Abstracts (Alphabetical by Speaker/Instructor/Presenter)

A Novel, Relative Potency-Based IFNγ Measurement Approach for CAR-T Potency Assessment

• Speaker: Agatha Feltus, Precision BioSciences
• Abstract: Measurement of IFNγ production is a widely used approach for CAR-T cell therapy potency assessment in clinical candidates and approved commercial products. Detection of cytokine release (specifically IFNγ) is an indicator of T-cell activation and is considered a surrogate measure for cytolytic function and CAR-T potency. For allogenic CAR-T products, a measure of absolute quantitation of IFNγ secretion as potency measurement can be misleading, with existing IFNγ quantitation approaches being difficult to validate for use in a QC environment to support lot release and stability testing supporting GMP manufacture. Here, we present a novel bioassay approach for the quantitation of IFNγ secretion in a relative potency format based on Promega’s Lumit™ technology, to support cell therapy process development and GMP manufacture, and show data around potency assay performance for validation in a QC environment.
• (Day 2: Potency Assay Development for Complex Products Session)

How to Deal with Assays with Partial Dose-Response Curves

• Speaker: John Dunn, Brendan Bioanalytics
• Abstract: In regression statistics, the similarity of regressions (parallelism) is often determined with the Extra Sum of Squares method used in the RSSE (Chi-Square) and F Test potency methods. These residual methods can use any appropriately weighted least squares regression model (e.g. linear, 3PL, 4PL, 5PL) that provide an adequate fit to the data, including partial dose-response curves and ill-behaved data. The difference in RSSEs between curves constrained to the same shape and their unconstrained curves (RSSEnonpar) is a direct measure of the amount of non-parallelism between the assayed data points of the two curves and becomes progressively larger the more nonparallel the curves. Dividing the RSSEnonpar by the unconstrained RSSEs in an F Test provides a hypothesis test for parallelism. A RSSE threshold can be established empirically that includes an acceptable amount of non-similarity. This talk will show how these residual methods utilizing different regression models can analyze potency assays with partial curves and ill-behaved data.
• (Day 3: RAPID-FIRE TALK, Statistical Tools Session)

A Homogenous PBMC ADCC Bioassay Enables Bridging Studies with ADCC Reporter Bioassays in Immunotherapy Monoclonal Antibody Development

• Presenter: Steven Edenson, Promega Corporation
• Abstract: Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action for therapeutic antibodies. Previously, we developed a surrogate ADCC Reporter Bioassay using engineered reporter effector cells and demonstrated its suitability for antibody product release and stability study. Here we developed an improved ADCC assay using PBMC and engineered HiBiT-target cells for use in antibody early characterization and enable ADCC method bridging studies.
• (Poster)

Bioassay Critical Reagent vs Reference Standards – How are they the same, how are they different?

• Speaker: Seth Foltz, Eli Lilly and Company
• Abstract: Reference standards are vitally important for the execution of bioassay methods as the unknown potency of the test sample is determined by relative comparison to the known, certified property value of the reference standard. Often overlooked is the role of critical reagents in the execution of these bioassays. Critical reagents can have a large impact on assay performance, including causing shifts in measured relative potency over time. An overview of how critical reagents are selected, qualified, defined, maintained, and monitored thru the lifecycle of a project will be discussed.
• (Day 1: Assay Lifecycle Session)

Bioluminescence Reporters for In-process Potency Testing of T Cell Redirecting Viral Vectors

• Speaker: Julia Gilden, Promega Corporation
• Abstract: T cell therapies, such as CAR-T and TCR-T, are a promising frontier in the treatment of cancer. These therapies depend on genetic modification of T cells that redirects their activity toward a tumor-associated antigen. The modified T cells may be derived from the individual patient or a healthy donor, and engineering is often performed via viral vectors. Lentiviral vectors are increasingly the gene transfer tool of choice because of their safety and efficacy, but in-process potency testing of lentiviral preps is a challenge for this new treatment modality. We will present several new bioluminescent cell-based bioassays that allow for rapid, simple potency quantification of T cell redirecting viral vectors. The bioassays are built in biologically relevant cellular models and the readouts reflect true T cell effector functions, allowing for in-process potency testing that reflects the mode of action of the final drug product.
• (Day 2: RAPID-FIRE TALK, Potency Assay Development for Complex Products Session)

