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 Third Annual Optimizing Bioassays for Biologics will showcase strategies for biologics with multiple modes of action while bridging multiple perspectives on bioassay development. 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.
Who should attend: Optimizing Bioassays for Biologics will bring together key individuals from bioanalytical R&D, cell and antibody engineering, immunology, pharmacology, and preclinical and clinical development.
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WEDNESDAY, NOVEMBER 18
2:00 pm Chairperson’s Opening Remarks
Max L. Tejada, Ph.D., Senior Research Scientist II, Analytical Operations, Gilead Sciences
2:05 Back to the Future, Envisioning the Next Generation Bioassay Development
Han Li, Ph.D., Principal Scientist, Lead Discovery and Optimization, Bristol Myers Squibb
Bioassay development requires a unique combination of techniques, from cell line selection and generation, to assay design, validation and final testing. An ideal bioassay needs to reflect true MOA, robust, sensitive and reproducible. Our thoughts on
a bioassay development incorporating advance technologies from in house and vendors will be presented. Our ultimate goal is to develop a system that will have broad and long lasting applications.
2:35 Development of Luminex as a Platform for the Detection of Anti-Drug Antibody IgE Isotypes in Human Serum
LiNa Loo, Ph.D., Senior Scientist, Bioanalytical Development, Merck
Since biotherapeutic drugs such as monoclonal antibodies (mAbs) have the potential to induce immunogenicity, it is critical to perform an immunogenicity assessment to ensure drug efficacy and patient safety. Here, Luminex and Mesoscale were evaluated
as platforms for detection of anti-drug antibody IgE isotype in human serum. By using a mouse-human chimeric drug-specific monoclonal IgE antibody as the positive control, the assay characteristics were compared for the two platforms.
3:05 Ready-to-Use Potency Assays for Anti-VEGF Drugs Like Bevacizumab
Abhishek Saharia, Ph.D., Director, Biologics, DiscoveRx
Cell-based bioassays often pose a hurdle during a rapidly moving biologics development program. High standards for assay accuracy, precision, reproducibility and robustness are additionally put to the test by the use of continuous culture cells that can
add to variability and increase the cost and complexity of each assay. This is particularly challenging for anti-VEGF drugs, as the prevalent assay is the proliferation of human umbilical vein endothelial cells (HUVECs), which requires 72-96 hours
to run, utilizes cells that are difficult to culture and introduces performance variability due to changes in passage number, culture conditions and operator. Here, we describe the PathHunter® bioassay that has been developed as a fit-for-purpose
QC Lot Release assay for anti-VEGF drugs. The assay quantifies inhibition of VEGF-A-induced VEGFR2 receptor activation, by measuring receptor dimerization as an early event in the receptor activation cascade. With its shorter assay time (<24 hours),
simple ‘add and read’ protocol and use of cryopreserved ready-to-assay cells, the PathHunter assay has many advantages over the standard HUVEC assay. Data will be presented comparing the performance and reproducibility of the PathHunter
Bevacizumab bioassay to the standard HUVEC proliferation assay.
3:25 A Cell-Based Flow Cytometry Assay to Detect and Titer Neutralizing Antibodies that Block Uptake of Enzyme Replacement Therapies Utilizing CI-M6PR
Andrew Melton, Ph.D., Scientist, Biomarin
Enzyme replacement therapies (ERTs) commonly utilize the cation-independent mannose-6-phosphate receptor (CI-M6PR) on the cell surface to target delivery of enzymes to the lysosome. However, patients receiving ERTs often produce an antibody response that
may prevent ERT uptake into the cell. To evaluate the impact of antibodies on ERT uptake through CI-M6PR, we developed a cell-based flow cytometry assay for detecting antibodies that neutralize uptake of VIMIZIM® (elosulfase alfa, rhGALNS). VIMIZIM®
is an FDA-approved ERT for patients with Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome). Jurkat cells, a non-adherent human T lymphocyte-derived cell line, expressed CI-M6PR on the cell surface and internalized Alexa488-labeled rhGALNS
with optimal uptake after 4 hours incubation at 37⁰C. Experiments with Trypan Blue quenching, incubation of cells with Alexa488-rhGALNS at 4⁰C instead of 37⁰C, or treatment of cells with Cytochalasin D, indicated that the median fluorescence intensity
(MFI) signal measured by the flow cytometer results from internalized Alexa488-rhGALNS and not Alexa488-rhGALNS bound to the cell surface. A positive control polyclonal goat anti-rhGALNS antibody reduced uptake of 0.4 μg/mL rhGALNS at concentrations
≥ 0.97 µg/mL. Assay precision, selectivity, drug tolerance and specificity were determined, and cut points were calculated to set criteria for assessing positive samples. Clinical trial samples tested in this cell-based platform were comparable
with data from an in vitro CI-M6PR binding ELISA-based platform. These results demonstrate the utility of a cell-based flow cytometry assay for detection and measurement of antibodies that interfere with ERT uptake.
