13 Steps To Successful Potency Assays

Today, we feature an article from BioProcess International magazine co-authored by Biological Assays event chair. Laureen E. Little, Ph.D., Principal Consultant, Quality Services.

13 Steps To Successful Potency Assays

1. 1. Acquire as broad and in-depth as possible an understanding of the biological and other properties and actions of the cellular therapy product(s) for which you wish to develop potency methods. You should collect sufficient product characterization data (molecular, biochemical, immunologic, phenotypic, hysical, and biological properties) throughout preclinical and clinical development to inform and refine your approach to measuring potency.
2. 2. Acquire broad and in-depth knowledge of the process by which your cellular product is manufactured.
3. 3. Familiarize yourself with the way in which the product is administered and the events that follow administration.
4. 4. Start designing and developing two (or possibly more) potency assays early — as early as during preclinical development is desirable. Developing at least two different methods increases your chances that one of them will be suitable for both your company and the regulators.
5. 5. Realize that biological potency assay design and development for GMP use requires considerable, specialized, and varied knowledge and experience. If you are not already an expert, either become one (for suggestions, see box, Gaining Expertise in Biological Potency Method Development”), find and apprentice yourself to one in your organization, hire one, or engage the services of an expert consultant. Be sure to confirm that any prospective employee or consultant has specific knowledge and experience in developing and validating biological potency assays.
6. 6. Initiate and maintain a discussion with your CBER Reviewers so that you have agency feedback to assist in your development decision making.
7. 7. Familiarize yourself with all relevant regulations and guidance and get current and stay current with the technical literature.
8. 8. Thoroughly characterize all rare and other reagents, reference materials, standards, and controls.
9. 9. Thoroughly characterize and qualify your potency assay(s).
10. 10. Give regular technical presentations to gain input from your colleagues.
11. 11. Be diligent in creating a formal Method Development Plan and in keeping a Development Report and refer to them often.
12. 12. Determine the robustness of, and validate your potency method(s) before your end of phase 2/pre-pivotal trial meeting with FDA
13. 13. If you outsource development of your potency assay(s), always have a knowledgeable and experienced employee or consultant, whose services your company has engaged, guide and manage the product development.

Would you like to meet with Dr. Little?  Dr. Little will be hosting the workshop A Practical Guide to the Development of Assays for Evaluation of Biologics and Biotherapeutics – a Systematic Approach to Supporting Product Development in the Regulatory Environment on Monday, May 5 in Berkeley, California at Biological Assays.  For more information on her workshop and the rest of the event, which takes place May 5-7, download the agenda. As a reader of this blog, you are eligible save 20% off the standard rate when you register to join us and mention code B14177JT.

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Recommendations for Cell Banks Used in GXP Assays: Preparation, Characterization, and Storage

Today, we take a look Recommendations for Cell Banks Used in GXP Assays according to Ana T. Menendez, Nadine Ritter, Jonathan Zmuda, Darshana Jani, and Jaya Goyal.   This appeared in a recent issue of BioProcess International.

Special Report: Recommendations for Cell Banks Used in GXP Assay

Cells and cell-derived reagents form the basis of an operationally challenging class of test methods used in execution of product potency testing (stability and lot release), assessments of pharmacokinetic/pharmacodynamic (PK/PD) profiles, detection of antidrug antibodies (ADAs) or neutralizing antibodies (NAB), and characterization and comparability testing of biopharmaceutical products. Frequently, cell-based assays provide the only measurement of the tertiary/quaternary structure of each batch of product at the time of lot release and during stability testing to assist in determining product shelf-life. Cultured cells themselves are often used to generate monoclonal antibodies (MAbs), enzymes, or substrates for use as critical reagents in other types of tests, including ligand binding and enzymatic assays. In all these applications, the cells serve as highly critical, highly complex “reagents” that require distinct characterization and control measures to ensure operational consistency over time.

Cells are sensitive to innumerable chemical and physical elements that can alter expression of their cellular proteome and change growth characteristics or responsiveness to ligands/biopharmaceuticals. Establishment and characterization of homogeneous, stable cell banks are necessary to ensure that starting cellular material for each assay is as consistent as possible. Appropriately established and stored master and working cell banks (MCBs and WCBs) provide a continuous supply of viable cells to generate accurate, reliable results within specified test methods or provide the cell-derived reagents used in those methods.

Although the strategy for preparing cell banks is clearly defined by regulators for cells used to produce biotechnology products, it is less clear what strategies should be applied to cells that are used solely as part of analytical or bioanalytical test methods. An early FDA points-to-consider (PTC) guidance on characterization of cell lines for producing biologicals (1) outlined many MCB and WCB characterization requirements that were later adopted into ICH Q5D for production cell lines (2). But it is still common to find the phrase PTC testing used in relation to the list of tests applied to nonproduction cell banks even though that guidance was not intended to apply to such banks.

