Abstracts (Alphabetical by Speaker/Presenter)

In Vitro Potency Assay for a Novel Molecule Reflecting Intracellular Target Activation

• Speaker: Sonia Bastidas, Novartis Pharma AG
• Abstract: AB-X is an immune-stimulatory protein conjugate consisting of a monoclonal antibody AB and an agonist X against an intracellular target receptor. The molecule has a complex MoA and the activation of the intracellular receptor represents a major part of this MoA.
  Here, we present the development of a bioassay, reflecting the activation of the intracellular target receptor. Starting with an assay with low sensitivity, we developed a highly sensitive assay suited for release and stability (QC) and reflecting expected quality differences between different molecular properties.
• (Day 2: Product Specific Bioassays Session)

Impact of Pipette Choice on Bioassay Performance in GMP QC

• Presenter: Lisa-Marie Bauhofer, Eurofins BioPharma Product Testing Munich GmbH
• Abstract: Manual and electronic (multi-channel) pipettes are commonly used in biopharmaceutical industry and a key component in Bioassays which are not run on a liquid handler. In general, electronic pipettes are thought to be less error prone and lead to less variation between analysts.
  We have made the observation in several cases that a change of pipette types (manual vs. electronic) or brand, can influence the assay performance especially at the extremes of assay range leading even to assay acceptance criteria failure.
  The results from two cases, one manual vs. electronic and one brand specific, will be shared in more detail to visualize the issue.
• Contributing Authors: Lisa-Marie Bauhofer, Sebastian Königsberger, Zdenka Cicova, Frances Brauer, Andrea Heinze, Alexander Knorre
  Eurofins BioPharma Product Testing Munich GmbH
• (Poster)

Equivalence Bounds for Similarity: Where and What is the Limit?

• Speaker: Erica Bortolotto, UCB Pharma
• Abstract: Similarity is the key assumption for relative potency assays. Failure to assess parallelism generates a meaningless relative potency that cannot be reported or interpreted. Different parameters can be used to assess the similarity but which one is the most appropriate parameter in the real life? Where is the limit to discriminate between a good or a bad result in routine?
• (Day 1: Determining Similarity of Reference Vs. Test Sample Dose-Response Curve Session)

Optimization of Cell-Based Relative Potency Assay for Recombinant FSH

• Speaker: Emma Bowen, Covance
• Abstract: Rekovelle™ (Ferring) is a recombinant human follicle stimulating hormone (rFSH) used for fertility treatment. A cell-based relative potency assay in a 96-well plate format was developed and optimised at Covance prior to release testing. Development of the assay involved simplification of the initial method to reduce within assay variation. Optimisation then involved assessments of cell seeding concentrations, incubation times and plate layouts as well as the setting of system and sample acceptance criteria. After optimisation a DOE-designed robustness study was performed prior to a comprehensive, successful, validation. Here we will look at the results and lessons learnt during optimisation, robustness assessment and validation of a manual 96-well plate potency assay.
• (Day 2: Product Specific Bioassays Session)

Secure and Traceable Acoustic Liquid Handling — An Integrated System for Regulated Laboratory Environments

• Presenter: Russell Burge, Labcyte Inc.
• Abstract: The Labcyte Key to bioassay success is implementation of multifactorial optimization approaches such as Design of Experiment (DOE) and sample randomization to control for inherent experimental biases. Both methodologies benefit greatly from automated liquid handling solutions that are reproducible, accurate and enable any-well to any-well liquid transfers. Acoustic liquid handlers utilize sound energy for rapid, non-contact any-well to any-well liquid transfer thus making them ideally suited to the needs of bioassay development and deployment in pharmaceutical, biotech and CRO organizations. Data quality improvements over manual or automated tip based liquid handling systems are readily measurable at significantly lower transfer volumes. Lower transfer volumes lead to reagent savings and enable direct dilution for dose-response testing eliminating dilution error propagation. Compared to automated tip-based systems, the complex transfer protocols required for DOE and randomization approaches are readily accommodated on acoustic liquid handlers and non-contact dispensing removes any sample/tip interactions eliminating sample loss due to adhesion to or contamination from the tip plastic itself. Within the listed organizations, bioassays are often run in FDA regulated environments and, as such, benefit from instrumentation that supports user access control, protocol traceability and versioning, protocol approval enforcement and controlled reporting. A barrier to adoption of acoustic liquid handling for bioassay work has been the lack of such controls. Now, Echo® Liquid Handlers can meet FDA 21CFR Part 11 requirements. Newly available software is tightly integrated with Echo® systems and software applications for a seamless experience. We will provide details on how such acoustic liquid handlers can be deployed in regulated labs.
• Contributing Authors: Iain Russell, Russell Burge, and Harry Vlahos
  Labcyte Inc., San Jose CA, USA
• (Poster)

Influence of Galactosylation on Fc-Mediated Binding and Functional Properties of Adalimumab