3:50 Refreshment Break with Exhibit and Poster Viewing
4:30 Challenges in Potency Assay Development for a Non-Replicating Lentiviral Vector
Brenna Kelley-Clarke, Ph.D., Scientist II, Assay Development, Immune Design
Immune Design has developed an HIV-1-based integration-deficient lentiviral vector currently being evaluated in cancer immunotherapy (LV305). LV305 is designed to transduce human dendritic cells, triggering presentation of an encoded antigen via the MHC
Class I pathway; this is proposed to elicit an effective CD8-T lymphocyte response towards malignancies that over-express that antigen. The biological complexity of LV305 and its proposed MOA pose unique challenges in developing a potency assay matrix.
5:00 A Regulatory Perspective for Development of Potency Assay for Cellular and Gene Therapy Products: A Product Lifecycle Approach
Xiaobin (Victor) Lu, Ph.D., CMC Reviewer, Division of Cellular & Gene Therapies, OCTGT, CBER, FDA
The challenges of developing a potency assay for a CGT are multi-factorial including complexity of assay procedures, inherent variability due to use of living cells, tissues or live organisms, and variable reagents. Furthermore, a bioassay based
potency assay can be impacted by changes made in the product manufacturing processes. This presentation will outline a product life-cycle approach for developing potency assays for a CGT product from preclinical studies to the Biological License
Application. It will conclude with general recommendations for addressing some of the challenges in potency assay development and implementation for CGT during product and clinical development programs.
5:30 Assay Acceptance Criteria for Multiwell-Plate–Based Biological Potency Assays
C. Jane Robinson, Scientific Liaison, Biopharmaceutical Emerging Best Practices Association
Following preliminary consultation, a draft white paper “Assay Acceptance Criteria for Multiwell-Plate–Based Biological Potency Assays” was published in 2014. In the subsequent extensive consultation process, a variety of issues
were raised and discussed, including a range of current practices. Some of these issues and the recommendations of the white paper, including the two-level sequential assessment of acceptance criteria and use of an assay control sample, will be
6:00 End of Day One of Optimizing Bioassays for Biologics
6:00 Dinner Short Course Registration*
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THURSDAY, NOVEMBER 19
7:30 am Registration and Morning Coffee
8:00 am Chairperson’s Remarks
Liming Shi, MS, MA, Senior Research Scientist, Bioassay Development, Eli Lilly and Company
8:05 Bioassay Development for Bispecific Antibodies: A Different Ball Game
Piyush M. Vyas, Ph.D., Research Scientist, Bioassay Development, Eli Lilly and Company
Bispecific Antibodies are evolving rapidly and are already in stages for clinical trials. Some of these Bispecific Antibodies have shown synergy in their biological activity as compared to the combination of their individual counterparts. Bioassay
development and Data analyses for such Bispecific Antibodies to show the synergy, is a complex process. Traditional data analyses approaches might not be suitable enough in order to analyze such data. Data analyses of such Bispecific Antibodies
need to be approached in a different way.
8:35 Regulated ADC Bioanalysis Using Ligand Binding Assays: Challenges and Strategies
Seema Kumar, Ph.D., Principal Scientist, Pfizer, Inc.
The complex multi-component structure in combination with heterogeneous and dynamically evolving behavior presents unique challenges in ADC bioanalysis. The challenges may vary depending on the objective of ADC bioanalysis. The case studies showcasing
various bioanalytical strategies that could be employed in developing and validating successful ligand binding assays for ADC characterization will be presented.
Critical Success Factors for Cell-Based Assay Development and Transfer
John Kamerud, Ph.D., Scientific Director, Eurofins
Method development or transfer must occur before a CRO validates an assay. The development / transfer of complex methods that involve the use of cell lines require specific criteria to be evaluated to ensure the success of the assay. In particular,
the growth characteristics of the cell line, culture conditions, cell banking requirements, assay format, readout, reagents and data interpretation should be evaluated and controlled to ensure a robust path to validation.
9:35 Problem Solving Roundtable Discussions
These interactive discussion groups are open to all attendees, speakers, sponsors, & exhibitors. Participants choose a specific breakout discussion group to join. Each group has a moderator to ensure focused discussions around key issues within
the topic. This format allows participants to meet potential collaborators, share examples from their work, vet ideas with peers, and be part of a group problem-solving endeavor. The discussions provide an informal exchange of ideas and are
not meant to be a corporate or specific product discussion.
Table 5: Incorporating New Technologies into Bioassay Development
Moderator: Han Li, Ph.D., Principal Scientist, Lead Discovery and Optimization, Bristol Myers Squibb
- Is automation an option for bioassay development?
- High density plates meet precision and accuracy, what is the criteria now?
- Platforms matters---reporter assay, affinity binding assay, HTRF assay, BRET assay, or your thoughts?
- New areas, new requirements---how bioassay meets gene therapy and CAR-T technologies?
