Development of a Suite of Assays to Characterize Omalizumab

• Presenter: Debbie Allan, Sartorius Stedim Biotech
• Abstract: Under normal circumstances, IgE forms a very small proportion of circulating immunoglobulins, however, atypical abundance results in hypersensitivity. During allergic asthama an allergen is inhaled and IgE is produced in response. Upon second exposure IgE attaches to the allergen and to the surface of the inflammatory cells releasing mediators, often resulting in airway narrowing. Omalizumab is a humanized murine monoclonal antibody targeting the Fc region of IgE. Thus, omalizumab specifically recognizes free, unbound (to FcRI) IgE in circulating interstitial blood and IgE expressing B-cells, resulting in sequestration and reducing illicit activation of the immune response to non-threatening stimuli. Omalizumab does not bind IgE which has already bound the FcRI receptor on inflammatory cells. This is critical characteristic of omalizumab as other anti-IgE antibodies which bind the IgE-FcRI complex aggregate the receptor and potentiate the inflammatory response. Omalizumab, therefore, avoids the anaphylactic effects caused by other anti IgE antibodies.
  To fully characterize a biosimilar molecule both the Fab and Fc functions need to be assessed. To characterize the Fab functions a direct IgE ELISA has been developed whereby omalizumab and the FceRI receptor compete for IgE. A histamine release assay is also in development. It combines whole blood, serum from allergic donors and omalizumab which are then exposed to allergen after which histamine is quantified using a commercially available histamine ELISA kit. Although Fc function is not anticipated it still needs explored to fully characterize the biosimilar. To determine the presence of Fc functions we have a direct C1q ELISA to show the potential for binding C1q and thereby the possibility of complement activation. It was also important to provide evidence that omalizumab did not have ADCC , ADCP and CDC activity thus negative assays for these methodologies are underway.
• Contributing Authors: Debbie Allan¹, Lisa Blackwood¹, and Laura McAleer^{1
  1} Sartorius Stedim BioOutsource Limited, Glasgow, UK
• (Poster)

A Single Immunogenicity Assay for Testing Antigen Potency in Combination DTaP Vaccines: Simultaneous Quantitation of Anti-DT, anti-TT, Anti-PTxd and Anti-FHA Antibodies in Guinea-Pig Serum with a Luminex®-xMAP® Bead-Based Serological Assay

• Speaker: Silvio Bandiera, Sanofi Pasteur
• Abstract: The diphtheria toxoid (DT), tetanus toxoid (TT), and acellular pertussis (aP) single immunogenicity assay (DTaP SIA) is a Luminex®-xMAP®-bead-based multiplex immunoassay for estimating the potency of DTaP pediatric combination vaccines in guinea pigs. This manuscript describes the validation of this assay for the simultaneous quantitation of anti-diphtheria toxoid (anti-DT), anti-tetanus toxoid (anti-TT), anti-pertussis toxoid (anti-PTxd), and anti-filamentous hemagglutinin (anti-FHA) antibodies in guinea pig serum following injection of a DTaP vaccine formulation. The results were expressed in arbitrary units/mL (AU/mL) using reference serum for comparison. Specificity was demonstrated by ≥75% homologous and ≤25% heterologous inhibition for all the antigens. The results were linear for anti-DT, anti-TT, anti-PTxd and anti-FHA antibodies. Accuracy was demonstrated with recovery of between 80% and 120% for all four antibodies. The relative standard deviation of repeatability was ≤20%. The results demonstrate that this SIA can be used for the linear, accurate, and precise simultaneous detection of all four antibodies, based on both the ICH Q2 and the EMA guidelines on bioanalytical method validation.
• Contributing Authors: Silvio Bandiera, Annabelle Lebas, Livia Canizares-Martinello, Fabien Guinchard, Corinne Lyonnais, Sophie Perrin, Marine Nicolas, Sylvie Uhlrich, Martine Chabaud-Riou
• Affiliations: Sanofi Pasteur, Campus Mérieux, 1541 Avenue Marcel Mérieux, 69280, Marcy l’Etoile, France
• (Day 1: Vaccine Product Bioassays Session)

Vaccine Clinical Serologies: Making the Bridge Between Analytical and Clinical Biases

• Speaker: Anne Benoit, GSK Vaccines
• Abstract: Clinical assays are developed and maintained to ensure that clinicians can rely upon the measurements to make appropriate decisions. In the context of vaccine trials, clinical assays support immunogenicity and efficacy endpoints and systematic measurement errors may compromise clinical decisions.We derive the mathematical relationships between proportional or constant systematic measurement errors and three typical clinical endpoints: geometric means concentrations ratio, seroprotection and seroconversion. We assess the impact of those errors on the estimation of the clinical endpoints but also on the decision making in clinical trial.Knowledge of this information allows for a fit-for-purpose risk-based approach to the development and maintenance of clinical assays and therefore to better connect the clinical laboratories and trials objectives.
• Contributing Authors: Anne Benoit, Swetlana Berger, Andrea Callegaro, GSK Vaccines, Belgium
• (Day 1: Vaccine Product Bioassays Session)

Experience with a Novel Statistical Approach to Plate Based Methods

• Speakers: Alka Bishop and Sarah Watts, Porton BioPharma Ltd
• Abstract: Porton Biopharma Ltd. has manufactured Anthrax Vaccine since the 1950s for UK Government. Production processes have not changed since that time and employ traditional methodology. A mouse potency test, which employs a plate-based Toxin Neuralisation Assay (TNA), has been developed to replace the current challenge assay. In addition, to support improvements in the manufacture of the vaccine from Thompson Bottles to Wave Bags a plate-based ELISA was developed. In collaboration with Quantics, data analysis for both of these tests was conducted using the company’s newly-developed statistical package QuBAS. QuBAS analysis was compared to traditional practices of data analysis, generating similar results to SoftMax Pro. It was found that using QuBAS, precision was improved; it was possible to use data that would have been previously rejected by SoftMax Pro; and skewing of data was prevented which resulted in fewer repeat tests. QuBAS shows promise for analysis of plate-based methods.
• (Day 2: Calculating Potency Session)

