Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Balancing Process mAb Modifications with Effects on Potency

• Speaker: Mimmi Ballard, Vir Biotechnology, Inc.
• Abstract: (Pending)
• (Day 1: Potency Assay Development Case Studies Session)

Development and Optimization of a TNFα Neutralising Assay Using a Quality by Design Approach

• Presenter: Pierre Baudoux, Quality Assistance
• Abstract: Development and optimisation steps are of major importance to ensure the delivery of reliable results in the routine use of a bioassay. Quality by Design (QbD) approach requires a combination of thorough knowledge of the assay and the use of proper statistical tools to achieve best results. .
  Development and optimisation steps are of major importance to ensure the delivery of reliable results in the routine use of a bioassay. Quality by Design (QbD) approach requires a combination of thorough knowledge of the scientific background of the assay and the use of proper statistical tools to achieve best results.This is a stepwise process including analytical target profile (ATP) definition, critical quality attributes (CQA) setting, candidate method analysis, risk assessment of any parameter that may influence assay results, screening experiments to identify the most important parameters, optimisation experiments on the major parameters.In the end, the “design space” of the assay is obtained defining the range of operating conditions that ensures, with a given probability of success, that results will meet CQA.In this study, we developed a TNFα neutralising assay for Adalimumab (Humira) testing using a QbD approach and finally validate it according to guideline.
• (Poster)

Development and Validation of an Antibody-Drug Conjugate Bioassay

• Presenter: Pierre Baudoux, Quality Assistance
• Abstract: Antibody-drug conjugates (ADCs) are a class of drugs used in the treatment of different cancers. Unlike chemotherapy, ADCs are designed to only target and kill tumour cells. Several tests are requested by the regulatory authorities in order to assess the quality, safety and efficacy of ADCs. Among these tests, a bioassay must be developed as a potency assay to demonstrate the cytotoxic effect of ADCs on target cells (specific cytotoxicity) as well as on cells that do not express the antigen (non-specific cytotoxicity).
  In this context, Quality Assistance has developed a cytotoxicity assay using trastuzumab emtansine (T-DM1) as a model compound. T-DM1 is an ADC that combines the humanised trastuzumab antibody (anti-HER-2) and the potent anti-microtubule agent DM1 (derivative of maytansine) using a highly stable linker. This ADC is used as treatment for patients diagnosed with a HER-2 positive breast cancer.This study demonstrates the successful development and validation of a cytotoxicity bioassay using trastuzumab emtansine as a model antibody-drug conjugate and also highlights the importance of the revelator agent choice. Agents measuring the metabolic activity of the cells are not always the best choice depending of the cell culture conditions. It is therefore more appropriate to use agents that directly measure the cytotoxicity based on exclusion dyes, DNA intercalant agents or change of the mitochondria membrane potential.The method was finally validated according to USP <1033> guideline in terms of specificity, linearity, precision (repeatability and intermediate precision) and relative accuracy. Moreover, the method was demonstrated to be stability-indicating and therefore allows for the detection of degraded samples.
• (Poster)

Implementing MOA-Reflective ADCC Assays using Ready-to-Use KILR Target and Effector Cells from Screening to Lot Release

• Presenter: Alexander Baumann, Eurofins DiscoverX
• Abstract: The clinical success of an ever-increasing array of biologics has led to the development of a wide spectrum of immunomodulatory agents with distinct mechanisms-of-action (MOA) targeting novel antigens. During development of antibody-based biologics, evaluation of antibody (Fc) effector functions is required by regulators as is needed for antibody dependent cell-mediated cytotoxicity (ADCC). Increasingly regulators are requiring that ADCC assays, especially those used for lot release applications, measure immune cell-mediated killing rather than a surrogate endpoint for antibody engagement of antigen on target cells.
  Eurofins DiscoverX has developed a novel cytotoxicity assay platform technology (KILR®) that specifically measures killing of antigen-expressing target cells in a co-culture with immune cells, in an easy-to-use, dye-free, and radioactivity-free assay format. Based on the industry-validated Enzyme Fragment Complementation (EFC) technology, the KILR platform can be applied to multiple immune cell-mediated killing events, including ADCC, CDC, ADCP, and T-cell redirection.In this poster, we share examples of the universality of the KILR platform for development of ADCC assays for multiple antigens (e.g., HER2, CD20, CD19, CD38, CD33), and with a variety of effector cell types. Importantly, to ensure long-term assay reproducibility many KILR target cell models have now been developed in an assay-ready format. When used in combination with our engineered KILR CD16 Effector Cells, these KILR assay-ready target cells produce robust assay windows with excellent assay repeatability. We share phase-appropriate qualification data for the KILR Raji bioassay model that demonstrates that these assays are fit-for-purpose for screening and relative potency applications in lot-release testing.
• Contributing Authors: Debatri Chatterjee, Hyna Dotimas, Surekha Bonasu, Gaurav Agrawal, Alexander Baumann, and Jane E. Lamerdin
• (Poster)