HiBiT: A Tiny Tag to Assess MOA-based CAR-T Cell Potency

• Presenter: Julia Gilden, Promega Corporation
• Abstract: Despite significant clinical advances in CAR-T cell therapies for cancer, precisely and reproducibly analyzing the potency of these drugs remains a challenge. Single point cytokine release has been widely used as CAR-T functional lot release assay, but whether this is a reliable method truly reflecting the Mode of Action (MoA) has been challenged in recent analytical method develop forums. As new CAR-T products and production methodologies are developed, it is becoming increasingly important to be able to rigorously evaluate the relative potencies of each batch. Here we describe a live cell HiBiT detection system that can be used to measure CAR-T induced target cell killing (TCK) by a simple add-mix-read format in just a few hours. Instead of radioactive or fluorescent markers, the 11 amino acid HiBiT peptide is engineered into physiological relevant target cells through expression of an intracellular HiBiT fusion protein. Upon exposing HiBiT target cells to CAR-T, the lysis of target cells releases HiBiT fusion protein into the medium. The HiBiT fusion protein then binds to the cell impermeable LgBiT in the detection reagent and reconstitutes functional NanoBiT™ luciferase to emit luminescent light. This HiBiT assay provides killing signal specifically from target cells. The simple, homogeneous, highly sensitive and robust live cell assay performance will be demonstrated in CAR-T potency assessment.
• (Poster)

Analytical Transfer of Cell-Based Potency Assays: Challenges from Experience

• Presenter: Tobey Gooding, Labcorp
• Abstract: As a CRO, Labcorp Drug Development serves clients in a broad range of disciplines within the scientific community. This experience gives us broad exposure to the practices and approaches of the Biopharmaceutical Industry to meet regulatory requirements for development as well as transfer and support Biopotency Methods, whether they be Cell-Based Assays, In Vivo designs or basic Ligand Binding Assays. Even so, biological assays are some of the most complex and challenging techniques in the CMC space of product development and achieving a smooth technical transfer is no less of a challenge. Here we describe a standard process for a staged approach to transfers with the goal of early identification of potential issues, a phase for demonstration of capability by the receiving lab, and formal assay establishment within our operations team. Additionally, we describe some of the more common issues observed as an initial checklist for evaluation. While no process is perfect, many potential issues can be mitigated through solid planning to provide conditions which are conducive to successful method transfer.
• Author: Stuart Dunn, PhD, Labcorp Drug Development, Harrogate, UK
• (Poster)

Lesson Learned for Potency Assays for Bispecific mAbs for Accelerated Programs

• Speaker: Marianne Hayes, Janssen Research & Development, LLC R&D
• Abstract: Phase based potency assay development often includes binding assays for potency assays for antibody therapeutics in early phase development and support rapid progress to FIH. Cell-based assay(s) are phased in in later development to support full biological characterization and to develop the potency control strategy for commercialization. Extensive biological characterization based on knowledge of MOA, degradation pathways and structure function relationships is required and will likely require multiple bioassay(s). Complex MOAs and bi/multispecific therapeutics have increased complexity of the bioassays required in an environment where rapid late development is occurring to support accelerated approvals pathways to bring innovative medicines to patients. Examples illustrating the challenges and lessons learned in development, implementation and transfer with Bispecific Mabs on accelerated pathways will be discussed.
• (Day 1: Assay Lifecycle Session)

Development of Bioluminescent No-Wash FcBGamma Receptor Binding Immunoassay to Guide the Development of Antibody Therapeutics

