Briefing Document

 

Cellular, Tissue, and Gene Therapies Advisory Committee

 

Meeting # 41

 

February 9, 2006

 

 

Potency Measurements for Cell and Gene Therapy Products


 

Briefing Package

Cellular, Tissue, and Gene Therapies Advisory Committee Meeting #41

Feb, 9, 2006

Potency Measurements for Cell and Gene Therapy Products

 

 

CONTENTS

 

INTRODUCTION……………………………………………………………….......…3

 

MEETING GOALS…………………………………………………………..........…...3

 

BACKGROUND…………………………………………………………………..…....3

Potency Definitions………………….…………………………….…………………........3

Products regulated by OCTGT………………………………………………………...….4

Cellular Therapy Products…………………………………………………...……….……4

Gene Therapy Products………………………………………………………………'.......5

Tumor Vaccines……………………………………………………………………...'...…5

Challenges for measuring potency for C&GT products ……………………………....…….5

 

TECHNIQUES FOR POTENCY MEASUREMENT ……………….……………..…6

Biological Assay Methods…………………….……………………………………….…..6

Analytical Assay Methods………………………………...…………………………….…7

Immunochemical Procedures………………………………………….……….....….7

Molecular and Biochemical Procedures……………………………………….....…..8

 

CONSIDERATIONS FOR POTENCY MEASUREMENTS ……………...........…....8

 

SUMMARY……………………………………………………………………….….…10 

 

REFERENCES……………………………………………………………………….…11

 

QUESTIONS ………….……..…………………………………………………………12

 

 

 

 


INTRODUCTION

 

Successful development of biological therapeutics requires full product characterization to ensure safety and efficacy.  Product characterization involves quality control testing programs consisting of a meaningful array of in-process and final product tests that provide reasonable assurance of product safety, quality, and purity. Potency measurements are a critical part of product characterization, as they are intended to measure the activity of the product within established limits.  As such, potency measurements are required as part of lot release testing (1, 2). As a measure of the biological activity of the product, potency results can provide valuable information about product consistency, stability, and comparability. The types of assays used to measure potency can vary greatly and are dependent, in large part, on the nature of the product. Cellular and gene therapy manufacturers (hereafter referred to as sponsors) should develop potency assay(s) that are based on scientific principles related to the specific properties of the product (e.g. stimulation of lymphocyte proliferation by a recombinant cytokine).

 

The complexity of C&GT products presents a significant and often novel set of challenges to sponsors in meeting the requirements for developing, validating and performing potency assays. Many, but not all, of these challenges will be presented in this briefing document prepared for the CTGTAC potency discussion. We look forward to scientific input from the advisory committee to help us (Office of Cellular, Tissue and Gene Therapies) and sponsors advance the development of C&GT products.

 

 

MEETING GOALS

 

This meeting of the Cellular, Tissue and Gene Therapies Advisory Committee (CTGTAC) is being organized to achieve the following goals:

 

·        To discuss challenges related to the development of meaningful and relevant potency assay measurements for C&GT products.

·        To obtain perspectives and advice from members of the CTGTAC regarding the implementation of scientifically valid assays for measuring potency of C&GT products.

 

 

BACKGROUND

 

Potency:

 

All licensed biological products must be demonstrated to be potent for licensure (1, 2). The word potency is defined as “the specific ability or capacity of the product, .…, to effect a given result” (21 CFR 600.3[s])(3a). With this in mind, potency measurements have been defined to include: “…either in vitro or in vivo tests, or both, which have been specifically designed for each product…” (US 21 CFR 610.10) (3b). These definitions provide considerable freedom to both sponsors and regulators when evaluating suitable potency assay design(s). Nevertheless, regulators have generally accepted that an appropriately designed potency assay for biologics should be a quantitative measure of biological activity specific to the product that is linked to the relevant biological properties (4).  Thus, potency assay measurements should reflect particular biological characteristics of the individual product.

 

Potency measurements are performed as part of product release testing to ensure that all lots of the product exhibit activity within established limits.  These limits, referred to as acceptance criteria (5a), are established based on data collected during product development. Demonstrating product consistency by meeting acceptance criteria for potency is important in order to obtain meaningful efficacy data from the clinical trial, and provide assurance that all product lots should perform as expected at a given dose in patients.

