Guidance for Industry 

Potency Tests for Cellular and Gene 

Therapy Products 

DRAFT GUIDANCE

This guidance document is for comment purposes only.

Submit comments on this draft guidance by the date provided in the Federal Register notice 

announcing the availability of the draft guidance. Submit written comments to the Division of 

Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, 

Rockville, MD 20852. Submit electronic comments to http://www.regulations.gov. You should 

identify all comments with the docket number listed in the notice of availability that publishes in 

the Federal Register. 

Additional copies of this draft guidance are available from the Office of Communication, 

Training and Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, 

MD 20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact the Office of Communication, Training 

and Manufacturers Assistance at 1-800-835-4709 or 301-827-1800. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

October 2008
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

Table of Contents 

I. INTRODUCTION................................................................................................................1

II. BACKGROUND ..................................................................................................................2

A. What Are the Regulatory Requirements for Potency of Licensed Biological 

Products? ..................................................................................................................2

B. What are the Potency Requirements for Investigational CGT Products? .........3

C. What Is the Relationship Between Potency and Clinical Effectiveness for CGT 

Products? ..................................................................................................................4

III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS ...................................5

A. How to Determine What to Measure for Potency?...............................................5

B. What Methods May be Used to Measure Potency? ..............................................6

1. Biological assays................................................................................................. 6 

2. Non-biological analytical assays......................................................................... 6 

3. Multiple assays (assay matrix)............................................................................ 7 

C. What is Necessary to Correlate an Analytical Assay with Biological Activity?.7

D. When Should Potency Assay Development Initiate?............................................8

E. What is Progressive Potency Assay Implementation?..........................................8

1. Early product development:................................................................................ 8 

2. Later phase product development:...................................................................... 9 

3. Biological License .............................................................................................. 9 

IV. ASSAY DESIGN AND VALIDATION .............................................................................9

A. What Should be Considered During Assay Design?.............................................9

B. How Should Reference Materials and Controls be Utilized? ............................10

C. What Should be Considered for an Assay Validation Plan? .............................11

1. Regulations ....................................................................................................... 11 

2. Statistical design and analysis........................................................................... 11 

3. Validation of qualitative assays........................................................................ 12 

4. Assay evaluation and modification................................................................... 12 

V. REFERENCES...................................................................................................................14

i 
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

Guidance for Industry 

Potency Tests for Cellular and Gene Therapy Products 

This draft guidance, when finalized, will represent the Food and Drug Administration’s (FDA’s) 

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 

criteria for product release. This guidance is intended to supplement related documents (Refs. 1 

through 11, and 15) and does not replace or supersede any existing documents. 

This guidance applies only to CGT products5 reviewed by FDA’s Office of Cellular, Tissue and 

Gene Therapies (OCTGT), CBER under Section 351 of the Public Health Service Act (PHS Act) 

(42 U.S.C. 262) (Refs. 1 and 2). This guidance does not apply to human cells, tissues, and 

cellular and tissue products (HCT/Ps), which are regulated solely 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. This guidance also does not apply to biological products 

reviewed by CDER or by CBER’s Office of Vaccine Research and Review (OVRR) or Office of 

Blood Research and Review (OBRR). 

 

1

 For the purpose of this document, “tests” are used interchangeably with “assays” and “measurement.” 

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

Information pertaining to the transfer of some of the therapeutic biological products that had been reviewed and 

regulated by the Center for Biologics Evaluation and Research (CBER) to Center for Drug Evaluation and Research 

(CDER) can be found at: http://www.fda.gov/cber/transfer/transfer.htm. 

1
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s 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 FDA’s guidances means that something is suggested or 

recommended, but not required. 

II. BACKGROUND 

 

A. What Are the Regulatory Requirements for Potency of Licensed Biological 

Products? 

All biological products must meet prescribed requirements of safety, purity and potency 

for BLA approval (42 U.S.C. 262, Federal Food, Drug and Cosmetic Act, (FDC Act) (21 

U.S.C. 321 et seq.); 21 CFR 601.2). For CGTs, product conformance testing (21 CFR 

601.20(a)) and control of the manufacturing process (21 CFR 601.20(c)) are required to 

comply with FDA’s Current Good Manufacturing Practice (CGMP) For Finished 

Pharmaceuticals regulations (21 CFR Parts 210 and 2116

) as well as the biologics 

regulations (21 CFR Part 600 et seq.). No lot of any licensed product may 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. 

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)). Strength7

 is defined as “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)(ii)). Regulations stipulate 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 definition in § 600.3(s) of this 

chapter.” (21 CFR 610.10). 