• Presenter: Kai Hillman, Promega Corporation
• Abstract: Therapeutic antibodies and Fc fusion proteins are effective against a variety of diseases because of their exquisite specificity in binding to an antigen and ability to activate an immune response through effector functions. The effector functions are triggered when antibody Fc domain interact with Fc gamma receptors present on immune effector cells like natural killer cells and macrophages. Multiple factors can impact the interactions between the Fc domain and Fc gamma receptors and careful optimization and monitoring of these interactions are necessary to maintain the efficacy and safety of the antibody drugs. To meet the need for reliable, simple to use assays, we have developed a suite of bioluminescent biochemical assays to measure and compare the affinities of Fc gamma receptors for the Fc domains.
• (Poster)

State of the Cell & Gene Therapy Industry

• Speaker: Michael Lehmicke, Alliance for Regenerative Medicine
• Abstract: In laying out the state of the industry and future perspectives in the cell and gene therapy sector, the Alliance for Regenerative Medicine will walk through regulatory and scientific highlights, trends in investments and clinical trials, and opportunities in policy and manufacturing. The talk will also include reflections on the current state of CMC regulations in Cell and Gene Therapy, specifically with respect to potency assays.
• (Day 2: Potency Assay Development for Complex Products Session)

Combination of SPR-Based Binding Kinetic and SEC-Based Conformational Assay to Evaluate mAb-Target Antigen Physicochemical Properties Important for ADCs Therapeutic Function

• Speaker: Alessandra Mariani, Menarini Biotech
• Abstract: CD205/Ly75, was selected as target antigen for a humanized anti-CD205 IgG1 conjugated to the DM4 toxin, MEN1309 (ref. 1). The over expression of CD205 in different solid cancers, combined with its capability to induce ligand internalization, were key factor in the election of CD205 to excellent target for an Antibody Drug Conjugate (ADC) (ref. 2-3). On the other side, the CD205 physiological expression on Antigen Presenting Cells (APCs) and leucocytes, represents a risk factor for toxic side effect induced by MEN1309 binding on healthy blood cells, a phenomenon frequent for ADCs and known as ”on-target but off-tumor” binding (ref. 4). In the present study, the previously described pH dependent CD205 conformational change (ref. 5) impact on the binding of MEN1309 was evaluated, looking for possible consequences on MEN1309 therapeutic function. SPR analyses not only confirmed the effective impact of the pH in the complexity of MEN1309-CD205 interaction, but also showed that MEN1309 binds the target antigen with different kinetic in response to pH 7.4, or pH 6.5 , which represent the pH conditions of CD205 binding occurring respectively on healthy or malignant cells. In particular, a significantly faster binding kinetic was observed at pH 7.4 and these results were confirmed by SEC RALS analysis of MBH1309:CD205 mixtures, which showed only at pH 7.4 a separation of the two molecules, while at pH 6.5 they remained combined in immune-complexes. Overall, these data demonstrate a strong impact of pH on MEN1309 binding kinetic to the CD205 target antigen and thus to the mAb therapeutic function. At pH 7.4 (healthy cells environment) the MBH1309- CD205 binding kinetic is faster, indeed they associate and dissociate more rapidly, while complexes at pH 6.5 (malignant cells microenvironment) associate a bit slower and not dissociate as fast as under pH7.4 conditions, resulting in a more stable MEN1309-CD205 binding. Finally the present study identify in vitro analytical methods able to support the development of new ADCs. The combination between SPR-based binding kinetic and SEC-based conformational assays allows to evaluate mAb-target antigen physiochemical properties which could enhance the selectivity of ADCs for the malignant cells.
• Contributing Authors: Alessandra Mariani, Sabrina Novelli, Meri Gabrielli and Thoralf Keller
• Bibliography:
  1. Merlino at al. “MEN1309/OBT076, a first-in-class Antibody–Drug Conjugate targeting CD205 in solid tumors”. Molecular cancer therapeutics (2019) 18:1533–432. K. Mahnke et al. “The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments” The journal of cell biology (2000) 151(3):673-843. GV. Canzonieri et al. “CD205, a target antigen for a novel antibody drug conjugate (ADC): Evaluation of antigen expression on non-Hodgkin lymphoma (NHL)”. Journal of Clinical Oncology (2017)4. J.M. Lambert and C.Q. Morris. “Antibody–Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review”. Advances in Therapy (2017) 34:1015–10355. J. Lin and J. Sagert “Targeting drug conjugates to the Tumor Microenvironment: probody drug conjugates” Innovations for Next-Generation Antibody-Drug conjugates (2018) 281-298
• (Day 3: Antibody Product Potency Assay Development Session)

