Guidance for Industry 
 
Potency Tests for Cellular and 
Gene Therapy Products 
 
 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852-
1448, or by calling 1-800-835-4709 or 301-827-1800, or email ocod@fda.hhs.gov, or from the 
Internet at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/default.htm. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers listed above.  
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
January 2011 Contains Nonbinding Recommendations 
 
 
 
Table of Contents 
 
I.  INTRODUCTION................................................................................................................1 
II.  BACKGROUND..................................................................................................................2 
A.  What is Potency Testing?........................................................................................2 
B.  What Are the Regulatory Requirements for Potency of Licensed Biological 
Products?..................................................................................................................2 
C.  What are the Potency Requirements for Investigational CGT Products?.........4 
D.  What is the Relationship Between Potency and Clinical Effectiveness for CGT 
Products?..................................................................................................................5 
III.  RECOMMENDATIONS FOR POTENCY MEASUREMENTS...................................6 
A.  What Should be Measured for Potency?...............................................................6 
1.  Product Characterization.....................................................................................6 
2.  Mechanism of Action (MOA).............................................................................6 
B.   What Analytical Methods May be Used to Measure Potency?............................7 
1.   Biological assays.................................................................................................7 
2.   Non-biological analytical assays.........................................................................7 
3.   Multiple assays (assay matrix)............................................................................8 
C.   What is Necessary to Establish a Correlation between Biological Activity and 
a Non-Biological Analytical Assay(s)?...................................................................8 
D.  When Should Potency Assay Development Begin?...............................................9 
E.  What is Progressive Potency Assay Implementation?..........................................9 
1.  Early product development:................................................................................9 
2.  Later phase product development:....................................................................10 
3.  Biologics License..............................................................................................10 
4.   Potency Assay Evaluation and Modification....................................................11 
IV.  POTENCY ASSAY DESIGN AND VALIDATION.......................................................11 
A.  What Should be Considered During Potency Assay Design?............................11 
B.   How Should Reference Materials and Controls be Used?.................................12 
C.  What Should be Considered for a Potency Assay Validation Plan?.................13 
1.   Regulations.......................................................................................................13 
2.   Statistical design and analysis...........................................................................14 
3.   Validation of qualitative assays........................................................................14 
V.  REFERENCES...................................................................................................................16 
i  Contains Nonbinding Recommendations 
 
 
Guidance for Industry 
 
Potency Tests for Cellular and Gene Therapy Products 
 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this 
topic.  It does not create or confer any rights for or on any person and does not operate to bind FDA 
or the public.  You can use an alternative approach if the approach satisfies the requirements of the 
applicable statutes and regulations.  If you want to discuss an alternative approach, contact the 
appropriate FDA staff.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance. 
 
 
I.  INTRODUCTION 
  
We, FDA, are issuing this guidance to provide you, manufacturers of cellular and gene therapy 
(CGT) products, with recommendations for developing tests1 to measure potency.2  These 
recommendations are intended to clarify the potency information that could support an 
Investigational New Drug Application3 (IND) or a Biologics License Application4 (BLA).  Because 
potency measurements are designed specifically for a particular product, this guidance does not 
make recommendations regarding specific types of potency assays, nor does it propose acceptance 
criteria for product release.  This guidance is intended to supplement related documents (Refs. 1 
through 12) and does not replace or supersede any currently published guidance documents, with the 
exception that this guidance finalizes the draft guidance entitled Guidance for Industry:  Potency 
Tests for Cellular and Gene Therapy Products dated October 2008 (October 9, 2008, 73 FR 59635).   
 
This guidance applies only to CGT products5 reviewed by FDAs Office of Cellular, Tissue and 
Gene Therapies (OCTGT), Center for Biologics Evaluation and Research (CBER), under section 
351 of the Public Health Service Act (PHS Act) (42 U.S.C. 262) (Refs. 1 and 2).  Note that this 
guidance applies to therapeutic vaccines that are CGT products; however, it does not apply to 
therapeutic vaccines that are not CGT products.    
 
This guidance also does not apply to products regulated under section 361 of the PHS Act (42 U.S.C. 
264) as described under 21 CFR 1271.10 or to products regulated as medical devices under  
21 CFR Part 820.  Furthermore, this guidance does not apply to biological products reviewed by 
CDER or by CBERs Office of Vaccine Research and Review (OVRR) or CBERs Office of Blood 
Research and Review (OBRR).   
 
                                                 
1 For the purpose of this guidance, the term tests is used interchangeably with the terms assays and measurements. 
2 As defined in 21 CFR 600.3(s), and discussed in Section II.A of this guidance.  
3 See 21 CFR Part 312.  
4 See 21 CFR Part 601. 
5 For information on therapeutic biological products that are reviewed and regulated by the Center for Drug Evaluation 
and Research (CDER) see:  http://www.fda.gov/AboutFDA/CentersOffices/CBER/ucm133463.htm, accessed August 17, 
2010.     
1  Contains Nonbinding Recommendations 
 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  The 
use of the word should in FDAs guidances means that something is suggested or recommended, but 
not required. 
 