• Presenter: Agata Burzawa, BioReliance
• Abstract: The N-glycan profile of an antibody plays a critical role in product stability, immunogenicity or pharmacokinetics and can also significantly influence the Fc-region mediated effector functions of antibodies, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). β-galactosylation is known to be crucial for CDC activity, since terminal galactose enhances binding of antibody Fc regions to the C1q complement protein, which is required for CDC. β-galactosylated glycans can also increase antibody Fc-region binding to the FcɣIIIa receptor, which can give rise to enhanced ADCC activity.
  Product characterization is key to successful biological drug development. Comprehensive characterization of new therapeutic monoclonal antibodies requires a deep understanding of their structural and functional critical quality attributes (CQAs), which greatly impact product potency, stability and safety.
  We evaluated the criticality of glycan structure on antibody Fc-region mediated effector functions through the application of a highly resolving N-glycan assay, in combination with binding and cell-based assays. In order to demonstrate the influence of terminal galactose on the effector functions of adalimumab, we removed β-galactose from N-glycans using β-1,4 galactosidase and evaluated the impact of the changes in glycosylation profile on C1q and FcɣIIIa receptor binding properties and subsequent CDC and ADCC activity.
• (Poster)

Application of MOA-based Reporter Bioassays for Immunotherapy Drug Development

• Speaker: Jey Cheng, Promega Corporation
• Abstract: Developing a robust, sensitive, and relevant potency assay represents a substantive challenge especially for those products with complex or multiple MOA. We will present the latest reporter gene technology advancement and demonstrate how they can be used for a broad range of applications during antibody drug development in single and combination immunotherapy. In the case studies, we will present data on assay development and qualification for a Tim-3 Bioassay for anti-Tim-3 monoclonal antibody and combination bioassay development for bispecific antibody targeting PD-1 and a second immune checkpoint receptor.
• (Day 2: Product Characterization with Biological Assays Session)

Validating Multi-Plate Format Bioassays

• Speaker: Jette Christensen, Novo Nordisk
• Abstract: (Pending)
• (Day 1: Bioassay Lifecycle Activities Session)

Novel C1q Affinity Chromatography for Extended Characterization of IgG-type mAbs

• Speaker: Florian Cymer, Roche
• Abstract: Complement component C1q represents the IgG-binding receptor in the classical complement pathway and can eventually induce effector functions such as complement dependent cytotoxicity (CDC) as well as complement dependent cellular cytotoxicity (CDCC). Therapeutic monoclonal antibodies (mAbs) can induce complement activity as part of their mode of action and for mAbs lacking complement activity, absence of complement activity might have to be demonstrated in extended characterization experiments to assess safety aspects. C1q binding of therapeutic monoclonal antibodies (mAbs) is often used as a surrogate assay for CDC to characterize mAbs in early development, during comparability studies and during the assessment of critical quality attributes (CQA). Currently an ELISA format is widely used in the pharmaceutical industry to assess IgG-C1q interactions. Given the complex requirements for complement activation via C1q, the relative long time needed to generate ELISA results and the inability of the ELISA data to identify different binding-species in heterogeneous samples, we explored additional methods to assess C1q interaction. Here, we describe the development of a prototype C1q affinity column using engineered C1q headgroups. We use a wide panel of IgG1 mutants that are known to spontaneously form hexamers in solution, IgG1 mutants that do not interact with C1q and in vitro glycoengineered samples in several C1q interaction assays and compare the results to published data, which includes data from cell based assays. Our results demonstrate that C1q affinity chromatography can complement other binding assays and cover a wide range of C1q interactions, not possible by current methods. Furthermore, the method allows more rapid analysis of samples compared to an ELISA based binding assay and allows to identify different C1q binding species. The advantages and limitations of the different methods will be discussed.
• (Day 2: Product Characterization with Biological Assays Session)

From Cradle to Commercial:The Life of a Bioassay

• Speaker: Peter Day, Genentech
• Abstract: This presentation spans the life of a cell-based potency assay for a mAb, from phase I through preparation for BLA filing. As the molecule progressed through the clinical phases, method enhancements were made to improve performance, increase efficiency and meet regulatory expectations. Finally, the method was validated for testing of commercial lots.
• (Day 1: Bioassay Lifecycle Activities Session)

Why Bioassayists are Even More Important than Statistical Software

• Speaker: Stan Deming, Statistical Designs
• Abstract: I have always been of the opinion that bioassayists should be trained well, be treated well, and be paid well … and take pride in their work. Bioassayists provide the bedrock of information that drives the pharmaceutical industry.In my experience, the old saying “GIGO — garbage in, garbage out” holds especially true for bioassays. It is the responsibility of bioassayists to avoid blunders that generate errors in the assay results. If a bioassayist has “messed up,” the results should not be trusted as they float up the chain of information to the top-level decision makers. Unfortunately, these blunders often go unnoticed, or are thought to be “correctable” by proper statistical analysis. Yet Gerald Hahn reminds us that we should not rely on statistics to save us from these blunders: “… the world’s best statistical analysis cannot rescue a poorly planned [or executed] experiment….”This talk will illustrate some of the subtle blunders that can be made when a bioassay is carried out, blunders that should be avoided by a well-trained bioassayist.
• (Day 1: Statistical Analysis: Why and How Much? Session)

