Welcome to Bioassays 2015: Scientific Approaches & Regulatory Strategies On behalf of the Scientific Organizing Committee and CASSS, we are excited to welcome you to Bioassays 2015: Scientific Approaches & Regulatory Strategies and look forward to your participation and input March 23 - 24, 2015 at the Sheraton Hotel in Silver Spring, Maryland. The CASSS Bioassays meeting has established itself as a premier conference and unique opportunity for participants and opinion leaders to discuss and debate current regulatory and industry topics regarding bioassays. Bioassays are a critical component of the analytical control strategies for biologics and other complex molecules. The ability of an assay to characterize and demonstrate biological activity is essential and developing such bioassays is becoming more difficult as biologic drugs are engineered to be more complex and/or have multiple modes of action. Companies are continuously challenged with developing assays that are biologically relevant for the analysis of multiple potential mechanisms. Bioassays are also used for lot release, stability, comparability and characterization studies, which requires that the assays be robust and, in most cases, suitable for a QC lab. Bioassays 2015 is structured to encourage attendee interaction. Each session includes case study presentations followed by a panel discussion allowing for lively dialogue between attendees from academia, industry and regulatory agencies. As in previous years, we expect this format to result in additional focus on the technical and regulatory details of the topic. Regulatory participation from the US FDA, Health Canada and various European agencies has been strong each year. In addition, an exhibitor showcase and poster reception at the end of Day One will give attendees the opportunity to present additional topics and continue the day's discussion in an informal setting. We would like to thank the speakers and the panel members who are giving generously of their time and resources and to you for your attendance. We would also like to acknowledge the generosity of our program partners for the continued support of the CASSS Bioassays meeting: AbbVie, Inc.; Biogen Idec and MedImmune. We are grateful for the expert management from CASSS and the audio-visual expertise of Michael Johnstone from MJ Audio-Visual Productions. Their experience and guidance in the preparation of this meeting has been invaluable. We are sure you will find Bioassays 2015 to be informative and productive, and that it will provide you with current perspectives on bioassays. Scientific Organizing Committee: Thomas Arroll, Seattle Genetics, Inc., USA Evangelos Bakopanos, Health Canada, Canada Katrin Buss, Federal Institute for Drugs and Medical Devices, BfArM, Germany Chana Fuchs, CDER, FDA, USA (Co-chair) Denise Gavin, CBER, FDA, USA (Co-chair) Hélène Gazzano-Santoro, Genentech, a Member of the Roche Group, USA Stephen Hartman, AbbVie Inc., USA Xu-Rong Jiang, MedImmune, A member of the AstraZeneca Group, USA Helena Madden, Biogen Idec, USA (Co-chair) Bruce Meiklejohn, Eli Lilly and Company, USA (Co-chair) Thomas Anders Millward, Novartis Pharma AG, Switzerland Noel Rieder, Amgen Inc., USA Sally Seaver, Seaver Associates LLC, USA

The Scientific Organizing Committee gratefully acknowledges the following program partners for their generous support of Bioassays 2015:

SUSTAINING PLATINUM PROGRAM PARTNERS

AbbVie, Inc. Biogen Idec

MedImmune, A member of the AstraZeneca Group

SUSTAINING SILVER PROGRAM PARTNER

Pfizer, Inc. PROGRAM PARTNERS

Amgen Inc. Genentech, a Member of the

Roche Group

EXHIBITOR PROGRAM PARTNERS

Catalent Pharma Solutions DiscoveRx Corporation

Eurofins Lancaster Laboratories

PPD Promega Corporation

Stegmann Systems GmbH

The Scientific Organizing Committee gratefully acknowledges the following media for their promotional consideration of Bioassays 2015:

MEDIA PROGRAM PARTNERS American Laboratory

American Pharmaceutical Review BioProcess International

BioProcessing Journal Genetic Engineering & Biotechnology News

International Pharmaceutical Quality Pharmaceutical Outsourcing

RSC Advances SeparationsNOW.com

Technology Networks Limited

Bioassays 2015: Scientific Approaches & Regulatory Strategies Scientific Program Summary

Monday, March 23, 2015

07:30 – 17:30 Registration in the Cypress Foyer 07:30 – 08:30 Continental Breakfast in the Magnolia Ballroom 08:30 – 08:45 CASSS Welcome and Introductory Comments in the Cypress Ballroom Helena Madden, Biogen Idec, Cambridge, MA USA

Bioassays 2015 Welcome and Introductory Comments in the Cypress Ballroom

Helena Madden, Biogen Idec, Cambridge, MA USA

Potency Tests for Cell and Gene Therapy Products: Overcoming the Challenges Workshop Session One in the Cypress Ballroom

Session Chairs: Denise Gavin, CBER, FDA and Sally Seaver, Seaver Associates LLC 08:45 – 08:50 Introduction 08:50 – 09:15 Bioassays in the Lifecycle of Cell & Gene Therapy Products: A Regulatory