 
II.  BACKGROUND  
   
A.  What is Potency Testing?  
 
Potency is defined as the specific ability or capacity of the product, as indicated by 
appropriate laboratory tests or by adequately controlled clinical data obtained through the 
administration of the product in the manner intended, to effect a given result. (21 CFR 
600.3(s)).  Strength6 is defined as [t]he potency, that is, the therapeutic activity of the drug 
product as indicated by appropriate laboratory tests or by adequately developed and 
controlled clinical data. . . . (21 CFR 210.3(b)(16)).  Regulations require that [t]ests for 
potency shall consist of either in vitro or in vivo tests, or both, which have been specifically 
designed for each product so as to indicate its potency in a manner adequate to satisfy the 
interpretation of potency given by the definition in  600.3(s) of this chapter.  
(21 CFR 610.10).  
 
Potency tests, along with a number of other tests, are performed as part of product 
conformance testing,7 comparability studies (Ref. 3), and stability testing (Ref. 4).  These 
tests are used to measure product attributes associated with product quality and 
manufacturing controls, and are performed to assure identity, purity, strength (potency), and 
stability of products used during all phases of clinical study.  Similarly, potency 
measurements are used to demonstrate that only product lots that meet defined specifications 
or acceptance criteria are administered during all phases of clinical investigation and 
following market approval.   
 
B.  What Are the Regulatory Requirements for Potency of Licensed Biological 
Products? 
 
All biological products regulated under section 351 of the PHS Act must meet prescribed 
requirements of safety, purity and potency for BLA approval; Federal Food, Drug and 
Cosmetic Act, (FDC Act), (21 U.S.C. 321 et seq.); (21 CFR 601.2).  For CGT, product 
conformance testing (21 CFR 601.20(a)) and control of the manufacturing process (21 CFR 
601.20(c)) are required to comply with FDAs Current Good Manufacturing Practice 
(CGMP) For Finished Pharmaceuticals regulations (21 CFR Parts 210 and 2118) as well as 
                                                 
6  For purposes of this guidance, strength is the equivalent of potency. 
7 For the purpose of this guidance, product conformance testing includes in-process, drug substance and final product 
tests.  
8 The drug CGMP regulations contain the minimum current good manufacturing practice for methods to be used in, and 
the facilities or controls to be used for, the manufacture, processing, packing, or holding of a drug to assure that the drug 
2  Contains Nonbinding Recommendations 
 
 
the biologics regulations (21 CFR Part 600).  No lot of any licensed product shall be 
released by the manufacturer prior to the completion of tests for conformity with standards 
applicable to such product, (21 CFR 610.1), which include tests for potency, sterility, purity, 
and identity (21 CFR Part 610, Subpart B).  These requirements apply to all biological 
products, including autologous and single patient allogeneic products, where a lot may be 
defined as a single dose. 
 
Some CGT products may also contain, in addition to the active ingredient 9, one or more 
substances commonly referred to in the scientific literature as an adjuvant10.  A complete 
discussion of the requirements for adjuvant testing is beyond the scope of this document.  
However, it should be noted that an adjuvant shall not be introduced into a product unless 
there is satisfactory evidence that it does not affect adversely the safety or potency of the 
product (21 CFR 610.15(a)).  We recommend that you consult11 with your CBER review 
team for additional information regarding potency testing for products that include one or 
more adjuvants. 
 
FDA regulations allow for considerable flexibility in determining the appropriate 
measurement(s) of potency for each product.  Potency is determined based on individual 
product attributes; therefore, the adequacy of potency tests is evaluated on a case-by-case 
basis.  However, all potency assays used for release testing of licensed biological drug 
products must comply with applicable biologics and CGMP regulations including: 
 
  Indicate potency (biological activity/activities12) specific to the product (21 CFR 600.3(s) 
and 610.10; and 21 CFR 210.3(b)(16)(ii));  
  Provide test results for release of the product (21 CFR 610.1; 21 CFR 211.165(a)); 
  Provide quantitative data (21 CFR 211.194; see also 21 CFR 600.3(kk); 21 CFR 
211.165(d); 211.165(e);  
  Meet pre-defined acceptance and/or rejection criteria (21 CFR 211.165(d); see also  
21 CFR 600.3(kk); and 21 CFR 210.3(b)(20)); 
  Include appropriate reference materials, standards, and/or controls (see  
21 CFR 210.3(b)(16)(ii) and 211.160); 
                                                                                                                                                                   
meets the requirements of the FDC Act as to safety, and has the identity and strength and meets the quality and purity 
characteristics that it purports or is represented to possess. 
9 Active ingredient means any component that is intended to furnish pharmacologic activity or other direct effects in the 
diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body of 
man or other animals (21 CFR 210.3(b)(7)). 
10 There is currently no regulatory definition for adjuvant.  Therefore, for the purpose of this guidance, an adjuvant is 
defined as any agent or combination of agents, added to or used in conjunction with a CGT product, to augment or 
potentiate the specific activity of the CGT product. 
11 These discussions can be in the form of IND amendments, informal and formal regulatory meetings.  Please see FDA 
Guidance:  Formal Meetings Between the FDA and Sponsors or Applicants at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM153222.pdf  or see 
CBERs Standard Operating Procedures and Policy (SOPP) 8101.1 (Scheduling and Conduct of Regulatory Review 
Meetings with Sponsors and Applicants - 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/ProceduresSOPPs/ucm07944
8.htm) and 21 CFR 312.47 (Meetings - http://www.access.gpo.gov/nara/cfr/waisidx_08/21cfr312_08.html), which 
describe the procedures for meetings to address issues relating to product development, all accessed August 17, 2010. 
12 Biological activity is the specific ability or capacity of the product to achieve a defined biological effect (Ref. 5) 
3  Contains Nonbinding Recommendations 
 