Cell-Based Potency Assays for QC: Considerations for Development and Analytical Transfer

• Presenter: Stu Dunn, Covance
• Abstract: Potency is the only critical quality attribute (CQA) that can demonstrate biological function. Assays used for QC release must be developed such that they are relevant to the intended mechanism of action (MOA) and relate to clinical response where possible. Potency assays are notoriously difficult to develop and transfer, requiring considerable expertise and understanding to both perform and troubleshoot. Furthermore, assays required for advanced therapeutic medicinal products add another level of complexity over that of large molecules due to several potential or even unknown mechanisms of action. This poster informs on key considerations, from over a decade of experience, in developing cell-based potency assays for QC release and to highlight challenges associated with the analytical transfer of cell-based potency assays.
• (Poster)

Determination of Biological Potencies of Antibody Drug Conjugates (ADCs)

• Presenter: Cornelia Fabritius, Eurofins BioPharma Product Testing Munich GmbH
• Abstract: Antibody-drug conjugates (ADCs) are an important class of highly potent biotherapeutics. The possibility of a targeted therapy of cancer increased the interest of research in this kind of immunoconjugates over the past decade.Simultaneously, testing the bifunctional mechanisms of action challenges the testing of these biopharmaceuticals. On the one hand bioassays have to address testing the specificity for the interaction of the unconjugated and conjugated antibody and its target. On the other hand the toxicity to target expressing cells has to be monitored. Additionally, focusing on different Drug Antibody Ratios (DARs) is crucial at early development stages as drug loading can impact the therapeutic efficiency of an ADC.
  Beside these scientific aspects handling of potential instable cytostatic material requires exceptionally safety instructions as well.Different perspectives to address these compounds in a bioassay laboratory were given on the basis of exemplary dose-response curves.
• Contributing Authors: Cornelia Fabritius, Andrea Heinze and Alexander Knorre
• (Poster)

Designing a MOA Reflective Reporter Gene Assay for an Immunomodulatory Antibody that Requires Fc Cross-linking for Agonism

• Speaker: Matt Gothard, MedImmune
• Abstract: T-cells require two types of signals from Antigen Presenting Cells (APCs) in order to become activated and initiate an immune response. The first signal is via the presentation of specific antigen on the MHC of the APCs to T-cell receptors. The second is a co-stimulatory signal that is mediated by the interaction of surface ligands on the APCs and specific co-stimulatory receptors on T-cells. In the absence of this co-stimulatory signal, T-cells that recognise antigen will either die or become anergic. There is currently great interest in manipulating this co-stimulatory signalling in order to enhance the functions of APCs and thereby increase the T-cell immune response. This could be a particularly useful way to amplify the anti-tumour immune response for cancer patients. We describe an immunomodulatory agonist antibody targeting a co-stimulatory receptor on T-cells. The Fc portion of this antibody binds to Fc receptors on the surface of APCs. This Fc crosslinking is essential to the function of the antibody. Whilst there is great therapeutic potential in such antibodies, they do present certain challenges when developing bioassays. In order to be truly reflective of the mechanism of action, both the Fc and Fab interactions should be demonstrated in a single bioassay. This presentation will discuss the design and development of a reporter gene assay for the detection and quantification of such an immunomodulatory antibody.
• (Day 2: Product Specific Bioassays Session)

Introduction to Design, Monitoring and Validating Cell-Based Bioassays

• Instructors: Bassam Hallis, Public Health England and Jane Robinson, BEBPA
• Abstract: This workshop is intended for those new to the field of bioassays for potency testing of biopharmaceuticals.
  Bioassays differ in important ways from other analytical techniques, presenting challenges for those new to the field. All too often, experienced bioassayists forget to explain the reasons behind the “obvious” practices they take for granted. An additional problem for many novice bioassayists is that initially they are involved in only one step of the process of assay development, validation and routine usage: understanding the full process can help in recognizing issues that may impact subsequent steps or suggest where to look for previous evidence of a problem.
  The workshop will concentrate on cell-based assays as these are widely used and illustrate the majority of issues encountered across all types of bioassay. We will cover the basic principles of bioassays, showing how appropriate assay design and analysis are essential to obtain meaningful results. We will discuss assay variability, the concept of relative potency and its practical implications, use and development of reference standards, setting assay acceptance criteria, monitoring, trending, and validation, all the time highlighting common problems and ways of avoiding them. Cell-based assays will be compared with other types of bioassay, and the advantages and problems of changing from one cell-based assay to another, or to a different type of bioassay, will be addressed.
  There will be opportunities for discussion of the workshop topics and participants are encouraged to raise their own bioassay questions and problems, hopefully leaving with solutions or helpful suggestions.
• (Workshop 2)

