2019 US Bioassay Conference
3rd Annual | San Pedro, CA | March 13-15, 2019
2019 USB Speaker Abstracts
Leonard Blackwell, Biogen
Title: The New Laboratory Paradigm for Pharmaceutical Development
Abstract: In today’s market, drug development requires agility and efficiency to effectively develop products at a lower cost and reduce financial risk. At Biogen we accomplish this in part by automation of laboratory processes from sample to result using a combination of robotics and automated data analysis solutions. These tools simplify routine tests by reducing the amount of specialized skill and expertise required for a given method allowing flexibility to full time staff and effective use of the temporary workforce. Robotic liquid handling is designed to automate our most common and high demand analytical platforms. This is especially effective in the bioassay space where there is much time spent on manual liquid handling which limits the analyst’s capability in sample turnaround and requires a high degree of training to develop the expertise required to produce acceptable accuracy and precision. Automated data analysis allows the flow of information into an integrated platform that pulls together the sample management database, robotics, and instrumentation. Analytical data is automatically calculated reducing time spent on crunching numbers and moving information by transcription. This new laboratory paradigm makes our work force more efficient by focusing specific high-level expertise for our most strategic work. The presentation will use case studies highlighting our automated technology used to support our new work model and provide an overview of this industry trend.
Tam Bui, Custom Biologics
Title: Development and Qualification of an ACT-101 Competitive Cell-Based Ligand-binding Assay
Abstract: ACT-101 is a non-glycosylated recombinant form of human alpha-fetoprotein (AFP) produced by recombinant DNA technology in a transgenic goat system. AFP is naturally synthesized primarily by the yolk sac and foetal liver and is abundantly found in the plasma of embryonic vertebrates and maternal circulation during pregnancy. In humans, AFP is rapidly depleted after birth. AFP receptors are present in embryonic and most tumour cells, but are absent in normal adult cells, making them an attractive target for delivery of chemotherapeutic payloads to selectively kill cancer cells. A competitive binding cell-based potency assay using the U937 human lymphoblast cell line was developed and qualified to compare the ability of ACT-101 to competitively bind to U937 cells in the presence of a fixed concentration of fluorescently labelled ACT-101. ACT-101 binding is then determined by measuring the fluorescent signal of treated cells. The optimized assay was qualified to assess intermediate precision, accuracy, specificity and linearity.
Contributing Authors: María Fabiana De Rosa, Custom Biologics™, Igor Sherman, Alpha Cancer Technologies Inc. and Tam Bui, Custom Biologics™
Janice Callahan, Callahan Associates
Title: The Log Transformation: A Biologist’s Best Friend
Abstract: All assays have variability that must be quantified in order to report test measurement uncertainty. The distribution of data from biological assays often does not follow a bell-shaped curve. This is evidenced during data analysis. For example, a constant coefficient of variation (CV) or relative standard deviation (RSD) indicates an increasing standard deviation with increasing mean values across the range of an assay. With few exceptions, statistical analyses require a constant standard deviation. Although commonly used statistical techniques might be considered robust to deviations from such assumptions, applying them will result in wrong, even if slightly so, answers. A better solution is to simply log transform the data before analysis. In this presentation, examples will be used to show how variability problems can be detected, how applying a log transformation fixes them and will illustrate the impact of back transformation on outcomes when data are returned to their units of scale.
Joseph Callahan, Genentech (Roche)
Title: Development of a Reporter Gene Potency Assay for Bispecifics
Abstract: Bispecific antibodies bind 2 unique antigens. Here, we present a case study for a bispecific antibody reporter gene potency assay that uses 2 cell-types with a target cell-dependent luminescent readout. The assay was demonstrated to be MOA-reflective, robust, QC friendly, and sensitive to molecular changes in the drug. Strategies for optimization and qualification, stressed sample testing results, and efforts to enable future multi-product application will be discussed.