FDA regulations allow for considerable flexibility in determining the appropriate 

measurement(s) of potency for each product. Potency is determined based on individual 

product characteristics; therefore, the adequacy of potency assays is evaluated on a case6

 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 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. 7

 For purposes of this guidance, strength is the equivalent of potency. 

2
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

by-case basis. 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/activities) 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); 

• Establish and document the accuracy, sensitivity, specificity and reproducibility 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)) 

• Meet labeling requirements (21 CFR 610.61(g)(3) and 610.61(r)) 

B. What are the Potency Requirements for Investigational CGT Products? 

In early clinical phase investigations, it may not be possible to meet all of the 

requirements described above for licensed biological products (Refs. 3, 4, 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)). 

Potency measurements are necessary for product characterization testing,8

 comparability 

studies (Ref. 6), and stability protocols (Ref. 7), which are used to establish that a 

consistently manufactured product is administered during all phases of clinical 

investigation. However, 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 product characterization 

testing, including the development of potency assays. General recommendations for 

progressive potency assay implementation are outlined in Section III.E. As described in 

8

 For the purpose of this guidance, product characterization testing includes in-process, drug substance and final 

product tests that measure product attributes associated with product consistency and quality in order to assure 

identity, purity, strength (potency) and stability of products used during all phases of clinical study. 

 

3
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

Sections III.A, III.E, and IV.C.4 of this document, your potency measurement will evolve 

and may change significantly as you develop your product. 

Table 1:

Challenges to Potency Assay 

Development for CGT products: 

Examples: 

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 

active ingredients 

• Multiple genes expressed by the same vector 

• 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 

C. What Is the Relationship Between Potency and Clinical Effectiveness for 

CGT Products? 

There is no single test that can measure adequately those product attributes that predict 

clinical efficacy. Clinical effectivenessis demonstrated by adequate and well-controlled 

clinical investigations conducted with a consistently manufactured quality product. 

Clinical effectiveness may be correlated to product potency, but clinical study data is not 

a practicable quantitative measure of potency to release a lot. Rather, clinical study 

results may be used to establish a correlation(s) 9

 between the product’s clinical efficacy 

and a potency measurement(s), which can be used for lot release, stability, and/or 

comparability studies (see Section III.C for more details related to correlation studies). 

 

9 Correlation means a statistical relationship between two or more variables such that systematic changes in the 

value of one variable are accompanied by systematic changes in the other. 

4
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

III. RECOMMENDATIONS FOR POTENCY MEASUREMENTS

A. How to Determine What to Measure for Potency? 

Because of the complexity of CGT products, 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 data 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 (see Section III.C). This information may provide you 

with a basic understanding about product characteristics 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 Sections III.E 

and IV.C.4). As you develop your product(s), you should measure a wide range of 

product properties in addition to those performed for routine lot release. This may help 

you to assess which product attribute(s) best correlate(s) with potency. Although some of 

the assays you evaluate may not be practical for lot release (e.g., difficult to consistently 

obtain quantitative results, time-consuming), most properly designed assays (see Section 

IV.A) have the potential to provide valuable information about product attributes related 

to biological activity or clinical effectiveness, or both. 

CGT products may present challenges for developing assays to measure specific 

biological attributes that quantitatively demonstrate potency (see Table 1). CGT products 

often have complex and/or poorly defined mechanism(s) of action (i.e., relevant 

therapeutic or clinical functional activity), making it difficult to determine which product 

attribute is most relevant to measuring potency. Nonetheless, potency measurements 

should reflect the relevant biological attributes. For example, a gene therapy vector 

should rely 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 assay should incorporate both a measure of the gene transfer 

frequency and the biological effect of the transferred gene. 

In addition, the proposed mechanism(s) of action for CGT products may be dependent on 

more than one active ingredient10 (e.g., multiple cell types, multiple vectors, multiepitope vaccines). For some complex products (e.g., cellular tumor vaccine) there could 

be ambiguity about which ingredients contribute to potency. For products that contain 

10 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)).

5
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

more than one known active ingredient, you should design potency measurement(s) to 

determine the biological activity (strength) of all active ingredients (see 21 CFR 

211.165(a)). 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 for 

interference or synergy between active ingredients. 

B. What 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.A. Bioassays measure potency by evaluating a 

product’s active ingredients 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. 12). 