Perspective Xiaobin (Victor) Lu, CBER, FDA, Silver Spring, MD USA 09:15 – 09:40 Developing a Potency Assay Matrix for a Viral Vector Gene Therapy

Product: MYDICAR®: AAV1/SERCA2a for Heart Failure Barbara Thorne, Celladon Corporation, San Diego, CA USA

09:40 – 10:05 Strategies and Challenges in Selecting Bioassays for Quality Control of Cell and Gene Therapy Products Erik Rutjens, Novartis Pharma AG, Basel, Switzerland

10:05 – 10:30 Bioassay Development for Human Stem Cell-derived Retinal Pigment

Epithelium: Progress and Challenges Irina Klimanskaya, Ocata Therapeutics, Inc., Marlborough, MA USA 10:30 – 11:00 AM Break – Visit the Exhibits and Posters in the Magnolia Ballroom 11:00 – 12:15 PANEL DISCUSSION – Questions and Answers Irina Klimanskaya, Ocata Therapeutics, Inc., USA Xiaobin (Victor) Lu, CBER, FDA, USA

Anthony Ridgway, Health Canada, Canada Erik Rutjens, Novartis Pharma AG, Switzerland Barbara Thorne, Celladon Corporation, USA

Monday, March 23 continued… 12:15 – 13:30 Hosted Lunch in the Magnolia Ballroom

Advances in Bioassay Technologies and Platforms Workshop Session Two in the Cypress Ballroom

Session Chairs: Thomas Arroll, Seattle Genetics, Inc., Stephen Hartman, AbbVie, Inc. and Xu-Rong Jiang, MedImmune, A member of the AstraZeneca Group

13:30 – 13:35 Introduction 13:35 – 14:00 Development and Validation of a To-be Marketed Cancer Immunotherapy

Antibody Max Tejada, Genentech, a Member of the Roche Group, South San Francisco, CA USA

14:00 – 14:25 An ADCP Reporter Bioassay for Assessing Fc Effector Function

Shihua Lin, MedImmune, A member of the AstraZeneca Group, Gaithersburg, MD USA

14:25 - 14:50 Development and Qualification of a Robust and Easily Executable Bioassay

Using mRNA Transcription, Assessed with Dual RT-qPCR as Assay Response

Michael Sadick, Catalent Pharma Solutions, Kansas City, MO USA 14:50 - 15:15 Expectations and Limitations of Bioassays: An EU Regulator’s View

Christian Mayer, AGES – Austrian Agency for Health & Food Safety, Vienna, Austria

15:15 - 15:45 PM Break – Visit the Exhibits and Posters in the Magnolia Ballroom 15:45 - 17:00 PANEL DISCUSSION – Questions and Answers

Evangelos Bakopanos, Health Canada, Canada Shihua Lin, MedImmune, A member of the AstraZeneca Group, USA Christian Mayer, AGES – Austrian Agency for Health & Food Safety, Austria Michael Sadick, Catalent Pharma Solutions, USA Jennifer Swisher, CDER, FDA, USA Max Tejada, Genentech, a Member of the Roche Group, USA

17:00 – 17:15 Break

Monday, March 23 continued…

Exhibitor Partner Scientific Showcase in the Cypress Ballroom Session Chairs: Thomas Arroll, Seattle Genetics, Inc., Stephen Hartman, AbbVie, Inc. and Xu-Rong

Jiang, MedImmune, A member of the AstraZeneca Group 17:15 – 17:30 Introduction 17:30 – 17:45 Novel PD-1 Blockade Bioassay to Assess Therapeutic Antibodies in PD-1 and

PD-L1 Immunotherapy Programs Frank Fan, Promega Corporation, Madison, WI USA

17:45 – 18:00 Simple Bioassays for Potency & Nab Testing of Biologics Such as

Bevacizumab Abhishek Saharia, DiscoveRx Corporation, Fremont, CA USA 18:00 – 18:15 PLA 3.0 from Plate to Process

Ralf Stegmann, Stegmann Systems GmbH, Rodgau, Germany 18:15 – 18:30 Critical Reagent Qualification for QC Bioassays

Rita Stiemke, PPD, Inc. Middleton, WI USA 18:30 – 18:45 DISCUSSION – Questions and Answers 18:45 – 20:30 Exhibitor and Poster Reception in the Magnolia Ballroom 20:30 Adjourn Day One

Tuesday, March 24, 2015 08:00 – 17:00 Registration in the Cypress Foyer 07:30 – 08:30 Continental Breakfast in the Magnolia Ballroom

Bioassays to Support Commercialization of Drug Products Workshop Session Three in the Cypress Ballroom

Session Chairs: Hélène Gazzano-Santoro, Genentech, a Member of the Roche Group, Bruce Meiklejohn, Eli Lilly and Company and Thomas Millward, Novartis Pharma AG

08:30 – 08:35 Introduction 08:35 – 09:00 Health Canada’s Experience with Bioassays Used for Consistency Testing of