 
  Establish and document the accuracy, sensitivity, specificity and reproducibility13 of the 
test methods employed through validation (21 CFR 211.165(e) and 211.194(a)(2)); 
  Measure identity and strength (activity) of all active ingredients (21 CFR 211.165(a); 
see also 21 CFR 210.3(b)(7));  
  Provide data to establish dating periods (see 21 CFR 600.3(l) and 610.53(a)); and  
  Meet labeling requirements (21 CFR 610.61(g)(3) and 610.61(r)) 
 
C.  What are the Potency Requirements for Investigational CGT Products?  
 
In early phase clinical investigations, it may not be possible to meet all of the requirements 
described above for licensed biological products (Refs. 6 through 8).  Nonetheless, you must 
submit data to assure the identity, quality, purity and strength (21 CFR 312.23(a)(7)(i)) as 
well as stability (21 CFR 312.23(a)(7)(ii)) of products used during all phases of clinical 
study.  [T]he amount of information needed to make that assurance will vary with the phase 
of the investigation, the proposed duration of the investigation, the dosage form, and the 
amount of information otherwise available. (21 CFR 312.23(a)(7)(i)).  
 
The complexity of CGT products can present significant challenge(s) to establishing potency 
assays (see Table 1).  To facilitate the development of CGT products, we recommend an 
incremental approach to the implementation of potency tests.  General recommendations for 
progressive potency assay implementation are outlined in Section III.E.  As described in 
Section III of this document, your potency measurement(s) will evolve and may change 
significantly as you develop your product.  We recommend that you have timely discussions 
with your CBER review team as you design, evaluate and validate your potency 
measurement. 
 
Table 114: 
 
Challenges to Potency Assay  Examples: 
Development for CGT products: 
Inherent variability of starting materials   Autologous and allogeneic donor variability 
 Cell line heterogeneity 
 Error-prone replicating viruses 
Limited lot size and limited material for testing   Single dose therapy using autologous cells suspended in a 
small volume 
Limited stability   Viability of cellular products 
Lack of appropriate reference standards   Autologous cellular material 
 Novel gene therapy vectors 
Multiple active ingredients   Multiple cell lines combined in final product 
 Heterogeneous mixtures of peptide pulsed tumor and/or 
immune-modulatory cells 
 Multiple vectors used in combination 
The potential for interference or synergy between   Multiple genes expressed by the same vector 
                                                 
13 Reproducibility as used in this guidance is cited from 21 CFR 211.165(e), and is not meant to be consistent with the 
guidance document ICH Q2(R1), where it is defined by inter-laboratory studies. 
14 The items listed in Table 1 are examples of the types of challenges that you may encounter when developing potency 
assays for these products.  The table does not list all possible challenges that you may encounter.   
4  Contains Nonbinding Recommendations 
 
 
Challenges to Potency Assay  Examples: 
Development for CGT products: 
active ingredients   Multiple cell types in autologous/allogeneic cell 
preparations 
Complex mechanism of action(s)    Multiple potential effector functions of cells 
   Multiple steps required for function such as infection,  
integration, and expression of a transgene 
 Vectors containing multiple genes 
In vivo fate of product   Migration from site of administration 
 Cellular differentiation into the desired cell type 
 Viral or cellular replication 
 Viral vector infection, uncoating, and transgene expression 
 
 
D.  What is the Relationship Between Potency and Clinical Effectiveness for CGT 
Products?  
There is no single test that can adequately measure those product attributes that predict 
clinical efficacy.  Manufacturers demonstrate clinical effectiveness by substantial 
evidence, i.e., evidence that the product will have the effect it purports or is represented to 
have under the conditions of use prescribed, recommended, or suggested in the labeling or 
proposed labeling thereof (section 505(d) of the FDC Act).  As a general matter, substantial 
evidence of clinical efficacy is obtained from adequate and well-controlled investigations 
conducted with a consistently manufactured product, as specified in 21 CFR 314.126(d) (Ref. 
12).  Potency measurements are a necessary part of product characterization testing, 
comparability studies, and stability protocols, which are used to establish that a consistently 
manufactured product is administered during all phases of clinical investigation.   
Efficacy data from well controlled clinical investigations can provide evidence that a product 
has biological activity, and thus is potent.  However, use of clinical study data may not be a 
practicable method to quantitatively test for potency to release a lot (i.e., clinical data may 
not be available prior to release of individual product lots; clinical data may not be coupled to 
individual lots).  That said, this guidance document will discuss ways to develop a release 
assay(s) to measure your products potency.  As discussed below, clinical data may be used 
to establish a correlation(s)15 between biological activity and a more practical potency 
measurement(s) that can be used for lot release, stability, and/or comparability studies (see 
Section III).  
 
                                                 
15 As used in this document, correlation means a statistical and biological relationship between two or more variables 
such that systematic changes in the value of one variable are accompanied by systematic changes in the other.   
5  Contains Nonbinding Recommendations 
 
 
 
III.  RECOMMENDATIONS FOR POTENCY MEASUREMENTS 
 
A.  What Should be Measured for Potency? 
 
1.  Product Characterization 
 
CGT products are complex, thus you need to acquire an appropriate understanding of 
the biological properties of your product in order to develop relevant and meaningful 
potency measurements.  You should collect sufficient product characterization data 
(i.e., molecular, biochemical, immunologic, phenotypic, physical and biological 
properties) throughout preclinical and clinical development to inform and refine your 
approach to measuring potency.  
 