Development of a Suite of Assays to Characterize an Immuno-Oncology Target

• Speaker: Lisa Blackwood, Sartorius Stedim Biotech
• Abstract: Immuno-oncology is an expanding field as the world of science looks to treat different types of cancer. One of the most promising approaches is monoclonal antibodies (mAbs) directed against checkpoint inhibitors. These mAbs act by triggering the immune system to defend itself against cancer cells by blocking the immune systems normal checkpoints. These pathways in a healthy person are critical for maintaining self-tolerance and modulating our immune response to minimize damage to neighbouring tissue. However, cancer cells express checkpoint inhibitor molecules to create a suppressive environment as an act of immune resistance. These inhibitors can be readily blocked by antibodies, for example nivolumab and ipilimumab, which bind to PD-1 and CTLA-4, respectively, inhibiting their interactions with their ligands, arresting any further inhibitory signals.
  Sartorius Stedim Biotech have developed a suite of assays to characterize immuno-oncology targets, from Fab binding to target antigen by ELISA and SPR through to more complex MLR bioassays. Using nivolumab as an example we will demonstrate the flexibility of our platform methods to adapt to other immuno-oncology targets from early clone selection through to functional characterization of the mAb.
• (Day 2: Product Specific Case Studies Session)

How to Select and Optimize Characterization Criteria for Assay-Ready Cells – A Case Study

• Speaker: Karin Blume, Svar Life Science
• Abstract: Characterization of cultured cell lines for production of biologic material and cell lines used for potency applications are outlined in various guidance documents – but there is an absence of specific instructive information about the requirements for genetically modified cell lines, such as modified cell banks used for production of assay ready cells utilized for e.g. in bioassays, ADCC assays and neutralization assays in GxP applications.
  Here we offer examples from 10+ years working in development, validation and production of custom genetically modified cell lines and assay ready cells used in reporter gen assays. Examples will showcase critical aspects of characterization and include lessons learned as well as recommendations on what can be considered as an appropriate level for assay read cells.
• (Day 1: Hot Topics: Ready-to-Use Cells Session)

Evaluation of iLite ADCC Effector Cells Using Different CD20 Target Cells

• Presenter: Karin Blume, Svar Life Science AB
• Abstract: The clinical activity of numerous monoclonal antibodies is to some extent mediated by antibody-dependent cell-mediated cytotoxicity (ADCC). Traditional methods for quantification of ADCC activity are labour intensive and show a high level of inherent variability due to the use of primary human NK cells from different donors as effector cells. The use of an engineered effector cell line has shown a significantly lower variation between assays. However, there is a need for a controlled set of target cells to allow differences in ADCC activity to be determined with precision and a high degree of specificity.
  Here, we present a comparative case study using a novel ADCC effector cell line expressing the firefly luciferase reporter gene under control of the V-variant of the FcγIIIa receptor, to compare a novel target cell line overexpressing a constant high level of a specific antigen and the homologous wild type target cell line. Our comparative data show results for typical bioassay application characterization such as sensitivity, specificity, accuracy, precision and potency.
• Contributing Authors: A Charles River & Svar Life Science Collaboration
• (Poster)

Parallelism Assessment in Bioassay from Development to Transfer

• Presenter: Claire Borel, Sanofi
• Abstract:
  • The F-test is a statistical test applied on reference and sample curves and based on variance analysis without taking bioassay method variability into account. The main weakness of the F-test lies in its inability to reject non-similar curves obtained with high variability assays. The risk to accept data coming from high variability assays is better controlled with an equivalence approach.
  • Establishment of equivalence limits requires a large dataset not available at the beginning of the development. For early phases, the parallelism between standard and test curves is established using a combination of F-test p-value and R². This allows to minimize the risks of rejecting very precise assays due to small differences and accepting non-parallel assays due to high variability.
  • The statistical evaluation of parallelism can be changed for late phase to an equivalence approach (with equivalence limits) for better assay control. The equivalence limits are defined from historical data reflecting bioassay variability. They provide an added advantage to the F-test by identifying the parameters of the curves (bottom asymptote, top asymptote or slope) responsible of the non-parallelism.
• Contributing Authors: BOREL Claire, CREPIN Ronan, ALEXANDRE Sylvie, PERIC Delphine, VARRICCHIO Sara, GEORGES Pauline, BISCH Grégoire, LECHERE Karine, CHEREL Monique, BRAULT Dominique, SOUAIFI-AMARA Hajer
• (Poster)

Automation of ELISA Platform by Liquid Handling Station Accelerates Analysis of Therapeutic Antibodies

• Presenter: Anke Breckner, AbbVie Deutschland GmbH & Co.KG
• Abstract: ( An expanding pipeline increases the demand for rapid assay development, which can be met by the use of platform methods. As a first step we established a standardized assay set-up for the Enzyme-linked Immunosorbent Assay (ELISA), which facilitates global cross-functional method development as well as the implementation of automated systems for sample analysis. By automation of the standardized ELISA platform we will be able to increase sample throughput and improve efficiency for analyzing therapeutic antibodies. The Hamilton liquid handling system automatizes the ELISA platform method, which offers the direct measurement of the monoclonal antibody’s binding capability to its intended target, an important method for potency testing. The liquid handling station performs the complete ELISA binding assay in a fully-automated fashion due to the integration of a microplate washer and absorption plate reader. The joint control of this workstation allows an efficient scheduling of all devices and working steps. Dilutions, washing steps and incubation times are carried out for up to three microplates simultaneously with only a minimum of manual preparation of the workstation. Due to the standardized and easy-to-operate system set-up, the implementation of upcoming platform ELISAs will be facilitated.)
• Contributing Authors: Anke Breckner¹, Nicolai Nitsche², Lisa Schroeder¹, Stephanie Voehl², Dr. Gleb Konotop², Dr. Thorsten Pflanzner², and Renate Kron^{2
  1} Development Sciences, AbbVie Deutschland GmbH & Co. KG, Ludwigshafen Germany
• (Poster)

