# Presentation: 425493

## Chemistry, Manufacturing and Control Issues in Production of Therapeutic Biologic Protein Products

**Ingrid Markovic, Ph.D., **Biologist
- Laboratory of Biochemistry, Division of Therapeutic Proteins
- Office of Biotechnology Products, Office of Pharmaceutical Science
- Center for Drug Evaluation and Research
- FDA

## OBP/OPS/CDER

- Division of Therapeutic Proteins
- Amy Rosenberg, M.D.*, Director*
- Barry Cherney, Ph.D.,* Deputy*

- Office of the Director
- Steven Kozlowski, M.D.,* Director*
- Wendy Shores, Ph.D*., Deputy*

- Division of Monoclonal Antibodies
- Kathleen Clouse, Ph.D.*, Director*
- Patrick Swann, Ph.D., *Deputy*

- Fc-Fusion Proteins

- Monoclonal
- Antibodies

- Enzymes

- Cytokines

- Growth factors

- Toxins

**Fabrazyme**

**Interferons**
**Interleukins**

**Herceptin**
**Avastin**
**Erbitux**

**Enbrel**

**G-CSF**
**Epo**

**Botox**

***Parallel Office to ONDQA, which also reviews proteins (e.g., insulin, HGH, etc.)***
**(courtesy of Dr. S. Kozlowski)**

## Majority of Biotech Products Use Living Cells to Produce a Protein Product

- Insert gene encoding the protein of interest
- Cells require proper conditions for optimal growth (temp, pH, oxygen, feeds, etc.)
- Culture and fermentation can take weeks
- Complex Purification Steps
- Safe product with desired potency

- Bar Charts, Inc. 2003

## Protein Therapeutics

- Protein Therapeutics are licensed through **Biologics License Application (BLA)** under provisions of both Public Health Service (PHS) and Food Drug & Cosmetic (FD&C) Acts
- Protein therapeutics are also regulated through **New Drug Application (NDA)** under provisions of FD&C Act (e.g., insulin & HGH)
- BLA under PHS act lacks an abbreviated pathway for follow-on biologics or biosimilars

## How are Protein Therapeutics different from Small Molecule Drugs?

- Contain intrinsic infectious agents
- Aseptic techniques required during production (terminal heat or gamma sterilization rarely applied)
- Usually have heterogeneous composition
- Numerous process and product-related impurities
- Change in the manufacturing process can cause change in product composition
- Exact structure may be unknown (e.g., all possible variants often not fully characterized)

## Structure of Small Molecule vs. Protein Drugs

**Structure of Small Molecule vs. ****Protein Drugs**

- Proteins have expected:
- Size, charge, hydrophobicity
- Correct folding (S-S bonds)
- Subunits
- Glycosylation
- Bioactivity
- & Unexpected:
- Aggregation (side effects)
-  Incorrect folding
- Amino acid modifications
- ox, deam, cys
- Truncation, proteolysis

- Statin

**Therapeutic protein ~5,000 - 300,000 Da**

- ~400 Da

*****

**x**

**Notes:**
This slide uses animation when in slide mode.  Try to fix.

## Manufacturing Process

## Components of the Manufacturing Process

-  Expression vector (plasmid)
-  Cell banking system
-  Master Cell Bank (MCB)
-  Working Cell Bank (WCB)
-  End of Production Cells (EOP)
- Drug substance manufacturing and release
- Drug product formulation and release

## Expression Vector and Cell Banking System

## Source Materials

**Mice**

**Humans**

**Mammalian cell-culture**

**Yeast**

**Bacteria**

**Transgenics**

**Viruses**
**TSE**
**agents**

**Bacteria**
**Mycoplasma**
**Fungi**

## Expression Vectors (Plasmids)

- Used for transfer of genes from one organism to another
- Used for production of large amounts of protein
- Description of origin of the construct
- Plasmid mapping (e.g., restriction sites, integration sites, promoter, copy number etc.) and stability
- Sequencing of gene of interest

## MCB and WCB

- A working cell bank (WCB) is derived from the master cell bank (MCB) and is used to initiate a production batch

## Characterization of Cell Banks 

**dependent upon cell substrate and manufacturing process*

## Characterization of Cell Banks (cont.)

## Sources of Adventitious Agents

- Cell Substrate
- Endogenous viruses
- Exogenous microbial contamination
- Source material screening:
- Human (HIV, HBV, HCV, CJD, etc.)
- Animal (TSE sources, species-specific viruses)
- Raw Materials
- Cell culture reagents (animal and non-animal derived)
- Environment
- Water
- Air
- Humans/technicians

## Viral Clearance for Phase 1 IND 

- Demonstration of viral clearance may be required. Exceptions: certain source materials (e.g., E. coli, yeast) or in the event of unmet medical need
- Perform small scale clearance study that mimics the clinical purification process
- Spike Drug Substance with a model virus to demonstrate viral removal by several logs beyond the potential load
- CHO cell substrate – demonstrate retroviral clearance
- Human cell substrate - demonstrate clearance of enveloped and non-enveloped viruses (e.g., parvoviruses)
- Design the process upfront to adequately assess potential risks
- Two orthogonal robust steps (e.g., low pH, nano-filtration, solvent/detergent treatment, heat) typically included in the purification process

