the risk to processes,
products, and patients. However, it is important to keep
in mind that, because of a relative lack of high-precisioncounting methods and limited sample volumes, monitoring is a semi-quantitative exercise and cannot identify andquantify all contaminants that might be present.
The most common practice to assess the risk of contaminants introduced by personnel is to use agar contact
plates to take samples from gowns and gloves worn bypersons as they leave the aseptic area. Typical locations

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼251

monitored include the fingertips, the top of the head,
the left and right arms, the torso, and the left and rightlegs. This is carried out at defined intervals as part of the
training and gowning qualification for personnel entering the controlled manufacturing areas. To account forthe occurrence of coughing or sneezing, it is also typicalto sample the facemask. Most frequently, 55 mm plateswith TDA/SCDA media are used. The plate is pressedagainst the sampling location for a specified time period(typically 3-5 seconds). Afterwards, the gown must be
discarded due to the potential reduction of gown integrity upon contact with the plates. For Grade B/ISO Class 7
areas, the same limits should be applied to finger-sample
plates as to gown-sample plates. In addition to gowning
control, procedures should be established for personnel
to be able to notify staff about health conditions. Staff displaying signs of illness, open lesions, shedding skin conditions, or gastric upsets should not enter cleanrooms.
Appropriate job role duplication/mirroring must be inplace to establish a sustained labor force for continual
and uninterrupted processing.

Process simulation and aseptic process validation
For cell-based therapy manufacturing processes, contamination control risk assessment should be informedby the performance of process simulations, in which aliquid sterile microbiological nutrient medium replaces
product material in the process and final filled units are
incubated for the purpose of detecting microbial contamination. The process is run as closely as possible to
the routine process, taking into account interventions
known to occur during normal production, as well as
projected worst-case contamination risk conditions.
It is important that process simulations include anassessment of the worst-case processing parametersthat might lead to microbial contamination events.
Process simulations should be performed as part ofthe initial validation of a facility to establish baseline
expectations for risk and should be performed routinely
thereafter at intervals of a frequency determined by risk
management assessment to monitor the effectivenessof contamination controls in the facility. Process simulations should also be performed after any significant
modification to the HVAC system, facility, equipment,or manufacturing process.

Line clearanceLine clearance refers to the clearance of residue after agiven manufacturing process step and before initiation ofa new manufacturing process step. The primary purposeof line clearance is to prevent mix-ups and cross-contamination between manufacturing operations. In the context
of cell-based therapy product manufacturing facilities,
line clearance of workstations and manufacturing rooms
is to be done whether the process change-over is batchto-batch or product-to-product. All materials and waste
associated with the previous lot should be cleared, withattention given to the particularities of the equipment
and with respect to quality assurance, materials labelling,
and documentation. This is particularly important in
autologous cell-based therapy manufacturing settings,
where each lot corresponds to an individual patient.
In such cases, though multiple patient batches can be
concurrently manufactured at different workstations inthe same room, only one patient batch can be processed
at a time in a workstation-based BSC; thus, proper line
clearance must be done after each batch is processed.
All aspects of line clearance should be supported byquality risk assessments and validation based on facility
and cleanroom-specific microbial flora profiles. Line
clearance procedures should be performed by trained
manufacturing operators and independently verified.

Cleaning and disinfection
Cleaning and disinfection of equipment and areas usedwithin the facility should be conducted according to a defined schedule. Disinfectants should be capable of destroying bacterial endospores and fungal spores and, according
to common practice, at least two different disinfectants
should be used in rotation. Disinfectants and detergentsused for cleaning must be validated prior to use throughmeasures such as surface challenge testing. The frequencyof cleaning and disinfection should be commensurate withthe manufacturing environmental requirements and production risk assessment established at the time of facility
start-up and should be regularly reviewed and updated asneeded. Personnel responsible for cleaning and disinfection should be trained to competently work in a manner
to minimize the risk of contamination.
Additionally, protocols should also be in place forcases of viral vector or cell product spillages. Viral

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼252

vector spillages are of particular concern (especially inmulti-product facilities) due to the presence of infection-competent and potentially replication-competentviral particles (which may lead to undesired genetictransduction events), and the potential for contaminationof the final product lot, while product spillages (especially in the context of autologous CAR-T manufacturing)
may result in inter-patient product contamination.
When closed processes are employed, leakages ofprocess material may also occur due to compromised
integrity of single-use plastic components of the productapparatuses, which pose similar risks as spillages during
more conventional open operations.

