Development and Selection of Potency Assays for a Cell Therapy Platform

• Speaker: Sarah Busch, Athersys, Inc.
• Abstract: This presentation will include an overview of the identification and use of several potency assays during development of the MultiStem cell therapy platform, with a focus on the neurological disease and injury space.
• (Day 1: Bioassays for Complex Products Session)

Making the Switch: Potency Method Bridging Considerations and Challenges

• Presenter: Delana Butz, PPD Inc.
• Abstract: Over the course of a product’s lifecycle, it may become necessary to replace an existing potency method with an improved or new method. Making this switch comes with both technical and logistical considerations. Strengths and limitations of the associated technologies, reference material evaluation, method performance capabilities, critical reagent needs, size/composition/comparison strategies for associated bridging studies, implications of production process or formulation changes, and impact to on-going stability studies are just a few of the technical considerations for changing methods. Logistical considerations can also be significant and include: timeline considerations for training staff at the sponsor site and any relevant CROs, variation in hands-on laboratory time required for the methods, critical reagent burn rates, sample test forecasting associated with changes in through-put, and maintenance of the existing method and critical reagents for informational testing at a future date.
• (Poster)

Case Studies Of Product CQA Assessment With Bioanalytical Strategy for CMC Biologics Development

• Speaker: Ping Carlson, Bristol-Myers Squibb
• Abstract: A CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.
  Assessment of critical quality attributes (CQA) forms the foundation of CMC development of biotherapeutics, including process, formulation and bioanalytical development as well as the design of an integrated control strategy for robust manufacturing.
  Bioanalytical results from stability storage, stressed and forced degradation demonstrate the primary degradation mechanisms of the biotherapeutic and provide understanding of product CQAs during routine storage as well as excursions.
  Here we present case studies of structure-function based bioanalytical strategy for CQA assessment.
• (Day 2: Product Specific Method Development Session)

Past and Present Procedures Used to Develop Cell-Based Potency Assays with Single Digit Precision

• Speaker: Anthony Chen, Genentech
• Abstract: Cell-based potency assays were well known for high imprecision rates (up to 100% CV) in the early 90’s.
  This presentation will highlight the collective procedures of the past and present used to improve assay precision (% CV). These consisted of the use of better reagents; consistent number of cells and stage of cell cycle; the employment of assay and product controls, and reference standards; and better training and technique. The precision was further improved by increasing the number of. These changes resulted in assays with precision in the mid to low teens.
  Today, current procedures utilizing new technologies such as robotics, engineered cell lines and reporter genes, and more sensitive detection systems have further enhanced cell-based assays to that of single digit precision. In all, these improvements have enabled the use of cell-based assays for CoA release and Stability testing and biological characterization of recombinant proteins for product development.
• Contributing Authors: Anthony B Chen¹, Catherine Cruz², and Guoying Jiang^{2
  1} Departments of IMP Quality Operations; ² ADQC-Biological Technologies
  Genentech Inc., So San Francisco, CA 94080
• (Day 1: Keynote Presentation)

Considerations for Establishing Patient Centric Commercial Potency Specifications

• Speaker: David Cirelli, Pfizer
• Abstract: (Pending)
• (Day 2: Keynote Presentation)

Developing QC Friendly and MoA Reflective Potency Assays for Clinical and Commercial Products

• Speaker: Catherine Cruz, Genentech
• Abstract: (Pending)
• (Day 1: Lifecycle of Potency Assays Session)

Outliers in Bioassay 2: The Effects of a Single Blunder in Down-The-Plate Dilution

• Speaker: Stan Deming, Statistical Designs
• Abstract: As John K. Taylor has pointed out, “There are three sources of error in measurements and they can be classed as systematic, random, and just plain mistakes … The third kind of uncertainty may be called blunders. They happen when mistakes are made, knowingly or unknowingly. Errors resulting from them are not statistically manageable and, in fact, they can invalidate an otherwise good set of data. A measurement system that is unstable and fraught with blunders is not in statistical control and cannot be relied upon to produce useful data.”
  Many existing outlier approaches in bioassay assume that all errors are random. These approaches ignore the effects of blunders. Interestingly, a blunder can be a kind of outlier event.
  One effect of a single blunder in down-the-plate dilutions is to mask the presence of traditional outliers. A single blunder in down-the-plate dilutions can distort the reported relative potencies of samples on the plate.
  NOTE: If you choose to attend this talk, you must promise to use the information for good and not for evil.
• (Day 2: Outliers in Potency Assays Session)

