Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Handling Product References for Potency Assays: A Review of the BEBPA White Paper

• Speaker: BEBPA Expert Working Group – Represented by Laureen Little, President, BEBPA
• Abstract: The potency of a product is considered a critical quality attribute (CQA). As such, sponsors are responsible for developing a quantitative method to ascertain the potency of each manufactured commercial batch. Potency assays are unique in several aspects; however, two aspects are critically important when considering how to handle the product reference for these assays. Firstly, most potency assays are relative potency assays. This signifies that reference material defines the ultimate unitage assigned to test samples. Therefore, the potency assignment of reference material for primary and secondary references must be undertaken with great care both statistically and practically. Additionally, these references should be routinely monitored, and their potency verified in an on-going fashion. Secondly, the entire dose-response of the reference material is compared to the entire dose-response of the test material. It is a critical method system suitability criterion that these two dose-response be determined to be similar prior to calculating the batch test sample potency. Therefore, it is important that product reference have a representative biological response and that the dose-response curve be an appropriate one for comparison. These are two examples of the complexity of selecting and qualifying product references which are suitable for complex biotechnology products. BEBPA has convened an expert working group to work on a white paper suggesting various approaches and concerns regarding the selection and maintenance of product reference material. This talk will cover the highlights of this white paper.
• Contributing Authors: Laureen Little, Jane Robinson, Mike Sadick, Mike Merges, Sian Estdale, Seth Foltz, Matt Borer, Peter Rigsby, Marie Gottar-Gullier, Zeban Kolen, Stan Deming, Perceval Sondag, Dorota Bulik
• (Day 3: Reference Standards for Potency Assays Session)

CEPI Centralized Laboratory Network for Measurement of Immune Responses Elicited by SARS-CoV-2 Vaccines

• Speaker: Valentina Bernasconi, CEPI
• Abstract: At present, more than 300 vaccine developers are active worldwide to develop a vaccine against SARS-CoV-2. Comparing immune responses against different vaccine candidates intended for the prevention of SARS-CoV-2 infection is challenging. To improve immunological assay standardization across different vaccine candidates and meaningful comparison of results, the Coalition for Epidemic Preparedness Innovations (CEPI) has recently created a Centralized Laboratory Network for COVID-19 vaccine immunogenicity testing selecting laboratories with high quality standards worldwide, picking the most advanced assays to be used across the Network and providing all the laboratories with harmonized of protocols and key reagents.
  The Centralized Laboratory Network today counts 8 laboratories worldwide: 2 in North America (Q2 in US; Nexelis in Canada); Public Health England (PHE) Porton Down in the UK; 3 in Europe (NIBSC in the UK; Vismederi in Italy; Viroclinics in the Netherlands) and 2 in Asia (Translational Health Science and Technology Institute, THSTI, in India; International Centre for Diarrhoeal Disease Research, icddrb, in Bangladesh). The CEPI Centralized Laboratory Network is open to all vaccine developers worldwide to apply for testing samples from pre-clinical to Phase II clinical studies to facilitate rapid evaluation, approval, and dissemination of the most effective vaccine candidates and supports their pathway towards licensure, increasing the chances of finding a successful SARS-CoV-2 vaccine.
• Contributing Author: Valentina Bernasconi, Scientist, CEPI
• (Day 3: Reference Standards for Potency Assays Session)

Evaluation of a Cell-Based Potency Assay to Support Clinical Development of an Amniotic-Derived Biologic for the Treatment of Osteoarthritis

• Speaker: Miranda Burnette, Organogenesis
• Abstract: Amniotic suspension allograft (ASA) is a biologic comprised of a mixture of cells derived from amniotic fluid and milled amniotic membrane, derived from the same donor, stored frozen at -80°C in a cryopreservation solution to support clinical development for the treatment of knee osteoarthritis. After more than a decade on the market regulated by FDA as a human cell, tissue, or cellular and tissue-based product (361 HCT/P), Organogenesis is pursuing an Investigational New Drug (IND) and Biologics License Application (BLA) for ASA. This talk will focus on the development of a cell-based potency bioassay for lot release of ASA—building from model justification and characterization to target selection, control of assay variability, and development of release specifications before culminating in a qualified cell-based potency assay based on a single-target ELISA readout.
• Contributing Authors: Miranda Burnette PhD, Kelly Kimmerling PhD, MaryRose Kammer MS, Daniel Lesnoy, Zorina Pitkin PhD, and Katie Mowry PhD
• (Day 4: Potency Assay Development for Complex Products Session)