First Steps and Experiences with Liquid Handling in a GMP QC Bioassay Laboratory

• Presenter: Andrea Heinze, Eurofins BioPharma Product Testing Munich GmbH
• Abstract: Liquid handler instruments are already widely used by R&D groups in the biopharmaceutical industry. Also in a GMP Quality Control environment automation is getting more important.
  In parallel to our instrument validation, we investigated the liquid handler performance for one of our routine bioassays. Experiments were performed to automate steps of a bioassay, which are currently performed manually, as changing to an electronic pipet did not pass all acceptance criteria. Potencies at 100%, the lower potency limit and upper potency limit were tested.
  Cell seeding with a liquid handler as being one of the more variable steps of cell-based bioassays was investigated for different cell densities, over several plates and compared to analyst performance.
• Contributing Authors: Andrea Heinze, Frances Brauer, Sebastian Warnecke and Alexander Knorre
• (Poster)

The Challenges in Testing ADCs Including Characterization and Release Assays

• Speaker: Ulrike Herbrand, Charles River Laboratories
• Abstract: Antibody-drug conjugates (ADCs) add an additional level of challenge to testing of biotherapeutics. Besides the antibody, which needs to be evaluated including the potential and known mechanisms of action (MoA), there is a cytostatic compound conjugated, that alters the behavior of the antibody-vehicle within the typical assays. Assays to measure proliferation, apoptosis, ADCC, ADCP, internalization and cell cycle arrest for the model ADC Trastuzumab emtansine will be addressed.
• (Day 1: Bioassays for Product Release and Product Characterization Session)

Hitting the Gas: Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Co-Stimulatory Immune Checkpoint Receptors

• Presenter: Gopal Krishnan, Promega Corporation
• 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 autoimmunemediated 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, TIGIT, TIM-3) and co-stimulatory (e.g. GITR, 4-1BB, OX40, CD40, CD27, HVEM) 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 line-based bioluminescent reporter bioassays for co-stimulatory immune checkpoint targets including GITR, 4-1BB, OX40, CD40, CD27, HVEM and more. These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of intracellular signals mediated by each immune co-stimulatory receptor. These bioassays reflect mechanisms of action for the drug candidates designed for each co-stimulatory 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: Jun Wang, Michael Beck, Jamison Grailer, Jim Hartnett, Gopal Krishnan, Frank Fan, Mei Cong and Zhi-jie Jey Cheng Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• (Poster)

Novel, Improved Cell-Based Assays to Enable Immunotherapy Drug Development for Checkpoint Receptors

• Presenter: Jane Lamerdin, Eurofins DiscoverX
• Abstract: Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therefore, therapeutics that block inhibitory receptors or activate immuno-stimulatory checkpoint receptors have proved to be powerful agents to restore anti-tumor immune responses. However, developing drugs targeting these checkpoint proteins has proved to be quite challenging as cell-based assays used to screen for functional drugs are often difficult to create, involve the use of human primary cells, and have long, complicated protocols. Here, we present data for several new PathHunter® Checkpoint assays that target clinically relevant co-inhibitory and co-stimulatory checkpoint receptors using the industry-validated Enzyme Fragment Complementation (EFC) technology. The PathHunter assays measure receptor activation and signaling for co-stimulatory receptors and co-inhibitory receptors. These assays facilitate the development of relevant therapeutics, enabling rapid and sensitive screening of biologics and small molecules. Furthermore, the robustness and reproducibility of these assays lend themselves well to use in lead optimization, relative potency, and QC lot release testing of immunotherapy drugs. These Mechanism of Action-based, biologically-relevant, cell-based assays do not require human primary cells and have an easy-to-use protocol, providing a highly sensitive response in less than 5 hours for the PD-1 Signaling assay. Here, we present data for assays targeting a number of clinically important immunotherapy targets, including PD-1 (with PD-L1 and PD-L2), SIRPα, and OX40.
• Contributing Authors: Jane E. Lamerdin, Hanako Daino-Laizure, Mimi Nguyen, Vicki Liu, Alexander Baumann, and Jennifer Lin-Jones. Affiliations Eurofins DiscoverX, USA
• (Poster)

Standardization and Automation of QC Bioassays by Acoustic Dispensing

• Speaker: Aurore Lejeune-Dodge, Labcyte (UK)
• Abstract: (Pending)
• (Day 1: Handling Outliers for Bioassays Session)

Bioassay Town Hall: A Discussion of Practical Issues with Establishing Assay and Test Sample Criteria for Bioassays