TOST, Perfect TOST, and Alternatives

• Speaker: Stan Deming, Statistical Designs and David LeBlond, Robert Singer Consulting
• Abstract: In our view, statistical tools are often used to answer six common questions about a true mean based on a statistical sample of data: Is “greater than,” “less than,” “different from,” “outside,” “between,” or “the same as,” specifications (reference values)? The Frequentist Confidence Distribution and the Bayesian Posterior Distribution offer simple frameworks for answering these questions. Because it is not possible to show that things are truly the same, the first question must become a “not-different-by-more-than-a-certain-amount-?” question (a “between” question). The “outside” question seldom makes business sense, and it arises only rarely. The “different from,” “greater than,” and “less than” questions are discussed in most statistics books, but the “between” question has received less attention. One current approach to the “between” question is to use two one-sided tests (TOST), but there does not appear to be agreement on how to use or combine the results of the two individual tests. We make the case that TOST gives a correct level of confidence only when it is based on the included area between specifications under a Confidence Distribution or a Posterior Distribution – a “perfect TOST.”
• (Day 1: Statistical Tools for Method Development Session)

Multiplex Binding Assays as a Powerful Tool for Potency Testing of Multispecific Molecules

• Speaker: Özlem Demirel, Sanofi
• Abstract: Multispecific molecules displace more and more monospecific ones. Developing potency assays for multispecific molecules are more challenging and labor intensive. Meso Scale Discovery (MSD) technology is a powerful tool for multiplexing binding assays which reduces time and resources needed for potency assay development tremendously. The U-Plex platform of MSD was employed successfully for multiplexing binding assays for trispecific antibodies. By this, three targets are measured in one single well and one binding assay is sufficient to report relative potency for three targets.
  Multiplex binding assays can be established as platform assays for the same molecule class, again significantly decreasing workload for potency assay development. Furthermore, assay design can be transferred to other molecule classes.
• Contributing Authors: Özlem Demirel, Ana Serchinger, Noura Nouri and Dirk Usener
  Sanofi, CMC Development, BioAnalytics Germany, Bioassays
• (Day 2: Potency Assay Development Session)

Analytical Transfer of Cell-Based Assays: Observations from Experience

• Presenter: Stuar Dunn, Labcorp Drug Development
• Abstract: (Pending)
• (Poster)

Application of Novel Luminescent Bioassays for Redirected T Cell Therapies: From Discovery to Lot Release

• Presenter: Steve Edenson, Promega Corporation
• Abstract: Cell therapy using genetically engineered immune cells has been demonstrated as a powerful therapeutic modality and is showing promising clinical outcomes in patients with cancer. In adoptive T cell therapy, T cells are genetically modified to express chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR) that redirect T cell specificity towards a tumor-associated antigen (TAA). Here, we present novel bioassay platforms for the discovery, characterization, and lot release of redirected T cell therapies. To facilitate the discovery and characterization of transgenic TCRs, we developed a suite of TCR-KO reporter T cell lines. Reintroduction of peptide-specific TCR α and β chains into the TCR-KO reporter cells results in sensitive antigen-dependent TCR activation and luciferase reporter expression. The selective expression of CD4 and/or CD8 in the TCR-KO cell lines enables the discovery of TCRs for MHCI or MHCII-restricted antigens, or without MHC bias. Stable TCR-expressing reporter cell lines can be engineered for quantitative antigen ranking and safety profiling of lead TCRs. To quantitatively measure the effector function of redirected primary T cells, we have used split NanoBiT luciferase technology to develop a HiBiT target cell killing (TCK) assay. Co-culture of CAR-expressing T cells with target cells stably expressing HiBiT results in lysis of the target cell and release of HiBiT. HiBiT then binds LgBiT in the detection reagent and forms a functional NanoBiT luciferase to generate luminescence. We developed a suite of TCK cell lines expressing clinically-relevant TAAs (e.g., CD19, BCMA, HER2, etc.). The HiBiT TCK cell lines have the potential to support GMP QC release testing for late-stage clinical and commercial T cell therapies. These luminescent bioassays are robust, highly sensitive, and easy-to-use, and represent novel tools for the discovery, development, and lot release of redirected T cell therapies.
• Contributing Authors: Julia Gilden, Jamison Grailer, Pete Stecha, Brock Binkowski, Jim Hartnett, Michael Slater, Frank Fan and Mei Cong
• (Poster)

Assay Monitoring and Trending for Multi-Valent ELISA: Challenges and Considerations

• Speaker: Kate English, MSD Brinny
• Abstract: It is a business need and a regulatory requirement to track and trend data, particularly for drug Product ELISA testing for release and stability testing. Large volumes of data need sophisticated software to allow for tracking and trending of multi-valent assays, while still being useable enough to visualise data at a high level across the attributes being trended. MSD have recently implemented Assay monitoring program for a multi-valent ELISA via exporting ELISA data directly from SoftMax Pro through data integrity compliant pathways to a Tibco Spotfire dashboard for tracking and trending. This allows quick, direct and thorough assay monitoring and makes monitoring complicated data sets much easier. Challenges met during this project include ensuring consistency in ELISA metadata input, developing a data flow pathway, and ensuring a robust, compliant process was developed for long term assay monitoring.
• (Day 2: Potency Assay Development Session)