Biotherapeutic Products Evangelos Bakopanos, Health Canada, Ottawa, ON Canada 09:00 – 09:25 Lifecycle of an Enzyme Activity Assay for BMN 110: Development and

Validation to Post-marketing Commitments Loc Vo, BioMarin Pharmaceutical Inc., Novato, CA USA 09:25 – 09:50 Bioassay Strategies for Assessment of Co-stimulation Inhibitors in Immuno-

oncology: Considerations from Development to Commercialization Cynthia Inzano, Bristol-Myers Squibb Company, Princeton, NJ USA

09:50 – 10:15 Bioassay Evolution and Lessons Learned Thomas Luntz, Catalent Pharma Solutions, Morrisville, NC USA 10:15 - 10:45 AM Break – Visit the Exhibits and Posters in the Magnolia Ballroom 10:45 – 12:00 PANEL DISCUSSION – Questions and Answers Evangelos Bakopanos, Health Canada, Canada

Jeffrey Glenn, Bristol-Myers Squibb Company, USA Thomas Lunz, Catalent Pharma Solutions, USA

Emily Shacter, ThinkFDA, USA Craig Thelwell, National Institute for Biological Standards and Control (NIBSC), United Kingdom Loc Vo, BioMarin Pharmaceutical Inc., USA

12:00 – 13:15 Hosted Lunch in the Magnolia Ballroom

Tuesday, March 24 continued…

Bioassay Challenges during Product Globalization Workshop Session Four in the Cypress Ballroom

Session Chairs: Katrin Buss, Federal Institute for Drugs and Medical Devices, BfArM, Helena Madden, Biogen Idec and Noel Rieder, Amgen Inc.

13:15 – 13:20 Introduction 13:20 – 13:45 Global Company, Global Products, Local Laboratories

Camille Dycke, F. Hoffmann-La Roche Ltd., Basel, Switzerland 13:45 – 14:10 Development of the 1st International Standard for Pegylated G-CSF (PEG-

G-CSF) Meenu Wadhwa, National Institute for Biological Standards and Control (NIBSC), South Mimms, United Kingdom

14:10 – 14:35 Bioassay Strategy to Support the Transfer of a Therapeutic Protein for

Manufacturing and Filing in China Justin Jia, WuXi AppTec, Shanghai, China 14:35 – 15:00 Strategies to Assist Global Regulatory Acceptance of Assurance of Potency Anthony Mire-Sluis, Amgen Inc., Thousand Oaks, CA USA 15:00 – 15:30 PM Break – Visit the Exhibits and Posters in the Magnolia Ballroom 15:30 – 16:45 PANEL DISCUSSION – Questions and Answers

Katrin Buss, Federal Institute for Drugs and Medical Devices, BfArM, Germany Camille Dycke, F. Hoffmann-La Roche Ltd., Switzerland

Chana Fuchs, CDER, FDA, USA Justin Jia, WuXi AppTec, China

Anthony Mire-Sluis, Amgen Inc., USA Meenu Wadhwa, National Institute for Biological Standards and Control (NIBSC), United Kingdom

16:45 – 17:00 Bioassays Workshop Recap Closing Remarks and Invitation to Bioassays 2016 Bruce Meiklejohn, Eli Lilly and Company, Indianapolis, IN USA 17:00 Adjournment

Potency Tests for Cell and Gene Therapy Products: Overcoming the Challenges

Session Abstract Session Chairs: Denise Gavin, CBER, FDA and Sally Seaver, Seaver Associates LLC Cell and Gene therapies (CGT) are an evolving class of promising biological products. However, despite over two decades of investigation there are still few cell and gene therapies (CGT) that are commercially available or in late stage clinical development. Successful development of any biological therapeutic requires full product characterization to ensure safety and efficacy. Animal or cell based bioassays are a critical part of this product characterization. Usually several bioassay candidates are developed as potential potency tests, and the most robust, quantitative and QC friendly method that can be validated is selected to become the potency test for lot release and monitoring lot stability. The other bioassays are reserved for product characterization and comparability assessment after process changes including scale-up. To date few CGT have had robust and meaningful potency tests. Some of the challenges faced by developers of potency tests for CGT products include (1) extremely short product shelf life (hours to days), (2) the need to express a GT vector in one cell type and to quantitate its biological activity in a second cell type, (3) developing bioassays for all of the active ingredients, especially for multi-gene or multi-cell products, (4) developing bioassays that measure relevant biological activities (5) defining and banking a “reference standard” for these products, esp. if the product is autologous, and (6) determining which bioassays need to be executed for routine lot release and stability assessments. In addition, implementing a potency assay early in CGT product development is critical for understanding the product attributes related to biological activity and clinical efficacy. This session will provide an overview of regulatory expectations for measuring potency for CGT products as well as several case studies describing how these challenges were overcome to develop potency tests for lot release and stability and comparability assessments. NOTES:

Presenter’s Abstracts Bioassays in the Lifecycle of Cell & Gene Therapy Products: A Regulatory Perspective Xiaobin (Victor) Lu CBER, FDA, Silver Spring, MD USA Abstract and slides were not available at the time of printing. NOTES:

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Developing a Potency Assay Matrix for a Viral Vector Gene Therapy Product: MYDICAR®: AAV1/SERCA2a for Heart Failure Barbara Thorne Celladon Corporation, San Diego, CA USA Heart failure affects millions of people annually, and impaired calcium cycling and lower than normal levels of the cardiac calcium pump SERCA2a are key abnormalities. AAV1/SERCA2a is an investigational gene therapy product in clinical development as an enzyme replacement therapy to treat advanced heart failure patients with reduced ejection fraction (HFREF) by restoring SERCA2a expression. Results of a phase 1 / 2a clinical trial, CUPID 1 (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease), showed a favorable safety profile and suggested benefit of AAV1/SERCA2a. A phase 2b trial (CUPID 2) is ongoing, and the program has been granted Breakthrough Therapy status by the FDA. Potency assays for viral vectored gene therapy products can be challenging and complex. For a genetic enzyme-replacement therapy, the mechanism of action includes transduction by the viral particle to deliver the therapeutic gene to the cell, expression of the recombinant protein in cells, which in the case of SERCA2a is an intracellular integral membrane protein, and biological function of the expressed protein. A case study on AAV1/SERCA2a will be presented, describing the potency matrix developed based on the FDA Guidance for Industry on Potency Tests for Cellular and Gene Therapy Products. The matrix includes three quantitative assays used for product lot release and stability testing, which measure complementary characteristics of the product (genome concentration, viral infectivity, and SERCA2a protein expression). A qualitative SERCA2a enzymatic activity assay for product characterization is also an essential component of the overall potency matrix. NOTES:

Strategies and Challenges in Selecting Bioassays for Quality Control of Cell and Gene Therapy Products Erik Rutjens Novartis Pharma AG, Basel, Switzerland Quality control of Cell and Gene Therapy (CGT) products provides new challenges due to the different nature and sometimes patient-specific properties of the therapeutic product. Bioassays are important components for controlling quality attributes like purity, identity and potency for biologicals and even more for CGT therapeutics. Developing a comprehensive testing strategy which often may consist of several different assays is important. As an example, the role of bioassays in the quality control strategy of CTL019, a cell therapy product using T cells modified to express a chimeric antigen receptor against CD19, is presented. This strategy uses several different bioanalytical assays for both release testing and characterization. Current challenges like validation of assays and developing potency assays for such a complex product are discussed. Slides were not available at the time of printing. NOTES:

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Bioassay Development for Human Stem Cell-derived Retinal Pigment Epithelium: Progress and Challenges Irina Klimanskaya Ocata Therapeutics, Inc., Marlborough, MA USA Retinal pigment epithelium (RPE) is a highly specialized tissue underlying the retina and forming a part of the blood/retina barrier. The photoreceptor layer, which has no blood supply of its own, depends on RPE for “life support” including nutrient delivery, maintaining visual cycle of retinal, absorption of stray light, removal of shed photoreceptor segments by phagocytosis. Malfunctions of RPE can lead to various pathologies of the retina and subsequent blindness. RPE derived from differentiated human embryonic stem cells (hESC) has shown acceptable safety and evidence of efficacy in preserving vision in animal models of retinal degeneration. More recently, hESC-derived RPE cells have been transplanted into patients afflicted with age-related macular degeneration (AMD) and Stargardt’s disease in the course of Phase I clinical trials Proliferation of RPE in culture is accompanied by de-differentiation characterized by such phenotypic changes as: loss of epithelial morphology, pigment, and certain molecular markers. However, after reaching confluence, the original morphology is re-established, and during further maturation, the cells become highly pigmented and restore the pattern of expression of molecular markers of fully differentiated RPE. Cells are harvested and cryopreserved for clinical use when they have re-differentiated but are not fully mature, and we have established an array of in vitro assays to confirm their potential to fully mature and perform anticipated functions upon engraftment. Each RPE bulk lot is assessed for the ability of the cells to establish a monolayer of polarized cuboidal epithelia with tight junctions, a high degree of pigmentation, RPE specific-protein deposition, up-regulation of RPE-specific gene expression, and the absence of non-RPE or senescent cells. The challenges and future directions in developing a physiologically relevant, reproducible, quantitative, stability-indicating RPE potency assay based on phagocytosis are discussed. NOTES:

Potency Tests for Cell and Gene Therapy Products: Overcoming the Challenges

Workshop Session PANEL DISCUSSION – Questions and Answers Irina Klimanskaya, Ocata Therapeutics, Inc., USA Xiaobin (Victor) Lu, CBER, FDA, USA Anthony Ridgway, Health Canada, Canada Erik Rutjens, Novartis Pharma AG, Switzerland Barbara Thorne, Celladon Corporation, USA The following questions will guide the panel discussion:

• What are some of the attributes of CGT products that hinder/obstruct bioassay development and choice of potency test?