SPR For Tomorrow’s Biotherapeutics

• Presenter: Jennifer Clark, Antibody Analytics
• Abstract: (Pending)
• Contributing Authors: Jennifer Clark, Gillian Goodwin, Stuart Knowling
• (Poster)

Bridging Study of ADCC Reporter Bioassay for Product Lot Release with Improved PBMC-Based ADCC Cytotoxicity Assay during Monoclonal Antibody Development

• Speaker: Mei Cong, Promega
• Abstract: Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important and common Mode of Action (MOA) of antibody drugs, especially of those targeting cancers. Previously, we have established surrogate ADCC reporter bioassays for product release using engineered effector cells stably expressing FcRIIIa and luciferase reporter. The assay demonstrated specificity and is suitable for stability study in a quality-controlled environment with appropriate precision, accuracy, linearity, and robustness. However, classic ADCC assay measuring target cell killing is still required to demonstrate drug’s MOA. Here we will discuss an improved ADCC cytotoxicity bioassay using ADCC-prequalified PBMCs and engineered target cells with NanoBiT® Technology. The Nano-Glo® HiBiT Extracellular Detection System can accurately quantify any HiBiT-Tagged protein released from the target cells due to ADCC. The assay is simple, homogenous, highly sensitive, and demonstrates antibody potency comparable with the ADCC Reporter Bioassay in ADCC bridging studies.
• (Day 1: Bridges Over Troubled Water Session)

Bridging MOA-based ADCC Reporter Bioassay with an Improved PBMC-based ADCC Cytotoxicity Assay for Immunotherapy mAb Development

• Presenter: Mei Cong, Promega
• Abstract: Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody-bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the use of primary effector cells makes them vulnerable to high assay variability and therefore not suitable for use in a quality-controlled environment during product manufacture and development.
  Previously, we developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing PBMC-based ADCC cytotoxicity assays and ADCC reporter bioassays, we recently developed an improved ADCC cytotoxicity assay using PBMCs and engineered HiBiT target cells. When HiBiT-expressing target cells are incubated with an antibody and PBMCs, the target cells are lysed and release HiBiT peptides, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate luminescence. The assay is simple, homogenous, highly sensitive, and gives a robust assay window. It shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies.
  Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• Contributing Authors: Pete Stecha, Denise Garvin, Jim Hartnett, Aileen Paguio, Brock Binkowski, Frank Fan, Mei Cong, and Zhijie Jey Chent
• (Poster)

Development of an ELISA for Use in Retrospective Studies Using Dried Blood Spot Cards

• Speaker: Hannah Cuthbertson, Public Health England
• Abstract: Neonatal blood samples are routinely taken onto dried blood spot cards for the diagnosis of a range of health conditions shortly after birth. After these cards have served their primary purpose they could be used to answer health-related questions for the individual or a cohort. In the UK dried blood spot cards are stored in uncontrolled conditions. This poses a problem for retrospective studies because temperature and humidity impact the quality of this sample type. We have previously developed and validated an ELISA for the quantification of antibodies in fresh human venous or cord blood samples for the use in vaccine clinical trials. We have now adapted this ELISA so that it can be used with blood extracted from dried blood spot cards. In addition to the normal assessment of an ELISA’s performance we also present the challenges of sample extraction and sample condition.
• Contributing Authors: Hannah Cuthbertson, Mary Matheson, Bassam Hallis
• (Day 1: Vaccine Product Bioassays Session)

Fixed and Random Effects in Non-Linear Models for ELISA Assay Calibration

• Presenter: Lorraine Dawirs, GSK
• Abstract: The four-parameter logistic model is usually applied for fitting the standard curve in the case of ELISA assays. The objective of the calibration analysis is, indeed, to define an appropriate statistical model and afterwards to define the reading zone. Calibration analysis is therefore performed only on standard curves. The experiments are typically performed on different days and by multiple operators on different plates. The different curves are then usually modeled independently per plate by using four parameter logistic models.
  In this work, this simple approach will be compared to a mixed model fitted on ELISA assay data, where effects of multiple days and operators are taken into account in a non-linear random effects model. Homoscedastic and heteroscedastic models (with a power of the mean variance function) will be compared. Different procedures in SAS (i.e. proc nlin, proc nlmixed) and different R package (i.e. drc, medrc, minpack.lm) will be compared on a case study.
• Contributing Authors: Bernard Francq, Lorraine Dawirs & Dhliwayo Farai
• (Poster)

Development of a Cell-Based Bioassay for a Mixture of Two HexaBody Molecules Targeting DR5

• Speaker: Simone De Haij, Genmab
• Abstract: Genmab is an international biotechnology company specialized in the development of innovative antibody therapeutics for the treatment of cancer. Genmab’s proprietary HexaBody® technology allows for the creation of potent therapeutics by inducing assembly of antibody hexamers (clusters of six) after target binding at the cell surface. This results in enhanced C1q binding and complement dependent cytotoxicity (CDC), as well as antibody-mediated clustering of cell surface receptors. Hyperclustering of death receptor 5 (DR5) after binding of its ligand induces apoptosis. The HexaBody technology was applied to improve antibody-mediated DR5 clustering. HexaBody-DR5/DR5 (GEN1029) is a 1:1 mixture of two non-competing DR5-specific antibodies and shows potent DR5 agonist activity on cancer cells. A Phase I/II study in solid cancers is ongoing. Cytotoxicity by HexaBody-DR5/DR5 requires dual epitope targeting, enhanced hexamerization and is most optimal in the presence of the complement component C1q. This presentation will discuss the selection and development of a cell-based potency assay that reflects the mechanism of action of this novel type of antibody therapeutic.
• (Day 2: Product Specific Case Studies Session)

