Optimizing Bioassays for Biologics

Bioassays, at their core, spring from a fusion of biological and statistical sciences, and are used to measure activity or function of a compound or group of compounds in samples. The biological aspect is based on finding or creating a living system that responds to the compound with most development effort focused on how to make the response larger, more consistent, and more selective. The statistical aspects involve the design of the assay system, techniques for constructing measures of the relative activity (or potency) of preparations of the stimulant, tools for improving the assay system, and methods for demonstrating the properties of potency estimates. The health authorities and USP have provided guidance for the design and validation of a bioassay; however, they do not discuss solutions to common problems. At Cambridge Healthtech Institute's Fifth Annual Optimizing Bioassays for Biologics, leaders working in bioanalytical and bioassay development will come together to provide case studies and best practices for handling the most common issues in biological assay development, validation, transfer, and maintenance. Health authorities will weigh in on new guidelines as well as provide insight into what they consider a well-characterized and well-controlled bioassay. Finally, new technologies and bioassay formats will be presented along with recommendations for implementation to ensure a steady drug development pipeline.

Final Agenda

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Recommended Pre-Conference Symposium*

• Bioassays for Immuno-Oncology

*Separate registration required

Recommended Pre-Conference Training Seminar*

• TS2: Introduction to Design of Experiments

*Separate registration required

WEDNESDAY October 25

12:45 pm Conference Registration

STRATEGIC BIOASSAY DESIGN

1:40 Chairperson's Opening Remarks

Erica Bortolotto, Ph.D., Senior Scientist, Bioassay Development, Analytical Sciences for Biologicals, UCB PHARMA

1:45 KEYNOTE: Essentials in Bioassay Design

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

Of all critical quality attributes (CQAs) for drug product and drug substance the bioassay is the most important as it is the only CQA that measures drug activity. Bioassays are a unique combination of scientific understanding to generate a signal that tests the mechanism of the drug to generate a specific signal and the associated statistical manipulation to generate a reliable and accurate measure of relative potency. Immunological assays are similar in the combination of science and statistical methods for determination of signal and decision cut points for PK response detection.

2:15 Setting Realistic Product Specifications to Avoid OOS

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

Regulators advise: ‘Set product specifications based on clinical and production experience.’ Far too often, the capabilities of a biological assay drive product specifications for a biotechnology product. Out of specification results are very costly. This talk will describe strategies for using limited early product production experience, limited clinical experience, and limited knowledge about assay capabilities to set specifications that the process can be expected to reliably meet.

2:45 Development of a Beads-Based Multiplex Assay for the Evaluation of Vaccine Immunogenicity Following a DoE Approach

Rachid Marhaba, Ph.D., Senior Scientist, Clinical Laboratory Sciences, GSK Vaccines GmbH

Design of Experiments (DoE) is a statistical approach for process development and optimization. The influence and most importantly the interaction of several factors will be evaluated in order to define optimal test conditions. We used the DoE approach in order to set-up and optimize a beads-based Multiplex ELISA on Luminex® platform that will be used to evaluate immunogenicity of a multivalent vaccine product in clinical trials. We present here the methodology we used and discuss the evaluation approach.

3:15 Robust and Simple Potency Bioassays for Biologics and Biosimilar Development

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

We will share examples of simple, quantitative cell-based bioassays for potency determination and stability testing of biotherapeutics. These quantitative and robust assays are based on receptor’s native biology and readout reflective of drug’s MOA, and are scalable utilizing thaw-and-use cryopreserved cells that deliver superior performance with high reproducibility (3-7% RSD).

3:30 Refreshment Break in the Exhibit Hall with Poster Viewing

SETTING EQUIVALENCE BOUNDS FOR SIMILARITY

4:10 Equivalence Bounds for Similarity: Where and What Is the Limit?

Erica Bortolotto, Ph.D., Senior Scientist, Bioassay Development, Analytical Sciences for Biologicals, UCB PHARMA

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 an acceptable and non-acceptable results in routine?