Trim the Fat from your Bioassay

• Speaker: Brooke Franklin, Quantics Biostatistics
• Abstract: Manufacturing is all about “Lean” (i.e. efficient) how do we apply the same disciplines to bioassay? During development many assays evolve in a complex way that is not necessarily “Lean”. It doesn’t have to stay that way! Once the biology is stable and well characterized it is time to reevaluate the design, before validation.We will explore practical ways to revisit assay design to increase throughput (efficiency) whilst retaining precision (effectiveness). Using case studies, we will illustrate these principles to show how outcomes can be greatly improved by embracing the principles of “Lean”.
• (Day 2: Potency Assay Development Session)

Development of a Potency Assay for Low Affinity Insulin Analogues

• Speaker: Stine Frederiksen, Novo Nordisk A/S
• Abstract: (Pending)
• (Day 1: Potency Assay Development Case Studies Session)

Improvement of an Early-Stage Developed Potency Assay to the Standards of a Late-Stage Assay

• Speaker: Gergö Fülöp
• Abstract: In this talk a case study is presented where we walk around the following topics:
  • The difference between early and late-stage project assays.
  • Transfer of a potency assay between sites and its issues.
  • Troubleshooting to improve assay performance.
  • Improvements to make the assay more reliable, high-throughput and time-efficient.
• (Day 2: Potency Assay Development Session)

From the Well-Beaten Path into the Unknown: Bioassay Development, Characterization, and Automation Today

• Speaker: Caterina Giorno, Boehringer Ingelheim Pharma GmbH & Co. KG
• Abstract: Development and characterization of the Mode of Action using cell-based bioassays is a well-known and established process during the development and commercial phase of biopharmaceuticals.During the development phase of biopharmaceuticals, a detailed understanding of the mode of action is required. This understanding increases with multiple investigations. Therefore, usually complex and time-consuming assays, including cell-based bioassays, are used.The insight into the mode of action is one side of the coin – another is the different behaviors of the molecule itself. The comprehension of effector-function relationships, glycoengineering, as well as intense bioassay analytics can highlight critical, quality attributes that need to be addressed and considered during a product life cycle.Gaining in-depth knowledge about molecules requires resilient, hands-on analytics for which high capacities are required.While standardized cell-based bioassays are extensively established, automated processes are still a challenge that require careful attention and a deep understanding in robotic systems.During the talk, we will show the outcome of a comprehensive characterization of an agonistic antibody as well as the benefits and hurdles of automating bioassays.
• (Day 2: Automating Potency Assays Session)

Cell-Based Luminescent Reporter Bioassays for Immunotherapies Targeting Macrophage Effector Functions

• Presenter: Jamison Grailer, Promega Corporation
• Abstract: Macrophages play a key role in the elimination of tumor cells via phagocytosis and presentation of tumor antigens to T cells. Antibody-dependent phagocytosis (ADCP) is an important mechanism-of-action (MoA) for antibody-based cancer immunotherapies. Therapeutic strategies that enhance ADCP can augment direct tumor destruction and enable anti-tumor immunity. ADCP is initiated by binding of antibody Fc domains to Fc gamma receptors (FcγRs), and FcγR crosslinking results in signal transduction that drives inflammatory cytokine production and phagocytosis. Simultaneously, phagocytosis is regulated by immune checkpoints that serve to facilitate the removal of damaged cells while preserving healthy cells. Modulation of these immune checkpoints (e.g., SIRPα, ILT4) can enhance macrophage effector functions, such as phagocytosis. Current assays to measure ADCP are tedious, low throughput, and highly variable. Here, we present a suite of cell-based luminescent reporter bioassays to measure ADCP function and modulation: 1) Bioluminescent reporter cell lines expressing individual FcγRs provide robust potency measurement of single FcγR activation. 2) A monocytic cell background was used to measure ADCP via endogenous expression of multiple FcγRs and integration of a FcγR-activation dependent luminescent reporter. 3) Direct detection of cellular phagocytosis can be measured using the HiBiT split luciferase system. 4) Modulation of macrophage function can be determined using cell-based reporter bioassays for SIRPα/CD47 and ILT4/HLA-G. These bioassays are robust and easy-to-use, and most are pre-qualified according to ICH guidelines. Together, these novel reporter bioassays provide a robust, high-throughput toolbox to facilitate discovery and development of immunotherapies targeting macrophage effector functions.
• Contributing Authors: Jonathan Mitchell, Denise Garvin, Julia Gilden, Pete Stecha, Frank Fan, Mei Cong and Jamison Grailer
• (Poster)

Turbocharging Potency Assays – Challenges and Opportunities from Multi-Specific Drug Modalities