2. Non-biological analytical assays11

Development of a quantitative bioassay for some CGT products may be 

complicated by properties of the product and/or technical limitations (see Table 

1). In cases where bioassay development is not feasible, it may be necessary to 

identify a surrogate of biological activity. For example, you may need to use an 

analytical assay(s) that is practical and reliable 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 correlation to a relevant product-specific biological 

activity(s) (see Section III.C, Refs. 13 and 14). To establish meaningful 

correlations, you should conduct rigorous product characterization testing, as 

recommended throughout this document. 

11 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. Furthermore, we acknowledge 

that in other contexts a bioassay may be considered an analytical assay. 

6
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

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 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 to develop multiple 

complementary assays that measure different product characteristics 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. 13 and 14). The assay 

matrix may include assays that give a quantitative readout (e.g., units of activity) 

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.A). 

C. What is Necessary to Correlate an Analytical Assay with Biological Activity? 

To demonstrate potency using an analytical assay as a surrogate measurement of 

biological activity, you should provide sufficient data to establish a correlation between 

the surrogate measurement(s) and the biological activity(ies) that is related to potency. 

The relationship between the surrogate measurement and biological activity may be 

established using various approaches, including comparison to preclinical/proof of 

concept data, in vivo animal or clinical data, 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 should meet criteria 

listed above in Section II.A. This could necessitate that you stress the product (i.e., show 

that the assay can detect an inactive or degraded product) and perform sufficiently 

controlled studies (see Section IV.). 

The suitability of data used to support surrogate assays for biological activity 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; 

7
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

• How well the biological activity of the product is understood; and 

• How well the surrogate measurement(s) reflects biological activity. 

If you intend to demonstrate potency by correlating a surrogate assay(s) to a relevant 

biological activity, you should start collecting product and assay characterization data 

during early investigational phases. 

D. When Should Potency Assay Development Initiate? 

As discussed throughout this document, thorough product characterization is necessary to 

understand the product parameter(s) that affect quality, 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 an effective product. Thus, because 

the ability to measure potency is essential to product characterization, you should initiate 

potency assay development 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 bioactivity may be sufficient. 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. Nevertheless, as 

clinical studies progress 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)). 

8
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

 2. Later phase product development: 

The primary objective of later phase investigational studies (i.e., Phase 3, 

pivotal12) is to gather meaningful data about product efficacy. Efficacy is 

determined by adequate and well-controlled clinical study(ies). Therefore, your 

potency assay design and acceptance criteria should be sufficient to assure that a 

well-characterized, consistently manufactured product was administered during 

your pivotal study(ies). Conformance to established limits for potency should 

thus provide reasonable confidence that future product lots will perform as 

expected at a given dose in patients. 

In addition, you should use a well-characterized potency assay with established 

limits during stability testing of conformance lots used to establish expiry dating 

for licensure (see 21 CFR 610.53; Ref. 7). 

3. Biological License 

To market a biological product, a validated potency assay 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.A). 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 depict 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). 

IV. ASSAY DESIGN AND VALIDATION 

A. What Should be Considered During Assay Design? 

In accordance with CGMP regulations, assay design should allow you to collect data that 

will permit you to evaluate your assay(s). 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 variability. Therefore, you should consider sources of variability in 

12 For the purpose of this document the term “pivotal” study is 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). 

9
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

the assay method and take steps to limit them in your assay design. General principles 

for reducing variability include using well-defined reagents, well-calibrated equipment, 

and adequately trained 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. 13 through 20). 

 

B. How Should Reference Materials and Controls be Utilized? 

As with all well designed experiments, developing a potency assay should include 

appropriate controls and a comparison to an appropriate reference material, when 

available. Running a 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 reproducible. 

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. For instance, there are fluorescent 

bead/antibodies and particle size standards13 and guidelines14 available to help calibrate 

equipment and help define acceptable parameters for quantitative flow cytometry analysis 

(Ref. 18). Reference materials are also currently available for adenovirus type 5 (Ref. 

19)15 and retrovirus16 vectors. A reference material for adeno-associated virus type 2 

vectors17 is under development. Standard materials and controls for lentivirus vectors 

have also been described (Ref. 20). 

In the event that a universal standard or reference material is not available, you should 

develop your own “in house” reference material(s) (Refs. 9 through 11). 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 

13 National Institute of Standards and Technology. Available at 

http://ts.nist.gov/MeasurementServices/ReferenceMaterials/232.cfm. 14 Fluorescence Calibration and Quantitative Measurement of Fluorescence Intensity; Approved Guideline. NCCLS: 

ILA24 Vol 24 No 26. Available at http://www.nccls.org. 15 Adenovirus Type 5 Reference Material (ARM) available at http://www.atcc.org/common/documents/pdf/VR1516text2.pdf. 