Jey Cheng, Promega
Title: Case Study: Characterization and Qualification of Thaw-and-use Cell Banks for Use in Bioassays
Abstract: (Pending)
Jill Crouse-Zeineddini, Amgen
Title: Case Study: Use of Acoustic Droplet Ejection Technology in Development and Regulated Testing Laboratories
Abstract: (Pending)
Stan Deming, Statistical Designs
Title: Statistics for Quantal Assays – They’re Different
Abstract: Rates and proportions arise from quantal measurements. Two examples are the fraction of 10 guinea pigs that withstand a viral challenge dose (e.g., 7/10 = 0.7) and the extent of polyploidy in cells (e.g., 2%). Rates and proportions are expressed on a bounded scale that necessarily ranges from 0 to 1, or from 0% to 100%. Statistics for measurements expressed as rates and proportions are different from the more familiar statistics for measurements expressed on a continuous scale where the measured values are unbounded. When the latter statistics are misused in the former context, disturbing things happen
— for example, confidence intervals might arise for which the lower boundary lies below 0, clearly nonsensical. This talk will review (introduce) simple statistical calculations and tests that are appropriate for working with rates and proportions.
Kimberly Flecke, Pfizer, Inc.
Title: Case Study: Process to Assure Potency from Clinical Phases Through Lifecycle of a Product
Abstract: (Pending)
Gerald Feldman, US Food & Drug Administration
Title: To Err is Human: Common Errors Observed in Bioassay Development
Abstract: (Pending)
Guoying Jiang, Genentech
Title: Reporter Cell-based Assay: Connecting to the Biological Relevance
Abstract: Bioassays reflecting mechanisms of action (MoA) are indispensable for assessing the potency of therapeutic antibodies for lot release and stability testing, comparability assessment, and biological characterization. New formats of therapeutic antibodies or complex MoAs often pose substantial challenges to develop and install a suitable bioassay in quality control (QC) laboratories. In later years, bioassays using reporter cell lines have gained increasingly wider application. Provided that the reporter cell-based assay closely mimics the in vivo MoA (i.e. the biological relevance), it is highly valuable for the ease of execution, accuracy, precision, and feasible implementation in QC. This presentation will describe case studies to demonstrate the biological relevance of several reporter cell-based bioassays and strategies to support their use as the potency assay on the control system.
Xu-Rong Jiang, AstraZeneca BioVentures
Title: Scientific Considerations and Case Studies on Bioassay Changes
Abstract: (Pending)
David Lansky, Precision Bioassay, Inc.
Title: Near-Universal Bounds for Similarity in Bioassays
Abstract: A major goal of bioassay development, validation, and monitoring is to minimize bias of potency. Testing for similarity via equivalence tests has become an essential part of modern bioassay analyses. Sensitivity analyses, reported here, show that scaled shifts in parameters measure non-similarity in ways that are assay-independent. We show that well-chosen similarity equivalence bounds limit bias in potency due to non-similarity. Hence, equivalence bounds for non-similarity can be informed by bias limits based on product specifications and the analytic target profile.
Nancy Niemuth, Battelle
Title: Your Statistician is Your Friend: Optimizing Reagent Qualification Using Equivalence Tests
Abstract: (Pending)
Mike Sadick, Catalent Biologics
Title: “I CAN Let You Do That, Dave.” Solutions for Automation of GMP Assays, Including Bioassays
Abstract: For many reasons, there has been increasing interest in automation of GMP assays for bio-, molecular and immuno-assays. Of these types of assays, automation of cellular bioassays has been the most challenging, and often, elusive. What are the drivers to automate these kinds of assays? What options exist for automating different types of biologics assays? What are the challenges, and their potential solutions, associated with the automation of these assays? Several strategies, representing Catalent thinking concerning automation of laboratory assays, will be presented. Case studies will be presented that exemplify the benefits of assay automation and detail the strategies by which challenges, including bioassays, were met and overcome.
Contributing Authors: Chris Hepler, Jeff Dixon, Brian Woodrow and Michael Sadick
Nancy Sajjadi, Independent Consultant
Title: Understanding Critical Fold Difference and Its Application in Reporting Assay Precision
Abstract: Characterization of biopharmaceutical products in early phase development is critical for developing product knowledge and identifying candidate assays to measure potency. Methods for quantifying biological properties typically have higher levels of variability than methods used to assess other product attributes. Critical quality attributes including dose and potency must be quantified with a high degree of confidence for product lots intended for use in preclinical and early phase clinical studies, as well as to evaluate product stability. Although the effects of high variability may be mitigated by higher replication in testing, and the reporting of mean values with an associated confidence interval, it can be useful to document the capacity of the assay to reliably discriminate differences in dose or potency levels between lots or within a lot over time. An introduction to the theoretical framework of critical fold difference and example calculations are intended to serve as guidance for non-statisticians involved in generating or reviewing assay precision data.