Development and Implementation of a Semi-Automated Bioassay for GMP Use

• Presenter: Maja Fedorowicz, REGENERON Pharmaceuticals, Inc.
• Abstract: Potency assays typically require extensive hands-on time and frequently exhibit a high degree of variability due to the use of live cells or enzymes, multiple dilutions, extensive sample manipulations and small pipetting volumes. Several automated platforms have been developed to address bioassay variability and increase throughput. However, the investment required to implement these technologies, coupled with data integrity and compliance considerations, have limited their incorporation into a GMP environment. Herein, we describe the development and implementation of a GMP bioassay with multiple pipetting steps on the Hamilton Microlab STARlet robotic liquid handler platform. Complex pipetting steps in the bioassay were targeted for automation, while the remainder of the assay was performed manually, drastically reducing analyst hands-on time. Data from side-by-side, manual versus semi-automated, assays were used to demonstrate the readiness of the semi-automated program for assay validation. A novel bioassay validation protocol was developed that supports the validation of the manual, as well as the semi-automated assay on the Hamilton at the same time. The validation data demonstrated that the semi-automated bioassay exhibits comparable performance versus the manual method with respect to invalid rates, accuracy, and precision, while achieving significant reductions in analyst hands-on time and pipetting. This semi-automated bioassay is ready to be used in the GMP routine testing laboratory at Regeneron. Taken together, this semi-automated bioassay represents a viable replacement for manual assay performance in a GMP environment and provides proof of concept for future, end-to-end bioassay automation, using an integrated robotic system.
• Contributing Authors: Maja A. Fedorowicz, Brandi M. Lewis, Hayley P. Mattice, Xiulian Du, John Lambropoulos, Miteshkumar Acharya
  REGENERON Pharmaceuticals, Inc.
  81 Columbia Turnpike, Rensselaer NY 12144
• (Poster)

Bioassay Potency Bias: Potential Routes to Address Method Bias in Reference Standard Characterization

• Speaker: Seth Foltz, Pfizer
• Abstract: Reference standards are critically important for the execution of bioassay methods as the potency of the test sample is determined by relative comparison to the potency of the reference standard. The determination of reference standard potency throughout a product’s lifecycle must be accurate, precise, reliable, and consistent across time and across multiple batches of reference standard materials. A new approach called sample-standard ratio (SSR) can help mitigate the potential impact of bias on the assigned potency of a reference standard. Key elements such as plate layout, plate reader, analyst, testing laboratories, and/or combination can all contribute to assay bias. The SSR approach helps address these elements during standard qualification and helps better define where any key bias might be during this testing. This approach is especially useful during the qualification of standards which have very tight acceptance criteria.
• (Day 2: Problematic Reference Material Issues Session)

Bridging MOA-based ADCC Reporter Bioassay with an Improved PBMC-based ADCC Cytotoxicity Assay for Immunotherapy mAb Development

• Presenter: Jamison Grailer, Promega
• Abstract: Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody and primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the use of primary effector cells makes them vulnerable to high assay variability and therefore not suitable for use in a quality-controlled environment during product manufacture and development.
  Previously, we developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure an ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing PBMC-based ADCC cytotoxicity assays and ADCC reporter bioassays, we recently developed an improved ADCC cytotoxicity assay using PBMCs and engineered HiBiT target cells. When HiBiT-expressing target cells are incubated with an antibody and PBMCs, the target cells are lysed and release HiBiT peptides, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate luminescence. The assay is simple, homogenous, highly sensitive, and gives a robust assay window. It shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies.
• Contributing Authors: Pete Stecha, Denise Garvin, Jim Hartnett, Aileen Paguio, Brock Binkowski, Frank Fan, Mei Cong, and Zhijie Jey Cheng
  Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• (Poster)

Conversion of a Potency Assay from Cell-Based ELISA to AlphaLISA: Taming a Disturbance in the Force