Potency Matrix for AAV Gene Therapies

• Speaker: Cullen Mason, Biogen
• Abstract: Potency for gene therapies requires transduction of the viral material into the target cells, transcription and translation of the viral transgene and proper localization and function of the protein product. Therefore, multiple cell-based methods are required to fully characterize and control for the functional attributes of an AAV gene therapy. Our phase-based strategy for method development and product release will be discussed. Case studies demonstrating the correlation between the different methods in the potency matrix will also be presented.
• (Day 2: Potency Assay Development for Complex Products Session)

Speed Root Cause Analysis with Data Exploration Tools

• Speaker: Tara Scherder, SynoloStats
• Abstract: The first question during root cause analysis of unexpected process performance is often: Is this an analytical or process issue? The use of simple data visualization tools combined with analytical subject matter expertise can provide rapid insights to answer this question and provide clues to the most likely root cause.
• (Day 3: Statistical Tools Session)

Outline for Characterization of Reporter Cell Lines – Critical Reagents for Robust Cell-based Reporter-Gene Assays

• Presenter: Therese Segerstein, Svar Life Science AB
• Abstract: Over the last decades, biopharmaceuticals have revolutionized the treatment of a wide range of diseases in all areas of medicine. The development of such drugs has led to a need for bioassays that can be used to accurately characterize various aspects of the drug. Developing a bioassay is a complex, rigorous undertaking that needs to address several challenges including modeling the mechanisms of action associated with the biotherapeutic and ensuring that robust and validatable assays can be achieved.
  Assay-ready reporter-gene assays have proven to be a valuable tool in assessing both functionality and MOA-representing assays, but given their importance in drug development, reporter-gene assays need to be designed, produced, and validated in a way that ensures that they behave consistently over time, in varying experimental setting and subsequent pivotal assays.Here, we present an outline for a suggested set of assays by which the performance of cell-based reporter gene assays can be determined and validated.This is an essential step in the quality assessment to ensure that the assays are reliable, accurate, and reproducible and that errors resulting from biological variation and methodology are kept to a minimum.
• Download full poster HERE!
• (Poster)

Functional Bioassays measuring the Potency of Complement Therapeutics

• Presenter: Therese Segerstein, Svar Life Science AB
• Abstract: In the battle against complement-driven pathogenesis, complement-associated proteins have become promising targets for new therapeutics, providing hope to patients suffering from the destructive effects of a dysfunctional complement system. The complexity and diversity of complement dysfunction and associated conditions have resulted in a multitude of creative complement therapeutic approaches, many of which are currently in clinical trials. This plethora of candidate drugs provides tools for blocking complement initiation, interfering with the amplification, or neutralizing the damaging effectors of complement activity (Fig. 1), yet challenges the standard analytical packages in place today. A pillar stone when testing a biologically active drug, is a relevant functional assay, a bioassay. The ideal bioassay is based on the mechanism of action (MOA) and has a functional and quantitative readout and can be utilized for several purposes, where Potency measurement is a key parameter.
  In this poster, we showcase an example of how the Svar Life Science complement-specific assays can be used to analyze complement functionality, activation, and dysregulation. This approach can be mimicked for all three complement pathways, screened with Wieslab® functional assays, and several complement activation biomarkers, such as TCC, C4d, and Properdin.Complementary to the above-mentioned Immuno-assays, we also offer iLite® cell-based assays for cell-based reporter gene analysis of complement anaphylatoxins and their receptors.
• (Poster)

Overview of Statistical Software for Bioassay Scientists

• Speaker: Perceval Sondag, Novo Nordisk
• Abstract: Bioassay Scientist who wish to perform some statistical analysis themselves are often left with the decision of which software to use. This talk will present an overview of the different software that are on the market, their pros and cons, skill level/learning curve required, and the type of analysis they are best fit for.
• (Day 3: Statistical Tools Session)