• Speaker: Barbara Hebeis, MedImmune
• Abstract: Biotherapeutics have revolutionised the therapeutic options available to patients, however, in order to address increasing complexity in disease pathologies, biotherapeutics will themselves have to increase in complexity.One option to achieve this is addressing several disease modulating targets within one drug molecule. The resulting multi active or multi specific drugs provide novel challenges to CMC, aside from manufacturing consistency and stability, they pose a particular challenge for the development of appropriate Potency Assays.This talk will describe some of the biotherapeutic modalities currently in development as well as provide some insights into opportunities and challenges for the development of Potency Assays aiming to cover the complex modes of action arising from multi activity or specificity.
• (Day 1: Potency Assay Development Case Studies Session)

End-to-End vs. Partial Bioassay Automation in a GMP Environment: Evaluation of the Benefits of Different Bioassay Automation Degrees

• Speaker: Patrick Heim, Hoffmann-La Roche Ltd.
• Abstract: Sample preparation for bioassays is time extensive, requiring many pipetting motions and a high focus by analysts. Therefore, it is beneficial to automate sample preparation to reduce hands-ontimes, to prevent ergonomic injuries and to save the analysts’ mental resources for other tasks, respectively. End-to-end Automation starting from sample preparation including all steps to the assay readout seems to be even more beneficial. This showcase will demonstrate that it is worth to evaluate the degree of automation for different assays individually and for best usage of the automation system holistically. The presentation will further provide a summary of additional real life benefits of bioassay automation as right-first-time-rate, lead-time reductions and potential bioassay shift work through End-to-End automation.
• (Day 2: Automating Potency Assays Session)

Bioluminescence Technology for AAV Characterization

• Speaker: Trish Hoang, Promega Corporation
• Abstract: Recombinant adeno-associated viruses (rAAV) are a widely used delivery vehicle for transgene expression for treatment of monogenic diseases. Key features that make rAAV a leading platform in gene therapy are tissue tropism, long-term transgene expression, and low immunogenicity. During early development and optimization of novel rAAVs, surrogate protein expression (e.g., fluorescent proteins such as GFP) is used to track if desired endpoint has been achieved. Despite its broad use, GFP expression is only measurable 48 hours post transduction due to its limited stability and detection sensitivity. In this work, we describe a highly sensitive assay using NanoLuc® luciferase as a surrogate to monitor rAAV transduction. NanoLuc® luciferase is extremely bright, and therefore offers a very sensitive detection of gene expression. Moreover, assays can be performed on a standard luminometer. We generated a panel of AAV serotypes 1-10 expressing NanoLuc® reporter gene. We then transduced each serotype into 10 human cell lines representing different tissue types, at various multiplicity of infection (MOI) ranging from 1 to 10,000. Luminescence signals were dependent on MOI and displayed a large differential transduction efficiency among serotypes and cell types.
  The sensitivity of the AAV-NanoLuc® reporter and its tolerance to matrix interference led to the development of a novel neutralization antibody (NAb) assay. Individuals with pre-existing immunity to AAVs might be less likely to benefit from AAV therapies due to reduced rAAV uptake or cytotoxic T cell response resulting in loss of transduced cells and affecting efficacy or safety outcomes. Sensitive NAb assays are required to determine cut off for inclusion of qualified individuals in clinical trials. Furthermore, NAb assays can support studies of immunomodulating or suppressive regimes to block immune response in administration of rAAV. Hence, we developed a NanoLuc® luciferase cell-based transduction inhibition assay to measure neutralizing antibodies against AAV. The assay requires low MOI of 100 to transduce cells and less than 24 hours to detect. As a proof of concept, we screened 100 human serum samples to determine the assay cut off points for pre-existing immunity to AAV2 or AAV9. NAb titers were further assessed for positive samples. Our rapid, highly sensitive and reliable system offers a universal, transferrable platform across laboratories to assess the prevalence of AAV neutralizing antibodies in intended patient populations to help inform clinical development strategies.
• Contributing Authors: Trish Hoang, Rod Flemming, Jeff Nelson, Vanessa Ott, Kara Machleidt, and Marjeta Urh
  Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
  E-mail: trish.hoang@promega.com
• (Day 2: Cell and Gene Therapy Session)

Comments and Insights on ICH Q2(R2)

• Speaker: Pierre Lebrun, Pharmalex Belgium
• Abstract: ICH Q2(R2) finally bridges validation recommendation to biological assay. This is a great news for the pharmaceutical industry in general.In this presentation, the general structure of ICH Q2 and Q14 will be covered, defining what’s an analytical procedure life-cycle and what are the regulatory recommendations and expectations about analytical quality by design (aQbD).Then, the focus of this presentation is to present an in-depth review of the new ICH Q2(R2) draft issued in March 2022. Definitions and analytical procedure qualification (validation) will be covered. Then a discussion will be conducted on what changed from ICH Q2 (R1), what most people are happy with, and what could be improved during the last revision cycle of the document.
• (Day 1: Regulatory Insights Session)