16 Retrovirus Reference Material. Available at 

http://www.atcc.org/common/catalog/numSearch/numResults.cfm?atccNum=VR-1450. 17 Information related to the Adeno-Associated Virus Reference Material is available at 

http:\\www.wilbio.com\ReferenceMaterials\aav2.htm. 

10
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

(including “in house” reference material/control) was developed. We encourage you to 

consult with your CBER review team when developing or obtaining reference materials. 

Because you will use reference materials at various stages of product development and 

characterization, you should subject them to stability studies in parallel with your product 

stability studies (Ref. 7). 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 retain samples (Refs. 6 through 8) of each lot of reference material for 

comparison with newly manufactured reference material and prepare in advance for 

depletion or expiration of reference materials. 

 

C. What Should be Considered for an 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 with 

predefined acceptance criteria (see 21 CFR 211.165(e)). The validation process 

identifies potential sources of errors and quantifies them within the assay method. 

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, Reproducibility) 

• Sensitivity (Limit Of Detection/Quantitation) 

• Specificity 

• Linearity and Range 

• System Suitability 

• Robustness/Ruggedness 

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. Data collected from 

potency assay validation studies, when provided in electronic format, can 

facilitate statistical evaluations by the CBER review committee. The results of 

11
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

validation studies should address the targeted validation parameters and their 

conformance to acceptance criteria. We encourage you to initiate early 

discussions with the review team to receive feedback on the design and analysis 

of potency experiments. (See 21 CFR 211.194 for requirements pertaining to the 

laboratory records you must keep.) 

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 and sensitivity 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 

might be able to demonstrate reproducibility. 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 reproducibility. Also, limits of detection and/or quantitation may 

be built into the assay design suitability criteria. For example, if a reasonable 

amount of the control or reference material does not exhibit the desired activity 

with sufficient statistical justification, the assay would not generally be 

considered acceptable. Importantly, 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. 

4. Assay evaluation and modification 

Manufacturing and testing practices evolve during product development or postlicensure, 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 to 

demonstrate that the modified/new assay continues to be an appropriate measure 

of potency (21 CFR 211.165(e)). These changes must be submitted as a 

supplement to an approved application (21 CFR 601.12(b)(3)(vi)). 

12
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

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) 

• Whether the proposed assay meets assay criteria outlined above (see above 

and Section II.A) 

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 further 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. 

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. 14), and your assay(s) might 

change over time as you develop your product and learn new information and methods. We 

recommend that you have timely discussions with your review team as you design, evaluate and 

validate your potency measurement. 

13
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

 

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/cber/genadmin/fr101493.pdf.

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/cber/gdlns/clinxeno.htm.

3. 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/cber/gdlns/gtindcmc.htm.

4. 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/cber/gdlns/cmcsomcell.htm.

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/cber/gdlns/ichtest.pdf. 

6. 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/cder/guidance/6677fnl.pdf.

7. 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/cber/gdlns/ichq5c071096.pdf.

8. Guidance for Industry: CGMP for Phase 1 Investigational Drugs. (July 2008). Available at 

http://www.fda.gov/cber/gdlns/indcgmp.htm.

9. Draft Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, 

Manufacturing, and Controls Documentation. (August 2000). Available at 

http://www.fda.gov/cber/gdlns/methval.htm.

∗

10. International Conference on Harmonisation Guideline Validation of Analytical Procedures: 

Text and Methodology Q2(R1). (November 2005). Available at 

http://www.ich.org/LOB/media/MEDIA417.pdf.

11. Chapter <1225> Validation of Compendial Methods. US Pharmacopeia 28, United States 

Pharmacopeia Convention, Inc., Rockville, MD: 2005. 

12. 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/about/ni_Mission.htm.

13. 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. 

∗

 When finalized, this guidance will represent FDA’s current thinking on this topic. 

14
Contains Nonbinding Recommendations 

Draft – Not for Implementation 

15

14. 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.

15. Chapter <111> Design and Analysis of Biological Assays. US Pharmacopeia 28, United 

States Pharmacopeia Convention, Inc., Rockville, MD: 2005.

16. 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).

17. Montgomery, D. C. Design and Analysis of Experiments. John Wiley & Sons; 6th edition 

(2005). 

18. 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). 

19. Hutchins, B., et al., Working Toward an Adenovirus Vector Testing Standard. Molecular 

Therapy Vol. 2, No. 6, (December 2000).

20. 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.