 

Potency assessment(s) for biologics are meant to measure the active component(s) within a complex mixture by quantifying the product’s activity in a biological system. However, the biological assays used to measure biological activity can yield highly variable results.  Assay variability should be characterized and its effect minimized through appropriate assay design. Suitable reference standards should also be included in potency assay design to identify and monitor variability (4, 5b).

 

To market a biological product a validated potency assay must be described and justified in the Biological Licensing Application (BLA) (2, 5c). Assay validation is the process of determining suitability of a given methodology for providing useful analytical data: does the assay measure what it is intended to measure? The validation process identifies potential sources of variability and addresses the quantitation of these variables in the assay method. A valid potency assay(s) should have defined ranges and values for relevant assay parameters such as: accuracy, precision, sensitivity [limit of detection/quantitation], specificity, linearity, reproducibility and robustness (5c, 6, 7).

 

 

Products regulated by OCTGT:

 

CBER’s Office of Cellular, Tissue, and Gene Therapies (OCTGT) is responsible for the regulation of cellular therapies, gene therapies, tissues, tissue engineered products (including cells/vectors with scaffolds/matrices), xenotransplantation products, tumor vaccines and other novel products. Since a comprehensive discussion of all of these products is beyond the scope of one meeting, we will focus on potency assay measurements for cellular and gene therapy (C&GT) products, including C&GT-based tumor vaccines. Common to all cellular and gene therapies is the intention to intervene in a disease course or clinical outcome by means of introducing new genetic or cellular material into humans. 

 

Cellular Therapy Products

 

Examples of cellular therapy (CT) products include: differentiated cells (e.g. pancreatic, chondrocytes, myocytes, neural, stromal), immune cells [e.g. dendritic cells, lymphocytes, macrophages]), and stem cells (hematopoietic, neural, embryonic, cord blood, mesenchymal). The source material for CT products may be cells or tissue obtained from individual subjects (autologous), allogeneic donors or established cell lines. Substantial inherent biological variability is unavoidable when a mixed cell populations and/or individual donors are involved. 

 

Gene Therapy Products

 

GT products may include: viral vectors (e.g. adeno-, adeno-associated, retro-, paramyxo-, lenti-, pox- viruses) and non-viral vectors (e.g. plasmids, chromatin). Viral vectors may be replication competent (e.g. oncolytic) or replication incompetent. Non-viral vectors can be administered directly or as a complex lipid-containing mixture. The genetic information inserted into viral or non-viral vectors can include cassettes for protein expression, anti-sense RNA expression, or expression of short hairpin RNAs for RNA interference.  GT vectors may be administered directly to patients or may be administered as cellular products that are modified with viral or non-viral vectors ex vivo.

 

Tumor Vaccines

 

Many C&GT products are also being developed as tumor vaccines (8). Examples of tumor vaccines might include: tumor and/or immune cells alone or pulsed with peptides, proteins or gene transfer vectors; tumor cell lysates; and GT vectors expressing tumor antigens, cytokines and/or other immune-modulatory genes. Tumor vaccines may also include addition of an adjuvant.

 

 

Challenges to Measuring Potency of C&GT Products:

 

C&GT products can be very complex, presenting significant and often unique set(s) of challenges to sponsors for designing, validating and performing potency assays. The following table summarizes some of the challenges that may exist for many C&GT products.

 

Challenges

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 vector

Multiple components

·   Viral vectors containing multiple genes

·   Heterogeneous mixtures of peptide pulsed tumor and/or immune-modulatory cells

·   Multiple cell lines combined in final product

The potential for interference or synergy between components

·   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

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

 

 

 

TECHNIQUES FOR POTENCY MEASUREMENTS

 

For the purpose of this discussion, potency assessments will be broken down into two broad categories of assays, bioassays (e.g. functional assays) and analytical assays (e.g. immunochemical, biochemical, molecular assays). Bioassays can directly measure a relevant biological activity. However, since bioassays may not always be feasible for C&GT products, analytical assay methods may also be suitable when they are correlated to or directly reflect a specified biological activity (8). Correlation studies are discussed in the section titled “Considerations for Potency Measurements”. The following section includes a discussion of numerous techniques that could be used to assess potency, however, this should be considered neither a comprehensive nor a preferred list.