In the absence of clarifying information about the significant differences in intended use between production cell lines and those used for analytical/bioanalytical methods, some laboratories choose to apply the entirety of the PTC guidance to both. Conversely, others fail to establish cell banks at all for analytical use, severely jeopardizing the desired state of assay control. The US Pharmacopeia recently published a suite of chapters — <1032> Development (3), <1033> Validation (4), and <1034> Assay Analysis (5) — providing guidance on good manufacturing practice (GMP) potency bioassays. Several paragraphs in USP <1032> provide a general outline for MCB and WCB preparation; however, specific details are not provided to assist laboratories with the less obvious but still critical aspects of creating, characterizing, and storing MCBs and WCBs.

Recommendations presented herein support and significantly elaborate on principles noted in USP <1032> for establishment and characterization of mammalian and bacterial cell banks used to support analytical/bioanalytical testing. These approaches may also be extrapolated (when applicable) to include cells used for reagent production, for growing viruses used in test methods, and so on. These strategies represent our combined opinions on best practices in establishment, characterization, and maintenance of controlled and consistent cell sources using a risk-based and product-phase–appropriate approach. Each sponsor should determine which recommendations to adopt and when each will be performed based on the level of risk acceptable in developing and validating methods that use cells or cell-derived reagents.



References
(1) CBER. Points to Consider in the Characterization of Cell Lines Used to Produce Biological Products. US Food and Drug Administration: Rockville, MD, 1993; www.fda.gov/downloads/biologicsbloodvaccines/safetyavailability/ucm162863.pdf.
(2) ICH Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products. US. Fed. Reg. 63(182) 1998: 50244–50249
(3) USP <1032> Development and Design of Biological Assays. Pharmacop. Forum 36(4) 2010: 956.
(4) USP <1033> Validation of Biological Assays. Pharmacop. Forum 36(4) 2010: 986.
(5) USP <1034> Analysis of Biological Assays. Pharmacop. Forum 36(4) 2010: 1005.

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Hard Cell: Potency Testing for Cellular Therapy Products

Today, we hear from Tom Pritchett and Laureen Little who published the article "Hard Cell": Potency Testing for Cellular Therapy Products and look at the Regulatory Requirements and Expectations in BioProcess International.  

Regulatory Requirements and Expectation

Human cells are regulated as biologics if any of the following criteria are met: They are more than minimally manipulated, they are combined with an article other than a preservation or storage agent, they are used in a way that is not homologous to their normal function, or they have a systemic effect and depend on the metabolic activity of living cells for their primary function.

As biological products, cellular therapies are regulated under section 351 of the Public Health Services Act (42 U.S.C. 262) and the Food, Drug and Cosmetics Act (21 U.S.C. 321 et seq.) They also must comply with FDA’s current good manufacturing Practice (CGMP) regulations for Finished Pharmaceuticals (21 CFR Parts 210 and 211) and with relevant sections of the biologics regulations at 21 CFR 600. Compliance with these requirements applies to all biological products, including autologous and single patient allogeneic products, in which a lot may be defined as a single dose(2). In addition, cell therapy products must comply with applicable parts of the current good tissue practice (CGTP) rules in 21 CFR part 1271. The FDA pairs cellular therapy products with gene therapy products for regulatory purposes and refers to them as CGT (cellular and gene therapy) products. Regulatory submissions for CT products are reviewed by FDA’s Office of Cellular, Tissue and Gene Therapies (OCTGT) in the Center for Biologics Evaluation and Research (CBER).

The FDA feels that “These regulations provide considerable freedom when evaluating suitable potency assays” (6). For approval of a biologics license application (BLA), CT products must meet the requirements of safety, purity, and potency prescribed in the above regulations. However, as stated in FDA’s guidance on CGT potency testing, “FDA regulations allow for considerable flexibility in determining the appropriate measurement(s) of potency for each product.” As is typical, the FDA evaluates the adequacy of potency tests for such cutting-edge products case by case. (2).

In reviewing a potency assay to be used for release testing of a licensed product, the agency considers whether the method meets the following regulatory requirements and expectations (2, 6).

Lauren Little will be joining us at Biological Assays  to present the white paper report What are We Monitoring to Maintain our Potency Assays?.  To find out more about her presentation and the rest of the program, download the agenda.  Would you like to join Lauren?  As a reader of this blog,when you register to join us this May 5-7, 2014 in Berkeley, California and mention code B14177JT, you can save 20% off the standard rate.