June Saunders, Emergent BioSolutions
Title: Bioassay Transfer and Comparative Testing for a Commercial Antibody
Abstract: (Pending)
Tilman Schlothauer, Roche
Title: Appropriate Fc Functionality Assessment – Control of the Effector Functions of Therapeutic Antibodies
Abstract: For antibody characterization an assay platform has been established to assess Antibody-Fc-Receptor interaction. This platform, comprised of a broad panel of reagent tools and assay formats, is utilized for mechanistic studies towards the understanding of Fc functionality in pre-clinical development. In later clinical phases each antibody Fc engineering requires an appropriate assessment of Fc functionality during process development, comparability exercises and reporting to Health Authorities. For enhanced Fc functionality, another intensity and method setup is required compared to effector silenced antibody formats. Within the Fc receptor platform, we have developed several tools for the different purposes of early and late stage therapeutic mAb development.
Perceval Sondag, Pharmalex
Title: Similarity (or Parallelism) Testing is Mandatory Prior to Calculate Relative Potency
Abstract: Many tests are assessing similarity between two 4PL curves, but all are flawed. Difference tests, such as F-Ratio tests and Chi-squared test, have long been shown to increase the acceptance rate of highly variable assays, while rejecting very precise assays. The literature now suggests equivalence tests, but the derivation of the equivalence margins has been challenging for bioassay scientists and statisticians. This talk proposes a way to assess similarity in a way that controls the bias in relative potency estimation. Instead of focusing on the mathematical formulae behind the tests (which can be tedious for non-statisticians), this talk explains how it actually works, and how to easily implement it in practice.
Anton Stetsenko, ADC Therapeutics
Title: A Bioassay Comparability Assessment Between Two Contract Labs (Case Study)
Abstract: During the life cycle of a product, bioassay procedure can be developed, qualified or validated at different Contract Testing Laboratories (CTL) and procedural changes are inevitable when an opportunity for improvement occurred. It creates a challenge for the Sponsor to bridge existing results and assess comparability between “quite similar, but not identical” methods. “What type of samples?” and “How many measurements/replicates?” are the most critical questions to be answered during such exercise. A risk assessment is another important part of the bridging. This case study is about methods comparability assessment for cytotoxicity bioassay between two CTLs.
Atul Tiwari, Syngene International Ltd
Title: Cellular Functional Bioassay for Insulin Analogues-Challenges & Learnings
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 will 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.
Leslie Wagner, US Food & Drug Administration
Title: Test Method Replacement: A Regulators Perspective
Abstract: (Pending)
Steven Walfish, USP
Title: USP Bioassay Chapter Revision Journey
Abstract: (Pending)
2019 HCP Workshops
Sian Estdale, Covance
Title: Reference Standards – Challenges and Opportunities
Abstract: (Pending)
Bassam Hallis, Public Health England
Title: Reference Standards – Challenges and Opportunities
Abstract: (Pending)
Jane Robinson, BEBPA
Title: Reference Standards – Challenges and Opportunities
Abstract: (Pending)
2019 HCP Posters
Jared Bailey, Labcyte Inc.
Title: Host Cell Residual DNA Testing in Reduced Volume qPCR Reactions Using Acoustic Liquid Handling
Abstract: The removal of host cell impurities is a critical step in the production of biopharmaceutical products. One impurity targeted for clearance during the purification process is residual DNA arising from host cells. In addition to potential safety issues associated with extraneous host cell DNA, the regulatory guidance for products produced in cell culture specifies that DNA content in the final product should be as low as possible, as determined by a highly sensitive method. Traditional methods of quantitating residual host cell DNA have been limited by laborious sample preparation protocols, and lack of sensitivity. The Applied Biosystems resDNASEQ™ Quantitative System is a quantitative PCR (qPCR)-based system for the detection of residual DNA from a variety of host cell lines. These results are specific and highly sensitive with the included TaqMan® probes being capable of detecting as little as 0.03 pg of host cell DNA. Kits have been made with probes specific for a wide range of cells lines including those from human, E. coli, and Chinese hamster Ovary (CHO) backgrounds.