4:40 Defining Equivalence Criteria for a Parallelism Experiment: A Case Study

Anne Benoit, Ph.D., Expert Biostatistician, Biostatistics and Statistical Programming, Non-Clinical R&D Statistics – CLS, Clinical Evidence Generation, GSK Vaccines

During the development and lifecycle of an ELISA to be used for IgG measurement in clinical testing of vaccines studies, new standards need to be prepared. To ensure the continuum with the results already generated, the parallelism between the current and the candidate standards needs to be assessed. Following the USP recommendations, we derive fit-for-purpose equivalence criteria from historical data for the current standard through the use of tolerance intervals.

5:10 Panel Discussion: Optimizing Assays Using Statistics and Rational Design

Moderator:
Perceval Sondag, Principal Statistician, Non-Clinical Statistics, Arlenda

• Avoiding out of specification results
• DoE approaches
• Selecting equivalence criteria
• Keys to developing MoA-reflective potency assays

Panelists:
Anne Benoit, Ph.D., Expert Biostatistician, Biostatistics and Statistical Programming, Non-Clinical R&D Statistics – CLS, Clinical Evidence Generation, GSK Vaccines

Martin Kane, MS, CRE, Managing Data Scientist, Statistical and Data Sciences Practice, Exponent

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

5:40 Dinner Short Course Registration

Recommended Dinner Short Course*

• SC4: Advice on Putting Together an Integrated Summary of Immunogenicity

*Separate registration required

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THURSDAY, October 26

7:30 am Morning Coffee

PARALLEL LINE ANALYSIS TO DETERMINE RELATIVE POTENCY

7:55 Chairperson's Opening Remarks

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

8:00 Parallelism and Constraining Curves: The 4-Parameter Logistic Function

Martin Kane, MS, CRE, Managing Data Scientist, Statistical and Data Sciences Practice, Exponent

It is often necessary to fit a mathematical curve to biologic/assay data for the determination of relative potency. This process usually involves constraining one or more asymptotes in order to perform the pertinent relative analysis. This talk will describe the constraint requirements and the process of fitting these curves, and an example will be demonstrated.

8:30 Extensive Comparison of Parallelism Tests for Potency Bioassays

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

The USP requires to test for parallelism before estimating the relative potency. Several tests for parallelism have been proposed, but at no point, a neutral comparison of all these tests has been conducted. This talk presents an extensive simulation study to assess what test works best in what situation. The simulation study explores realistic scenarios: precise and imprecise assays, optimal and suboptimal assay designs, etc.

9:00 The Interface of the CAP/CLIA and GCLP Divide

Jennifer Ohayon, Ph.D., Scientific Director, Eurofins Bioanalytical Services

The regulatory boundaries of the GLP-compliant bioanalytical lab, conventionally seen a separate regulatory entity from the CAP/CLIA regulated clinical chemistry lab, are beginning to merge in the pursuit of biomarker-supported clinical studies and personalized medicine.

9:30 Problem Solving Roundtable Discussions

Design of Experiments (DoE) for Development of Biological Assays

Moderators: David Lansky, Ph.D., President, Precision Bioassay, Inc.
Martin Kane, MS, CRE, Managing Data Scientist, Statistical and Data Sciences Practice, Exponent

• Why DoE?
• What responses to focus on, and why, during early bioassay development
• Design techniques and strategies for adapting bioassays to DoE
• Simple graphical analysis methods for DoE on bioassays
• Strategies for learning more about DoE

USP General Chapters for Bioassays

Moderator: Maura Kibbey, Ph.D., Director, Global Biologics, U.S. Pharmacopeia

• Discussion of benefits and challenges for implementation of chapter guidance
• Sharing ideas for future chapter revisions
• Recommending new USP guidance that would be helpful

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

AUTOMATION TECHNOLOGIES

11:10 Shrinking Potency Assays: Development of an Automated Low Volume Dispensing System

Robyn Beckwith, Ph.D., Associate Scientist, Analytical Development and Quality Control, Product Technical Development, Genentech

Analytical testing environments commonly face constraints on sample throughput and cost for complex procedures such as potency assays. We present a strategy to mitigate these issues using a novel Low Volume Dispensing System (LVDS) with a case study of an automated homogeneous enzymatic potency assay in miniaturized 384-well format. The LVDS leverages contactless liquid handling technologies, including acoustic ejection and air-pressurized microvalves, to dispense nanoliter- and microliter-scale volumes in an automated assay workflow. We assess comparability of the 384-well LVDS and 96-well manual assay formats, as well as demonstrate suitable assay performance for the LVDS potency method. Automation of miniaturized potency assays using the LVDS platform increases sample throughput, reduces hands-on assay time, and provides significant cost savings.