When initially determining the biological activity or activities that will guide your 
potency assay design, you should consider relevant pre-clinical investigations, proof 
of concept studies, early clinical studies, available historical experience, and available 
reference materials and controls related to your product class.  This information may 
provide you with a basic understanding regarding product attributes and biological 
activities that contribute to function.  Characterization data obtained during product 
development may provide support for the potency assay that you choose initially, or it 
may lead to an improved potency measurement as you prepare to market your product 
(see Section III.E for product lifecycle considerations).   
 
As part of product development, we recommend that you measure a wide range of 
product attributes in addition to the tests used for routine lot release.  Exploratory 
studies may help you to assess which product attribute(s) best correlate(s) with 
potency.  The purpose of exploratory studies is to gain product information, which 
will help you to design meaningful and relevant potency assays; although these 
studies may not necessarily help you to set specifications or assign acceptance criteria 
for assays that may or may not become a specification for lot release.  While some of 
the assays you evaluate may not be practical for lot release, they may provide you 
with helpful information about product attributes related to biological activity or 
clinical effectiveness, or both.  
 
2.  Mechanism of Action (MOA) 
 
Ideally, the potency assay will represent the product's mechanism of action (i.e., 
relevant therapeutic activity or intended biological effect).  However, many CGT 
products have complex (e.g., rely on multiple biological activities) and/or not fully 
characterized mechanisms of action (MOA), making it difficult to determine which 
product attributes are most relevant to measuring potency.  Nonetheless, all attempts 
should be made to develop potency measurements that reflect the products relevant 
biological properties.  For example, a gene therapy vector relies on at least two 
biological activities for its potency: the ability to transfer a genetic sequence to a cell; 
and the biological effect of the expressed genetic sequence.  Therefore, the potency 
6  Contains Nonbinding Recommendations 
 
 
assay should incorporate both a measure of gene transfer and the biological effect of 
the transferred gene (see Section III.E for product lifecycle considerations).  
 
In addition, the proposed MOA for CGT products may be dependent on more than 
one active ingredient (e.g., multiple cell types, multiple vectors, multi-epitope 
vaccines).  For some complex products (e.g., cellular tumor vaccines), there could be 
ambiguity about which ingredients contribute to potency.  Note that under  
21 CFR 211.165, for each batch of drug product, there must be appropriate laboratory 
determination of satisfactory conformance to final specifications for the drug product, 
including the identity and strength of each active ingredient prior to release.  Thus, if 
your product contains more than one active ingredient you might need more than one 
assay to measure potency of the product because one assay might be insufficient to 
measure the activity of each of the active ingredients (Section III.B.3).  Additionally, 
when designing your assay(s), you should also consider the potential non-additive 
effects between active ingredients, such as interference or synergy.   
 
B.  What Analytical Methods May be Used to Measure Potency? 
 
1.  Biological assays 
  
The traditional approach for assessing the potency of biological products is to develop 
a quantitative biological assay (bioassay) that measures the activity of the product 
related to its specific ability to effect a given result, and that also meets the criteria 
listed in Section II.B.  Bioassays can provide a measure of potency by evaluating a 
products active ingredient(s) within a living biological system.  Bioassays can 
include in vivo animal studies, in vitro organ, tissue or cell culture systems, or any 
combination of these.  You may use in vitro or in vivo assays; however, we 
encourage the responsible limitation of animal use whenever possible (Ref. 13). 
 
2.  Non-biological analytical assays16 
 
Development of a quantitative bioassay for some CGT products may be complicated 
by properties of the product and/or technical limitations of certain assays (see Table 
1).  In cases where development of a suitable bioassay is not feasible it may be 
necessary to identify a surrogate measurement of biological activity.  For example, 
you may need to use a non-biological analytical assay(s) that is practical and 
demonstrates adequate performance characteristics for lot release.  Analytical assays 
can provide extensive product characterization data by evaluating immunochemical, 
biochemical, and/or molecular attributes of the product.  These attributes may be used 
to demonstrate potency if the surrogate measurement(s) can be substantiated by 
                                                 
16 To distinguish traditional bioassay methods (performed in a living system) from non-bioassay methods (performed 
outside of living system), we use analytical assay to refer to methods that measure immunochemical (e.g., quantitative 
flow cytometry, enzyme-linked immunosorbant assay), molecular (e.g., reverse transcription polymerase chain reaction, 
quantitative polymerase chain reaction, microarray) or biochemical (e.g., protein binding, enzymatic reactions) 
properties of the product outside of a living system.  However, we acknowledge that in other contexts a bioassay may be 
considered an analytical assay. 
7  Contains Nonbinding Recommendations 
 
 
correlation to a relevant product-specific biological activity(s) (see Section III.C, 
Refs. 14 and 15).  To establish meaningful correlations, you should conduct rigorous 
product characterization testing, as recommended throughout this document.  
 