References
2 CBER. Guidance for Industry, Potency Tests for Cellular and Gene Therapy Products, January 2011.
6 Gavin DK (CBER Office of Cellular,Tissue and Gene Therapies, Division of Cellular and Gene Therapies). Presentation at the Cellular, Tissue, and Gene Therapies Advisory Committee Meeting, 9 February 2006. Slides and transcript available at www.fda.gov/ohrms/dockets/ac/cber06.html#CellularTissueGeneTherapies.

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Biological Assay Qualification Using Design of Experiments

Leading up to the Biological Assays event taking place this May, we'll be teaming up to showcase some of the latest articles and news from our partners at BioProcess International.  Today we feature a piece authored by Todd Coffey, Mike Grevenkamp, Annie Wilson, Mary Hu Biological Assay Qualification Using Design of Experiments.

In 2012, the United States Pharmacopeia (USP) published a complementary set of three guidance documents on the development, analysis, and validation of biological assays (1,2,3). USP chapter <1033> recommends a novel, systematic approach for bioassay validation using design of experiments (DoE) that incorporates robustness of critical parameters (2). Use of DoE to establish robustness has been reported (4,–5), but to our knowledge its use in qualification or validation protocols for assessing assay accuracy, precision, and linearity is not described in literature. A validation approach incorporating different levels of critical assay parameters provides a more realistic representation of how an assay will perform in routine testing. 

After initial development of a cell-based bioassay to enable an investigational new drug (IND) application, we used a DoE-based approach to perform a comprehensive qualification of that bioassay to estimate its accuracy, precision, linearity, and robustness. Such an approach to qualification requires experimental and statistical effort similar to that of traditional validation for cell-based bioassays (6,7,8), but the additional insights we gained have led us to adopt this new approach as our standard method for qualifying bioassays. Here we outline our approach and describe its benefits and challenges.

Head to BioProcess International to read the methods, results and conclusions of the report.

This May at Bioassays, we'll have have experts on hand from Genentech and Eli Lilly to take a deeper look into Establishing and Qualifying Product Reference for the Relative Potency Assay.  For more information on these sessions and the rest of the program, download the agenda.  Would you like to join us in Berkeley?  As a reader of this blog, when you register to join us and mention code B14177JT, you can save 20% off the standard rate!

References
1 Chapter <1032> Design and Development of Biological Assays. USP 35–NF 30, 2012. 
2 Chapter <1033> Biological Assay Validation. USP 35–NF 30, 2012. 
3 Chapter <1034> Analysis of Biological Assays. USP 35–NF 30, 2012.
4 Chen XC, et al. Implementation of Design of Experiments (DOE) in the Development and Validation of a Cell-Based Bioassay for the Detection of Anti-Drug Neutralizing Antibodies in Human Serum. J. Immunol. Meth. 376 (1–2) 2012: 32–45. 
5 Kutle L, et al. Robustness Testing of Live Attenuated Rubella Vaccine Potency Assay Using Fractional Factorial Design of Experiments. Vaccine 28(33) 2010: 5497–5502.
6 Cheng Z-J, et al. Development of a Bioluminescent Cell-Based Bioassay to Measure Fc Receptor Functionality in Antibody-Dependent Cellular Cytotoxicity. Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research, Mar 31–Apr 4, Chicago, IL. AACR Cancer Res. 72(8) 2012: supplement abstract #2840. 
7 Wei X, et al. Development and Validation of a Quantitative Cell-Based Bioassay for Comparing the Pharmacokinetic Profiles of Two Recombinant Erythropoietic Proteins in Serum. J. Pharm. Biomed. Anal. 43(2) 2007: 666–676.
8 Gazzano-Santoro H, et al. Validation of a Rat Pheochromocytoma (PC12)-Based Cell Survival Assay for Determining Biological Potency of Recombinant Human Nerve Growth Factor. J. Pharm. Biomed. Anal. 21(5) 1999: 945–959.

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Top 5 Reasons to Attend the Biological Assays Event

Why should you join us at Biological Assays this May in California?

1. Decrease your OOS problems and improve the precision of your assay by hearing the latest approaches to tame your noisy data from a statistical, biological and regulatory perspective
2. Practical truths about selecting and maintaining reference material which will keep you out of regulatory hot water; statistical plans, tracking and trending protocols and case studies
3. Avoid the pitfalls in your programs and hear the truth about the trials and triumphs of implementing USP recommendations for assessing similarity
4. Learn the tricks of the trade to run an efficient and compliant bioassay QC laboratory
5. Hear new trends & case studies about developing, qualifying and validating complex bioassays from your colleagues in bioassay development

Find out more about this year’s program by downloading the agenda.