 11:40 Optimization & Robustness Study of Potency Bioassays Using Thaw-and-Use Cells

Jey Cheng, Ph.D., R&D Group Leader, Bioassay Development, Promega

Vanessa Ott, Ph.D., Global Strategic Marketing Manager, Cellular Analysis and Proteomics, Promega Corporation

Cells are critical reagents in cell-based assays and the main sources of assay variation. Cryopreserved, Thaw-and-Use cells provide benefits of flexibility, run-to-run consistence, and easiness of global transfer. They have been gradually adopted in many GMP bioassays. Here, I will describe the procedures we have successfully established at Promega to implement Thaw-and-Use cells in potency bioassays. We will discuss cell line characterization, controlled cell culture and cell freezing, and process development for scale up production.

12:10 Sponsored Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:10 Dessert and Coffee Break in the Exhibit Hall with Poster Viewing

STUDY DESIGN FOR BIOASSAY VALIDATION

1:50 Chairperson's Remarks

Martin Kane, MS, CRE, Managing Data Scientist, Statistical and Data Sciences Practice, Exponent

1:55 The Use of Automation in Development and Validation of Cell-Based Bioassays

Natalia Kozhemyakina, Ph.D., Head, Bioassay Laboratory, Analytical Development Department, BIOCAD

This presentation highlights general considerations on the development and validation of mechanism-of-action reflective potency assays. Cell-based assays are complex, multistage, long-term studies that require aseptic techniques and special training. The use of an automated station greatly facilitates the process of their development and validation. In addition to improving the lab efficiency it allows to reduce significantly the variability and increase consistency of analysis. We demonstrate their advantages on real examples.

2:25 Design and Development of Cell-Based Potency Assays for Biologics

Ravish Patel, Ph.D., Manager, Quality Control, Epygen Biotech

Functional assays, such as ligand-binding assays (LBAs) and mechanism of action (MOA) cell-based assays (CBAs), must be developed to effectively assess biosimilars. In many cases, LBAs are crucial for determining PK and PD behavior, and they are also often used as orthogonal assays to CBAs. Bioassay plays an important role in the development of product and manufacture process. This presentation will highlight general consideration on the LBAs as a reflective of mechanism of action (MOA) and determination of Biosimilarity.

REFERENCE STABILITY AND BIOASSAY CONTROL

2:55 USP Reference Standards in Support of Relative Potency Measurements

Maura C. Kibbey, Ph.D., Director, Science & Standards, Global Biologics, U.S. Pharmacopeia

The United States Pharmacopeial Convention (USP) is an independent scientific organization that protects public health through standards for medicines and their ingredients. As biological science contributes to more advanced therapies, standards continue to play a critical role in drug development and manufacturing. This talk will highlight the importance of quality standards for relative potency and USP’s efforts to modernize compendial bioassays and bridging expectations for revision sponsors.

3:25 Use of Echo® Liquid Handlers to Improve the Efficiency and Reliability of Bioassay Laboratories

Tim Allison, Ph.D., Field Applications Manager, North America, Labcyte Inc.

Adoption of Acoustic Droplet Ejection (ADE) Technology presents an exciting opportunity for bioassay development and execution. Any-well-to-any-well acoustic dispensing of compounds, reagents, and samples in nanoliter volume increments affords bioassay development maximum flexibility with regards to experiment design and effective miniaturization of assays resulting in significant cost savings, improved data quality, minimal expenditure of precious samples, and improved reproducibility; and all while staying GMP compliant.

3:55 Close of Optimizing Bioassays for Biologics

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