Transferring Potency Assays

• Instructors: Laureen Little, BEBPA, Siân Estdale, Labcorp, and Alexander Knorre, Eurofins BioPharma Product Testing Munich GmbH
• Abstract: Industry experts will provide an overview of tools and approach es to use for smoother, faster and more efficient potency trans fers. Phase appropriate practices, SOP traps, software equivalen cy and when is similar good enough are all topics to be discussed.
• (Workshop 2: Transferring Potency Assays Workshop)

Set-Up and Validation of Tailored Antibody-Dependent Cellular Toxicity Method Using ADCC Reporter Bioassay

• Presenter: Giulia Mancini, Eurofins Biopharma Product Testing
• Abstract: Immunotherapy has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing, with numerous treatments in clinical and preclinical development. Various key immune cell subsets play an important role in mediating tumor cell death through several ways involving immune-mediated cell killing mechanisms such as T cell cytotoxicity-mediated killing and antibody-dependent cell cytotoxicity (ADCC), which result in the induction of target cell apoptosis.
  The ADCC reporter bioassay develop by Promega, is a bioluminescent reporter-gene assay for quantifying the biological activity of the antibody via FcγRIIIa-mediated pathway activation in an ADCC mechanism of action. When the antibody binds to the antigens on the target cells and the FcγRIIIa receptor on the ADCC bioassay effector cells, it leads to the activation of FcγRIIIa and the subsequent luciferase activation in the ADCC bioassay effector cells. The antibody-induced luciferase activity in the effector cell is quantified with a luminescence readout which is specific to the antibody and antigen-expressing target cells.The purpose of this study was to validate and to develop target models to study the effect of immunotherapy on breast and lung cancer cell lines by using the ADCC Reporter Bioassay complete kit. The first activity of the present study has been the validation of ADCC Reporter Bioassay complete kit. The validation has been performed following the ICH guidelines and the results obtained completely fulfiil those requirements. Indeed, the ADCC Reporter Bioassay resulted precise (with CV%≤ 11% for intraassay precision and ≤ 30% for intra- inter-assay), accurate (80% ≤ % expected relative potency tested ≤ 125%) and specific. Moreover, the bioassay confirmed its robustness by calculating the EC50 variation at three different reading times (5, 15 and 30 minutes after the end of the assay). The EC50 after 15 and 30 minutes resulted in ±10% of the value read at 5 minutes. Moreover, the accuracy has been analyzed by the comparison of the Control Ab curve to itself at 1µg/mL, here referred as 100%, 0.5µg/mL (50%) and 1.5µg/mL (150%). The recommended starting concentration of Control Ab anti-CD20 ranged from 1 and 3µg/mL, indeed as expected the 150% showed the same performance of the 100%. Additionally, no differences have been found for the 50% of concentration since, although the recommended concentration of CD20 was comprises between 1µg/mL and 3µg/mL, the 0.5µg/ mL has been proved to be already optimal (full-dose response range).The second part of the study will be aimed at developing a personalized ADCC Reporter Bioassay by including either breast or lung cancer cell lines and PD-1/PD-L1 antibody. Antibody therapy based on PD1/PD-L1 blocking or ADCC effector has produced significant clinical benefits for cancer patients. Indeed, PD-1/PD-L1 inhibitors have been approved by the US Food and Drug Administration (FDA) for the treatment of a wide spectrum of tumors, including Non-Small-Cell Lung cancer (NSCLC) and TrypleNegative Breast cancer.
• (Poster)

Combination of SPR-Based Binding Kinetic and SEC-Based Conformational Assay to Evaluate mAb-Target Antigen Physicochemical Properties Important for ADCs Therapeutic Function