Bioluminescent Cell-Based Assays for Cell Therapy Development

• Speaker: Jey Cheng, Promega Corporation
• Abstract: Cell therapy using genetically engineered immune cells have been demonstrated as powerful new medicines and showed promising clinical outcomes in patients with cancer. It involves genetic modification of immune effector cells (T cells, NK, microphages et al) with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). Here we will present latest data on how bioluminescent technology can be used to measure multiple mechanisms of action for cell therapy including T cell activation and cytokine production, and T cell dependent cell mediated cytotoxicity (TDCC) during cell therapy development. These assays are simple, fast, sensitive and require no-wash steps. We will discuss how these new technologies can be applied at different stage of cell therapy development workflow from lead identification and optimization to product release.
• (Day 1: Roundtable: Rapid Fire Talks: Complex Product Bioassays)

Implementation of Acoustic Droplet Ejection Technology in Quality Control Potency Testing

• Speaker: Jill Crouse-Zeineddini, Amgen
• Abstract: Liquid handling is an integral part of the execution of potency assays. The product dilution component of assay execution is particularly challenging and can be both labor intensive and time consuming. This is also the step that typically contributes the most to assay imprecision and can be impacted by human factors. Different approaches to preparing the individual concentration points on the dose response curve have also attempted to mitigate some of the assay imprecision. Amgen has implemented acoustic droplet ejection (ADE) as one means of improving potency assay performance. ADE is an automated tip less liquid handling technology that transfers liquid using sound waves to deliver a uniform, fixed amount of liquid to a desired destination.
  When a new technology is implemented in a regulated space, and in addition for an application that it was not originally designed for, multiple challenges can arise. At Amgen, we experienced some of these challenges and have ultimately been successful in implementing ADE into potency assay testing for clinical and commercial products. This presentation will describe our journey of implementation of ADE in performing potency assays for GMP testing.
• Contributing Author: Jill Crouse-Zeineddini, Scientific Director, Amgen Inc.
• (Day 1: Regulatory Insights and Potency Method Development Session)

Assessing Stability-Indicating Methods of a CAR T Cellular Therapy Product

• Speaker: Mel Davis-Pickett, Bristol Myers Squibb
• Abstract: Stability studies are critical to establish a retest period for a drug substance or a shelf life for a drug product and recommended storage conditions. In order to determine stability, analytical methods should be stability indicating. The case study highlighted in this presentation describes the strategy, data analysis, and determination of stability indicating properties for methods used in a cell therapy. Stability indicating properties of multiple product release methods as well as those for extended characterization testing are reviewed.
• (Day 4: Potency Assay Development for Complex Products Session)

Why We Like The 15PL

• Speaker: Stan Deming, Statistical Designs
• Abstract: It is still common practice to: (a) fit a four-parameter logistic (4PL) model to the reference standard; (b) fit a 4PL model to a test sample; (c) compare their estimated upper asymptotes, lower asymptotes, and slopes to assess similarity (parallelism); and (d) compare estimated ED50 values to obtain the relative potency for the test sample. Because of the effect of noise on parameter estimation, especially when the parameter estimates rely on extrapolation, this can lead to false positives when assessing dissimilarity (non-parallelism). In 1978, DeLean, Munson, and Rodbard showed that a common-parameters approach is often a more efficient use of information on an assay plate. Following DeLean et al., our preference is to treat each column on a plate as a separate sample of the same material with common upper asymptote, common lower asymptote, and common slope (three parameters), with one ED50 value per column (12 parameters) using a fifteen-parameter logistic (15PL) model. Advantages are: (a) better accuracy and precision of relative potency estimation; (b) detection of blunders in pre-plate dilutions and/or transfers to the top well; and (c) fewer false positives when assessing non-similarity (non-parallelism).
• Contributing Authors: Stan Deming, President, Statistical Designs and Anton Stetsenko, Associate Director, 4D Molecular Therapeutics
• (Day 2: Method Development Tools Session)