In this study, we show the utility of multiple resDNASEQ systems coupled with the newly released Echo® 655 Liquid Handler at reduced reaction volumes. The Echo 655 Liquid Handler uses a transducer to acoustically dispense in 2.5 nL increments without risking tip-based contamination or human to human variability. The nanoliter granularity of the Echo system enables greatly lowered reagent cost at the highest level of precision and accuracy. This allows for a greater number of technical replicates per assay to increase experimental confidence. By combining the Echo 655 Liquid Handler with the Applied Biosystems resDNASEQ™ system, we demonstrate an additional 10-fold sensitivity to the manufacturer’s quoted limit of quantitation, simplification of sample preparation, and a 6-fold reduction in reaction size.
Jared Bailey, Labcyte Inc.
Title: Echo® 525 Liquid Handler-enabled Bioassay: Establishing a Miniaturized Humira / TNFα L929 Cytotoxicity Bioassay
Abstract: TNFα is a pro-inflammatory cytokine overexpressed in autoimmune disease and is a target for drug development. Adalimumab (Humira™) is an anti-TNFα antibody which binds TNFα and suppresses the immune response. An established model for anti-TNFα therapeutics is the L929 mouse fibroblast cytotoxicity assay. Cell-based assays are widely used and illustrate issues encountered across many bioassays. This presentation will describe the deconstruction and optimization of the cell-based workflow to provide an automated 384 well bioassay. The result is a single instrument assay build which provides additional controls and replicates in a miniaturized reaction to reduce sample requirement, cost and potential human error.
Ping Carlson, Celgene Corporation
Title: Robustness Study Design and Data Analysis_ Case Study of Cell-Based Reporter Assay for a Therapeutic Antibody
Abstract: (Bioassay (particularly cell-based assay) is used for a late-stage release of a therapeutic antibody. It is important that the bioassay is sufficiently robust to determine manufacturing consistency throughout a product’s life cycle.
This cell-based reporter gene assay was developed at Celgene Corporation. The robustness study was conducted as part of method development. The robustness study consisted of a total of 8 variable factors were evaluated with a Design of Experiment (DOE). In addition to the multifactorial evaluations, individual parameters including impact of ligand concentration, assay incubation time, use of thaw-and-use versus cultured cells, cultured cell passages with younger and older cells, luciferase substrate from different vendors and plate / incubator position effect were also evaluated side-by-side for evaluation of assay performance. The method was demonstrated to be robust, where minor (20-30%) changes in assay factors showed minimal impact to potency determination, system suitability, or sample acceptance criteria. There were also minor interactions observed between factors on determination of relative potency (%RP). Comparison of the group means showed that the mean results of assay parameters evaluated were all well within the method historical trend and were found to not have any practical significance on assay performance.
Based on these findings, assay parameters were determined to be optimized within the design space model. Therefore, the method was robust within the variance levels of variable factors tested.)
Contributing Authors: Ping Carlson, Rajeev Boregowda, Kendall Carey
Celgene, Biologics Development, Bioassay, Summit, NJ
Jey Cheng, Promega Corporation
Title: Quantitative Cell-Based Reporter Gene Bioassays to Advance Individual or Combination Cancer Immunotherapy
Abstract: A major challenge in the development of antibody-based biologics drugs is access to quantitative and reproducible functional bioassays. In contrast to the cumbersome, variable methods currently used that rely on primary cells, we have developed a portfolio of functional cell-based reporter bioassays to easily measure the activity of biologics drugs designed to target immune checkpoint receptors including co-inhibitory (e.g. PD-1, CTLA-4, LAG-3) and co-stimulatory (e.g. 4-1BB, GITR, OX40) receptors. These bioassays consist of stable cell lines that express luciferase under the precise control of receptor-mediated intracellular signals. Here we describe the application of these MOA-based bioassays for biologics drug discovery, development, potency and stability studies.