- What other technologies can be brought in as a future bioassay platform?
Table 6: Challenges in Assay Bridging
Moderator: Maura C. Kibbey, Ph.D., Senior Scientific Liaison, Biologics & Biotechnology, United States Pharmacopeia
- Determining if product-related impurities contribute to potency
- Demonstrating that the method is stability-indicating
- Pros and cons of parallel assays during clinical development versus switching post-licensure
Table 7: Method Transfers
Moderator: Max L. Tejada, Ph.D., Senior Research Scientist II, Analytical Operations, Gilead Sciences
- Approaches to clinical, commercial phases
- Approaches to internal transfers (development to QC lab) and external transfers (different sites, partners and CMOs)
- Challenges, lessons learned
10:35 Coffee Break with Poster Viewing
11:15 Use of a Tiered Approach to Develop Robust Potency Assays in Support of Monoclonal Antibody Product Development
Laura Geagan, Staff Scientist, Analytical Development, Sanofi Global Biotherapeutics
Cell-based assays are often used for evaluating the potency of biological therapeutics during product development. The development of cell-based methods frequently coincides with the stage of development of the product they support. Early stage assays
are often required to provide meaningful data to support process, purification and formulation development at a time when optimization of the method is not complete. As the drug progresses through the development paradigm, the performance of the
cell-based potency assay improves as well. In this presentation we describe a tiered approach to develop a robust cell-based potency assay to support pre-clinical product development. The analysis paradigm was updated as the drug progressed into
Phase I for consistency with guidance provided in USP chapters 1032-1034. The use of the assay to support process development will be discussed.
11:45 FTiH Support of an ADC: Stability, Assay Development, and Clinical Experiences
John F. Kellie, Ph.D., Investigator, Bioanalytical Sciences and Toxicokinetics, GlaxoSmithKline
Characterization of circulating ADC species (conjugated antibody, total antibody, and payload) is critical to understanding the safety and efficacy of ADC therapeutics. Current methodology requires development and validation of immunoassays and liquid
chromatography-mass spectrometry assays. This presentation will share experiences from assay validation through first time in human bioanalytical study support along with an update and outlook for state-of-the-art technologies set to drive ADC
method development in the future.
12:15 pm Enjoy Lunch on Your Own
1:15 Cupcakes and Coffee in the Exhibit Hall with Poster Viewing
2:00 Chairperson’s Remarks
2:05 Transfer and Validation of a Cell-Based Neutralizing Antibody (NAb) Assay to a CRO
Florence Guilhot, Ph.D., Head, Translational Pharmacology Lab, NovImmune SA
Biotherapeutics can lead to the production of anti-drug antibodies (ADA) in treated subjects which may result in loss of efficacy or elicit adverse events. Standard immunoassays can detect ADA, but cannot differentiate between neutralizing and
non-neutralizing ADA. Development of a neutralizing antibody (NAb) assay is a key step to support clinical trials. This presentation will provide an overview of (i) assay characteristics of the functional reporter NAb cell-based assay (ii)
challenges for the transfer and validation of a NAb assay to support the clinical trial.
2:35 Leveraging Automated Liquid Handlers, High-Density Plates, and Multi-Dimensional Assay Optimization to Accelerate the Delivery of Neutralizing Antibody Bioassay
John M. Lehrach, Research Scientist II, Bristol-Meyers Squibb
BMS Core BioAssay Group (CBG) leveraged the cellular assay repertoire, cell inventory, and the detection technology platforms in Leads Discovery and Optimization (LDO) Department to perform automation assisted multi-dimentional optimization for
the development of a Nab Bioassay. BMS informatics tools enabled automated data analysis. The technology platform selected was a homogenous cAMP HTRF assay. CBG delivered a 96-well format Nab assay with excellent Nab sensitivity, assay reproducibility,
and serum tolerance utilizing assay-ready cryo cells.
3:05 Compendial Potency Assays and Associated Biological Reference Materials – Challenges in Assay Bridging
Maura C. Kibbey, Ph.D., Senior Scientific Liaison, Biologics & Biotechnology, United States
USP, regulators, and manufacturers share a common goal of reducing in vivo testing, yet replacing animal assays with suitable in vitro assays may be challenging. This presentation will highlight USP’s current efforts to include modern
bioassays in the USPNF as well as bridging expectations for revision sponsors who would like to propose a modern assay for the compendium.
3:35 Global Bioassay Transfers
Camille Dycke, Ph.D., F. Hoffmann-La Roche Ltd. / Genentech; Associate Director, Method Management
and Technologies, Bioassay, Global Biologics QC
To support global product release, the bioassay used in the commercial control system of the product is transferred to multiple QC testing laboratories around the globe. In order to facilitate these global method transfers, the implementation
of global processes, a global training plan and a solid method life cycle management program, are key elements to ensure success. A few case studies will be presented, illustrating product globalization.
4:05 Close of Conference
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