Optimizing Dilution Curves For Potency Assays

• Speaker: John Dunn, Brendan Bioanalytics
• Abstract: Developing a reliable cell-based potency test can be challenging. Some curves do not reach saturation plateaus, hooks can appear at different dilutions, curves can be severely asymmetric. And some tests are just ill-behaved. This talk will step through a series of analytical tools to determine optimal dilution doses and other factors to make potency tests more stable and reliable. Examples will be shown from two cell-based potency tests. The process involves collectively analyzing the behavior of a pool of assays. The distribution of responses and the intra-assay and inter-assay variability of the individual dilutions is the first step. Determining the distribution of accurately weighted residuals² from the regression curves is next, followed by computing precision error profiles to determine the effective biological activity range (LLOQ and ULOQ) of the potency test. Once the optimal assay protocol has been established, setting equivalence margins or direct measures of parallelism/similarity thresholds can be effectively determined from pooled assays after identifying and masking critically failed regression curves and nonparallel curve pairs. These methods will also be described.
• (Day 1 Roundtable: Rapid Fire Talks: Solutions for Potency Assays)

TOPIC: CDER Reviewer Perspective on Potency Assays

• Speaker: Gerald Feldman, FDA
• Abstract: Potency assays are a critical component of the control systems that monitor consistency of a manufactured biological product. Historically, assays used at the IND stage were not thoroughly optimized for precision, accuracy or throughput, and as the clinical trial progresses and regulatory filings for market approval approaches, there is often a need to make modifications to the assay to meet the validation requirements. Even after the market approval, changes in product demand, and advances in technology, availability of engineered cell lines and better detection methods to measure cellular responses often necessitate modifications to the potency assay. This talk will attempt to provide both some perspective on the current status of potency assays from a regulatory perspective, as well as discuss current developments that will impact potency assay programs now and in the future.
• Contributing Author: Gerald M Feldman, Ph.D.
  Chief, Laboratory of Immunobiology
  Office of Biotechnology Products, CDER
• (Day 1: Regulatory Insights and Potency Method Development Session)

Mixed Model Bioassay Analysis: Why & How

• Speaker: David Lansky, Precision Bioassay
• Abstract: Fitting statistical models to biological assays is challenging because of: non-additive effects; non-constant variance of the responses; non-normally distributed responses; occasional outliers, complex groupings caused by common practices of grouped dilutions (for example: by shared preliminary dilution steps or multichannel pipettes); and appreciable variation in different parts of the response curve associated with different grouping factors. Fitting models that address all these issues improves all of: assay and sample acceptance, assessment of similarity, estimation of potency, as well as assay and reference standard monitoring.
• (Day 2: Method Development Tools Session)

Translating the USP for Cell-Based Gene Therapy Methods

• Speaker: Catherine Liloia, PPD
• Abstract: As cell-based methods continue to evolve in support of gene therapies to assess product potency through gene or protein expression, the increased complexity and variation of assay readouts can bring questions on how to apply concepts from the USP Bioassay General Chapters from method development and analysis through validation. Various method development projects leveraging ELISA, Western Blot, flow cytometry, and qPCR readouts will be reviewed with focus on key steps in development and data analysis that explore how concepts detailed in the guidance were applied. Subsequent method qualifications and validations will be covered to reflect success of these development exercises in leading to robust analytical methods.
• (Day 4: Potency Assay Development for Complex Products Session)

Development, Qualification, and Bridging of a QC Potency Method for a CAR T cell Therapy

• Speaker: Kathy Matsuda, Bristol Myers Squibb
• Abstract: (Pending)
• (Day 1: Roundtable: Rapid Fire Talks: Complex Product Bioassays)

Why the Reference Standard is so Important to Potency Assays

• Speaker: Mena Odocayen, Genentech
• Abstract: Potency assays assess the biological activity of a drug relative to a reference standard. The importance of choosing a reference standard and the strategies used to introduce new reference standards is key to maintaining a connection from Phase I to pivotal material. Therefore, it is crucial when choosing a reference standard to understand the changes in the manufacturing process and how that could impact critical quality attributes and therefore activity in the potency assay. In this case study, we present the effects of terminal sialic acid (SA), which are known to affect both biological activity and serum half-life (i.e. PK), on activity in potency assays. Specifically, we observed a strong negative correlation between SA content and in vitro potency of an Fc fusion protein (i.e. higher SA content results in lower potency). The strategy used to address this challenge will be discussed with focus on the impact of the reference standard implementation.
• (Day 3: Reference Standards for Potency Assays Session)