• Speaker: Christopher Hepler, Catalent Biologics
• Abstract: A relative potency assay was based upon inhibition of a mAb binding to a cell-surface expressed antigen on viable cells and assessed via flow cytometry. The assay produced data that were variable and insufficiently accurate. To address the assay’s shortcomings, a new relative potency assay with similar molecular underpinnings was developed, based upon an AlphaLISA platform. The resulting assay was not only significantly more accurate and less variable than the original FACS assay, it was also overwhelmingly easier and faster to execute.
• Contributing Authors: Michael Sadick, Tami Conley, Ramsey Connor, Lakshmi Botta, Laura Black, Mayur Gajera and Lauren Parker
  Catalent Biologics, Morrisville, NC 27560
• (Day 2: Product Specific Method Development Session)

From a Phase I Ligand Binding to a Late Phase Cell-Based Potency Assay for a Ph Dependent Antibody with a Challenging Mode of Action

• Speaker: Melody Janssen, Scientia Potentia Consultancy
• Abstract: Potency assays are a pivotal part of the development of any biologics.
  Potency assays are bridging manufacturing with the clinics by translating properties and stability of a molecule with its biological activity.
  Traditionally, potency assays are cell based and as such set up early in the process. During the various stages of the clinical and process development, the assay is refined until it is finally ready to be validated close to the final clinical stages and market authorization.
  But there has been a shift in industry to start with ligand binding assays for early phases and have a cell-based assay for late phases when manufacturing is also close to be a validated process.
  During this talk I will take you along with me through the different potency assays that were designed for a pH dependent antibody with a challenging mode of action.
• (Day 2: Product Specific Method Development Session)

Immunogenicity Assays from Concept to the Practical Application

• Speaker: Cheikh Kane, Nexelis
• Abstract: Biologics CMC properties, clinical trial design and reagents quality in general are critical for generating appropriate Immunogenicity assays. This presentation will discuss the impact of those factors on the Immunogenicity assays as well as discuss the QC trending during the clinical samples testing.
• (Day 1: Lifecycle of Potency Assays Session)

An Introduction to Strategic Bioassay Design, Development, Analysis, Validation, and Monitoring

• Instructor: David Lansky, Precision Bioassay, Inc.
• Abstract: This workshop will start with an introduction to the statistical concepts needed for bioassays (all illustrated with useful and relevant examples) and some investigation into the properties of bioassays. These inform the choices we make in applying design of experiments (DOE) to bioassay development, validation, and monitoring. Examples (mostly from cell-based bioassays, some using robotics) will illustrate strategic ways to design bioassays to make it (relatively) easy to use DOE for: early bioassay development, improving an existing bioassay, demonstrating robustness, qualification, and validation. We will cover ways that these strategic assay design considerations, when combined with good assay analysis methods, also support good assay monitoring (with both graphical and quantitative tools) and adaptation to changes in assay capabilities as well as analytic requirements throughout the life cycle of an assay.

Who should attend?
Those who are involved with the design, development, analysis, validation, monitoring, transfer, and updating of biological assays will benefit from this course. This includes bench scientists, assay managers, those responsible for planning and analyzing bioassay validation experiments, statisticians who are new to bioassay, as well as senior and upper level managers who have responsibility for bioassay development and validation. The course focuses on key concepts, properties of bioassays, with enough details to understand how medical needs drive product specifications, which drive the requirements for assay capabilities, which give us targets for assay development. We will not focus on formulae or calculations. We will discuss the strengths and weaknesses of various big picture approaches to bioassay development and validation.

• (Workshop: Track 2)

Design Space Considerations for The Validation of Potency Method

• Speaker: Ruojia Li, Bristol-Myers Squibb
• Abstract: This talk offers an overview of potency method validation considerations, and insights into a scientific/statistically driven approach for defining validation study design and for setting meaningful validation acceptance criteria. The approach uses expected specification as a starting point to define required method performance parameters. A sequence of statistical assessments are then applied to establish validation study design and acceptance criteria that provide sufficient confidence that the validated method is suitable for intended purpose, and that a suitable method will pass the validation criteria. Case studies of potency method validation are used to illustrate the approach. The concept introduced in this talk can be applied across methodologies beyond potency assays.
• (Day 1: Statistical Tools Session)