• Presenter: Alessandra Mariani, Menarini Biotech
• Abstract: CD205/Ly75, was selected as target antigen for a humanized anti-CD205 IgG1 conjugated to the DM4 toxin, MEN1309 (ref. 1). The over expression of CD205 in different solid cancers, combined with its capability to induce ligand internalization, were key factor in the election of CD205 to excellent target for an Antibody Drug Conjugate (ADC) (ref. 2-3). On the other side, the CD205 physiological expression on Antigen Presenting Cells (APCs) and leucocytes, represents a risk factor for toxic side effect induced by MEN1309 binding on healthy blood cells, a phenomenon frequent for ADCs and known as ”on-target but off-tumor” binding (ref. 4).
  In the present study, the previously described pH dependent CD205 conformational change (ref. 5) impact on the binding of MEN1309 was evaluated, looking for possible consequences on MEN1309 therapeutic function.
  SPR analyses not only confirmed the effective impact of the pH in the complexity of MEN1309-CD205 interaction, but also showed that MEN1309 binds the target antigen with different kinetic in response to pH 7.4, or pH 6.5 , which represent the pH conditions of CD205 binding occurring respectively on healthy or malignant cells. In particular, a significantly faster binding kinetic was observed at pH 7.4 and these results were confirmed by SEC RALS analysis of MBH1309:CD205 mixtures, which showed only at pH 7.4 a separation of the two molecules, while at pH 6.5 they remained combined in immune-complexes.Overall, these data demonstrate a strong impact of pH on MEN1309 binding kinetic to the CD205 target antigen and thus to the mAb therapeutic function. At pH 7.4 (healthy cells environment) the MBH1309- CD205 binding kinetic is faster, indeed they associate and dissociate more rapidly, while complexes at pH 6.5 (malignant cells microenvironment) associate a bit slower and not dissociate as fast as under pH7.4 conditions, resulting in a more stable MEN1309-CD205 binding.Finally, the present study identify in vitro analytical methods able to support the development of new ADCs. The combination between SPR-based binding kinetic and SEC-based conformational assays allows to evaluate mAb-target antigen physiochemical properties which could enhance the selectivity of ADCs for the malignant cells.
• Contributing Authors: Alessandra Mariani, Sabrina Novelli, Meri Gabrielli and Thoralf Keller
• Bibliography: 1. Merlino at al. “MEN1309/OBT076, a first-in-class Antibody–Drug Conjugate targeting CD205 in solid tumors”. Molecular cancer therapeutics (2019) 18:1533–43
  2. K. Mahnke et al. “The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments” The journal of cell biology (2000) 151(3):673-84
  3. GV. Canzonieri et al. “CD205, a target antigen for a novel antibody drug conjugate (ADC): Evaluation of antigen expression on non-Hodgkin lymphoma (NHL)”. Journal of Clinical Oncology (2017)
  4. J.M. Lambert and C.Q. Morris. “Antibody–Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review”. Advances in Therapy (2017) 34:1015–1035
  5. J. Lin and J. Sagert “Targeting drug conjugates to the Tumor Microenvironment: probody drug conjugates” Innovations for Next-Generation Antibody-Drug conjugates (2018) 281-298
• (Poster)

Fast and Efficient Evaluation of the Impact of Fc N-glycosylation on Antibody-dependent Cellular Cytotoxicity

• Presenter: Ulrich Mayer, Svar Life Science
• Abstract: Determine the relationship between Fc N-glycan structure and ADCC activity using fast, efficient, and accurate workflows. A two-step enzymatic transglycosylation technology was used to generate human IgG with defined and homogeneous glycoforms and a reporter gene bioassay based on engineered target and effector cells was used to determine the ADCC activities of the antibodies.
• (Poster)

Development of a Highly Sensitive ADCC iLite® Reporter Gene Assay with True MoA-Reflecting Properties

• Presenter: Ulrich Mayer, Svar Life Science
• Abstract: We have created a sensitive proprietary iLite ADCC assay, consisting of a gene-reporter effector cell line, combined with custom-engineered target cell lines for commonly used receptors. The iLite ADCC assays offer superior sensitivity and dynamic range compared to assays from a leading competitor and correlate well with a PBMC-mediated assay, indicating that the iLite assay can fully capture the ADCC MoA.
• (Poster)

Development of an Automated Transgene Expression ELISA for Testing Potency of a Monovalent Adenovector COVID-19 Vaccine

• Speaker: Paul Noordhuis, Janssen Vaccines & Prevention B.V.
• Abstract: The Ad26.COV2.S (COVID-19) vaccine developed by Janssen Vaccines and Prevention is a monovalent Drug Product (DP) consisting of an adenoviral vector containing the COVID-19 spike protein sequence. A cell-based quantitative (relative) transgene expression ELISA is used for the characterization of the vaccine since considered to be reflective to the mode of action (Potency) of the adenovector based COVID-19 vaccine. Transgene expression can be measured with different types of cell-based ELISA using absorbance, luminescence or fluorescence detection techniques. The colorimetric detection ELISA was developed as a platform method in which the relative expression of the COVID-19 transgene could be determined after infection of A549 cells with drug substance (DS) or DP samples using DS as a reference standard. However, the throughput and detection range of an absorbance detection ELISA is low resulting in the need for an ELISA method with a higher throughput for high amounts of samples. As a result, an automated transgene expression ELISA based on luminescence detection was developed.During the development of the automated method, steps in the colorimetric method that were not automation compatible and those that can be optimized were identified first. Identified factors for optimization were primary and secondary antibody concentrations; cell seeding format (fixed cells in plates versus cell suspension); infection time; plate layout and volume; and data interpretation (statistical analysis). Equivalency to the colorimetric method was performed and afterwards a qualification according to ICHQ2R1 was performed.All the factors identified were successfully optimized to increase sample throughput significantly (105 samples vs 12 samples previously). Material and reagent use was reduced by 50%, also assay run time was shortened. Lastly, the assay was successfully qualified for testing of DS and DP samples according to ICHQ2R1 and the automated method was shown to be equivalent to the colorimetric method.In conclusion, the developed and qualified automated transgene expression ELISA reflects that the method is suitable for high throughput testing of the potency of DS/DP samples and can be leveraged to other vaccine products as well.
• (Day 2: Cell and Gene Therapy Session)