 

 

Biological Assay Methods:

 

Biological assays evaluate a product’s activity within a living system; and can be based on in vivo animal studies, in vitro organ or tissue based studies and/or in vitro cell culture based assays. The readout of a bioassay may include: a measure of an immune response in an appropriate animal model, evaluation of cell proliferation or activation in response to a stimulus, expression of antigens in response to stimulus, secretion of factors, or quantitation of a downstream signaling event. In some instances, a biochemical assay that measures enzymatic activity of the product may be a relevant readout.

 

Early approaches for measuring potency of CT products have often been limited to visual assessment of morphology, cell count, and assessment of cell viability. These methods do not provide information regarding the properties of the cells that reflect their functional activity. Product specific bioassays such as: biochemical analyses; release of specific bioactive molecules and/or cellular metabolism are more reflective of a product’s functional activity and are more likely to be predictive of a product’s activity once administered in vivo.

 

Biological assays used to measure potency for a GT product might include analysis of transgene activity in vivo in an appropriate animal model and in vitro functional assays (e.g. analysis of downstream signaling events, cell killing). These assays are generally specific to the transgene(s) or other element(s) encoded by the vector. The potency of C&GT products developed as tumor vaccines has been assessed by evaluating immune responses in appropriate animal models in vivo (e.g. antigen specific T-cell responses or antibody quantitation) or in vitro (e.g. proliferation of allogenic responder cells, T-cell activation, cytokine release, and cytotoxicity).

 

The major benefit of a bioassay is the ability to directly measure a product’s functional activity. However, the results of these assays are often variable making it difficult to obtain reproducible and robust data. In addition, these assays may take a considerable amount of time to perform, limiting their feasibility for routine lot release testing.

 

 

Analytical Assay Methods:

Assays that measure immunochemical (e.g. flow cytometry, ELISA, Elispot) and molecular (e.g. RT-PCR, Q-PCR, microarray, genomics, proteomics) properties are referred to here as analytical assays to distinguish them from biological assays. Analytical assays can provide wide-ranging information about product characteristics, but do not directly measure function of the product. Analytical assays may be used for potency measurements when correlated to a relevant biological property of the product. Use of technologies such as quantitative flow cytometry, microarray and proteomics analyses may also have the potential to provide a more complete understanding of the functional activities of C&GT products and sets the stage for predicting their fate once placed in vivo.

 

Immunochemical Procedures:

 

Immunochemical procedures utilizing antibodies that recognize specific epitopes of a protein molecule can be used to quantitate protein concentration (e.g. ELISA) and cell surface markers (e.g. flow cytometry). These assays may be used to quantify the readout from bioassays or may be used to measure the product itself (e.g. CT product).

 

Molecules expressed on cell surfaces provide important markers of the identity and differentiation state of cells. Monoclonal and polyclonal antibody reagents are available for detection of a wide variety of cell surface markers.  These markers can be used to distinguish cells of diverse lineage and differentiation states. Cell surface markers may in some instances also be used to distinguish tumor cells from normal cells. Such markers can provide information about the functional status of the cells, which could be correlated to other activity measures. The technology for detection of cell surface antigens has evolved to the point that it can be used to sensitively and quantitatively characterize cellular products (including ex vivo transduced cells). When properly controlled by use of internal reference materials and/or standards, flow cytometry can provide reproducible results.  Thus, it may provide one option for potency tests to be used for lot release and also for stability testing. CBER has participated actively in a NIST-FDA-CDC federal standardization consortium to develop reference standards for quantitative flow cytometry (9, 10).  To date, quantitative flow cytometry standard protocols, standard fluorescent microbeads and a standard fluorescein solution have been prepared and characterized, and are publicly available (9, 10, 11). Availability of these standards will supplement the use of internal reference materials for comparing tests performed at different times, whether for patient monitoring or for product testing. 