Bioassay Strategies for the Development of Therapeutic Oncolytic Viruses

• Instructors: Shihua Lin, AstraZeneca
• Abstract: Oncolytic virotherapy using a genetically engineered oncolytic virus (OV) has become a promising approach for cancer treatment. Development and manufacture of therapeutic OV requires a thorough analytical assessment of the product to ensure the desired efficacy, safety and quality. Due to the complexities of the manufacturing process, virus structure, and the potential mechanism(s) of action (MOA) which involves direct cancer cell killing via infection and indirect cell killing via transgene-driven immunomodulation, it is critical to develop appropriate biological assays to enable process development and optimize manufacturing, product characterization, CQA assessment and quality control.
  This presentation will outline the strategy and rationale that been used for developing bioassays for an OV product using recombinant Newcastle disease virus (NDV). These bioassays are used for lot release, stability and characterization assessing the identity, potency (infectivity, transgene expression and functionality), quality, safety and other biological properties.
• (Day 1: Bioassays for Complex Products Session)

Case Studies About Unexpected Reference Material Instabilities

• Speaker: Laureen Little, Quality Services
• Abstract: Throughout the life cycle of a potency bioassay there is probably no more critical rare reagent than the product reference. During product development we strive to select a representative product batch to utilize for clinical material release and we work diligently to formulate and store the reference material in a stable fashion. Once a product is commercialized the reference material is tightly controlled and monitored. These steps are described with formal protocols which are highly scrutinized during the approval process. Despite these efforts the stories about unstable reference material abound. This talk will step through the development and caring of product references for relative potency assays and discuss the signs and symptoms that the reference is losing or gaining potency. Case studies will be utilized whenever possible to explain more clearly the issue.
• (Day 2: Problematic Reference Material Issues Session)

Advanced Topics of Bioassays: Phase Appropriate Validation of Bioassays

• Instructors: Mike Merges, Emergent BioSolutions and Mike Sadick, Precision BioSciences
• Abstract: The M² workshop, presented by Mike Merges and Mike Sadick, will be an interactive session designed to address issues associated with taking bioassays to the next level, that of “phase appropriate validation” for use in GMP sample testing. We will cover the definitions and regulatory expectations that underpin many of the goals for bioassay validation. Among the topics that the workshop will cover are the definition and determination of optimal assay parameters such as accuracy, precision and linearity. Further, the workshop will provide insights as to phase-appropriate validation, as well as the differences between the approaches of ICH Q2 (RI) and USP <1033>. The session will, further, address the roles, determination and assignment of system suitability criteria and assay/sample acceptance criteria. The workshop instructors each have greater than 30 years of experience in the industry.
• (Workshops: Track 1)

Best Practices in Bioassay Development to Support Registration of Biopharmaceuticals

• Speaker: Debbie Meyer, Pfizer
• Abstract: The talk will be a presentation of the recently published white paper from the BioPhorum Development Group Bioassay Point Share industry consortium on best practices in the development, validation, and control of bioassays throughout the drug lifecycle.
• (Day 1: Lifecycle of Potency Assays Session)

Your Statistician is Your Friend: Parallelism in Immunoassays

• Speaker: Nancy Niemuth, Battelle
• Abstract: In theory, parallelism/similarity is the same for immunoassays as relative potency assays. The intended use of immunoassays and the need to work with biological sample matrices make the evaluation of parallelism between reference and test samples challenging in immunoassays. A broad range may be required for testing unknown samples, so that very few concentrations are available in the linear range of the assay where parallelism can be assessed. This talk considers several approaches to evaluating parallelism in immunoassays both for the assay and on a per sample basis.
• (Day 1: Statistical Tools Session)

Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Immune Checkpoint Receptors

• Presenter: Vanessa Ott, Promega
• Abstract: The human immune system is comprised of a complex network of immune checkpoint receptors that are promising new immunotherapy targets for the treatment of a variety of cancers and autoimmune-mediated disorders. Immunotherapies designed to block co-inhibitory receptors (e.g. PD-1, CTLA-4) are showing unprecedented efficacy in the treatment of cancer. However, not all patients and tumor types respond to this approach. This has resulted in broadening of immunotherapy research programs to target additional co-inhibitory (e.g. LAG-3, TIM-3, SIRPα) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) receptors individually and in combination.
  A major challenge in the development of biologics is access to quantitative and reproducible functional bioassays. Existing methods rely on primary cells and measurement of complex functional endpoints. These assays are cumbersome, highly variable and fail to yield data quality required for drug development in a quality-controlled environment. To address this need, we have developed a suite of cell-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. SIRPα, PD-1, CTLA-4, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals. Thus, the bioassays reflect mechanisms of action for the drug candidates designed for each immune checkpoint receptor and demonstrate high specificity, sensitivity and reproducibility. In summary, these reporter-based bioassays can serve as powerful tools in immunotherapy drug development for antibody screening, potency testing and stability studies.
• Contributing Authors: Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Frank Fan, Mei Cong and Zhi-jie Jey Cheng
  Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• (Poster)