The Life Cycle of a Potency Method: How Lessons Learned on the Road from Assay Development Towards Commercial Validation Lead to a High-Quality Bioassay

• Speaker: Femke de Keijzer-van de Water, Janssen Vaccines & Prevention B.V.
• Abstract: At Janssen Vaccines and Prevention BV, vaccines are developed that utilize the Janssen’s AdVac® adenovirus vector platform. For this purpose, Ad26 AdVac® vectors are engineered to contain small genetic fragments of a pathogen. Upon immunization, these small fragments are expressed, ultimately leading to immunity towards the pathogen in scope.
  Potency testing of these vaccines consists of a combination of measurement of infectivity and transgene expression, both attributes being part of the vaccine mode of action. For measurement of infectivity a quantitative PCR based potency assay (QPA) has been implemented, which is based on a previously published method.
  In 2007, first steps were taken to implement a QPA assay to measure infectivity of Ad26 vectors as an alternative to conventional infectivity methods like TCID50. The advantages of the QPA assay are higher throughput, shorter assay duration, and better precision than TCID50. In the QPA assay, real-time quantitative PCR is used to measure newly synthesized viral DNA after inoculation of a HEK293 cell monolayer with the test article or reference material, which is used as calibration curve. Post infection, excessive virus particles are washed away, and virus particles taken up by the cells are further incubated to allow for virus replication. Finally, cells are lysed, and quantitative PCR is performed using specific primers and probes. The potency of test articles is then determined relative to the calibration curve, to which a titer was assigned by TCID50 analysis.
  Initial assay implementation activities focused on optimization of assay conditions and assay format by testing of critical method parameters, implementation of specific primer-probe sets, assuring stability indicating potential of the assay, and initial robustness testing. For in process monitoring a robotized version of the method was developed which supports high sample throughput (currently >10000 samples/year), while in GMP release and stability testing a manual version was implemented.
  In the following years, the manual QPA assay was qualified to support Phase I and II clinical trials for multiple projects. In this stage, most improvement activities focused on inclusion of measurement of multivalent products in addition to monovalent products and its specific challenges and assessment of matrix effects by changes in product formulation. In addition, observations made during routine testing resulted in further improvement of assay and sample acceptance criteria.
  Heading further towards late stage clinical development (Phase III and preparation for process performance qualification (PPQ)), focus has been on a more structured approach of robustness testing and sample hold times using analytical quality by design and design of experiments. In addition, forced degradation studies were performed to assess stability indicating potential for different stress conditions and assay validation for PPQ was performed for our first product.
  For two other vaccines, preparations for analytical co-validation together with a Janssen commercial testing site are currently ongoing. This brings its own new challenges, with focus on anticipation for future. Examples are setting up strategies for continuous critical reagents supply and adapting to the advancing world of equipment and software availability in combination with data integrity.
  In summary, a bioassay potency method is continuously changing throughout its life cycle, each stage having its own challenges with as goal a high-quality method.
• (Day 1: Bioassay Lifecycles Session)

T-Cell Redirecting Antibodies Based Bioassay for Evaluation of PD-1/PD-L1 Inhibitors Activity

• Presenter: Alexander Doronin, BIOCAD
• Abstract: PD-1/PD-L1-based therapy has been named a revolution in cancer treatment. By the end of 2018, more than 100 anti-PD-1 and anti-PD-L1 antibodies were in various stages of development, and more than 2000 clinical trials with their use have been registered. Characterization of such antibodies requires a bioassay to determine their biological activity. In this study, we developed a cell-based bioassay for analyzing the activity of anti-PD-1 and anti-PD-L1 antibodies. We chose reporter system consisting of two cell lines and compared several approaches for activation of effector cell line based on superantigens, soluble anti-CD3 antibodies, transmembrane anti-CD3 antibodies, chimeric antigenic receptors and bispecific T-cell redirecting antibodies. The bispecific T-cell redirecting antibodies offer several advantages over the other approaches. We characterized the bioassay and demonstrated its applicability for analyzing the activity of anti-PD-1 and anti-PD-L1 antibodies. The proposed bioassay can be useful in the development of new therapeutic agents and methods for their characterization.
• Contributing Authors: A. N. Doronin, A. A. Gordeev, A. E. Kozlov, Ya. A. Smirnova, M. Yu. Puchkova, V. M. Ekimova, Yu. I. Basovskiy, V. V. Solovyev
• (Poster)

Bench to Bedside: Efpeglenatide Bioassays

• Speakers: Stuart Dunn, Covance and Christine Graf, Sanofi-Aventis Deutschland GmbH
• Abstract: Efpeglenatide is a new GLP1 agonist compound coupled to an Fc part. In order to determine relative potency, cAMP levels are measured in a cell-based assay. Here we report the development of a new state of the art bioassay to replace a time-consuming variable cAMP assay system, as well as challenges during assay transfer to a CRO / CMP. Failure to understand the developed assay or the importance of the right level of training can potentially lead to lengthy delays and even switching to another provider. However, the risk can be significantly reduced if a strong collaboration, meticulous planning and the right approach are implemented. A real-life example of a potency assay is presented, developed by Sanofi – BD Ger and successfully established within the CRO laboratory at Covance.
• (Day 2: Bioassays for Product Characterization Session)

T-cell Exhaustion: An in vitro Model that Emulates a Complex Biological Phenomenon