 

Molecular and Biochemical Procedures:

 

Procedures such as RT-PCR and Q-PCR can also supply information about the molecular characteristics of C&GT products, which could potentially be used for assessment of potency, provided the information can be correlated to a specified biological activity.  These assays can be quantitated, performed in a timely manner and appropriate controls are available or can be developed

 

Gene expression profiling by microarray technology could also be an excellent tool in the development of potency tests for C&GT products. Unlike conventional biochemical and molecular approaches, DNA microarray technology enables the discovery of unknown patterns and biological processes without a priori assumptions.  It can assess the status of thousands of genes at once, which is not possible by conventional biochemical/molecular biology methods (12). This information can help predict the functional status of complex biological systems which can then be correlated to a specified biological function(s). Microarray technology may likely be used for potency testing and could constitute an important part of the quality control data for C&GT products. 

 

To employ microarray technology for potency assessments it is critical to establish calibrated RNA samples and reference datasets. Substantiation of data will require objective assessment of the performance of different microarray platforms, the proficiency of individual laboratories in performing and interpreting data, as well as the merits of various data analysis procedures. Efforts have been focused in coordinating the MAQC project, a community-wide effort for microarray quality control (13). In addition, RNA standards are under development (External RNA Control Consortium (ERCC)) for their use in microarray analysis (14).

 

Recently developed proteomics techniques also provide a new approach to characterization of cellular products by identifying profiles of expressed proteins and their post-translationally modified states.  As this technology is further developed, it will also be useful in product testing, including potency.

 

 

CONSIDERATIONS FOR POTENCY MEASUREMENTS

 

Potency assays for C&GT products should be quantitative measurement(s) of a relevant biological function(s) or activity(s) (4). To encourage development of C&GT products, OCTGT has not strictly recommended a quantitative measurement of potency during early product development, opting instead for a progressive approach (15). This approach permits sponsors to initiate clinical trials while developing their potency assays. While many sponsors forego development of potency assays until the later stages of development, there are a number of incentives to early assay development such as: 1) a collection of data to support specifications for lot release; 2) a measure of product consistency necessary to demonstrate control of manufacturing process; 3) a basis for assessing manufacturing changes (comparability); 4) an evaluation of product stability: and 5) an evaluation of multiple assays. Considering the complexity of C&GT products, the challenges to development of potency measurements and the predicted need for considerable data (see techniques section) it is highly advisable to initiate potency assay design as early as possible. 

 

When possible, potency assay measurements may consist of a single biological assay (bioassay) that quantitatively determines the biological activity of the product (as described in the previous section).  However, considering the number of challenges with C&GT products, it may not always be feasible to develop a single, quantitative bioassay for product release. For instance, the limited stability of a CT product may prohibit the use of a time consuming, albeit direct, biological assay in an appropriate animal model. Instead, it may be necessary to find a balance between a bioassay that directly measures biological activity and an assay that is practical and reliable for lot release, such as the analytical assays described above (e.g. immunochemical, molecular and biochemical). However, analytical assays do not directly measure biological activity and it may be necessary to develop several assays (assay matrix) to measure the potency of the product. Such an assay matrix could consist of one or more quantitative analytical assays correlated to one or more bioassays.

 

Surrogate assays for biological activity may be based on various tests including evaluation of cell surface markers (flow cytometry), immunological detection of components (e.g. ELISA), and analysis of gene expression (genomics) or protein expression (proteomics) patterns (8). Examples of potential surrogate assays might include quantitative evaluation of phenotype biomarker(s) for a chondrocyte CT product correlated to an in vivo activity (e.g. structural repair of cartilage); or phenotypic marker(s) for dendritic cell activation correlated to biological activities in vitro such as antigen presentation and T-cell stimulation [proliferation].  For a GT product, a correlation might be made between quantitative expression in an in vitro culture system and assessment of a downstream signaling event, coupled with a measure of infectivity.

 

The suitability of a surrogate will depend on the ability of the surrogate to accurately predict biological activity. Suitability will also be influenced by other factors, including how well the functional activity of the product is understood and how well the original bioassay demonstrates that action by the product. In addition, the scientific advantages of the surrogate assay over the original biological assay (e.g. availability for release, assay consistency, quantitative) should also be considered when designing assays for potency.