Development of In Vitro Functional Bioassays for Potency and Immunogenicity Screening of Cell and Gene Therapy Products: Challenges and Opportunities

• Speaker: Sofie Pattijn, ImmunXperts
• Abstract: Cell and Gene Therapies have the potential to cure a broad spectrum of diseases and modify the progress of many other diseases. However, the development of new therapeutics comes with a series of challenges and risks.
  In contrast to traditional therapeutics large batch manufacturing and quality testing, these individual batches of cell products require specific potency assays showing the biological activity.
  Due to the complexity of most of these cell therapy products, the selection and development of a suitable in vitro potency bioassay comes with certain challenges. Identifying the specific function and mode of action of such a complex product is the first step. Additionally, representing the mode of action in an in vitro assay using a combination of different cell types, requires optimization and fine tuning on different levels. As an example, the development and optimization of a bioassay to screen for the immunosuppressive function of Mesenchymal Stem Cells will be shown.
  Next to the evaluation of the potency, in vitro bioassays can be used for the screening of the unwanted immunogenicity of Stem Cell Products. Especially with the increased use of allogenic ‘off the shelf’ therapies, in vitro bioassays to assess the Immunogenicity of Stem Cell products prior to administration in human can have significant value.
• Contributing Authors: Séverine Giltaire¹, Jana Schockaert¹, and Sofie Pattyn^{1
  1} ImmunXperts SA, rue Auguste Piccard 48, 6041 Gosselies, Belgium
• (Day 1: Bioassays for Complex Products Session)

Assessing Unwanted Immunogenicity of Cell and Gene Therapy Products

• Presenter: Sofie Pattijn, ImmunXperts
• Abstract: Cell and Gene Therapies have the potential to cure a broad spectrum of diseases and modify the progress of many other diseases, but this new class of drugs comes with a series of challenges and risks.
  Whether it is the presence of pre-existing antibodies, the induction of an innate immune response or allogenic reactivity of cellular products, an unwanted immune response can have a significant impact on the safety and efficacy of the treatment.
  Today, several tools are available to improve and accelerate therapeutic drug development in an early stage. A well balanced and rational development strategy can help to design less immunogenic drugs and reduce the number of clinical failures.
  Often used as a first step is an In silico T cell epitope prediction algorithm such as NetMHCpan which can be used to assess and compare the immunogenic potential of the lead candidates and guide de-immunization strategies.
  Further monitoring of the immunogenic risk can be performed using in vitro T cell proliferation assays, cytokine release assays and MAPPS assays to determine and rank the immunogenic risk of the test proteins or identify specific regions of concern.
• Contributing Authors: Séverine Giltaire¹, Jana Schockaert¹, Sofie Pattijn¹, and Amin Osmani^{1
  1} ImmunXperts SA, rue Auguste Piccard 48, 6041 Gosselies, Belgium
• (Poster)