## Production and Purification

## Downstream Processing

-    Isolation/Capture of protein
- Purification
- Drug substance
- 		Formulation
- Drug Product

**Downstream Processing**

**Notes:**
Protein produced in yeast, E.coli, mammalian cells (ex: 293, CHO)
Protein either secreted into media (ex: mammalian cells, yeast) or found in cells (ex: E.coli).
Centrifugation, clarification to remove insoluble debris, cells, etc.
Harvest is mixture of proteins, lipids, DNA, RNA, media components, etc

Can mix and match techniques throughout entire purification scheme

## Fermentation Process

**Fermentation Process**

## Purification Process

**Purification**** ****Process**

## Drug Substance and Drug Product Characterization

## Proteins Can be Heterogeneous Mixtures

**(9600)****2****≈ 10****8**

**O**

- Methionine oxidation (2 x 2)

**pyro-E**

- Pyro-Glu (2)

- High mannose,  G0, G1, G1, G2 (5)

- Sialylation (5)

- Glycation (2 x 2)

**K**

-    C-term Lys (2)

**(Courtesy of Dr. S. Kozlowski)**

**Proteins Can be Heterogeneous Mixtures**

- Deamidation (3 x 2)

**Notes:**
This slide has animation when in slide mode.  Try to fix

## Drug Substance Characterization

- Drug Substance should be positive for identity and have specified criteria for purity, potency and microbial contamination
- Acceptance criteria for release and stability attributes should be established
- Often broader early in the development and subject to revisions (e.g., narrowed down) as manufacturing process develops
- Results from release and stability testing should be provided in the IND
- Raw data supporting Drug Substance characterization should be provided in the IND

## Drug Substance Characterization (cont.)

**Safety**
- Ensured by the specified limits for bioburden and endotoxin, misc. process-related contaminants
**Purity & Characterization**
- Assesses capability of purification process to remove **process-related impurities** (e.g., endogenous viruses, host-cell proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy metals, etc.)
**Product-related impurities** (e.g., aggregates, breakdown products, product variants due to: oxidation, deamidation, denaturation, loss of C-term Lys in MAbs etc.)
**Product substances** (product variants that are active)
**Identity**
- Unique for protein of interest, especially relevant for closely related proteins manufactured in the same facility

## Drug Substance Characterization (cont.)

**Potency**
- Required to assess biological activity of the product
- Assay should be relevant for protein mechanism of action
- For MAb or Fc fusion proteins - a binding assay may be sufficient for early development, but a functional assay relevant for the mechanism of action should be developed
- If mechanism of action unknown - multiple bioactivities plus elucidating higher order structure may be required
**Strength**
- Protein content
**Stability**
- Drug Substance stability should be demonstrated with appropriate stability-indicating assays

## Drug Substance Characterization – Methodology

**Safety**
- LAL test, rabbit pyrogen test, bacterial culture methods
**Purity & Characterization including but not limited to:**
- Reversed-phase HPLC, Peptide mapping, MS
- SDS-PAGE, Western analysis, capillary electrophoresis
- SEC, AUC, FFF, light scattering
- Ion Exchange Chromatography
- Carbohydrate analysis (capillary electrophoresis, HPAEC = high-pH anion-exchange chromatography, IEF for sialic acid)
**Identity**
- N-terminal sequencing
- Peptide mapping
- Immunoassays (ELISA, Western blotting)
**Potency**
- Animal-based assays, cell-based assays, reporter gene, biochemical  (e.g., enzyme activity)
**Protein content**
- RIA, ELISA, UV absorbance, Bradford

## Drug Product Characterization

**Safety**
- Final Drug Product for injection should be sterile
- Within specified limits for endotoxin
- Immunogenicity should be screened and monitored
- Successfully reduced in MAb by replacing murine with human sequences
**Purity & Characterization **
- Product and process-related impurities & product-related substances should be within specified limits
**Identity**
- Unique for protein of interest, especially relevant for closely related proteins manufactured in the same facility

## Drug Product Characterization (cont.)

**Potency**
- Assay should be relevant for protein mechanism of action
- For MAb or Fc fusion proteins, a binding assay may be sufficient for early development, but a functional assay relevant for the mechanism of action should be developed
- If mechanism of action unknown - multiple bioactivities plus elucidation of higher order structure may be required
**Strength**
- Protein content
**Stability**
- Drug Product should maintain stability for the duration of the clinical trial
**Container closure compatibility**
- Primary function - barrier to microbial ingress
- Extractables/Leachables studies – requirement for licensure

## Extractables

- Migrate from a c/c system and/or other packaging components in DP vehicle or solvent under extreme T°C and time conditions	     			_*exaggerated conditions*_
- Helpful in the predicting potential leachables and in selecting the appropriate c/c system

## Leachables

- Migrate spontaneously from a c/c system and/or other packaging components  	   	  	 	 _*normal conditions of use and storage *_
- Often a subset of extractables, or derived  by their chemical modification