If you’d like to join us May 5-7, 2014 in Berkeley, California, as a reader of this blog, you’re eligible for 20% off the standard rate when you register to join us and mention code B14177JT. Have any questions or want to get involved? Feel free to reach out to me at jpereira@iirusa.com.

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Implementing Equivalence Testing for the Evaluation of Parallelism: Insights from Dr. Todd Coffey

In today's blog post, Biological Assays presenter Dr. Todd Coffey, CMC Statistician, Seattle Genetics, share with us a  little about his work in evaluate parallelism for bioassays.  Here's what he had to share.

One of the criticisms of the equivalence approach for assessing similarity is that development teams will inadvertently utilize compromised samples and implement a too-wide “zone of indifference”. How do you protect against this problem?
Compiling a set of historical data that includes the natural variability of parallel curves is not a trivial exercise. I offer three suggestions to protect inadvertently against including compromised samples: 1) Ensure the dataset is large enough to include all sources of assay variation. With an adequately large dataset, patterns can often be identified that may elucidate which samples are compromised and why they should not be included with the other parallel samples. 2) Carefully assess the data for unusual trends and patterns, both visually and with statistical analysis. 3) Compare the parallelism metric for degraded samples that are expected to be non-parallel to parallel samples that are suspected of being compromised.

The USP states that one can use the absolute difference of slopes or a ratio of slopes when using the equivalency approach for similarity. Do you have a preference and why?
Using ratios has the advantage of being generalizable across assays. However, calculating confidence intervals on the ratios is not always straight-forward because the ratio of two normally distributed variables is not normally distributed. Thus special care has to be used to correctly calculate confidence intervals on ratios. While not as generalizable across assays, calculating differences between standard and test is more straight-forward statistically and is also interpretable. For these reasons, I prefer to set equivalence limits using differences

How many reference vs. reference runs do you recommend using to establish a “zone of indifference”?
There are at least three issues to consider when discussing sample size. First, to be representative, the number of runs needs to include all sources of variation. Second, the sources of variation have a great impact on the relative value of measurements and the sample size is dependent on getting the right data. For example, if most of the assay variation comes from factors that vary between runs, then many measurements in the same run are of much less value than measurements from different assays. In this case, the sample size is dependent more on the number of times the assay is run after varying the factors that cause the variation. Finally, to provide accurate estimates of tolerance intervals, the sample size of independent measurements generally needs to be several dozen. When that sample size is not attainable, I recommend setting initial limits, monitoring the assay, and then modifying limits as new data emerge.

Many companies start with a difference approach to similarity during early product development when they have a small number of lots and the assay is still being developed. At what stage of development is it reasonable to implement an equivalency approach for similarity?
I recommend using the equivalence approach when there is an adequate set of representative historical data that contains all sources of variation. Sometimes this dataset is available during qualification or before the IND is submitted. If it is not available then, the next potential milestone may be when process characterization activities begin.

Dr. Coffey will be presenting Tips and Tricks for Implementing Equivalence Testing for the Evaluation of Parallelism this May 14-16, 2013 in Seattle, Washington at the Development, Validation and Maintenance of Biological Assays event.  For more information on his session and the rest of the program, download the agenda.  If you'd like to join him, as a reader of this blog when you register to join us and mention code IBA13JP and save 20% off the standard rate.  Have any questions?  Feel free to email Jennifer Pereira.

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Stay on the cutting edge of Bioassay Science

IBC’s Development, Validation & Maintenance of Biological Assays conference is the #1 place for you to keep up with the latest emerging trends. This informative, interactive conference is where bioassay professionals gather to discuss the real technical hurdles and how to overcome them. We have put together an amazing program for the 2013 Biological Assay conference to provide you with a one-stop industry resource to help you develop validate and maintain your bioassay.

This conference has in-depth technical tools, overview of regulatory trends, emerging technologies and multiple product case studies which will help you keep up with your crazy over-worked and “aggressive” schedule.

What You Will Take Away by Attending Bioassays 2013:

• - New Ideas & Approaches to Help You Accelerate Your Bioassay Projects
• - Case Studies Designed to Keep You at the Cutting Edge of Bioassay Science
• - The Latest Emerging Tools to Help You Identify Problematic Trends
• - Learn How to Get your Bioassays Assays Approved and Functioning in the Real World
• - How to Comply with Regulatory Requirements Efficiently without Wasting Resources
• - Assay Performance Benchmarks from Industry Leaders
• - Learn How to Develop Rugged and Robust Bioassays
• - And so much more!

For more information on this program, download the agenda.

If you’d like to join us in Seattle May 14-16, 2013, as a reader of this blog when you register to join us and mention code B13177JP, you’ll save 20% off the standard rate!* If you have any questions about the program visit email Jennifer Pereira.

*Rate applies to only new registratoins

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