 

Correlation of surrogate measures to biological activity can be determined through preclinical and proof of concept studies, clinical studies, cellular assays, and animal study results.  Acceptable correlation criteria will need to be evaluated on a case by case basis and will depend on a number of factors, including the type of product being analyzed and the types of correlations being made. It is the sponsor’s responsibility to perform the development work to demonstrate a correlation between the surrogate assay(s) and biological assay.

 

 

SUMMARY

 

We anticipate continued rapid development of cellular and gene therapy products that will warrant the use of available, as well as emerging, methods to assess potency. Whenever possible, reproducible, quantitative methods measuring biological activity should be used. Good assay design is needed whether developing a bioassay or analytical-based assay.  Appropriate assay design may be able to address some of the issues of bioassay variability and therefore make many bioassay(s) acceptable for potency measurement.  Input is requested of the advisory committee regarding assay design schemes that would be central to successfully validate biological assays and allow accurate quantification and interpretation of the results obtained.

 

Biological assays provide a direct measure of biological activity, but there are challenges for their use with C&GT products (e.g. inconsistent results, time consuming). When biological assays are not feasible, an alternative may be the use of analytical based assays (e.g. immunochemical [flow cytometry, ELISA] and molecular [Q-PCR, microarray]) that have the potential to provide more reproducible and timely results.  However, analytical measurements are generally considered an indirect measure of biological activity and will need to be correlated to specified biological function during product development.  The input of the advisory committee is requested on strategies and criteria for correlation studies between analytical based assays and biological activity.

 

Development of state of the art technologies are encouraged for use in potency measurements, however, further discussion is needed regarding how to implement these technologies. In particular, discussion is needed concerning how to validate the results obtained with genomic and proteomic analyses (e.g. by statistical analysis, confirmation by Q-PCR) and how to reconcile the differential data between RNA and protein analyses of the same sample set.  Additional consideration should also be given as to how and when to apply these technologies (e.g. during product development or for use as lot release).

 

There are a number of challenges to potency assay development for C&GT products which require scientific input from experts in the field. The questions presented at the end of this document are provided to stimulate discussion of some of these challenges.


REFERENCES

 

1.       Food Drug and Cosmetic Act, Section 351 of Public Health Service Act, 42 USC 262.

2.      US Code of Federal Regulations: Title 21, part 601 section 2 (21 CFR 601.2).

3.      (a) 21 CFR 600.3(s); (b) 21 CFR 610.10.

4.      International Conference on Harmonisation: Guidance on Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (ICH Q6B). August 1999.  http://www.fda.gov/cber/gdlns/ichtest.pdf.

5.      (a) 21 CFR 210.3(16)(i)(ii); (b) 21 CFR 211.165(d); (c) 21 CFR 211.165(e).

6.      US Food and Drug Administration. Guidance for Industry: Analytical Procedures and Methods Validation Chemistry, Manufacturing, and Controls Documentation 2000. Available at http://www.fda.gov/cber/gdlns/methval.htm.

7.      International Conference on Harmonisation (ICH) Guidance for Industry: Q2B Validation of Analytical Procedures: Methodology - 5/19/1997. Available at http://www.fda.gov/cber/gdlns/ichq2bmeth.pdf .

8.      Kawakami K, Puri, RK. 2004. Regulatory expectations during product development for tumour vaccines. In Brown F. Petricciani J editors. Development of therapeutic cancer vaccines. Dev. Biol, vol 116. Basel: Karger; pp. 53-9.

9.      Stelzer, GT., GE Marti, Anne Hurley, Phil McCoy, Jr., EL Lovett and Abe Schwartz: 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).

10.  NCCLS: ILA24 Vol 24 No 26: Fluorescence Calibration and Quantitative Measurement of Fluorescence  Intensity;  Approved Guideline. Available at www.nccls.org.

11.  National Institute of Standards and Technology. Available at www.NIST.gov

12.  Chee et al., 1996. Accessing Genetic Information with High Density DNA arrays. Science vol 274. no 5287, pp 610-614.

13.  Microarray Quality Control (MAQC) Project. Available at http://www.fda.gov/nctr/science/centers/toxicoinformatics/maqc/

14.  Warrington et al., 2005. The External RNA Controls Consortium: a progress report. Nature Methods, vol 2. no 10, pp 731-4.

15.  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). Available at http://www.fda.gov/cber/gdlns/gtindcmc.htm.