• Instructors: Laureen Little, BEBPA and Nancy Niemuth, Battelle
• Abstract: Featuring: “The Friendly Statistician” (Nancy Niemuth) and Your Overworked, Confused Bioassay Developer (Laureen Little)
  In small towns in the United States, when there is a problem that impacts those who live in town, they call a town meeting. Neighbors attend and discuss the problem, share ideas and sometimes just commiserate about the problem. It is a meeting of those affected by the problem, to openly discuss the issues and decide on a path forward. That is what this workshop aims to do. I (Laureen) have been actively establishing similarity criteria for assays that are currently being used to support commercial and soon to be approved products. I have run into a multitude of problems not covered in the USP, other publications or even in this conference. Some of these problems?
  Non-normally distributed data – some of which can be transformed, but some that can’t
  Problems with equivalence margins failing similarity because of low variability rather than lack of similarity
  Handling Chi Square Distributed data sets which are really “truncated” normal curves which are the result of a physical barrier. For example, the fit criteria for the dose-response curve – which can’t get a better fit than perfect
  How to establish criteria with a small dataset
  What about setting criteria for the confidence limits for the final reportable value? How do I do this when the format (multiple plates) is only established post validationMy go to friendly statistician this past year has been Nancy Niemuth, as I was enrolled in a statistics for bioassays course she taught. She provided paths forward for these and other problems and I wanted to share them with you.
  This workshop will start with a quick overview of some basic concepts. Then we will all be given some example data (not case studies – sorry those data aren’t mine to distribute, but the data sets will mimic the problems I have encountered) and we, as a group will work on problems at hand. Please bring your computers with some statistical software (even if only excel) and be prepared to jump in and give us your ideas.
• (Workshop 1)

Current Practices for Reference Materials

• Speaker: Laureen Little, BEBPA
• Abstract: (Pending)
• (Day 2: Interactive Survey)

Introduction to Outliers from a Regulatory Perspective

• Speaker: Laureen Little, BEBPA
• Abstract: (Pending)
• (Day 2: Handling Outliers for Bioassays Session)

Optimization and Qualification of an PO Biosimilar Bioassay Using a Commercial Tf-1 Reporter Gene Cell Line

• Presenter: Nena Lopez Lee, Intertek Manchester
• Abstract: A biosimilar product is a biological product that is approved based on the demonstration that it is highly similar to an already-approved biologic product (reference product). To ensure a biosimilar meets safety standards, rigorous testing needs to be completed looking at physicochemical, structural and potency attributes carried out to internationally recognized guidelines, such as ICH Q6B. Erythropoietin (EPO) is a glycoprotein hormone produced by the kidneys to stimulate the production of red blood cells (also known as erythropoiesis) and is an essential drug for patients with Chronic Kidney Disease (CKD) and cancers resulting in anaemia. When EPO binds to its receptor (EpoR) the Jak-Stat pathway is activated, resulting in red blood cell proliferation (Figure 1). The rising incidence of these diseases has been a major growth driver for the global EPO market, resulting in a surge in the need for EPO biosimilars. According to a report by Grand View Research, the EPO drug market is predicted to reach around 17.4 Billion USD by 2025 [1]. In this poster Intertek summarize the optimization and validation of a commercially available reporter gene assay for EPO, that was used to compare the biological activity of EPOGEN® and Eprex® Erythropoietin products. This commercially available cell line was chosen in order to accommodate a tight customer deadline and to reduce overall development costs. This comparison study was used to evaluate the future application of this bioassay in supporting EPO biosimilar development. These brands were chosen in order to cover the 2 main types of EPO formulation; HSA based and Tween based. A phased approach was adopted where the assay was initially run using the supplier’s protocol. The method was then optimized, with system suitability criteria being assessed and established. The optimized method was then validated to GMP standards.
• Contributing Author: Nena Lopez Lee, Senior Analyst, Intertek Manchester, UK
• (Poster)

The Development and Challenges of a Cell-Based Bioassay for a Bi-Specific Molecule Targeting a Novel Target Therapeutic Target

• Speaker: Thomas Maguire, Janssen Sciences Ireland UC
• Abstract: Janssen is a subsidiary of Johnson and Johnson and aims to prevent, treat and cure some of the most complex human diseases. Traditionally, the Janssen large molecule clinical portfolio has focused on developing monoclonal antibodies (mAbs). MAbs are bivalent, in that there are two binding sites per molecule capable of binding the same target antigen. More recently Janssen has been developing new therapeutic modalities beyond mAbs. As part of this portfolio diversification Janssen is evaluating Bi-specific antibodies (BsAbs) scaffolds which while bivalent, will target or bind two different antigens. This allows BsAbs to offer potential advantages over other monoclonal antibody formats, particularly in oncology, where they may target more than one tumour antigen on a cancer cell or engage the immune system by binding to a cancer cell and then recruiting cells of the immune system.
  However, with increased complexity in therapeutic modality comes greater analytical challenges particularly for potency assays to demonstrate the mode of action of the BsAbs. Potency assays are required to reflect the mechanism of action of the therapeutic agent, therefore for BsAbs more than one cell type may be required or multiple potency assays may have to be developed to demonstrate different mechanisms of action. This presentation gives an overview of the approach that Janssen has taken to developing a range of binding and cell based potency assays to monitor BsAbs in clinical development and the challenges faced in supporting a diverse BsAbs portfolio. Data from one such assay will be presented detailing the specific hurdles that were overcome such as the availability of commercial antigen, limited data sets for specification setting and correlation with functional assays.
• (Day 2: Product Specific Bioassays Session)

