Bioassays are a critical component of biologics drug discovery and development. With increasingly complex biologic modalities coming to market, including those with multiple mechanisms, accurate biological measurements are crucial. Cambridge Healthtech Institute’s Fourth Annual Optimizing Bioassays for Biologics conference will showcase strategies for biologics with multiple modes of action while bridging two perspectives on bioassay development: biology and statistics. At this event, industry leaders will showcase strategies for assay selection, validation, transfer, and maintenance with an emphasis on molecules with complex mechanisms. Health authorities will weigh in on new guidelines as well as provide insight into what they consider a well-characterized biologic. Finally, new technologies and bioassay formats will be presented along with recommendations for implementation to ensure a steady drug development pipeline.

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THURSDAY, OCTOBER 27

Assay Bridging, Transfer and Validation

2:00 pm Chairperson’s Remarks

Melody Sauerborn, Ph.D., Head, Research and Development, Mymetics BV

2:05 From Cell Line to Qualified/Validated Bioassay

Melody Sauerborn, Ph.D., Head, Research and Development, Mymetics BV

Cells are the pivotal part of any bioassay, being a Nab assay or a potency assay. But they are also a source of great variability. This presentation will focus on how to develop a cell line into an acceptable bioassay. We will look at early, intermediate and late cell responses, matrix effects and monitoring bioassay performance over time.

2:35 Essentials in Bioassay Design, Relative Potency Determination and Validation

Thomas Little, Ph.D., President, Bioassay Sciences, Thomas A. Little Consulting

This presentation covers essential concepts in bioassay design and validation. Topics include data transformation, curve fitting, weighting, masking, outlier detection and removal. A framework to control standards and reference standard stability will be presented including assay design space. Parallel line analysis for PLA linear models and 4PL Models will be discussed including equivalence testing, ratios and statistical tests will be compared. Acceptance criteria for bioassay validation will be discussed with rationale following USP 1033 and other guidance. A platform approach to bioassays will also be discussed to simplify development and validation.

3:05 Binding Assay as a Surrogate Assay for Functional Assay in Lot Release and Stability Testing: A Case Study

Ravish Patel, Ph.D., Scientist, Analytical Development Lab, EPR Centre for Cancer Research & Bioinformatics Pvt. Ltd. (A Vitane Group company)

Potency is considered a CQA for any biological product. The biological activity is the link between clinical response and activity measured in a bioassay. Functional assays vary in their underlying technology, their design and their complexity. Binding assays may be used as a surrogate for mAb potency testing if binding (Fab) is enough for MoA and if no effector functions (Fc) are involved in the antibody’s biological effect/binding activity.

3:35 Optimizing and Qualifying Bioassays for Biosimilars, Biobetters, and Innovator Drugs

Alpana Prasad, Ph.D., Product Manager, Marketing, DiscoverX Corporation

Developing robust cell-based bioassays for potency testing of biologic drugs is challenging due to the development and optimization required for each assay. We will discuss and demonstrate how DiscoverX cell-based assays accelerate the development of accurate and reproducible bioassays for biosimilar candidates and molecules with novel mechanisms of action.

3:50 Refreshment Break in the Exhibit Hall with Poster Viewing

Working with Multi-Specific Antibodies: ADCs, Bispecifics, and Beyond

4:30 Two Case Studies: Picking the Best Potency Assay for a Bispecific Antibody

Peter Day, Ph.D., Scientist, Genentech

Consideration of mechanism of action (MOA) is central to the design of any potency assay but the importance of MOA is multiplied in the case of bispecific antibodies. There are several factors, unique to bispecifics, which need to be considered when developing the potency assay(s). Does the MOA require bridging of the two targets? Is the binding cooperative or independent? Are the affinities for the targets similar? Here, we present two case studies on the unique challenges inherent in the development of potency assays for bispecifics.