3.  Multiple assays (assay matrix) 
 
In many cases, a single biological or analytical assay may not provide an adequate 
measure of potency.  The following are some potential reasons:  
 
  Product has complex and/or not fully characterized mechanism of action 
  Product has multiple active ingredients and/or multiple biological activities  
  Limited product stability 
  Biological assay is not quantitative, not sufficiently robust, or lacks precision 
 
If one assay is not sufficient to measure the product attribute(s) that indicates potency, 
then an alternative approach could be used, such as developing multiple 
complementary assays that measure different product attributes associated with 
quality, consistency and stability.  When used together and when results are 
correlated with a relevant biological activity, these complementary assays should 
provide an adequate measure of potency.  Such a collection of assays (referred to as 
an assay matrix) might consist of a combination of biological assays, biological and 
analytical assays, or analytical assays alone (Refs. 15 and 16).  The assay matrix may 
include assays that give a quantitative readout (e.g., units of activity) and/or 
qualitative readout (e.g., pass/fail).  If qualitative assays are used as part of an assay 
matrix to determine potency for lot release, stability or comparability studies, they 
should be accompanied by one or more quantitative assays (see Section II.B). 
 
C.  What is Necessary to Establish a Correlation between Biological Activity and a 
Non-Biological Analytical Assay(s)?  
 
To demonstrate potency using an analytical assay or assay matrix as a surrogate 
measurement of biological activity, you should provide sufficient, scientifically sound data 
(i.e., based on suitably qualified assays, an appropriate number of replicates, multiple lots or 
various patient samples, etc.) to establish a correlation between the surrogate measurement(s) 
and the biological activity related to potency.  We recommend that you consult with your 
CBER review team prior to design of correlative studies. 
 
The correlative relationship between the surrogate measurement and biological activity may 
be established using various approaches, including comparison to preclinical/proof of 
concept data, in vivo data (animal or clinical), or in vitro cellular or biochemical data.  If you 
choose to use an analytical assay as a surrogate measurement of biological activity to meet 
the potency requirements for licensed biological products, you will need to meet the criteria 
listed above in Section II.B.  You should also show that the assay can discriminate between 
active product and an inactive or degraded form of the product; and perform sufficiently 
controlled studies (see Section IV) and/or employ a validated analytical assay.  
 
8  Contains Nonbinding Recommendations 
 
 
The suitability of data used to support the correlative relationship between the surrogate 
assay and the biological activity of the product is evaluated on a case-by-case basis and 
depends on or is influenced by the following: 
 
  Type and relevance of the correlation(s) being made;   
  The amount of product information you have accumulated; 
  How well the biological activity of the product is understood; and  
  How well the surrogate measurement(s) reflects biological activity. 
 
As with any potency assay, you should start collecting product and assay characterization 
data to support your choice of assay during early investigational phases. 
 
D.  When Should Potency Assay Development Begin? 
 
As discussed throughout this document, thorough product characterization is necessary to 
understand the product parameter(s) that affect quality, potency, lot to lot consistency, and 
stability.  Moreover, understanding and controlling these parameters will be necessary to 
demonstrate consistency between production lots, to assess comparability of different 
manufacturing processes and/or various assays, and may also be necessary to allow you to 
determine which product attributes are related to biological activity and/or an effective 
product.  Because the ability to measure potency is fundamentally related to product 
characterization, you should initiate potency assay development by way of product 
characterization during preclinical and early clinical investigations to obtain as much product 
information as possible. 
 
In addition, measuring potency during early product development has a number of 
advantages, such as allowing you to: 
 
  Demonstrate product activity, quality and consistency throughout product 
development; 
  Generate a collection of data to support specifications for lot release;  
  Provide a basis for assessing manufacturing changes;  
  Evaluate product stability; 
  Recognize technical problems or reasons a different assay might be preferable; 
  Evaluate multiple assays; and 
  Collect sufficient data to support correlation studies, if necessary. 
 
E.  What is Progressive Potency Assay Implementation? 
 
   1.  Early product development: 
 
For some products in pre-clinical, Phase 1 and early Phase 2 studies, limited 
quantitative information on relevant biological attributes may be sufficient.  Assay 
acceptance criteria should be set as a numerical range and should be adjusted 
throughout the product development stages to reflect manufacturing and clinical 
9  Contains Nonbinding Recommendations 
 
 
experience.  Potency assays performed on product lots used for early clinical studies 
are likely to have wider acceptance ranges than assays used in later phase 
investigations.   
 
As clinical study progresses and product knowledge increases, you should develop 
and implement improved potency measurement(s) that quantitatively assesses 
relevant biological product attribute(s) (see 21 CFR 312.23(a)(7)).  
 
   2.  Later phase product development: 
 
The primary objective of later phase investigational studies (i.e., Phase 3, pivotal17) is 
to gather meaningful data about product efficacy, which is determined by adequate 
and well-controlled clinical trial(s).  One aspect of an adequate and well controlled 
trial is administering product lots with similar potency, in that conformance to 
established limits for potency is necessary to provide reasonable confidence that 
product lots will perform as expected at a given dose in patients.  Therefore, your 
potency assay or assay matrix design and acceptance criteria should establish 
appropriate limits for potency to assure that product lots are well-defined, biologically 
active, and consistently manufactured.  If you do not provide sufficient assurance of 
potency of product lots to be used in your pivotal trial(s), your trial may be 
considered deficient in design to meet its stated objectives and may be placed on 
clinical hold (21 CFR 312.42(b)(2)(ii)).    
 
In addition, you should use a potency assay or assay matrix, with established limits, 
during stability testing of conformance lots used to establish expiry dating for 
licensure (see 21 CFR 610.53; Ref. 4).  Assays used to establish stability and expiry 
dating should be demonstrated to be stability indicating.   
 