The Use of B and T cell Fluorospot to Assess Pre-Existing Immunity Towards Gene Therapy Products

• Speaker: Sofie Pattyn, ImmunXperts
• Abstract: Gene Therapy has the potential to cure a broad spectrum of diseases and modify the progress of many other diseases. However, pre-existing immunity towards AAV or Cas9 can be a major hurdle for therapeutic treatment as many patients developed immunity towards these naturally occurring viruses and bacteria.
  Assessing this pre-existing immunity can be done using single cell B and T cell Fluorospot assay, a variant of ELISpot utilizing fluorescent detection. B cell Fluorospot is used to quantify the number of specific immunoglobulin secreting cells. Multiple antigens can be screened at once in combination with the total number of immunoglobulin secreting B cells. T cell Fluorospot allows the simultaneous detection of up to four cytokines secreted by a single antigen-specific T cell.
  B and T cell Fluorospot assays were used to assess the pre-existing immunity towards different AAV vectors and CAS9 in healthy donors. The same assays can be used to pre-screen patients prior to treatment as this can impair the successful transduction of the vector and interfere with the therapeutic efficacy.)
• (Day 1 Roundtable: Rapid Fire Talks: Solutions for Potency Assays)

A Lesson from Monitoring Reference Standard Stability

• Speaker: Gareth Rees, Porton Biopharma Limited
• Abstract: (Changing the origin of animal sourcing in bioassays is fraught with difficulties both real and potential. There is little real-world information available; we have presented our experience in this poster.
  Porton Biopharma Limited (PBL) manufactures the UK’s anthrax vaccine and the monitoring of the freeze-dried reference standard in the vaccine potency assay has spanned a period when we used two different sources of guinea pigs. The same modelling techniques were used in the routine survival analysis for each release or stability batch tested. Survival analysis developed by Quantics Biostatistics (Quantics) has been used by PBL for a number of years and has resulted in a reduction in animal use greater than 25%. Quantics statisticians worked “blinded” on the routine data without knowledge of the colony source change.
  The effect of change in guinea pig supply was detected in the survival model reference standard response with slightly higher lethal dose 50% (LD50) prior to the colony change, and slightly lower intercept and slope of the model fit. These trends however did not meet the 5% significance level.
  Careful forward planning with the animal supply company and knowledge of the colony lineage has been confirmed to have minimal impact on the assay through trending results for the reference standard. The reference standard has been routinely monitored and has been demonstrated to be stable over a long period of time. The survival analysis is sensitive enough to detect a small although insignificant change in the guinea pig colony used)
• (Day 3: Reference Standards for Potency Assays Session)

AAV Gene Therapy a Complex Modality: Phase Appropriate Bioassay Strategies

• Speaker: Savita Sankar, Pfizer
• Abstract: Adeno-associated virus (AAV) has become a major modality for gene therapy programs in development. The complex mechanisms of action of these therapies require well thought-out and sometimes creative approaches to potency assay development. Multiple assays may be needed for a single program and the strategy for how these tools are used may evolve through the product lifecycle. Considerations for the development of robust, sensitive in vitro potency assays will be presented, as well as examples of how and when different methods could be implemented.
• (Day 4: Potency Assay Development for Complex Products Session)

Design of Experiments for the Validation of Potency Assays

• Speaker: Perceval Sondag, Merck
• Abstract: Imagine that you are going deer hunting. You need to recruit a team of hunters to come with you. They’ll bring guns, so you better trust their ability to shoot their target, and only their target. Accurately, precisely, and consistently. So you put candidate hunters to a test and take them target shooting. Now, if you could afford to have them shoot the target 1000 times each, it would be rather easy to know which hunter performs well. In the world of bioassays, where each assay is a hunter, each bullet costs a LOT of time, resources, and money. So you need to design your experiments well, in order to gather as much intel as you can, while using the smallest possible number of bullets.This talk will help you stop wasting bullets by discussing how to design your experiments efficiently to validate bioassays, and to fit robust dose-response curves.
• (Day 1: Regulatory Insights and Potency Method Development Session)

Quantification of Bevacizumab Activity Following Treatment of Patients with Ovarian Cancer or Glioblastoma