5:00 A High Throughput Platform for the Quantification of the Potency of Multifunctional Biologics

Michael Tovey, Ph.D., INSERM Director, Research, Laboratory of Biotechnology & Applied Pharmacology, Ecole Normale Supérieure de Cachan

Multifunctional biologics including bispecific monoclonal antibodies, antibody-drug conjugates (ADCs), & pegylated trans-signaling fusion proteins have been developed that have several functional epitopes and/or a multifunctional action. Such products present significant challenges for the development of assays that can assess the potency of each component. A novel molecular engineering approach has been used to resolve these challenges. A high throughput platform has been established based on the use of 384-well microtiter plates and unique multiple readout reporter gene assays for products including trans-signaling fusion proteins and bi-functional monoclonal antibodies that target FGF21, Her2, VEGFR2, and that exhibit ADCC activity.

5:30 The Development of LC-MS Based Bioassays for a Novel Class of Payloads

Raymond Xu, Ph.D., Associate Director, Clinical Pharmacology, Immunogen Inc.

Antibody–drug conjugates (ADCs) represent an increasingly important approach to cancer treatment by targeting the delivery of cytotoxic agent to tumor cell type of interest. ADCs are generally complex heterogeneous mixtures of multiple in-vivo drug species and present unique challenges to bioassay development. We will present some examples from our experience in the development of LC-MS based quantitative bioassays for the measurement of a new class of payloads from biological matrices.

6:00 End of Day One

6:30-9:30 Dinner Short Course: Strategic Bioassay Design and Analysis

Separate registration required.

FRIDAY, OCTOBER 28

8:00 am Chairperson’s Remarks

Liming Shi, MS, MA, Senior Group Leader, Bioassay Development, Hospira, a Pfizer Company

Emerging Formats and Technologies

8:05 Something Old, Something New: Strategies for Challenging Biological Assay Development

Matthew Roberts, Ph.D., Investigator, Biopharmaceutical Analytical Sciences, GlaxoSmithKline

Implementing assay formats that appropriately represent the mechanism of action or binding modality of recent biologic molecules poses many challenges to development. This is due to the increasingly complex biologic modalities under development and the biological processes these molecules affect. Non-cell based assay formats utilizing classic ELISA readout in combination with innovative commercial technologies are being used to overcome these challenges. Representative case studies highlighting recent progress will be presented.

8:35 In vitro Functional Bioassays for Immune Checkpoint and Immune Cell Metabolism Screening

Sofie Pattijn, CTO, ImmunXperts

With the new wave of candidate cancer therapies acting mainly on the immune system via immune checkpoint blockade and immune cell metabolism, the demand for in vitro characterization of these molecules has significantly increased. This presentation will give a technical overview of a series of in vitro assays using human primary cells used for the characterization of new leads. Also, the major benefits, limitations and challenges will be discussed.

  9:05 Immunogenicity Assay Optimization: Ways to Reduce Background and Increase Assay Sensitivity

Jamil Hantash, MSc, MBA, Vice President, Pharmaceutical Services, Intertek Pharmaceutical Services

Immunogenicity assays (ADA assays) are required for almost all biologics FDA submission packages. Such assays are required to have very low sensitivities to allow clinical directors to make decisions quickly during a clinical trial. Sensitivity is directly proportional to assay format and background. The three key reagents in any given format are the blocking buffer, the diluent used to perform the MSR and the buffer used to prepare detection reagents. In this short presentation we will examine few tricks and approaches to lower background of the assay resulting in an increase of the assay sensitivity.

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

11:15 Bioassay Strategies for Assessment of Co-Stimulation in Immuno-Oncology

Tara Stauffer, Senior Scientist, Bristol-Myers Squibb

Co-stimulatory immuno-oncology therapeutics present unique challenges when developing QC suitable bioassays due to complex mechanisms of action. Translation of these challenges into workable strategies spans the lifetime of a molecule from assay development through commercial transfer. In this talk, we will focus on how the unique nature of co-stimulatory immuno-oncology bioassays are addressed from a practical perspective.