Developing Bioassays for Qualification and Lot Release of Biologics: A Case Study Using the CD112/CD112R Blockade Bioassay for anti-CD112R Relative Potency Estimation

• Presenter: Caitlin Stallings, Promega Corporation
• Abstract: In the endeavor to generate novel therapeutics, it is essential to create bioassays which recapitulate the physiological mechanism of action of the target pathway. Ideal bioassays will allow for interrogation in a cell based model. These can be used for lot release of biologics to satisfy regulatory and scientific criteria. We have developed an assay specific to the CD112/CD112R pathway which can be used to estimate the relative potency of test antibodies against CD112R. With this bioassay we have evaluated a commercial anti-CD112R antibody and tested the relative accuracy, specificity, precision, dilution linearity, and range. The technique is simple and easy to use, employing thaw-and-use cells as a reagent to eliminate variability from cell propagation models. Overall, we have produced a relevant functional bioassay capable of being optimized to unique anti-CD112R antibodies for lot release.
• Contributing Authors: Caitlin Stallings, Irina Sedykh, Ngan Lam, Denise Garvin, James R. Hartnett, Jamison Grailer, Mei Cong
• (Poster)

Novel Bioluminescent Bioassays for the Discovery and Development of Molecular and Cellular T Cell Redirecting Therapy

• Presenter: Brad Swanson, Promega Corporation
• Abstract: Directing T cells to attack tumor cells is a proven approach for the treatment of cancer. There are various approaches to develop therapies that can activate or enhance the ability of the host immune system to recognize tumor cells and kill them. One approach is to engineer effector cells with receptors, traditional T cell receptors (TCRs) or chimeric antigen receptors (CARs), that recognize tumor-associated antigens (TAA). Robust assays are needed to facilitate development and manufacture of these therapies. We have developed a suite of TCR knock-out (TCR KO) bioluminescent reporter cell lines expressing either CD4 or CD8 co-receptors which can be used to measure activity of exogenously added antigen receptors, including assessments of antigen specificity and receptor potency. To measure T cell-mediated killing quantitatively and directly, we have used split NanoBiTTM luciferase technology to develop a homogenous target cell killing assay. Target cells stably express HiBiT intracellularly, which is released upon lysis. HiBiT then binds to LgBiT in the detection reagent and forms a functional NanoBiT luciferase to generate luminescence. Incubation of primary T cells with TAA-bearing HiBiT target cells and a titration of TAA-targeted bispecific T cell engager (BiTE) results in dose-dependent lysis of the target cells and increased luminescence. Both HiBiT target cells and TDCC-qualified T cells are provided in Thaw-and-Use format. Measurement of cytokine secretion is often used as a supporting assay in the assessment of effector cell activation leading to target cell killing. To streamline the quantitation of these secreted molecules, we have developed novel LumitTM luminescence-based immunoassays that have simple protocols with no wash steps and exhibit a broad assay window as demonstrated by the quantitation of IL-2 and IFN- from activated primary CD8+ T cells. These sensitive immunoassays are fast and well suited for high throughput assay formats.
  All of the luminescence-based assays presented in this report are homogenous, fast, highly sensitive, and have robust assay windows. They represent a new set of tools for the discovery and development of molecular and cellular T cell redirecting therapies.
• Contributing Authors: Julia Gilden, Jamison Grailer, Michael Slater, Pete Stecha, Jim Hartnett, Dan Lazar, Frank Fan, Mei Cong, Zhijie Cheng, Brad Swanson
• (Poster)

Defining a Lot Release Bioassay During Late-Stage Product Development

• Speaker: Victoria Swiss, Bristol Myers Squibb
• Abstract: Biological characterization of complex biotherapeutics often includes multiple assays which interrogate
  the relevant aspects of the biological activity. Selection of a suitable cell-based potency assay to support
  commercial lot release and stability is a critical strategic decision during late stage CMC development.
  Factors which influence this decision include how well the assay reflects the mechanism of action, assay
  performance and operational considerations. These critical assay qualities are considered and weighed
  during final assay selection. In addition, molecules with multiple MoAs must be evaluated against the
  relevant attributes which require control vs characterization. The assay type chosen and supporting
  characterization for the assay choice is critical to the success of control strategy. Case studies
  representing various scenarios of assay decisions and consideration of critical qualities will be discussed.
• (Day 1: Assay Lifecycle Session)