Improving Throughput by Optimizing Assay Design and Data Analysis

• Speaker: Mary Matheson, Public Health England
• Abstract: The traditional approach to bioassay design and data analysis often involves a complex set of suitability criteria designed to select data points suitable for back calculation from the reference curve to assign a result value. This method is reliant on using multiple dilution series and replicates. This presentation will describe an alternative approach that produces equivalent results to the traditional design, but with the advantages that the number and complexity of the suitability criteria can be greatly reduced. Using this analysis we found that the number of dilutions per test sample can be reduced from 8 to 4, with almost no effect on the results, thus doubling the throughput of the assay. Coupling this with analysis software that provides automated QC and QA will result in a major increase in laboratory capacity.
• (Day 1: Bioassay Lifecycle Activities Session)

Reproducible MOA-Reflecting Reporter-Based Bioassays to Enable Drug Development of Biosimilars and Biobetters

• Presenter: Reka Nagy, Promega Corporation
• Abstract: Cytokines and growth factors can be described as small immunomodulatory proteins secreted by a wide variety of cells including fibroblasts, endothelial and stromal cells, whose role it is to regulate surrounding cells in an autocrine, paracrine or endocrine fashion. Immunocytokines represent a promising class of activators of the immune system, with the potential to be used alone or in combination with other therapeutic modalities. Many are currently FDA approved therapy agents (e.g. IFN, IL-2 and Epo), while others are targets for approved biological blocking therapies to support treatment of a variety of diseases. Examples of cytokine blocking agents include basiliximab (IL-2R), tocilizumab and sarilumabad (IL-6R), siltuximab (IL-6), ustekinumab and its biosimilars (IL-12/IL-23 p40), secukinumab (IL-17A), bevasizumab (VEGF), and denosumab (RANKL). Many more are in development and trials as biosimilars and biobetters. IL-2 and IL-15 are still clinically important cytokines as researchers look to improve potency, patient tolerance and response by developing new molecules with sustained and targeted activities. We have developed luciferase reporter bioassays which individually can be used for the quantitation of a variety of cytokines and growth factors including IL-2, IL-6, IL-12, IL-15, IL-17, IL-23, VEGF and RANKL using respective mechanism of action pathways. The bioassay format is based on thaw-and use cells, eliminating the need to establish and pre-culture cytokine responsive cell lines which provides the benefits of convenience, reproducibility, and transferability. We demonstrate these assays measure cytokine response and inhibition with blocking drugs or potency changes from stressed samples. In summary, these reporter-based bioassays provides valuable tools for the development, stability testing, and potency determination in the manufacture of cytokine biosimilars and biobetters.
• Contributing Authors: Richard Moravac, Dun Li, Jennifer Wilkinson, Reka Nagy, Frank Fan and Mei Cong Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• (Poster)

Your Statistician is Your Friend: Optimizing Reagent Qualification using Equivalence Tests

• Speaker: Nancy Niemuth, Battelle
• Abstract: (Pending)
• (Day 1: Bioassay Lifecycle Activities Session)

Vaccine Potency Assays – Past, Present and Future. Influence of Statistics and Changing Methodologies/ Approaches

• Speaker: Gareth Rees, Porton Biopharma
• Abstract: (Pending)
• (Day 1: Bioassays for Product Release and Product Characterization Session)

Replacement of the In-Vivo Bioidentity Rabbit Blood Sugar Test (USP <121>) with an In-Vitro Bioidentity Cell- Based Assay

• Speaker: Sabrina Rüggeberg, Sanofi
• Abstract: Each insulin batch intended for the US market has to be released with an in-vivo bioidentity test according to USP chapter <121>. An in-vitro cell based assay (In-Cell Western – ICW) was developed and validated to substitute the in-vivo bioidentity assay for all batch release and stability tests. It was approved by FDA for Insulin Glargine, Lispro, and Glulisine. Furthermore, this in-vitro bioidentity assay (ICW) is planned to be implemented in USP chapter insulin <121> (pre-published in USP Pharmacopoeial Forum 43(4)). The ICW is a platform method applicable to (nearly) all insulins and insulin analogs. Insulin / analogs bind to the human insulin receptor, transfected into CHO cells, leading to an auto-phosphorylation. This auto-phosphorylation is the first step of a signaling cascade which in the end decreases the blood glucose level. The ICW can measure three insulin test items per assay plate in a 96 well format, with two assay plates leading to one reportable result.
• (Day 2: Assay Replacement Session)

How Innovation of Drugs Shapes Modern Bioanalysis and Potency Assessment – The Rise of ATMPs and the Challenge of Appropriate Assays