## Sources of leachables in the product

**Syringes/prefilled syringes, ampoules, vials, bottles**
**IV bags**
**Storage bags for product intermediates**
**Closures (screw caps, rubber stoppers)**
**Container liners (e.g., tube liners)**
**Processing equipment:**
- stainless steel storage tanks/bioreactors
- tubing
- gaskets, valves, rings
- filters
- purification resins

##  Examples of leachables impacting on safety and product quality

_**Example #1 – Impact of patient safety:**_
- Change: from HSA formulation to a polysorbate
- Unchanged container closure system (pre-filled syringes with the uncoated rubber stoppers)
- Source: vulcanizing agents leached from rubber stopper over time
- Outcome:
- no detectable changes in product quality
- safety: serious adverse event (PRCA)
- Hypothesis: leachables acted as adjuvants triggering immunogenicity
_**Example #2 - Impact on product quality**_:
- Change from a lyophilized to a liquid formulation
- Divalent cation leached from the rubber stopper
- Caused activation of metalloprotease (a process-related impurity co-eluted with the API)
- Impact: product degradation at the N-terminal site (stability study)

## Stability Program

- Drug Substance and Drug Product, real-time and accelerated stability data with several time points under upright and inverted conditions used to establish the expiration period
- Stress studies (e.g., UV, exaggerated light, temperature and pH) useful to elucidate product degradation pathways and for defining acceptance criteria
- Limited time stability studies may be acceptable if short-term trial is anticipated
- Stability data generated from engineering lots also acceptable
- Failure to demonstrate product stability is a potential hold issue

## Stability Program (cont.)

- The following testing should be included at a minimum:
**Safety**
- Bioburden/sterility
**Purity**
- Product and process-related impurities & product-related substances
**Sialic acid** - if appropriate
**Potency**
**Protein content/strength**
**pH**
**Appearance**
**Leachables ****(separate study, not part of routine stability testing)**** **

**Notes:**
Deleted Identity from stability protocol.

## Good Manufacturing Practices

## Inspectional Activity

- Three types:
- Pre-licensed (PLI) - announced, generally required for approval
- Pre-approval (PAI) – announced, could be waived
- Surveillance (biennial post-licensure) – unannounced
**No formal inspection requirement for sites manufacturing biologics under clinical investigation**
- Manufacturing and testing sites are subject to inspection
- Inspection system undergoing revision for OBP products
- Currently inspections of facilities manufacturing CDER BLA products:
- PAI led by TFRB, Office of Compliance, with Product Reviewer(s) sometimes part of on-site team
- Biennial post-licensure inspections led by Team Biologics with Product Reviewers which can be part of the on-site team involved in the inspection
- NDA Products:
- Pre-approval and post-licensure inspections led by district personnel

## Facilities and Practices

- Closed systems whenever possible
- Aseptic Processing
- CIP/SIP
- Disposable Systems
- Environmental Monitoring
- Water/HVAC
- Good record-keeping and documentation (phase 1)

## ICH Q7: Good Manufacturing Practice Guide For Active Pharmaceutical Ingredients

**Provide greater assurance in linking product quality to commercial manufacture**

**ICH Q7: Good Manufacturing Practice Guide For Active Pharmaceutical Ingredients**

**Notes:**
I don’t see the value of the table that is shown in this slide

## Potential Show Stoppers?

## Potential CMC Hold Issues for Phase 1 IND

- Comparability between preclinical and clinical lots not demonstrated
- Insufficient characterization of cell banks (e.g., adventitious agents testing, identity, etc.)
- Inadequate product characterization with regards to purity, identity, potency and safety
- Lack of final product release testing
- Lacking or inappropriate specifications for release and stability testing
- Lacking or inadequate potency assay
- Data supporting product stability have not been shown for the planned duration of clinical studies
- Lack or inappropriate immunogenicity assays for high risk products
- Lack of evidence for final Drug Product sterility

## Guidance Documents

- Guidance for Industry: Content and Format of Investigational New Drug Applications for Phase I Studies of Drugs, including Well-Characterized, Therapeutic, Biotechnology derived Products (1995)
- Guidance for Industry for the Submission of CMC Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody Product for *In Vivo* Use (1996)
- Guidance for Industry: IND for Phase 2 and 3 studies of Drugs, including Specified Therapeutic Biotechnology-Derived Products – CMC Content and Format (Draft, 1999)
- FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, including Therapeutic Biotechnology-derived Products (1996)
- Guidance for Industry: INDs - Approaches to Complying with cGMP's for Phase 1 Drugs (Draft, 2006)
- Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997)
- Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
- International Conference on Harmonization (ICH) documents
- 21 CFR 200’s, 600’s
- PHS Act, FD&C Act

## Acknowledgments

- Emily Shacter
- Barry Cherney
- Steven Kozlowski
- Wendy Shores
- Susan Kirshner
- Emanuela Lacana
- Patricia Hughes
- Joe Kutza
- All of OBP

## Questions? Comments? 

**Questions? Comments? **