In Vitro Potency Analytical Development for Gene Therapies

• Speaker: Shewit Tekeste, Janssen
• Abstract: The complexity of gene therapies mechanism of action stipulates and complicates potency analytical development. The potency guidance requirements provide a general roadmap of what a matrix approach entails with room for interpretation. Hereby, we will review the fundamental parameters and strategies to consider in developing an analytical potency readout. Emphasis will be applied to AAV potency assay development and examples discussed.
• (Day 2: Potency Assay Development for Complex Products Session)

A Fab-ulous Live Cell Assay that Addresses a Dual Control System Potency Strategy

• Speaker: Jeanette Villarreal Kinney, Genentech
• Abstract: A two-part control system strategy was taken for potency determination of a molecule with complex modality and Mechanism of Action (MoA). The first approach, characterizes the drug intermediate using a direct binding ELISA assay to determine potency, followed by implementation of a cell-based assay with specificity for the Drug conjugate. The complexity of the Antibody’s MoA is captured using a reporter gene cell-based assay capable of detecting protein-protein interactions in live cells. Both assays include the emergence of automation and ergonomic friendly technologies, leading to successful validation and incorporation of the potency methods for a new molecule with complex modality.
• (Day 3: Antibody Product Potency Assay Development Session)

Warp-Speed Animal Testing Removal and Replacement in Human Vaccines’ Final Testing. Industry and Regulatory Work and Remaining Challenges

• Speaker: Laura Viviani, Humane Society International
• Abstract: The presentation aims to share the ongoing activities to accelerate the global harmonization of the regulatory acceptance and industry implementation of non-animal approaches to batch release of human vaccines. Every batch of a human vaccine is required to be tested by the manufacturer and by regulatory authorities, in many countries, to assess its identity, purity, safety and toxicity, potency and immunogenicity, and pyrogenicity before it can be released to the population. This is a fundamental regulatory requirement that serves to ensure safety and effectiveness of all vaccines being delivered to prevent life-threatening infectious diseases. Many of the batch release tests rely on animals, especially for the oldest vaccines in the market, like the Tetanus, Diphtheria, Pertussis, Rabies and Polio vaccines. The numbers of animals used for testing these products are very high, and often the tests fail due to the inherent variability of animal-based tests. Repetitions and, thereby, additional use of animals are common occurrences that also lead to increased costs and time to the market. The last decade has seen an increase in the investment from various stakeholders in both developed and developing countries in non-animal-based innovation in testing. As a result, many regulatory authorities are updating their requirements or considering to do so. However, to completely remove and replace animal testing with in vitro alternatives, manufacturers and regulatory authorities have to address technical, policy and cultural challenges. The speaker will present some of the ongoing efforts on the development, implementation and regulatory acceptance of non-animal-based methods. Furthermore, she will explain the challenges that the stakeholders face and to need to overcome and some of the successes obtained, as well as challenges that remain.
• (Day 1: Assay Lifecycle Session)

USP General Chapter <1033> Biological Assay Validation Update

• Speaker: Ann Yellowlees, Quantics Biostatistics
• Abstract: USP NF General Chapter <1033>: Biological Assay Validation has been available to industry and regulators since 2013. With its worked example it provides the tools for practitioners to conduct a validation study with confidence. The chapter has now been reviewed and updated. The main aims of the update were to provide clarification, and to add detail on some concepts, including choice of acceptance criteria, total error (as an approach to combining accuracy and precision), assay format and assessment of linearity.
  The commenting period closed on 31 January 2023 and the comments will be reviewed with a view to finalization after updates and reviews of the other USP bioassay chapters.
  This talk will focus on the changes to the guidance and their potential benefits for bioassay validation in practice.
• (Day 3: Statistical Tools Session)