• Presenter: Lynne Dunsford, Antibody Analytics
• Abstract: Antibody Analytics has developed an in vitro model of chronic antigen stimulation that generated T cells with phenotypical and functional exhaustion. These T cells persistently express high levels of co-inhibitory molecules such as PD-1, TIM-3, CTLA-4 and LAG-3, and have impaired ability to produce cytokines (IL-2 and IFNγ). Importantly, treatment with the checkpoint inhibitors Nivolumab and Pembrolizumab (anti-PD-1 antibodies) partially restores the functionality of these cells in terms of IFNγ production and cytotoxicity.
  Since most T cell exhaustion assays employ mouse models, using chronic viral infections, this difficult and expensive method has not allowed testing of human-targeting therapeutics. There has been an unmet need for a convenient and biologically relevant in vitro model of T cell exhaustion.
  Antibody Analytics is the first CRO to develop and characterise a human in vitro T cell exhaustion assay the effectively mimics the cells of the tumour microenvironment. This assay is applicable for various targeted therapeutics including checkpoint inhibitors, bispecifics and combination therapies.
• (Poster)

Quantitative Cell-Based Bioassays to Advance Immunotherapy Programs Targeting Immune Checkpoint Receptors

• Presenter: Steven Edenson, 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 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, TIM-3) and co-stimulatory (e.g. 4-1BB, GITR, OX40, ICOS) 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-based functional bioassays to interrogate modulation of immune checkpoint receptors individually (e.g. PD-1, CTLA-4, LAG-3, TIM-3, GITR, 4-1BB) and in combination (e.g. PD-1+CTLA-4, PD-1+LAG-3). These assays consist of stable cell lines that express luciferase reporters driven by response elements under the precise control of mechanistically relevant intracellular signals. Thus, the bioassays reflect mechanisms of action for the drug candidates designed for each 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.
  Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• Contributing Authors: Jamison Grailer, Julia Gilden, Pete Stecha, Denise Garvin, Jun Wang, Michael Beck, Jim Hartnett, Frank Fan, Mei Cong and Zhi-jie Jey Cheng
• (Poster)

Potency Assessment for Novel AAV-Based Drugs: Exploration of Different Biological Activities and Stability Indicating Properties of the Assays

• Speaker: Marina Feschenko, Biogen
• Abstract: Gene therapies, including AAV-based vectors, are novel types of drugs, which require development of many new analytical assays and implementation of additional regulatory guidelines. Potency of gene therapy drugs can be assessed in animal models and cell-based assays. The lack of relevant animal models for human diseases coupled with large variability and high cost of testing makes them a rare choice for drug release. The cell-based assays can measure three different activities of the viral vector: ability to penetrate cells (infectivity), target protein expression, and functional potency. Do we need to have all three or we can justify selecting one or two assays for release? Our approach for two different programs and exploration of stability indicating properties of several potency assays will be presented.
• (Day 2: Potency Assays for Complex Products Session)

Developing Robust Ligand-Binding Assays Through Key Antigen Characterization

• Speaker: Luc Gagnon, NEOMED-LABS
• Abstract: Ligand binding assays (LBA) are analytical procedures (e.g., ELISA, MSD, Gyros, EIA) used to quantify an analyte based on its affinity for a ligand such as antibodies, antigens, etc. In the vaccine field, LBA are frequently used to assess the immunogenicity and magnitude of the vaccine-induced humoral immune response. Moreover, some of these assays correspond to primary/secondary readouts and are critical in the success of the vaccine filing. To properly fulfill its role, LBA must remain stable and reliable to provide accurate and consistent results along all vaccine development phases. As such, appropriate tools must be implemented to mitigate the impact of changing critical reagents on the quality of the results being generated. We have developed an orthogonal approach to fully characterize the critical reagents used in the LBA assays. The characterization package allows to assess: size in solution, purity identity, affinity, folding, aggregation and multimerization state. Here we present two case studies where such antigens were produced and fully characterized prior to being used in LBA. But using this approach, we are able to accelerate the development LBA assay without compromising the quality.
• (Day 1: Vaccine Product Bioassays Session)

Reference Standard – Challenges and Opportunities

• Instructors: Sian Estdale, Covance; Bassam Hallis, Public Health England; Jane Robinson, BEBPA; Peter Rigsby, NIBSC
• Abstract: (Pending)
• (Workshops-Track 1: Reference Standard – Challenges and Opportunities)

Delivering the Message

• Speaker: Barbara Hebeis, AstraZeneca
• Abstract: This talk will describe the development of a bioassay strategy to assess critical quality attributes of three different gene therapy modalities delivering the same drug. This will address challenges arising from tissue tropism, biology of the delivery vehicle and correlation with orthogonal tests in order to generate the control strategy for phase 1 clinical assessment.
• (Day 2: Potency Assays for Complex Products Session)

Case Study: Different Estimators of Precision and Measures for the Goodness-if-Fit in a Cell-Based Potency Assay

• Presenters: Nicole Herzmann, Richter-Helm BioLogics GmbH & Co. KG
• Abstract: The cell-based potency assay outlined in this case study demands special accurateness during manual pipetting, due to several small volume additions in a short time frame. Thus, the setting of reasonable assay acceptance criteria is crucial to guarantee the validity of the final relative potency result. During different phases of assay life cycle the definition of acceptance criteria was improved and a variety of statistical measures were tested for their suitability. Within this case study, the focus was set on statistical measures for the precision of the raw data, goodness-of-fit of the reference standard dose-response curve and the outlier testing. In conclusion, a suitable outlier rule and new statistical measures for setting acceptance criteria could be identified.
• Contributing Authors: N. Herzmann¹, J. Hecker¹, D. Welsch², and S. Klingelhöfer^{1
  1} Richter-Helm Biologics, Biological Assays, Suhrenkamp 59, 22335 Hamburg, Germany;
  ² Rey Analytical Research, Große Kirchstr. 52, 51373 Leverkusen, Germany
• (Poster)