Production and Release of Parasite Oocysts for CHIM Study

• Speaker: Natko Nuber, Novartis Pharma AG
• Abstract: For some diseases, human challenge studies are required – involving the deliberate exposure of volunteers to infectious substances. Current guidelines demand that the challenge agent is produced under GMP, very similar to how a classical biopharmaceutical is produced. But what can be done if – in this special case – following the rules to the letters simply let us run against a wall? Big pharma is well capable running “normal” projects, cranking up their well-oiled machinery, guided by SOPs, rules and regulations. Here, a special case will be discussed where nearly nothing turned out to be “normal”: How can the pathogen Cryptosporidium Parvum be supplied for a clinical study when that pathogen cannot be cultivated ex vivo and cannot be stored long-term? How can something be characterized and released when it is not sufficiently stable to survive the typical timelines for QC testing and release? This case study demonstrates that we as big pharma need to learn how to adapt to the new challenges that we will face as the classical portfolio more and more moves away from the well-known molecules.
• (Day 2: Cell and Gene Therapy Session)

Industry Approaches for Implementation of Ready-to-Use (RtU) Cells in Bioassays

• Presenter: Esther Ong, Fresenius Kabi SwissBioSim (On behalf of BioPhorum)
• Abstract: BioPhorum is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. Bioassays are an important part of an overall CMC analytical control strategy and cell-based assays are widely used for this purpose. Development of robust and reliable bioassays places increased requirements on the analytical cell banks with low lot-to-lot variability to deliver high-quality potency data. Ready to use cells (RtU) offer many benefits including reduced assay variability and provides frozen bank of uniform cell stock for utilization directly in an assay. To gain an understanding of current industry approachs for RtU cells, and to recommend the best practices for preparation, testing and control strategy for RtU cells, the BioPhorum Development Group Bioassay workstream conducted an intercompany collaboration exercise, which included a benchmarking survey and group discussions around approaches for reliably making RtU cells for Bioassays. Selected results of this industry collaboration are presented on this poster to encourage discussion on common approaches to RtU cell manufacturing, testing and implementation across the industry.)
• (Poster)

Implementation of Equivalence Criteria for Parallelism Assessment in a Late-Phase Bioassay: Case Study

• Presenter: Delphine Peric, Sanofi
• Abstract: (Pending)
• (Poster)

Bridging the Gap Between In Vivo and In Vitro Assays for Vaccines

• Speaker: Tim Schofield, CMC Sciences, LLC
• Abstract: Animal (in vivo) tests have been utilized for over a century to facilitate vaccines development and for quality control. This is a legacy of the application of the best science at their time of introduction. Advances in science and technology have, however, provided opportunities to replace animal tests with in vitro alternatives. This is complicated, however, by the variability of in vivo methods and a perceived risk of moving away from animals. This talk will describe risks associated with continuing to use in vivo assays and a pathway to replacement of animal testing.
• (Day 2: Potency Assay Development Session)

Design of Experiments for Bioassay

• Instructors: Perceval Sondag, Novo Nordisk and Pierre Lebrun, Pharmalex Belgium
• Abstract: First, trainees will learn, using a simple example, the value of DoE and how it can drastically increase the amount of information provided by each experiment. Then, we’ll discuss how to choose the appropriate design for different situations. Trainees will have an overview of the DoE catalog, including the advantage of each type of design (Screening designs, Factorial designs, Response-surface designs, Optimal designs). Finally, attendees will gain an appreciation for the many ways output can be used to better understand and optimize processes.
• (Workshop 1: Design of Experiments for Bioassays Workshop)

Quantify Antibody-Dependent Cell Phagocytosis (ADCP): Application of a Robust, Non-Radioactive KILR Cytotoxicity Platform

• Presenter: Santosh Subramanian, Eurofins DiscoverX
• Abstract: Cell-based assays play an important role in determination of mechanism of action (MOA) for therapeutic antibodies, particularly when evaluating their potential for effector functions. Antibody-dependent cellular phagocytosis (ADCP) has gained prominence as an important MOA to evaluate, especially for IND-enabling studies of antibodies with modified Fc regions. ADCP is mediated by a variety of effector cells, namely monocytes, macrophages, dendritic cells, and neutrophils, through multiple FcγRs. While the Fab region of the antibody binds to a specific antigen on the surface of target cells, the Fc region of the antibody binds and activates various Fcγ receptors on effector cells. Activation of specific Fcγ receptors, like FcγRIIa, FcγRI, and FcγRIIIa, leads to activation of a complex pathway that results in phagocytosis and destruction of the target cells in the lysosome of the effector cells.
  ADCP assays can be particularly difficult to standardize due to the variability of primary human Fcγexpressing effector cells and the lengthy and difficult protocols required for immune cell differentiation. We have applied our industry-validated KILR® cytotoxicity assay platform, which specifically measures killing of target cells in co-culture with immune cells, to quantitation of ADCP in a homogeneous platebased assay format, serving as an inexpensive alternative to laborious and expensive FACS or imaging approaches. Measurement of ADCP activity with KILR cells is a simple, reproducible and scalable method that directly measures the physiologically relevant endpoint: target cell destruction inside the lysosomes of effector macrophages. KILR ADCP assays are fast, robust, and reproducible, supporting screening and characterization of antibody drugs during early phase bio-comparability as well as in late stage characterization work. The broad application of KILR assays along the drug development pipeline, together with the ability of each stable KILR model to quantify CDC, ADCC, as well as ADCP allows this assay platform to support the development of almost any antibody therapeutic.
• Contributing Authors: Surekha Bonasu, Vandana Kaul, Hanako Daino-Laizure, Debatri Chatterjee, Gaurav Agrawal, Santosh Subramanian, and Jane E. Lamerdin
• (Poster)