3.  Biologics License 
 
To obtain a biologics license, a validated potency assay or assay matrix with defined 
acceptance criteria must be described and justified in the BLA (21 CFR 601.2(a) and 
211.165(e), see also Section II.B).  The acceptance criteria should be based on 
knowledge gained through manufacturing experience and data collected from assays 
performed during all phases of product development and clinical investigation (Ref. 
5).  As you evaluate product conformance lots or lots manufactured explicitly for use 
in your pivotal clinical studies, acceptance criteria should be refined to reflect these 
data.  
 
The potency assay acceptance criteria defined in your BLA, which are intended for 
subsequent lot release testing, should reflect the potency limits established for product 
lots used in the pivotal clinical studies demonstrating clinical effectiveness (see FDC 
Act, Section 505(d), 21 U.S.C. 351).  
                                                 
17 For purposes of this guidance, the terms pivotal trial or pivotal clinical studies are used to represent any clinical 
study where the data obtained from that study will be used to support a clinical efficacy claim for the biologics license 
application (BLA). 
1 0 Contains Nonbinding Recommendations 
 
 
 
4.  Potency Assay Evaluation and Modification 
 
Manufacturing and testing practices evolve during product development or post-
licensure, or both, making it necessary and/or beneficial to re-evaluate your potency 
assay.  If you plan to modify an assay that is used in an approved application or 
propose a new assay, you must perform validation studies (see Section IV) to 
demonstrate that the modified or new assay continues to be an appropriate measure of 
potency (21 CFR 211.165(e)).  In addition, a study designed with statistical 
considerations for sample size and planned analysis, should be conducted to 
demonstrate comparability between the original assay and the proposed modified or 
new assay.  The study plan should include pre-determined acceptance criteria to 
demonstrate equivalence between the assays.  The proposed changes in the potency 
assay as well as appropriate comparability study data must be submitted as 
supplements to an approved application (21 CFR 601.12(b)(3)(vi)).    
 
The quantity of data needed to support changes to potency measurements(s) will 
depend upon a number of factors, including: 
 
  Stage of product development; 
  Type of change within an existing assay; 
  Whether the assay is being used to measure a different product attribute(s); 
and 
  Whether the proposed assay meets assay criteria outlined above (see above 
and Section II.B) 
 
If you modify the potency measurement used during an investigational study, you 
should qualify the assay and provide justification for the proposed change(s) (e.g., 
more relevant, more practical, more quantitative).  
 
These recommendations emphasize the importance of maintaining retention samples (e.g., 
product, reference materials, critical reagents) whenever possible.  It will be difficult to 
compare assays or determine if new assays are performing appropriately without analyzing 
appropriate retention samples. 
 
 
IV.  POTENCY ASSAY DESIGN AND VALIDATION 
 
A.  What Should be Considered During Potency Assay Design? 
 
In accordance with CGMP regulations, assay design should allow you to collect data that will 
permit you to evaluate if your assay(s) is suitable for its intended use (Refs. 9 through 11; see 
e.g., 21 CFR 211.165 and 21 CFR 211.194).  This includes incorporating a sufficient number 
of replicates to allow for statistical analysis, using sample randomization to reduce biases 
(e.g., sources of bias associated with placement in a 96-well plate), and including appropriate 
controls.  Assay design should also reflect knowledge of the factors that influence assay 
1 1 Contains Nonbinding Recommendations 
 
 
variability.  Therefore, you should consider sources of variability in the assay method and 
take steps to limit them in your assay design.  Some sources of variability, even when 
reduced, are unavoidable and so should be balanced, measured and modeled.  General 
principles for reducing variability include using qualified reagents, qualified and calibrated 
equipment, and adequately trained and qualified operators.  Assay variability can also be 
substantially reduced by following detailed standard operating procedures (SOPs) and having 
appropriate controls in place.  Assay-specific controls will depend on the product being 
analyzed as well as the assay used.  You should also consider the long-term availability of 
critical reagents, including reference materials and controls.  Manufacturers may refer to 
several resources for a more detailed discussion of assay design strategies (e.g., Refs. 14 
through 21). 
  
B.   How Should Reference Materials and Controls be Used? 
 
As with all well designed experiments, developing a potency assay should include 
appropriate assay controls and a comparison to an appropriate product-specific reference 
material, when available.  Running a product-specific reference material and/or control 
samples in parallel with the product helps ensure that the assay is performing as expected.  In 
addition, controls help establish that the equipment and reagents are working within 
established limits.  A well designed set of control samples can substantially increase 
confidence that results are meaningful and reliable.  
 
You should develop your own in house reference material(s) (Refs. 9 through 11) as part of 
product development when feasible.  These may include well characterized clinical lots or 
other well characterized materials prepared by you or another resource (e.g., a well 
characterized cell line with a profile similar to your product).  There should be a clear 
rationale for how and why the reference material (including in house product-specific 
reference material/control) was developed.  We encourage you to consult with your CBER 
review team when developing or obtaining reference materials. 
 