FANG ELISAs for Human and Nonhuman Primate Filovirus Vaccine Immune Response

• Speaker: Thomas Rudge, Battelle
• Abstract: An FDA/EMA licensed vaccine for prevention of filovirus hemorrhagic fever will significantly reduce the morbidity/mortality in the event of an outbreak of disease caused by EBOV, SUDV, or MARV, in addition to providing protection for laboratory workers. Correlate or surrogate markers of immunity/protection will likely be required to achieve licensure via the FDA Animal Rule. Well-characterized humoral and cellular immune response assays are required to bridge the relationship between the NHP immune response which provides protection from filovirus challenge and the vaccine provided human immune response. Enzyme-linked immunosorbent assays (ELISAs) have been developed, qualified, and validated to evaluate the immune response to filovirus vaccines for identification of the surrogate measure(s) to establish vaccine effectiveness/efficacy. This presentation will include the approach taken to develop, qualify, and validate the EBOV-specific GP IgG ELISA for use with both human and NHP serum on GLP studies; the generation of critical reagents (coating antigen, reference standard, QCs); and the maintenance of the assay for long-term use, including statistical process control charting to monitor tracking/trending over time. Additionally, the generation of blinded proficiency panels for monitoring assay performance both at Battelle and other labs where the assay was transferred to will be discussed. Finally, the development of the SUDV and MARV GP IgG ELISAs, including generation of critical reagents will be presented. Taken together, all three assays provide a suite of ELISAs for the evaluation of the humoral immune response in humans and NHPs to vaccination against or exposure to filoviruses.
• Contributing Authors: T.L. Rudge Jr.¹, K.A. Sankovich¹, A.L. Allen¹, M.S. Anderson¹, N.A. Niemuth¹, C.S. Badorrek², W.C. Florence², C.E. Bounds³, K.T. Taylor⁴, L.A. Wolfraim⁴, and C.L. Sabourin^{5
  1} Battelle, Columbus, OH; ² Contract support for JPM-CBRN Medical, Fort Detrick, MD; ³ JPM-CBRN Medical, Fort Detrick, MD; ⁴ DMID-NIAID, Rockville, MD; ⁵ Tunnell Government Serices, Washington D.C.
• (Day 2: Product Specific Method Development Session)

Outliers in Bioassay 1: The Effects of a Blunder in Top-Well-Transfers

• Speaker: Michael Sadick, Precision BioSciences
• Abstract: From a bioassay perspective, replicate columns of a sample contain replicate information. For example, wells A1, A5, and A9 might be replicates of the first dilution of triplicate subsamples of a reference standard. It is commonly believed that the response for these three wells should be identical, and any deviation from their “true” value is considered to be simple random error. This is the environment within which preliminary outlier tests are often applied (Dixon’s Q test, for example) to see if one of these triplicate responses is discordant with the other two.
  From a statistical perspective, this is an incorrect understanding. Because the amounts placed in each top well can vary, the results down each column can differ by a constant ratio from the results down other columns of the same material. As a result, when preliminary outlier tests are applied to replicates “across the row” (e.g., wells D2, D6, D10), it is often the case that going down the rows with this method results in wells from the same column being rejected consistently.
  Thus, from a statistical perspective, it is more appropriate to treat each column as a separate sample. The relative potencies of the triplicate columns can then be compared to assess the variability of the top-well transfers. If one of these relative potencies appears to be discordant with the others, it can then be investigated as a different kind of outlier.
• (Day 2: Outliers in Potency Assays Session)

Phase-Appropriate Bioassay Implementation Strategies: Case Studies for Method Replacement

• Speaker: Tara Stauffer, Bristol-Myers Squibb
• Abstract: As therapeutic biologics move through clinical development towards licensure, functional potency methods designed to provide additional insight into molecular attributes may be introduced. Often these methods provide the same or better understanding of control of these attributes and as such, are suitable replacements for existing potency methods. This presentation discusses strategies for implementation of bioassays to replace existing potency methods. Case studies will be presented to highlight these strategies.
• (Day 1: Lifecycle of Potency Assays Session)

Outliers in Bioassay 3: The Effects of a Consistent Blunder in Down-The-Plate Dilution

• Speaker: Anton Stetsenko, ADC Therapeutics
• Abstract: As John K. Taylor has pointed out, “There are three sources of error in measurements and they can be classed as systematic, random, and just plain mistakes … The third kind of uncertainty may be called blunders. They happen when mistakes are made, knowingly or unknowingly. Errors resulting from them are not statistically manageable and, in fact, they can invalidate an otherwise good set of data. A measurement system that is unstable and fraught with blunders is not in statistical control and cannot be relied upon to produce useful data.”
  Many existing outlier approaches in bioassay assume that all errors are random. These approaches ignore the effects of blunders. Interestingly, a blunder can be a kind of outlier event.
  One effect of a single blunder in down-the-plate dilutions is to mask the presence of traditional outliers. A single blunder in down-the-plate dilutions can distort the reported relative potencies of samples on the plate.
  NOTE: If you choose to attend this talk, you must promise to use the information for good and not for evil.
• (Day 2: Outliers in Potency Assays Session)