Novel Reporter-based Bioassays for Evaluating FcγR-dependent Functions of Therapeutic Antibodies

• Presenter: Axel Johann, Promega GmbH
• Abstract: Monoclonal antibodies (mAbs) as therapeutic modalities have had promising effectiveness for a wide variety of clinical applications including cancer immunotherapy. They are primarily designed to deliver therapy through variable domains to address target specificity, and, for many, antibody constant regions (Fc) also play crucial roles mediated through interaction with Fcγ receptors (FcγRs). mAb Fc domains can be engineered to enhance or diminish their activities. Sensitive and precise analysis is critical for understanding functional alterations due to Fc engineering. Here, we report on the development of a platform of cell-based reporter bioassays with engineered cell lines expressing various Fcγ receptors for quantitative measurement of antibody Fc functions. This includes bioassays for measuring antibody Fc effector functions (ADCC, ADCP, CDC) and for determining the Fc crosslinking-dependency of agonistic activities for immunomodulatory antibodies. These reporter gene bioassays are homogenous, easy to use, sensitive and robust. They are optimized and pre-qualified to show the specificity, accuracy, precision, linearity and robustness required for both screening workflows and quality control lot release environments. Together, they represent a comprehensive suite of tools to use for antibody drug development in immunotherapy programs.
  Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
• Contributing Authors: Jamison Grailer, Denise Garvin, Jun Wang, Michael Beck, Pete Stecha, Jim Hartnett, Frank Fan, Mei Cong, and Zhi-jie Jey Cheng
• (Poster)

Assessing the Potency of SPEAR T-Cells: Development and Validation of a Flow Cytometry-Based Cytotoxicity Assay

• Presenter: Rachel Kenneil, Adaptimmune
• Abstract: In adoptive T-cell therapy with SPEAR (specific peptide enhanced affinity receptor) T-cells, CD3+ cells from the patient are transduced with a lentiviral vector to express a T-cell receptor with enhanced affinity against a target antigen. The T-cells are then expanded to reach target dose and infused into the patient. SPEAR T-cells targeting the antigens MAGE-A4 (ADP-A2M4) and AFP (ADP-A2AFP) are currently in Phase 1 clinical studies in patients with multiple solid tumours.
  Pivotal trials of SPEAR T-cells will require testing in a validated potency assay to be included to release batches. To meet this need, we have developed a flow cytometry-based assay that measures target-specific T-cell cytotoxicity within our UK Development laboratories. The ADP-A2A4 SPEAR T-cell cytotoxicity assay has been successfully transferred to the QC laboratory of our US cell manufacturing site and validated in accordance with ICH guidelines. The validated assay will be used by our QC laboratory to release product for our upcoming Phase 2 trial. We will present an overview of the assay from development to validation.
• Contributing Authors: Rachel Kenneil, Charlotte Lucy, Angelica Cazaly, Shital Kakkar, Jackie Muto, Megan Walsh, Mark Tatlock
• (Poster)

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

• Speaker: 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. In this talk, we present data for bioassays targeting two inhibitory checkpoints: PD-1 and SIRPα. These new MOA-based PathHunter® Checkpoint bioassays utilize the industry-validated Enzyme Fragment Complementation (EFC) technology, with an easy-to- use protocol that delivers rapid results. These assays facilitate the development of relevant therapeutics, enabling rapid and sensitive screening of biologics and small molecules. These cell-based assays do not require human primary cells, and deliver a highly sensitive response. Furthermore, the robustness and reproducibility of these assays lend themselves well for use in characterization, relative potency, and QC lot release testing of immunotherapy drugs.
• (Day 2: Bioassays for Product Characterization Session)

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 the new PathHunter® Checkpoint assays that target clinically relevant co-inhibitory and co-stimulatory checkpoint receptors and measure receptor activation and signaling, using the industry-validated Enzyme Fragment Complementation (EFC) technology. 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 for use in characterization, relative potency, and QC lot release testing of immunotherapy drugs. These mechanism of action-based, cell-based assays do not require human primary cells, and provide a highly sensitive response, with an easy-to- use protocol that delivers results in a day. In this poster, we present data for bioassays targeting PD-1, and the emerging IO target: SIRPα/CD47 signaling axis.
• Contributing Authors: Jane Lamerdin, Mimi Nguyen, Hyna Dotimas, Ai Shih, Alpana Prasad, and Jennifer Lin-Jones
• (Poster)

Qualified, Fit-for-Purpose Bioassays for Liraglutide and Exenatide as Frozen Ready-to-Use Cells

• Presenter: Jane Lamerdin, Eurofins DiscoverX
• Abstract: With more than 25% of the world’s population diagnosed with type II diabetes and/or metabolic syndrome, the requirement for cost effective drugs to control patient disease is increasingly urgent. Agonist drugs of the Glucagon-like peptide-1 (GLP-1) receptor, such as Victoza® (Liraglutide) and Byetta® (Exenatide), modulate glucose-induced insulin secretion and have shown excellent results in the clinic. These molecules are highly sought after for biosimilar and biobetter development, however, the existing cell-based assays to support this development are complex and require extensive development time. Here, we describe the development and qualification of ready-to-use (RTU), fit-for-purpose potency bioassays for these two drugs. Based on the drug’s well-characterized mechanism of action, these bioassays measure production of cyclic AMP (cAMP) in response to activation of the GLP1 receptor with agonist drug. The assay relies on an easy-to-use homogenous protocol for rapid implementation at any lab globally, resulting in a chemiluminescent readout that can be read on any plate reader. The assay was qualified using the marketed innovator molecules through a multi-day qualification exercise with two analysts. The assays demonstrated high reproducibility, accuracy, and precision, with good linearity over the tested range of 50%-150%. Importantly, these assays have been developed into frozen RTU cells and the data presented here demonstrates the multiple technical and operational advantages of this approach over the traditional continuous culture assays.
• Contributing Authors: Gayatri Paranjpe, Rajini Bompeli, Alpana Prasad, Alexander Baumann, and Jane E. Lamerdin
• (Poster)