• Speaker: Michael Tovey, Svar Life Science France
• Abstract: Highly sensitive iLite® reporter-gene assays have been developed that allow both the direct VEGF neutralizing activity of bevacizumab and the ability of bevacizumab to activate antibody dependent cellular cytotoxicity (ADCC) to be quantified rapidly and in a highly specific manner. The use of these assays has shown that in 46 patients with ovarian cancer following four cycle of bevacizumab treatment, and in longitudinal samples from a small cohort of patients with glioblastoma treated with bevacizumab, that there is a good correlation between bevacizumab drug levels determined by ELISA and bevacizumab activity, determined using either the VEGF-responsive reporter gene or the ADCC assays. The results of this study suggest that ADCC activity may be correlated with the ability of some patients to respond to treatment with bevacizumab in contrast to other patients with reduced bevacizumab Fc engagement in vivo, while the use of the VEGF-responsive reporter-gene assay may allow the appearance of anti-bevacizumab neutralizing antibodies to be used as a surrogate maker of treatment failure prior to the clinical signs of disease progression.
• (Day 1 Roundtable: Rapid Fire Talks: Solutions for Potency Assays)

Switching from In-Vivo to In-Vitro Assays: A Reviewer’s Perspective

• Speaker: Leslie Wagner, US FDA
• Abstract: Potency tests are required in all phases of clinical development through licensure, however due to the complexity of biologic products, especially vaccines, and their matrices, the in vivo format has played a central role in the control of potency. Animal assays are inherently variable and complex to perform. Prompted by humane considerations and in accordance with the 3 R’s (Reduce, Refine, or Replace), there is a global commitment to reduce and replace animal-based testing. Recent efforts by vaccine manufacturers have been directed towards developing in vitro assays for use with new products from early development through replacement for licensed products.While in vitro assays tend to be less variable and, in many cases, more sensitive, one of the challenges is demonstrating correlation between the in vivo and in vitro methods. This is especially true when the mechanism of action is poorly understood or not known at all, like comparing apples to oranges. A strong scientific rationale should be made for the in vitro test(s) and, if possible, the replacement method should reflect the product mechanism of action. If this is not possible, a combination of tests could serve to provide control of key attributes if, in combination, could be demonstrated to ensure consistency in manufacturing.An important requirement for a replacement test is that the in new method and its specifications should not lead to a change in pass/fail rates and the methods should lead to the same outcomes. The in vitro test must have at least a comparable level of sensitivity to that of the in vivo test. If the selected in vitro test is more sensitive, the specifications should still not be changed as this could result in failed vaccine batches that would have otherwise passed the in vivo assay. In either case alert limits could be established to monitor consistency.
• (Day 1: Regulatory Insights and Potency Method Development Session)

Statistical Model Selection Using USP <1034>

• Speaker: Steven Walfish, USP
• Abstract: USP General Chapter <1034> includes statistics concepts and methods of analysis for the calculation of potency and confidence intervals for a variety of relative potency bioassays. Relative potency is a measure obtained from the comparison of a test sample to a standard based of the capacity to produce the expected biological activity. USP <1034> presents several different models and suitability criteria to determine the reliability of the estimate. This talk will cover traditional linear and non-linear bioassay models with an emphasis on model suitability including parallelism. This talk will focus on the quantitative analysis, thought qualitative models are presented in the chapter. A case study comparing a parallel line assay to a slope ratio assay is presented to highlight the differences.
• (Day 2: Method Development Tools Session)

Potency Assays for Phase I/II Cell and Gene Therapy Products: Experiences of a Small “CMO” Within an Academic Institution

• Speaker: Scott Witting, Cincinnati Children’s Hospital Medical Center, TTSDL
• Abstract: Potency assays are required for release of both drug substances (viral vectors) and drug products (the cell therapy). In support of Phase I/II manufacturing, potency assays are critical for process optimization, lot to lot comparisons, and stability studies. However, the lack of reference standards, in-house standards (often not available in Phase I/II), and the intrinsic variability of biologic assays complicates potency assay development and implementation. The current strategy of the Translational Core Laboratories for viral vector potency is to offer both a platform assay, universal to all vector constructs, and a secondary multiplicity of infection (MOI) assay that closely mimics the Sponsor’s process. Our experiences with the optimization and performance of these assays will be discussed.
• (Day 2: Method Development Tools Session)