Development of Potency Assays for the Quantification of AAV Encoded Transgenes Expressed Under the Control of Retina Specific Promoters

• Speaker: Michael Tovey, Svar Life Science France
• Abstract: Adeno associated virus (AAV) mediated gene therapy is currently being developed to address serious retinal disorders such as Leber’s congenital amaurosis and achromatopsia using transgenes expressed under the control of retina specific promoters. This poses a particular set of challenges for the development of potency assays for drug development and for monitoring patients for continued expression of the transgene. AAV is a small single stranded DNA virus of 4.7 kb that is replication defective and requires live adenovirus or another means of supplying the immediate-early proteins required to replicate the virus or to express a transgene. Potency assays, based on the iLite® reporter-gene technology have been used to quantify the activity of an AAV5 vector expressing GUCY2D under the control of the human rhodopsin kinase photoreceptor-specific promoter (hGRK1) without the need for live adenovirus or the addition of a proteasome inhibitor, and for the quantification of the activity of the proteins CNGA3 and CNGB3, that form the cyclic nucleotide gated (CNG) cation ion channel encoded by transgenes under the control of a cone photoreceptor specific promoter derived from the 5’ UTR of the human red cone opsin gene. Mutations in the genes encoding the CNGA3 and CNGB3 proteins lead to the development of achromatopsia. The relative advantages of different strategies used to provide the immediate early proteins and the specific set of transcription factors required to express these transgenes under the control of retina specific promoters will be discussed.
• Contributing Authors: Lue Huang, Christophe Lallemand and Michael G. Tovey
  Svar Life Science France, 1 mail du Professeur George Mathé 94800 Villejuif France
• (Day 1: Bioassays for Complex Products Session)

Cell-Based Potency Assays for Peptides: Challenges and Strategies

• Speaker: Dirk Usener, Sanofi
• Abstract: Potency tests are essential for biopharmaceutical compounds in order to understand and monitor their mode of action. Also, for peptides e.g. incretin mimetica, those potency assays have become more important during clinical development and potency data are meanwhile mandatory within the CMC package in dossiers (NDA).
  GLP1 receptor agonist activity is used for Sanofi´s therapeutic pipeline of synthetic peptides, e.g. for the already marketed products (Lyxumia, iGlarLixi) and for development projects being in clinical trials. The development and validation of a platform potency assay targeting GLP1R is reported with special emphasis on recent technologies, like assay ready cells or reporter gene read out technologies.
• (Day 2: Product Specific Method Development Session)

The USP Bioassay Chapters: Come Join the Journey

• Instructor: Stephen Walfish, United States Pharmacopeia
• Abstract: USP Chapters <111>, <1030>, <1032>, <1033> and <1034> were last updated nearly fifteen years ago. An Expert Panel formed to review and update the chapters. With oversight from the USP Statistics Expert Committee the panel will determine if there is any new advancement that would make the bioassay chapters more valuable to their stakeholders. More examples, and clarifying the definitions is proposed. Any proposed changes are vetted in USP’s Pharmaceutical Forum (PF).
  This presentation focuses on the USP suite of bioassay chapters. A discussion of the current state, and future plans will be shared with the participants. An open dialogue with participants is expected to gain valuable feedback on areas where the chapters can be improved to be more user-friendly. Do not miss your chance to be part of the change.
• (Workshops: Track 3)

Key to Success in Bioassay Data Analysis

• Presenter: Yat Yee (Amos) Wong, PPD
• Abstract: A carefully planned bioassay data analysis procedure during method development can reduce the time and money required to collect additional data for method qualification and method validation. Key elements such as plate layout, drug dilution scheme, statistic curve fitting model, data transformation, and outlier tests have a huge impact on data variation and how the data fits to a dose response curve. These key elements should be evaluated systematically with sufficient consideration of variation and bias sources to reduce the possibility of extensively changing the data analysis procedure after method qualification. A properly chosen curve fit during method development helps with trending of the required information to establish relevant system suitability criteria and sample acceptance criteria. Ultimately, a well-developed data analysis procedure improves the success rate in method validation and the passing rate of the ongoing sample testing.
• (Poster)