11:45 Design of Experiment (DOE) to Expedite Potency Assay Development for Biologics

Ashley Mullan, Associate Scientist I, MedImmune

Design of experiment (DOE) is a useful approach to evaluate multiple variables systematically. More specifically DOE may be used to develop potency assays for biologics in an expedited fashion. Demonstrating biological activity of novel molecules including novel non-mAb is challenging due to limited prior knowledge and structural complexity of such molecules. The traditional ‘one factor at a time’ approach to assay development is not ideal for novel molecules as it can fail to identify the most optimal settings and gives little information on the impact of changing assay parameters. This presentation will include case studies showing how a DOE approach was used during the development of potency assays for non-mAb molecules.

12:15 Hitting the Gas: Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Co-Stimulatory Immune Checkpoint Receptors

Richard Somberg, Ph.D., Strategic Collaborations Manager, Promega

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, TIGIT, Tim-3) and co-stimulatory (e.g. GITR, 4-1BB, OX40, CD40) 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 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. <br /><br />In summary, these reporter-based bioassays can serve as powerful tools in immunotherapy drug development for antibody screening, potency testing and stability studies.

12:45 Luncheon Presentation: Strategies for Overcoming Interference in Immunogenicity Assays

Sebastien Boridy, BSc, Ph.D., Research Scientist, Immunology, Charles River

While drug interference is a well-known phenomenon in immunogenicity assays and is well characterized during method development and validation, interference due to soluble targets or the nature of the drug occurs only in particular circumstances. As molecules being developed are more and more complex, method development groups are now being faced with increasing interference issues. During this presentation, the challenges associated with and different strategies used to overcome interference will be discussed in several case studies.

1:15 Awarding of Poster Prize PLUS Coffee and Cupcakes in the Exhibit Hall

Statistical Tools for Bioassays

2:00 Chairperson’s Remarks

David Lansky, Ph.D., President, Precision Bioassay, Inc.

2:05 Using Simulation to Derive Acceptance Criteria for Parallelism Tests in Bioassays

Perceval Sondag, Senior Statistician, Non-Clinical Statistics, Arlenda

Both US and European Pharmacopeia require that parallelism be shown to compute relative potency. Classic tests (Chi-squared, F-ratio) are known to have poor performance, while computation of equivalence margins can be both difficult and costly, due to the need for sufficient reference product data. A case study will be presented that describes the use of simulation to derive the equivalence margin without sacrificing accuracy, while substantially reducing the need for reference product experimentation.

2:35 Good Bioassay Designs: Practical, Statistically Sound, and Ready for DOE

David Lansky, Ph.D., President, Precision Bioassay, Inc.

Biologists and statisticians have different criteria for ‘simple’ bioassay design. Common laboratory practices (i.e.; multichannel pipettes) induce statistically complex designs. Good compromise designs and their statistically sound analyses are presented. Equivalence testing for similarity brings new performance requirements. Modular designs allow protocol adjustments to achieve adequate precision for equivalence tests, adequate precision of potency for various intended uses of the assay, or to meet block size requirements for DOE.

Design-of-Experiment in Support of Antibody Product Development

3:05 Accelerating the Speed of Optimization and Validation through Design of Experiments

Martin Kane, Managing Data Scientist, Statistical and Data Sciences, Exponent

Design of Experiments (DOE) are a powerful set of statistical tools that can be used in product and process development to increase knowledge, reduce waste, and innovate at an accelerated pace. This presentation will outline general DOE concepts for factorial and fractional factorial DOE's and describe several DOE’s that were used in a biopharmaceutical product. It will demonstrate how the DOE tool will enable the acceleration of getting product out of the lab and into clinical trials.

3:35 Applying Stepwise DOE Method to Optimize Neutralizing Antibody Bioassay with Different Formats

Jenny Hu, Scientist, PKDM, Amgen, Inc.

The design-of-experiment method has been adopted in the development and optimization of neutralizing antibody bioassays. Due to the complex nature of bioassays, many critical factors are involved and need to be assessed. In this presentation, two case studies representing two different assay formats will be presented to show how stepwise DOE helps to accelerate the optimization process of all critical factors to achieve the desired assay sensitivity.

4:05 Close of Conference

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