• Speaker: Melody Sauerborn, Scientia Potentia Consultancy
• Abstract: Innovation is a pivotal process in drug development to address unmet medical needs. Advanced therapy medicinal products (ATMPs) are amongst these innovations and the number of ATMPs entering clinical stages has been substantially increasing over the last few years. Bioanalysis and potency assessment has proven to be a challenge, as the mode of action for cellular therapeutics is often not fully characterized. Currently most companies apply a multi-assay approach, often consisting of cellular surface analysis, cytokine release and one form of functional cell-based assay. The challenge arises when these assays need to be validated. While walking you through some case studies, we will address these assay and regulatory challenges and how they can be handled.
• (Day 1: Bioassays for Product Release and Product Characterization Session)

iLite® ADCC Assay – A Novel System for the Quantification of ADCC Activity

• Presenter: Therese Segerstein, Svar Lifesciences
• Abstract: The clinical activity of a number of monoclonal antibodies is mediated in part by antibody-dependent cell-mediated cytotoxicity (ADCC). Traditional methods for quantifying ADCC activity are labor intensive and have a high level of inherent variability due to the use of primary human NK cells from different donors as the effector cells. These limitations can be overcome by part by the use of engineered effector cells line, however there is a need for an ADCC assay with improved characteristics in regard to sensitivity, specificity and tolerance to serum matrix effect such as human serum etc.
  Here we present a novel ADCC effector cell expressing the V-variant of the FcγIIIa receptor and the firefly luciferase (FL) reporter gene under the control of a chimeric promotor incorporating recognitions sequences for the principal transcription factors involved in the FcγIIIa signal transduction, together with novel target cells overexpressing a constant high level of a specific antigen. Secondly, homologues control target cells, in which the specific target gene has been invalidated by genome editing, provides an ideal control. Furthermore, the cells also contain a normalization gene rending ADCC activity independent of cell number and serum matrix effect.
• (Poster)

Improved T Cell Activation Bioassays to Advance the Development of Bispecific Antibodies and Engineered T Cell Immunotherapies

• Presenter: Richard Somberg, Promega Corporation
• Abstract: T cells play a central role in cell-mediated immunity and can mediate long-term, antigen-specific, effector and memory responses. In recent years, a variety of immunotherapy strategies aimed at inducing, strengthening or engineering T cell responses have emerged as promising approaches for the treatment of diseases such as cancer and autoimmunity. Current methods used to measure TCRmediated T cell proliferation and cytokine production rely on primary PBMCs as a source of T cells, which must be stimulated via co-culture with APCs or anti-TCR/CD3 antibodies. These assays are laborious and highly variable due to their reliance on donor primary cells, complex assay protocols and unqualified assay reagents. As a result, these assays are difficult to establish in quality-controlled drug development settings. To overcome this barrier, we developed two reporter-based bioluminescent T cell activation bioassays that can be used for the development of bispecific antibodies and engineered T cell immunotherapies. The assays consist of Jurkat T cells genetically engineered to express luciferase downstream of either NFAT or IL-2 response elements. The T cell activation bioassays reflect the mechanisms of action of biologics designed to induce TCR and/or CD28-mediated T cell activation, as demonstrated using antiCD3 and/or anti-CD28 antibodies as well as blinatumomab, a bispecific antibody that simultaneously binds CD3 expressed on T cells and CD19 expressed on malignant B cells. The bioassays are pre-qualified according to ICH guidelines and show assay specificity, precision, accuracy and linearity required for routine use in potency and stability studies. Finally, our data illustrate the use of reporter-based T cell activation bioassays for characterizing and measuring the activity of engineered chimeric antigen receptor T cells.
• Contributing Authors: Pete Stecha, Denise Garvin, Jim Hartnett, Richard Somberg, Frank Fan, Mei Cong and Zhi-jie Jey Cheng Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• (Poster)

Comparison of Outlier Tests for Potency Assays

• Speaker: Perceval Sondag, Pharmalex Development Services LLC
• Abstract: When statistical outliers are present in Potency Assays, similarity testing may be compromised and Relative Potency (RP) estimates may be biased. The USP chapter <1032> recommends to screen for outliers before RP analysis. In this talk, several prominent outlier detection methods are discussed and compared. Three types of outliers are examined:
  Single observation outlier: a single measurement is excessively distant from the other values.
  Concentration point outlier: all the replicates at one concentration level exhibit a different behavior than the values at the other concentration points.
  Whole curve outlier: one of the curve replicates is affected by a manipulation error.
  The latter is common but completely ignored by the current literature. This talks presents a test that would be easy to implement in Bioassay software, for that type of outlier analysis.
• (Day 2: Handling Outliers for Bioassays Session)

Case Studies for Establishing Equivalence Margins for Similarity Assessments

• Speaker: Lasse Stolzenbach Wæhrens, Novo Nordisk
• Abstract: Setting SST criteria is an important decision for any method. In potency assays a p-value for parallelism have been and are used as an SST criteria. However, using a p-value can be challenging; Experiments can fail, as it should, due to obvious nonparallel curves, however it can also fail when it should not. This could be due to a method with relatively low variation between duplicate measurements of concentrations. As experience were made within the potency community, a substitute for the p-value, the equivalence test, has been adapted. By implementation of equivalence test instead of a p-value new challenges have emerged. Even though USP chapters gives guidelines to how to set equivalence margins, it is still a new way of thinking thereby mediating discussions within labs on how to set the equivalence margins. Especially when developing a new assay. How much data do we need? When do we lock the margins and so forth. This talk will discuss setting equivalence margins using case studies. This will be done both from the perspective of starting a new method, but also for changing from p-value to equivalence test for existing methods.
• (Day 1: Determining Similarity of Reference Vs. Test Sample Dose-Response Curve Session)