Other reference materials and standards can help with assay development and can be used to 
develop and qualify more relevant in house reference materials and/or controls.  A number of 
reference materials, standards, and controls are available or are being developed for 
characterizing biologics and/or for potential read-out systems for potency assays.  For 
instance, there are fluorescent bead/antibodies and particle size standards18 and guidelines19 
available to help calibrate equipment and help define acceptable parameters for quantitative 
flow cytometry analysis (Ref. 19).  Reference materials are also currently available for 
adenovirus type 5 (Ref. 20),20 retrovirus21 vectors and adeno-associated virus type 2 
                                                 
18 National Institute of Standards and Technology, available at https://www-s.nist.gov/srmors/view_detail.cfm?srm=1932  
(Item number SRM 1932), accessed September 1, 2010. 
19 Fluorescence Calibration and Quantitative Measurement of Fluorescence Intensity; Approved Guideline. NCCLS: 
ILA24 Vol 24 No 26, available at 
http://www.clsi.org/source/orders/categories.cfm?section=Immunology_and_Ligand_Assay1&CAT=IL (item ILA24A), 
accessed August 31, 2010. 
20 Adenovirus Type 5 Reference Material (ARM), available at 
http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=VR-
1516&Template=animalVirology (item number VR-1516), accessed August 31, 2010. 
1 2 Contains Nonbinding Recommendations 
 
 
vectors22.  Standard materials and controls for lentivirus vectors have also been described 
(Ref. 21).  Development of universal or standardized reference materials for other CGT 
products is encouraged. 
 
Because you will use in house reference materials at various stages of product development 
and characterization, you should subject reference materials to stability studies in parallel 
with your product stability studies and assign appropriate retest or expiration dates (Ref. 4).  
Moreover, you should appropriately characterize each new batch of reference material, 
compare it with the original, and establish appropriate procedures to qualify and eventually 
validate new reference materials.  When possible, you should retain23 samples (Refs. 3, 4 and 
8) of each lot of in house reference material for comparison with newly manufactured 
reference material and prepare in advance for depletion or expiration of reference materials.  
The use of statistical control charts to map the ongoing performance and stability of reference 
material during routine assays can be a useful quality control tool allowing for early detection 
of adverse trends.  
  
C.  What Should be Considered for a Potency Assay Validation Plan? 
 
  1.  Regulations 
 
To obtain a biologics license, you must submit data in your BLA demonstrating, 
among other things, that your product meets prescribed requirements of potency  
(21 CFR 601.2), which requires that you validate your potency assay (see  
21 CFR 211.165(e)).  The validation process identifies potential sources of error and 
quantifies them within the assay method.  During assay development, you should 
evaluate assay performance and suitability for use.  Numerous resources are available 
for analytical methods validation (Refs. 9 through 11).  You should perform analysis 
and validation of all relevant assay parameters (Refs. 9 through 11), including:  
 
  Accuracy;  
  Precision (Repeatability, Intermediate Precision); 
  Specificity;  
  Linearity and Range; 
  System Suitability; and 
  Robustness. 
  
                                                                                                                                                                   
21 Retrovirus Reference Material, available at 
http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=VR-
1450&Template=animalVirology  (item number VR-1450), accessed August 31, 2010. 
22 Adeno-Associated Virus2 Reference Standard Material available at 
http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=VR-
1616&Template=animalVirology (Item # VR-1616).  Additional information related to the Adeno-Associated Virus 
Reference Standard Material is available at http://www.isbiotech.org/ReferenceMaterials/aav2.htm, both accessed 
August 31, 2010.  
23  Retained samples of reference samples should be stored in accordance with 21 CFR 211.160(a).  While we recognize 
that storage conditions for reference materials may vary from those for finished products, they should be stored in a 
manner to retain product quality.    
1 3 Contains Nonbinding Recommendations 
 
 
2.  Statistical design and analysis 
 
It is critically important to apply sound and appropriate statistical methods to the 
design and analysis of laboratory experiments for potency measurements.  Otherwise, 
inferences drawn from such experimental data might not be valid.  Potential sources 
of assay variability and variations from replicates should be taken into account when 
reporting results.  You should fully describe your methods of analysis, including your 
justification and rationale.  These descriptions should be sufficiently clear to permit 
independent statistical analysis and evaluation of the results presented in the study 
reports.  You may provide data collected from potency assay validation studies in 
electronic format to facilitate statistical evaluations by the CBER review committee, 
but supplying data in an electronic format is not currently a requirement.  The results 
of validation studies should address the targeted validation parameters and their 
conformance to acceptance criteria.  (Refs. 9 through 11).  You must maintain 
laboratory records that include complete data derived from all tests necessary to 
assure compliance with established specifications and standards (21 CFR 211.194).  
We encourage you to initiate early discussions with the review team to receive 
feedback on the design, data format and analysis of potency experiments.    
 
  3.  Validation of qualitative assays 
 
As discussed in Section III.B.3, qualitative assays may be used as part of an assay 
matrix to assess potency, provided that you conduct suitable correlation studies.  You 
should validate all parameters relevant to your qualitative assay and provide a 
rationale for those parameters that you determine are not relevant.  For example, 
although certain assay validation parameters (e.g., linearity) may not be applicable to 
a qualitative assay with a pass or fail readout, appropriate control samples should be 
used to characterize the assay for specificity as well as for other features of acceptable 
performance (e.g., robustness, system suitability).   
 
Without quantitative data, demonstrating accuracy and precision could be 
challenging; however, with proper assay design (e.g., sufficient replicates), you 
should be able to demonstrate adequate assay consistency.  For semi-quantitative 
assays (assays with highly variable quantitative readout, e.g., response in an animal 
model), broader acceptance ranges may be considered for determining assay 
robustness and assay consistency.  You should establish acceptance criteria for the 
control and/or reference material in your qualitative assay to determine if each assay 
is acceptable.  If the controls fail in many of the individual assays the assay would not 
be considered acceptable.  Additionally, because of the complex nature of CGT 
products, specific circumstances for determining assay suitability will vary from 
assay to assay.  Therefore, we encourage you to discuss planned experiments with 
your CBER review team before you initiate specific assay designs and/or detailed 
experimental analyses of potency measurements.  
  