Bioassay Automation under GMP Conditions: How to Develop and Qualify the Program

• Speaker: Ayumi Takayanagi, Chugai Pharmaceutical Co., Ltd.
• Abstract: Recently, we had successfully developed a bioassay-automation system, including cell loading step, and qualified automated method under GMP condition. Generally, there are several challenges for establishing and qualifying full-automation bioassay system. The first challenge you may face is establishing a method that indicates comparable results to a manual procedure. Especially for cell-based assay, it is difficult to load cell suspension uniformly to an assay plate due to a long duration time from cell preparation to cell loading. To overcome this issue, we had investigated cell suspension stirring method and found a suitable condition that enables to seed cell suspension uniformly after 3 hours standing time in a reservoir. Subsequently, qualification of the program is another big challenge, usually taking a huge effort when it comes to GMP-use phase. This presentation will include the results of investigation study during method development and our strategies and results of the method qualification including method comparability program.
• (Day 2: Automating Potency Assays Session)

Strategies for Creating Engineered Cell Lines for Difficult to Develop Bioassays

• Speaker: Nicholaus Trenton, Seagen
• Abstract: Cell-based potency assays are important components for regulatory compliance of biopharmaceuticals due to their ability to measure the mechanism of action (MOA). However, these assays can be challenging to develop due to low and stable endpoint assay signal and difficulty in sourcing relevant cell lines with robust growth properties. Increasingly, cell lines need to be engineered to express either the target antigen and/or a reporter gene, though this work is frequently outsourced to third-party vendors. However, sourcing engineered cell lines may not always be possible, due to lack of vendor availability of novel targets, limitations of use due to costs or intellectual property, and poor cell line robustness. This talk will showcase a platform process for efficiently developing in-house robust engineered cell lines. This process involves a cost-benefit analysis of the engineered line of interest and a series of steps to characterize and qualify the cell line prior to its use in a bioassay. A case study will also be presented outlining the benefits of this process in generating an in-house cell line, and improvements of this cell line against an outsourced cell line in a bioassay.
• (Day 1: Cells for Cell-Based Assays Session)

The First Horizontal Standard on TNF-Alpha Antagonist Bioassays in European Pharmacopoeia

• Speaker: Jaana Vesterinen, Finnish Medicines Agency
• Abstract: (Pending)
• (Day 1: Regulatory Insights Session)

Case Study: Solving a Replicate CV Issue in a Competition ELISA

• Speaker: Armelle Viornery, Lonza AG
• Abstract: A Competition ELISA is used for potency testing of DS or DP test samples against the designated reference standard. The replicate precision for duplicate data points of the dilution curve is part of the assay acceptance criteria. However frequently this parameter was failing, although no issue was observed during the performance of the assay. The approach used to investigate the origin of the issue and how it was solved will be explained.
• (Day 2: Potency Assay Development Session)

Standards and Statistical Tools in Support of Bioassay

• Speaker: Steven Walfish, USP
• Abstract: USP has been leading the way in standards since 1820, when it began as a non-profit organization dedicated to protecting public health. At the forefront are the documentary standards for bioassay potency measurements. This presentation will delve into the current state of the documentary standards and propose new recombinant protein reference standards that can be used to support product quality and consistency over time.
• (Day 1: Statistical Tools for Method Development Session)

Do You Control Your Cells or Do Your Cells Control Your Assay?

• Speaker: Oliver Wehmeier, acCELLerate GmbH
• Abstract: In a cell-based potency assay the cells are probably the most critical reagent. The precise performance of cells depends on multiple factors which can be user-depending operational aspects, technical factors as well as cellular or environmental influences. Some are less critical than others, some hard to control or even difficult to standardize.To take the high variability of a cell-based assay as given for a living system may result in an unnecessary loss of significance. The goal must be to understand what modulates the responsiveness of a cell and how theses modulators can be controlled. This also means not to regulate too much what has no significant impact on the cells response as this restricts flexibility in an unnecessary way.Different determinants of cell quality and ways how to control these will be presented. The instant use of prequalified, cryopreserved cells will be discussed as a possibility to turn cells into a standardized assay reagent.
• (Day 1: Cells for Cell-Based Assays Session)