Building Robust and Simple Cell-Based Assays for Interleukins and Their Receptors

• Presenter: Jane Lamerdin, Eurofins DiscoverX
• Abstract: Ligand-induced receptor dimerization is an early functional step in receptor activation, representing the most proximal, functional readout for Interleukin receptors. It is well understood that specific signaling receptor subunits can heterodimerize with multiple high affinity receptors from the same family leading to complicated signaling cascades that are implicated in auto-immune, inflammatory, and oncogenic diseases. Here we present a novel application of our PathHunter® technology to monitor receptor-receptor interactions at the surface of intact live cells, applicable to diverse receptor types, with a specific focus on the interleukin family of receptors. The high specificity and simplicity of the assay protocol, serum tolerance, and reproducibility of these assays has enabled their use in cell-based screening, functional characterization, and QC lot release assays. Examples from our menu of assays spanning ~80% of all human interleukins will be presented, including assays for Anakinra and Tocilizumab. These robust assays have excellent reproducibility, accuracy and precision, demonstrating their suitability for use as QC lot release assays.
• Contributing Authors: Hyna Dotimas, Sangeetha Gunthuri, Hanako Daino-Laizure, Alpana Prasad, and Jane Lamerdin
• (Poster)

A Comprehensive Re-Examination of Equivalence Methods for Similarity

• Speaker: David Lansky, Precision Bioassay
• Abstract: Using equivalence bounds to assess similarity in bioassays are, with virtually no doubt, a giant step forward compared to testing null hypotheses of perfect similarity. There are several pervasive misunderstandings about the theory and practical uses of equivalence bounds, how to set them, and how to use them; this talk will address these with practical solutions. Important take-aways include: 1) equivalence bounds should be set based on the impact of non-similarity (one important impact is bias of potency), 2) power and sample size calculations are just as important for equivalence tests as they are for any other tests, 3) for many practical cases, demonstrating sufficient similarity given the intended use of the assay is very demanding and will likely require more within assay replicates and likely also replicate assays.
• (Day 1: Assessing Similarity Session)

Your Statistician is Your Friend: Parallelism in Immunoassays

• Speaker: Nancy Niemuth, Battelle
• Abstract: In theory, parallelism/similarity is the same for immunoassays as relative potency assays. The intended use of immunoassays and the need to work with biological sample matrices make the evaluation of parallelism between reference and test samples challenging in immunoassays. A broad range may be required for testing unknown samples, so that very few concentrations are available in the linear range of the assay where parallelism can be assessed. This talk considers several approaches to evaluating parallelism in immunoassays both for the assay and on a per sample basis.
• (Day 1: Assessing Similarity Session)

Bioanalytical characterization of a highly active TP

• Speaker: Natko Nuber, Novartis Pharma AG
• Abstract: ~MoA characterization through various bioassay formats
  ~Bioanalytical assessment of drug’s quaternary structure
  ~Impact of aggregates on the potency in vitro
• (Day 2: Bioassays for Product Characterization Session)

What Does It Take to Calculate Potency and Which Data Do You Need?

• Speaker: Ásdís Pétursdottir, Novo Nordisk
• Abstract: As a critical quality attribute, the determination of biological potency by bioassays play a key role in the development and control of biological products. A bioassay needs to be designed and developed to be sufficiently robust to measure manufacturing consistency. Historically, assays have been developed to suit a 96 well plate with a capacity of 3-4 samples per plate. An assay with 3 plates thus gives 3-4 results per setup. With an assay time of 2-5 days, the capacity is clearly limited. As there is an increasing need for potency determinations, either a high throughput approach or manning up has to be implemented.In this study we have investigated the effect of a plate layout on potency determination by the use of reduced curves. We will show, that by omitting numbers of dilution of the sample investigated, we obtain the same results as if full curves were used. By this approach it will be possible to increase the number of samples analyzed per run from 3 to 8 or 9.
• (Day 2: Calculating Potency Session)

Smart Bioassay Development, Validation and Transfer

• Speaker: Eric Rozet, Pharmalex Belgium
• Abstract: Since the adoption of the ICH Q8 document concerning the development of pharmaceutical processes following a quality by design (QbD) approach, there have been many discussions on the opportunity for analytical procedure developments to follow a similar approach. Authorities such as US Pharmacopoeia and ICH are currently preparing tomorrows regulations to implement Analytical QbD and introducing the idea of Analytical or Assay life cycle. The life cycle steps of a bioassay, that are development, optimization, validation, transfer, comparisons and bridging, are all interconnected and should ensure the results produced remain of acceptable quality. Indeed, bioassay results are used to make crucial decisions about the development of the drug products to assess their efficacy and safety.
  This presentation aims at showing the different steps to smartly use all information gathered throughout the life cycle of the bioassay to ensure its fitness of purpose. Combining scientific knowledge with adequate statistical methodologies has a crucial role to play, such as design of experiments, statistical modeling, design space definitions and probabilistic statements. Accumulating the knowledge of the bioassay performances gathered at each step ensures the release of useful bioassays, that are under control and that allows making the right decisions with confidence.
• (Day 2: Product Specific Case Studies Session)

Quality Control for Cell and Gene Therapy Products: Case Study of an Autologous CAR T Cell Product