Steven Edenson, Promega Corporation
Title: Measurement of Fc-mediated ADCC and CDC of anti-TNFα and anti-VEGF Therapeutic Antibodies using Reporter-based Bioassays and Engineered TNFα+ and VEGF+ Target Cells
Abstract: Fc-mediated effector functions are critical to the efficacy and safety of therapeutic antibodies. Measurement of Fc-mediated antibody-mediated cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) during antibody drug discovery and development is not only important for antibodies that harness ADCC and/or CDC as their primary mechanism of action (e.g. rituximab, trastuzumab), but also for antibodies designed to target and block soluble ligands such as TNFα and VEGF. We previously reported the development of a cell-based reporter bioassay platform which has been used to measure ADCC and ADCP mediated through FcRI, FcRIIa and FcRIIIa. These reporter bioassays exhibit the specificity, accuracy, precision, and robustness necessary for qualification according to ICH guidelines and have been used extensively to characterize and measure the potency of antibody-based biologics drugs that target cell surface immune receptors. In the current study, we sought to evaluate Fc-mediated ADCC and CDC activities of therapeutic antibodies designed to target and block soluble ligands including TNFα and VEGF. To measure ADCC activity of anti-TNFα and anti-VEGF blocking antibodies, we developed engineered target cells that express either membrane-bound TNFα or VEGF. When used as target cells with reporter-based effector cells expressing a relevant FcR, ADCC activity of adalimumab (anti-TNFα) and bevacizumab (anti-VEGF) was detected in a specific and dose-dependent manner. Similarly, when used in a luminescence-based CDC assay, the engineered target cells elicited an appropriate FcR-mediated response. The assay signals demonstrated IgG isotype specificity as IgG4 variants showed minimal activity in both ADCC and CDC assays. Our results demonstrate that the combined use of cell-based reporter bioassays with target cells engineered to express membrane-bound soluble ligands can provide a simple, specific, and quantitative platform to measure Fc-mediated effector functions of therapeutic antibodies targeting soluble ligands.
Rajni Kalyandasani, Sutro Biopharma, Inc.
Title: Development, Qualification and Transfer of a “Thaw-and-Use” Bioassay for a Therapeutic Antibody Drug Conjugate Produced using Sutro’s Proprietary XpressCF+™ Cell Free Expression System
Abstract: In this study, we describe the development of a cytotoxicity bioassay using cells in “thaw-anduse” format to measure the potency of Sutro’s clinical phase antibody drug conjugate STRO001, which is produced using our novel cell-free expression system. We describe various challenges encountered during the development, qualification and transfer of the assay to a contract testing QC laboratory.
Contributing Authors: Rajni Kalyandasani, Khyati Shah and Maureen Bruhns
Jeff Nelson, Promega Corporation
Title: Reproducible, MoA-reflecting Reporter-based Bioassays to Enable Drug Development of Biosimilars and Biobetters
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, IL15, 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 provide valuable tools for the development, stability testing, and potency determination in the manufacture of cytokine biosimilars and biobetters.
Khyati Shah, Sutro Biopharma, Inc.
Title: Development, Qualification and Transfer of a “Thaw-and-Use” Bioassay for a Therapeutic Antibody Drug Conjugate Produced using Sutro’s Proprietary XpressCF+™ Cell Free Expression System
Abstract: In this study, we describe the development of a cytotoxicity bioassay using cells in “thaw-anduse” format to measure the potency of Sutro’s clinical phase antibody drug conjugate STRO001, which is produced using our novel cell-free expression system. We describe various challenges encountered during the development, qualification and transfer of the assay to a contract testing QC laboratory.
Contributing Authors: Rajni Kalyandasani, Khyati Shah and Maureen Bruhns
Sharon Young, Thermo Fisher
[TITLE]
Abstract: Cell-based methods are reputed to be notoriously difficult to transfer to a new lab. As a CDMO, we have successfully implemented a number of client cell-based potency assays, including ADCC, proliferation, cytotoxicity, and gene reporter assays. While some assays are well-developed for ease of execution, many are in early development stages and are not fully ready for quick implementation in a cGMP environment. Typical challenges include the cell culturing procedure, assay robustness and throughput, and lack of appropriate criteria to ensure a reliable result. Method optimization and criteria setting are often needed, typically under significant time constraints with limited data available. Here we present considerations, challenges, and recommendations for successfully implementing a bioassay in a CDMO environment.