Enzyme Assays Made Easy Using Artificial but Physiologically Relevant Substrates

• Presenter: Chao Su , Swedish Orphan Biovitrum AB
• Abstract: Enzymes represent the most diverse kind of all proteins, catalyzing most chemical reactions in all cells and organelles. They can occur as interfacial players such as lipases, or clusters like the lysosomal hydrolases acting in a concerted way on mucopolysacchrides that are structurally heterogeneous. With difficulty for natural substrates in drug discovery, artificial ones are frequently used in enzyme assays. This will facilitate the drug development enormously in terms of resources pooled. The status quo is one must demonstrate the physiological relevancy for regulatory requirements. Three examples in enzyme replacement therapy (ERT) are discussed: Bile-salt-stimulated lipase (Kiobrina) using p-nitrophenyl butyrate (PNPB), idursulfase (Elaprase) using 4-methylumbelliferyl-α-L-iduronide-2-sulphate (MU-αIdo2S) and cerebroside-sulfatase using p-nitrocatechol sulfate (PNCS).
• (Poster)

Development and Qualification of Cell-Based Functional Bioassays for Insulin Analogues for Comparability Studies

• Presenter: Atul Tiwari, Syngene
• Abstract: Insulin represents the mainstay of therapy for the treatment of Type I and Type II Diabetes. With a globally increasing incidence and prevalence of diabetes and associated complications, an increasing focus lies towards the development of insulin analogues with different duration of actions. However, the development of biosimilar drugs demands disease relevant and/or mechanism of action based functional bioassays, which are robust, rapid, reproducible and QC-complied for demonstrating the similarity and establishing equivalence to the originator. Based on EMA guidelines the biological activity of insulin analogues need to be compared at two levels- receptor autophosporylation and metabolic activity. The metabolic activity assays can be performed in different cell types for varying end points including glycogen synthesis, lipogenesis, inhibition of lipolysis, and glucose uptake. Likewise, mitogenic activity assay mediated by IGF-1R though this might not be relevant to human insulin and most of its analogues, is carried out for addressing any potential safety concern. Here, we present data on the development and qualification of cell based functional bioassays for insulin analogues for comparability studies. The bioassays for insulin are not only of long-term duration but are of low throughput. Data will be presented for the approaches used towards the optimizations of different assay conditions, associated challenges and troubleshooting to make the assays robust, reliable and reproducible with higher throughput and faster turnaround times.
• (Poster)

Adventures in Liquid Handling (96- to 384-well format)

• Speaker: Paula Urquhart, Covance
• Abstract: (Pending)
• (Day 1: Improving Assays Using Technology Session)

Picking Up Product Degradation by Bioassay – A Healthy Balance Between Function and Structure

• Speaker: Annemie Wielant, UCB Pharma
• Abstract: Changes in the protein structure of a biopharmaceutical molecule can impact its biological function and generate safety concerns for the patient. For this reason, the stability of the molecule is constantly monitored by a large pool of physico-chemical and bioassay methods.
  However, the use of a bioassay as “stability-indicating” method can be questioned as most of the time the bioactivity of the product is only affected by strong forced degradation conditions and not by the conditions used during regular stability studies.
  To date, correlating the changes in higher order structure and the functional activity of the molecule is quite a challenge. By analyzing the same forced degraded samples by both native peptide mapping (as physico-chemical technique) and bioassay and by correlating the conformational changes at the complementary determining regions of the molecule with the bioassay results, this potential relationship could be revealed.
• (Day 2: Product Characterization with Biological Assays Session)

More than the Mean of its Parts: Combining Run Results (and other variance components issues)

• Speaker: Ann Yellowlees, Quantics Biostatistics
• Abstract: We will discuss the European Pharmacopoeia and US Pharmacopeia guidelines for combining relative potencies from multiple assays. Each guideline discusses three approaches; we will outline the advantages and disadvantages of each approach. We will explore the use of historical data and Bayesian methods, and we will use examples and simulations to compare the approaches and to illustrate problems with the guidelines, and recommend alternative approaches which overcome these.
• (Day 1: Statistical Analysis: Why and How Much? Session)

BEBPA: Best Emerging Biomathematical Practice for bioAssay

• Speaker: Ian Yellowlees, Quantics Biostatistics
• Abstract: (Pending)
• (Day 1: Statistical Analysis: Why and How Much? Session)

Making Changes to Bioassays for Commercial Products

• Speaker: Wei Zhang, Biogen
• Abstract: This presentation will discuss implementing changes to bioassays already approved for commercial products as part of lifecycle management of analytical methods. The focus will be on how decisions have made in two case studies on whether to make modifications to the existing assay, or to develop a new, better assay.
• (Day 2: Assay Replacement Session)