• Speaker: Petra Schroeder, BioNTech IMFS GmbH
• Abstract: Gene Therapy represents an emerging concept in human health care with the potential to address unmet medical needs and improve patients’ lives. Here we provide an insight into the analytical activities required for manufacturing and releasing Advanced Therapeutic Medicinal Products (ATMPs) by the example of an autologous CAR T cell product. In addition to compendial methods applied for microbiological control, analytical assays characterizing the specific product will be presented, covering purity, identity and potency along with vector copy number and test for replication competent retrovirus (RCR). Furthermore, the potential for cost reduction in analytics of ATMPs will be discussed.
• (Day 2: Potency Assays for Complex Products Session)

Introduction to Statistics for Assay Validation

• Instructor: Perceval Sondag, MSD
• Abstract: (Pending)
• (Workshops-Track 2: Introduction to Statistics for Assay Validation)

A Bioassay Comparability Assessment Between Two Contract Labs (Case Study)

• Speaker: Anton Stetsenko, ADC Therapeutics
• 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.
• (Day 1: Bridges Over Troubled Water Session)

Cell-Based Acoustic Liquid Handling – Transfer from 96- to 384-well Assay

• Presenter: Paula Urquhart, Covance
• 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. This presentation will discuss identifying solutions to the challenges of automating a bioassay. We describe the deconstruction and optimization of the cell-based workflow to provide an automated small volume 384 well bioassay with many benefits compared to traditional methods. The result is a single instrument assay which provides additional replicates in a miniaturized reaction to reduce sample requirement and cost.
• Contributing Authors: Paula Urquhart, Stuart Dunn and Sian Estdale
• (Poster)

The USP Bioassay Chapters: Come Join the Journey

• Instructor: Steven Walfish, Principal Scientific Liaison, United States Pharmacopeia
• Abstract: USP Chapters <111>, <1030>, <1032>, <1033> and <1034> were last updated nearly fifteen years ago. An Expert Panel formed to review and update the chapters. With oversight from the USP Statistics Expert Committee the panel will determine if there is any new advancement that would make the bioassay chapters more valuable to their stakeholders. More examples, and clarifying the definitions is propsed. Any proposed changes are vetted in USP’s Pharmaceutical Forum (PF).
  This presentation focuses on the USP suite of bioassay chapters. A discussion of the current state, and future plans will be shared with the participants. An open dialogue with participants is expected to gain valuable feedback on areas where the chapters can be improved to be more user-friendly. Do not miss your chance to be part of the change.
• (Workshops-Track 3: USP Bioassay Chapter Update and Comment Session)

Turning Cells into Reagents – What Makes a Cell Assay Ready?

• Speaker: Oliver Wehmeier, acCELLerate GmbH
• Abstract: Assay ready cells are cryopreserved at a functional state and can be used in bioassays without prior cultivation or cell passaging. Basically, the cells are applied instantly after thawing like any other reagent.
  Introduced initially for the drug discovery process where assay ready cells help to streamline high-throughput screening, the approach is now widely accepted for other applications as well, like in vitro toxicology and the potency testing of biotherapeutics in GMP lot release bioassays.
  Although establish for quite some time now, there is still no clear guidance on what differentiates assay ready cells from a regular cell bank and how assay ready cells should be prepared and qualified. By giving examples the requirements on assay ready cells for different applications and assay types are demonstrated. Critical points to be considered when bridging an assay from continuous culture to assay ready cells will be discussed.
• (Day 1: Hot Topics: Ready-to-Use Cells Session)

Bridging Bioassay Methods … A Rough Sea to Cross!?

• Speaker: Annemie Wielant, UCB Pharma
• Abstract: During the lifecycle of biopharmaceuticals, the quality and the stability of the molecule is constantly monitored by a large pool of physico-chemical and bioassay methods.
  Over time, an analytical method (including bioassay) can be optimized or substituted by a more (clinical phase) appropriate method.
  In order to demonstrate comparability between methods and in order to show continuity of data across drug development, often a well-designed method-bridging exercise has to be conducted.
  Through real bridging data, we will show that demonstrating method equivalence is not always that straight forward.
  Therefore, the support from appropriate statistical tools and considering the data from another perspective (i.e in combination with other analytical techniques) can add value in better understanding the relationship (or the differences) between the 2 considered bioassay formats.
• (Day 1: Bridges Over Troubled Water Session)

Evaluation of a Novel Approach to Similarity Assessment in Surface Plasmon Resonance Spectroscopy

• Speaker: Stefan Winkler, Roche Pharmaceuticals AG
• Abstract: The assessment of similarity is a cornerstone of every potency assay and a prerequisite to calculate potency. Assessments of similarity based on components of a four-parameter logistic function as well as parallel-line models are common ways to address similarity in biological assays. However, it is unclear if different models account for changes in similarity in a comparable manner and how newer models like improved slope ratio analysis (iSRA) used in surface plasmon resonance (SPR) based assays relate to the aforementioned models. Here, we evaluated iSRA by sensorgram comparison of a wide range of well-characterized CQA samples from a 2+1 Crossmab with regard to performance of the same samples in assays using full curve four-parameter logistic fit component analysis as well as parallel line analysis. By measuring various CQA samples covering a wide spectrum of similarities with all 3 methods, we obtained a broad dataset to assess the quality and possible use of iSRA for future CQA assessments and comparability studies as well as extended characterization assays.
• (Day 1: Assessing Similarity Session)

To Finney and Beyond

• Speaker: Ann Yellowlees, Quantics Biostatistics
• Abstract: Professor David Finney was the father of bioassay statistics.His achievements in statistics included pioneering the development of systems monitoring drug safety, which still influence today’s monitoring systems. He worked alongside many notable statisticians of the 20th century, including Ronald Fisher and Frank Yates.
  His widely known books, Probit Analysis (1947) and Statistical Method in Biological Assay (1952), redefined those subjects.
  Finney died in Edinburgh last November aged 101. In this talk, we reflect upon his contributions to bioassay and consider what a 2020 edition of Statistical Method in Biological Assay might contain for the 21st Century.
• (Day 2: Professor David J. Finney Tribute Session)