Cleaning validation
In GMP facilities, all equipment and tools associatedwith the manufacturing process and the cleanrooms
themselves must be properly cleaned, sanitized, andmaintained to prevent contamination or process failuresthat could affect the quality, safety, or efficacy of the final
product. Cleaning validation should be performed for the
purpose of confirming the effectiveness of cleaning procedures employed at the facility. It should be carried out forsurfaces making direct contact with drug substances anddrug products and for indirect product-contact surfacesthat have the potential to come in contact with surface
contaminants. Single-use items such beakers, pipettes,
weigh boats, silicone tubing, sample tubes, storage andbioreactor bags, and normal-flow filtration filters are typically not subject to cleaning validation. Typical processcontaminants for which cleaning is necessary include
media, buffers, storage solutions, cellular material frompatients, and viral vector particles.
Cleaning may consist of automated, semi-automated, or
manual processes although the latter are more difficult tovalidate given the variability of manual cleaning processes.
Cleaning validation refers to establishing documented evidence that provides assurance that the specific cleaning
process being employed produces consistent and reproducible results that comply with set standards. To avoid
unnecessary troubleshooting and expenditures, cleaningvalidation should be preceded by extensive considerationof cleaning process design studies, cleaning sample assay
validation, suitability of sampling, and recovery studies forassay and sampling methods.

Cleaning validation should be focused on the specificareas where cleaning is to occur, the equipment to becleaned, the equipment to be used for cleaning, and any
detergents and decontaminants to be used. It should take
into account process parameters including substanceconcentrations, contact times, microbicidal efficacy of
disinfectants, methods for removal of detergents, andstandards for qualification and training of operators.
Additionally, manufacturers should account for whether cleaning agents used are caustic, acidic, neutral,or oxidizing. Before conducting cleaning validation,
installation qualification and operational qualificationmust be performed on the process components to becleaned and on the equipment to be used for cleaning.
For multi-product facilities, particular attention should
be focused on any equipment that is used to process
multiple products.
Common cleaning validation procedures include
active residue identification, active residue detection,
method selection, sampling method selection, theestablishment of residue acceptance criteria, methods
validation, and recovery studies. To directly demonstrate
the effectiveness of a cleaning process, the processes canbe challenged. Typically, a process challenge consists ofat least three consecutive successful cleaning processruns, after which residues are measured and results arecompared to predetermined acceptance criteria. A mocksoiling procedure can also be performed. Mock soiling is
the soiling of equipment by a process other than routine
manufacturing that dirties the equipment similarly toroutine manufacturing. Mock soiling procedures must
adequately simulate normal manufacturing processes. To
facilitate the conducting of validation, matrices and tables are often used as tools to ensure proper preparationand implementation of cleaning validation procedures.

Environmental monitoring program
An environmental monitoring program can be used tomonitor and determine the type and level of contamination present in manufacturing facility environments.
The purpose of an environmental monitoring programis to gather data that can be used to assess effectivenessof established contamination controls to identify trends
that can allow for proactive interventions to prevent a
contamination event from impacting product quality.

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼253

Environmental monitoring is commonly performedby exposing nutrient medium plates to sample areas in
the facility that pose the highest contamination risks.
The sampling locations should be selected using a
documented risk-based approach that is reviewed andrepeated periodically to account for any changes in the
manufacturing process or the facility.

References• BioPharma International Jul 2019
• Designing The Cell Therapy Facility Of The Future. BioProcess
Online 2017. https://www.bioprocessonline.com/doc/designing-the-cell-therapy-facility-of-the-future-0001• FDA CFR - Code of Federal Regulations Title 21 PART 600
Biological Products• FDA CFR - Code of Federal Regulations Title 21 PART 211 Cur-rent Good Manufacturing Practices for finished pharmaceuticals• EudraLex Volume 4, Good Manufacturing Practice for Medicinal
Products for Human & Veterinary Use• EudraLex Volume 4, Guidelines on Good Manufacturing Practicespecific to Advanced Therapy Medicinal Products• Biosafety in Microbiological and Biomedical Laboratories(BMBL) 6th Edition, Centers for Disease Control and Prevention, National Institutes of HealthAppendix: List of Acronyms
AHU
AOQ
BSC
CDMO
CGMP
COC
COI
CQV
EHS
GMP
HVAC
IQ
OQ
PAL
PQ
P&ID
QMS
QRM
SOP
URB
URS

Air Handling Unit
Average Outgoing Quality
Biological Safety Cabinet
Contract Development and Manufacturing
Organization
Current Good Manufacturing Practice
Chain of Custody
Chain of Identity
Commissioning, Qualification, and Validation
Environmental, Health, and Safety
Good Manufacturing Practice
Heating, Ventilation, and Air Conditioning
Installation Qualification
Operational Qualification
Personnel Air Lock
Performance Qualification
Piping and Instrumentation Diagram
Quality Management System
Quality Risk Management
Standard Operating Procedure
User Requirement Brief
User Requirements Specification

Chapter 11: Manufacturing Strategy, Facility Design, and Manufacturing Operations

A-CELL✼254