 
1 4 Contains Nonbinding Recommendations 
 
 
As this guidance indicates, a considerable amount of data might be necessary to 
develop a suitable measurement of potency for your product (see also Ref. 15).  In 
addition, your assay(s) might change over time in response to new information 
obtained as you develop your product.  Therefore, we recommend that you have 
timely discussions with your CBER review team as you design, evaluate and validate 
your potency measurement. 
1 5 Contains Nonbinding Recommendations 
 
 
 
V.  REFERENCES  
   
1.  Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and 
Gene Therapy Products. October 14, 1993; 58 FR 53248.  Available at 
http://www.fda.gov/downloads/BiologicsBloodVaccines/SafetyAvailability/UCM148113.pdf, 
accessed August 17, 2010. 
2.  Guidance for Industry: Source Animal, Preclinical, and Clinical Issues Concerning the Use of 
Xenotransplantation Products in Humans. (April 2003).  Available at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/Xenotransplantation/ucm074354.htm, accessed August 17, 2010. 
3.  International Conference on Harmonisation: Guidance for Industry: Q5E Comparability of 
Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process. (June 
2005).  Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm073476.pdf, accessed August 17, 2010. 
4.  International Conference on Harmonisation: Final Guidelines on Stability Testing of 
Biotechnological/Biological Products (ICH Q5C). 61 FR 36466, July 10, 1996.  Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm073466.pdf, accessed August 17, 2010. 
5.  International Conference on Harmonisation: Guidance on Specifications: Test Procedures and 
Acceptance Criteria for Biotechnological/Biological Products (ICH Q6B). 64 FR 44928, August 
18, 1999.  Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm073488.pdf, accessed August 17, 2010. 
6.  Guidance for FDA Review Staff and Sponsors: Content and Review of Chemistry, 
Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New 
Drug Applications (INDs). (April 2008).  Available at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/CellularandGeneTherapy/ucm072587.htm, accessed August 17, 2010. 
7.  Guidance for Reviewers: Instructions and Template for Chemistry, Manufacturing, and Control 
(CMC) Reviewers of Human Somatic Cell Therapy Investigational New Drug Applications 
(INDs). (April 2008).  Available at 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guida
nces/Xenotransplantation/ucm074131.htm, accessed August 17, 2010. 
8.  Guidance for Industry: CGMP for Phase 1 Investigational Drugs. (July 2008).  Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm070273.pdf, accessed August 17, 2010. 
9.  Draft Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, 
Manufacturing, and Controls Documentation. (August 2000).  Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm122858.pdf, accessed August 17, 2010.  
10. International Conference on Harmonisation Guideline Validation of Analytical Procedures: Text 
and Methodology Q2(R1). (November 2005).  Available at 
                                                 
  When finalized, this guidance will represent FDAs current thinking on this topic. 
1 6 Contains Nonbinding Recommendations 
 
 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm065005.h
tm, accessed August 17, 2010. 
11. Chapter <1225> Validation of Compendial Methods. US Pharmacopeia 28, United States 
Pharmacopeia Convention, Inc., Rockville, MD: 2005.  
12. Guidance for Industry: Providing Clinical Evidence of Effectiveness for Human Drugs and 
Biological Products (May 1998). Available at 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/u
cm078749.pdf, accessed August 17, 2010. 
13. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) 
Mission, Vision and Strategic Priorities. (February 2004).  Available at 
http://iccvam.niehs.nih.gov/docs/5yearplan.htm, accessed August 17, 2010. 
14. Kawakami K, Puri, RK. Regulatory Expectations During Product Development for Tumor 
Vaccines. Brown F, Petricciani J editors. Development of therapeutic cancer vaccines. Dev. Biol, 
Basel, Karger, 2004, vol 116, pp. 53-9.  
15. Potency Measurements for Cellular and Gene Therapy Products, Cellular, Tissue and Gene 
Therapies Advisory Committee (CTGTAC) Meeting. Gaithersburg Hilton, February 9, 2006. 
Available at http://www.fda.gov/ohrms/dockets/ac/cber06.html#CellularTissueGeneTherapies.  
16. Chapter <111> Design and Analysis of Biological Assays. US Pharmacopeia 28, United States 
Pharmacopeia Convention, Inc., Rockville, MD: 2005. 
17. Brown, F., Mire-Sluis A. (Eds.), The Design and Analysis of Potency Assays for Biotechnology 
Products. Brown, F. (ed.), Developments in Biologicals, Vol. 107, Basel: Karger (2002). 
18. Montgomery, D. C. Design and Analysis of Experiments. John Wiley & Sons; 6th edition 
(2005).  
19. Stelzer, GT., et al., U.S.-Canadian Consensus Recommendations on the Immunophenotypic 
Analysis of Hematologic Neoplasia by Flow Cytometry: Standardization and Validation of 
Laboratory Procedures. Cytometry (Comm Clin Cytometry) 30:214-230 (1997).  
20. Hutchins, B., et al., Working Toward an Adenovirus Vector Testing Standard. Molecular 
Therapy Vol. 2, No. 6, (December 2000). 
21. Kiermer, V., et al., Report from the Lentivirus Vector Working Group:  Issues for Developing 
Assays and Reference Materials for Detecting Replication-Competent Lentivirus in Production 
Lots of Lentivirus Vectors. BioProcessing Journal, March/April 2005: 39-42. 
  
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