• What type of data is needed to show a correlation between a physiochemical product attribute measured in a physical-chemical analytical test and a relevant biological activity /potency test?

• What does “QC friendly” really mean? • How are relevant biological activities determined? How many relevant biological activities need

be measured for routine lot release and stability? How many for comparability assessments? • How can semi-quantitative methods be validated?

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Advances in Bioassay Technologies and Platforms Session Abstract

Session Chairs: Thomas Arroll, Seattle Genetics, Inc., Stephen Hartman, AbbVie, Inc. and Xu-Rong Jiang, MedImmune, A member of the AstraZeneca Group Bioassays are the only test methods that quantify a drug’s biological activity and thus play a crucial role in the characterization of biologic therapies. The interplay of living cells, biologically active reagents, various quantifiable readouts, multi-step procedures, specific instrumentation, and complex data analysis makes the development of a robust, accurate, precise bioassay extremely challenging. Products with multiple MoAs pose an even greater challenge. The increasing complexity of large molecule modalities (such as ADCs, bispecifics/bifunctionals, and immunomodulators) and their multiple biological activities also greatly increases the complexity of bioassay development. Many new technologies, approaches, and platforms can facilitate bioassay development, as well as provide additional information or performance enhancements, but they may also introduce new challenges. This session will discuss some recent advances in technologies and platforms that have improved bioassay method performance, facilitated/accelerated development and implementation, and revealed new/additional information about the product. We will also address the challenges faced when introducing a new technology/approach and consider the risks and benefits to making these changes. We will learn about bioassay approaches used for cancer immunotherapy products and assessing effector functions, as well as learn about alternative bioassay readouts. We will also discuss regulatory agency experiences, opinions, and expectations regarding the use of new bioassay method technologies. NOTES:

Presenter’s Abstracts Development and Validation of a To-be Marketed Cancer Immunotherapy Antibody Max Tejada Genentech, a Member of the Roche Group There is a growing class of immunotherapeutics that are currently under development that restore the ability of the immune system to fight cancer, infections and other diseases. The field of cancer immunotherapy includes cell-, antibody-, and cytokine-based therapies which exploit tumor-specific antigens and provoke the immune system into attacking the tumor cells. A mechanism of action reflective cell based assay was developed to support the potency testing of a late-phase immunotherapeutic antibody. The development, characterization and implementation of this potency assay will be described. Slides were not available at the time of printing. NOTES:

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An ADCP Reporter Bioassay for Assessing Fc Effector Function Shihua Lin MedImmune, A member of the AstraZeneca Group, Gaithersburg, MD USA Therapeutic antibodies rely on two types of functionalities to achieve clinical efficacy: target-specific binding by the antigen-binding fragment (Fab) domain and immune-mediated effector functions by crystallizable fragment (Fc) domain such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). This presentation will highlight general considerations of ADCP cellular signaling pathways and an approach used to develop a cell-based reporter gene potency assays for assessing Fc effector function involved in ADCP activities. NOTES:

Development and Qualification of a Robust and Easily Executable Bioassay Using mRNA Transcription, Assessed with Dual RT-qPCR as Assay Response Michael Sadick Catalent Pharma Solutions, Kansas City, MO USA Transcriptional gene regulation network within cells is one of the most explored biological responses to study normal and/or disease biological mechanisms. However, using such responses as a biological read out for drug potency bioassays have been challenging. The challenges have been due, in part, to the usual strategy of extraction and purification of “good quality RNA” after cell stimulation. A potency bioassay using real-time quantitative reverse transcription assay (RT-PCR) has been developed by Catalent using the TaqMan® assay chemistry in a duplex format. Using a cell line, the assay was developed to assess neutralization (inhibition) of ligand-induced bioactivity, in terms of induction of an immediate response gene. Two different fluorescent dyes, with the greatest degree of spectral separation, are used, allowing real time monitoring of gene expression of both target and normalizer gene in each individual well of an assay plate. Additionally, the assay uses cell lysate preparation requiring no RNA extraction procedure. The result is a potency bioassay with an excellent precision. The resulting quantitative gene expression assay may be performed as a one or two day assay. Data will be presented and discussed that describe development and subsequent Qualification of an mRNA-based bioassay with excellent accuracy, assay range and reproducibility. NOTES:

Expectations and Limitations of Bioassays: An EU Regulator`s View Christian Mayer AGES - Austrian Agency for Health & Food Safety, Vienna, Austria The measurement of the biological activity using a reliable potency assay is an essential requirement throughout a biological medicinal product’s life-cycle in support of process development, product characterization, in-process and quality release control, stability, and post-marketing process changes. The set-up and implementation of a bioassay requires the detailed understanding of the mode of action, and assay developers are confronted with a number of challenges such as robustness, complexity, inherent high variability, resource intensity, and the need to provide a causative link of results from bioassays to clinical performance. In my presentation the European regulatory expectations concerning new approaches in the bioassay technology will be discussed. I will also provide case studies to highlight the current thinking on this topic from an assessor’s perspective. NOTES:

Advances in Bioassay Technologies and Platforms Workshop Session

PANEL DISCUSSION – Questions and Answers Evangelos Bakopanos, Health Canada, Canada Shihua Lin, MedImmune, A member of the AstraZeneca Group, USA Christian Mayer, AGES – Austrian Agency for Health & Food Safety, Austria Michael Sadick, Catalent Pharma Solutions, USA Jennifer Swisher, CDER, FDA, USA Max Tejada, Genentech, a Member of the Roche Group, USA The following questions will guide the panel discussion:

• From a sponsor perspective, what are the most important items to consider when evaluating a new technology or platform?

• Are there any examples where a new assay readout or approach was not deemed appropriate by regulatory authorities?

• Using a single-source vendor for reagents and instruments is often unavoidable when evaluating a new technology. How can we innovate and minimize risk?

• What is expected for molecules with complex MOAs such as immune enhancing drugs which may elicit many different activities in vivo?

• For molecules which have a primary predominant MOA and one or more secondary MOAs (eg. Fc effector functions) how does one decide when additional potency assays need to be included on the release specifications for these secondary MOAs?

• Implementing a new technology in QC is often challenging, and even more so in a contract QC labs. Recommendations? Approaches? Experiences?

• How much bridging/comparability evaluation is necessary to replace an existing bioassay with one based on new technology?

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Exhibitor Partner Scientific Showcase Session Chairs: Thomas Arroll, Seattle Genetics, Inc., Stephen Hartman, AbbVie, Inc. and Xu-Rong Jiang, MedImmune, A member of the AstraZeneca Group 17:30 – 17:45 Novel PD-1 Blockade Bioassay to Assess Therapeutic Antibodies in PD-1 and PD-L1 Immunotherapy Programs Frank Fan Promega Corporation, Madison, WI USA Programmed death receptor-1 (PD-1) and its ligand (PD-L1) are among the few important immunotherapy targets for cancer. Current PD1 assays measure cell proliferation or cytokine production in primary T cells which are tedious, have high assay variation and small assay window. To enable quantitative potency measurement for key anti-PD-1 drugs in the market or in clinical trials such as pembrolizumab and nivolumab, as well as anti-PD-L1 drugs in clinical trials such as MPDL3280A and BMS-936559, here we report the development of a robust bioluminescent cell-based PD1 blockade bioassay. For this, we built a PD-1 effector cells in Jurkat cells which stably express human PD-1 and a NFAT-RE-luciferase reporter, and a PD-L1 positive artificial Antigen Presenting Cells (PD-L1+ aAPC) in CHO-K1 cells which stably express PD-L1 and an engineered TCR activator. Once these two cell types were co-cultivated, transcriptional activation of NFAT pathway in PD-1 effector cells, mediated by binding of TCR complex with TCR activator in PD-L1+ aAPC, is significantly suppressed by PD-1/PD-L1 engagement. This inhibition can then be specifically reversed by co-incubation of PD-1 or PD-L1 blocking antibodies in dose-dependent manner, but not by the antibody for other immune checkpoint receptors such as anti-CTLA4 ipilimumab. We further developed both PD1 effector cells and PD-L1+ aAPC in Thaw-and-Use format so the cells can be plated for assay without the need of cell culture. The resultant PD1 assay using Thaw-and-Use cells brings the benefit of convenience, low day-to-day variation, and easy lab-to-lab assay transfer. We demonstrate the assay is able to measure relative potency for antibody biologics, and also can detect potency changes for stressed antibody samples. In summary, the reporter-based PD1 blockade assay provides a valuable tool for both drug screening and characterization in early drug discovery, and lot release and stability study in drug manufacture for therapeutic antibody drug candidates in PD-1 and PD-L1 immunotherapy programs. NOTES:

17:45 – 18:00 Simple Bioassays for Potency & NAb Testing of Biologics Such as Bevacizumab Abhishek Saharia DiscoveRx Corporation, Fremont, CA USA One of the major bottlenecks in the development of biologics & biosimilars is the lack of available, easy-to-use bioassays for lot release & stability testing. Ideal bioassays need to reflect the clinical mechanism of action (MOA) of the biopharmaceutical drug and should be accurate, precise, highly reproducible and robust according to the ICH guidelines. In the case of bevacizumab (an anti-VEGF-A antibody), this is particularly challenging as the relevant functional readouts are time consuming and require handling of finicky primary cells. Here, we discuss the development and application of PathHunter® cell-based assays that rely on the native biology of the VEGF receptor, thereby reflecting the clinical MOA of the originator drug. This chemiluminescent assay is highly specific, robust and utilizes a homogenous mix-and-read protocol, facilitating rapid and reproducible quantitation of drug potency. The technology also enables accurate and sensitive detection of neutralizing antibodies even in high concentrations of human serum. The assays are developed in a convenient frozen ready-to-assay format that minimizes assay variability and increases ease of use. Some information will be presented on how the technology was used to develop >750 bioassays for GPCRs, RTKs, TGFβ superfamily, Interleukins, Cytokine receptors and a variety of other receptors & their ligands. NOTES:

18:00 – 18:15 PLA 3.0 from Plate to Process Ralf Stegmann Stegmann Systems GmbH, Rodgau, Germany Transferring an established assay to a new software solution or from one software solution into another one can be a challenging task. The process of establishing routine work templates within PLA 3.0 is the topic of this showcase. Starting with measurement data from a microplate, the setup of a biological assay as a repeatable measurement operating procedure will be demonstrated. NOTES:

Critical Reagent Qualification for QC Bioassays Rita Stiemke; Manuela Grassi; Kelsey Zee; Syrus Soltaninassab; Joel Galang; Peter Wunderli PPD, Inc. Middleton, WI USA PPD Inc. cell based laboratories capabilities include bioassay method design, development, qualification/validation, and implementation for release and stability testing of all types of biological therapeutics. PPD cell lab services support all types of cell-based bioassay methods including: traditional proliferation and cytotoxicity assays, stimulation response determination assays, transfection efficiency analyses, reporter gene and receptor binding assays, as well as viral infectivity and response assays. The services provided by the PPD cell laboratory are performed per ICH, FDA and USP guidelines, as well as conform to the requirements for current Good Manufacturing Practices. One requirement for robust bioassay method development includes the identification and evaluation of critical reagents. Critical reagents can include cell lines, standards/controls, growth factors, reagents and even materials, and often includes the serum used in cell culture. A case study is presented involving qualification of fetal bovine serum (FBS) for use in a qualified reporter gene assay method. As part of the method requirements, each lot of fetal bovine serum is evaluated in the assay prior to use for routine sample testing. For the reagent qualification of this method, multiple serum lots were evaluated for assay performance and one lot was identified for use. However, after application to routine testing, an increased assay failure rate was observed that, upon evaluation of assay parameters, was linked to an impact of the qualified serum lot on cell performance at higher passage number. The case study demonstrates that qualification of reagents should be designed to include all aspects critical to assay performance. It supports inclusion of critical reagent information and other assay parameters as part of continuous assay trending, to best evaluate and confirm adequate assay performance throughout the assay life cycle. NOTES:

Bioassays to Support Commercialization of Drug Products

Session Abstract Session Chairs: Hélène Gazzano-Santoro, Genentech, a Member of the Roche Group, Bruce Meiklejohn, Eli Lilly and Company and Thomas Millward, Novartis Pharma AG As a product progresses through clinical development towards commercialization, the requirements and expectations of the bioassays used to release and characterize the product evolve. By the time the product is approaching registration, the bioassay needs to be fully validated, and specifications need to be defined and justified. But in addition, the relationship between the bioassay and the clinical mode of action needs to be clearly established and reagents that are critical for the bioassay need to be under control and their supply guaranteed. For some products more than one bioassay may be needed for commercial release, and these bioassay(s) may need to be transferred to one or more QC testing labs. This session will discuss these and other aspects relating to bioassays during product commercialization. NOTES:

Presenter’s Abstracts Health Canada’s Experience with Bioassays Used for Consistency Testing of Biotherapeutic Products Evangelos Bakopanos Health Canada, Ottawa, ON Canada Health Canada's Biologics and Genetic Therapies Directorate (BGTD) is the Canadian federal authority that regulates biological drugs and radiopharmaceuticals for human use in Canada, whether manufactured in Canada or elsewhere. Some of the products regulated by BGTD include: blood and blood products, hemostatic agents, bacterial and viral vaccines, biotherapeutics (i.e. hormones, enzymes, cytokines & monoclonal antibodies), allergenic extracts, gene and cell therapies, tissues, and organs. Furthermore, BGTD is organized into two Evaluation Centres: the Centre for Evaluation of Radiopharmaceuticals and Biotherapeutics (CERB) & the Centre for Biologics Evaluation (CBE). A Manufacturer/Sponsor seeking to market a biologic drug in Canada must file a New Drug Submission (NDS) with BGTD. The NDS contains information and data regarding the drug's safety, effectiveness and quality. BGTD evaluates this information in order to assess the potential benefits and risks of the drug and to decide whether or not the Manufacturer/Sponsor is allowed to market the drug in Canada. In addition to reviewing the information submitted in the NDS dossier, BGTD laboratories may also test samples of the biologic drug under review. This so-called Consistency Testing is done in accordance with Health Canada’s Lot Release Program. This talk will provide a general overview Health Canada’s Lot Release Program and will specifically focus on Consistency Testing activities performed by CERB’s Potency Laboratory during the review of a New Drug Submission. Furthermore, this talk will highlight CERB’s Potency Laboratory experience with bioassays and will present some case studies. NOTES:

Lifecycle of an Enzyme Activity Assay for BMN 110: Development and Validation to Post-marketing Commitments Loc Vo BioMarin Pharmaceutical Inc., Novato, CA USA An enzyme activity method is a critical assessment of potency for an enzyme replacement drug product (BMN 110). Methods can undergo drastic changes from Phase 1/2 to Phase 3 / commercialization. Methods must also accommodate formulation changes during drug development. Following method validation, sufficient controls need to be implemented to assure assay performance. For BMN 110, the enzyme activity assay became the key test method for establishing comparability between clinical and commercial manufacturing processes and setting product expiry. During commercialization, regulatory interactions also affected specification development, which resulted into the evolution of a two-tiered specification for release and stability. Regulatory expectations may also alter assay format such as the addition of Km/Kcat determination or require an additional/orthogonal assay to satisfy concerns of potency. NOTES:

Bioassay Strategies for Assessment of Co-stimulation Inhibitors in Immuno-oncology: Considerations from Development to Commercialization Cynthia Inzano Bristol-Myers Squibb Company, Princeton, NJ USA The last ten years has produced a considerable increase in understanding the role of immunomodulatory checkpoints, responsible for maintenance of self-tolerance and level of immune response, in the ability of tumors to use these pathways for immune resistance. This knowledge has led to the development of immunomodulatory antibody products for use in antitumor therapy that target inhibitory pathways of effector cells requiring co-stimulatory signaling. Two examples of these antibodies, Nivolumab (Opdivo®) and Ipilimumab (Yervoy®), are currently approved for this purpose. However, the complex in vivo signaling systems used by this class of drugs pose unique challenges in the development of in vitro bioassays for quality control. Examples of bioassays, issues related to these methods, and data will be discussed further in this presentation. NOTES:

Bioassay Evolution and Lessons Learned Thomas Luntz Catalent Pharma Solutions, Morrisville, NC USA Bioassays are very challenging to develop and maintain along the pathway to drug product commercialization. The bioassay(s) must reflect the mechanism of action (MOA) and then, as expectations rise through clinical development, the bioassay must be suitable for future lot release and stability testing. This discussion will focus on the development, evolution and validation of bioassays, QC readiness, management and control of reagents and samples and considerations for change once a drug product is approved. Practical examples will be shared along with lessons learned. NOTES:

Bioassays to Support Commercialization of Drug Products

Workshop Session PANEL DISCUSSION – Questions and Answers Evangelos Bakopanos, Health Canada, Canada Jeffrey Glenn, Bristol-Myers Squibb Company, USA Thomas Lunz, Catalent Pharma Solutions, USA Emily Shacter, ThinkFDA, USA Craig Thelwell, National Institute for Biological Standards and Control (NIBSC), United Kingdom Loc Vo, BioMarin Pharmaceutical Inc., USA The following questions will guide the panel discussion:

• When developing a bioassay, which considerations are most important with respect to later commercialization of the product?

• How do the requirements of a bioassay for commercial product release differ from a bioassay used during product development? What are the critical characteristics of bioassays supporting commercial product release?

• Which, if any, intellectual property considerations apply to bioassay procedures or reagents (e.g. cell lines, plasmids or recombinant proteins) that may become relevant upon commercialization?

• What are the best approaches to manage and control bioassay reagents, and to ensure their supply? • How should potency specifications be managed during the progression from product development

to commercialization? What is the best approach to link potency specifications to clinical experience?

• What is the role of international reference standards in bioassays for commercial products?

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Bioassay Challenges during Product Globalization Session Abstract

Session Chairs: Katrin Buss, Federal Institute for Drugs and Medical Devices, BfArM, Helena Madden, Biogen Idec and Noel Rieder, Amgen Inc. Global drug registration enables the biopharmaceutical industry to enter markets all over the world and to provide life-changing medicines to patients everywhere. This requires a strategic approach to analytics from early drug development throughout post-approval lifecycle management. Analytical control strategies are influenced by country and region specific expectations and testing requirements that result in added complexity. Scientists and regulators must work together to understand regional requirements and best approaches to ensure successful licensure and assurance of product quality. Considerations for globalization of bioassays include: instrument and software selection, critical reagent availability, method robustness and reproducibility, and the requirement for registration and import testing. These challenges are more daunting when considered in the face of likely language, time, and cultural differences. This session will explore and contrast requirements from several areas of the world. We will also discuss strategies for selection, development, transfer, performance, and maintenance of a bioassay to support successful biopharmaceutical globalization. NOTES:

Presenter’s Abstracts Global Company, Global Products, Local Laboratories Camille Dycke F. Hoffmann-La Roche Ltd., Basel, Switzerland To support global product release, the bioassay used in the commercial control system of the product is transferred to multiple QC testing laboratories around the globe. Local laboratories practices such as method translation, local equipment specifications, lab practices for cell handling, and local health authorities’ expectations are regular challenges faced during these global method transfers. In order to facilitate them, it is important to ensure method robustness. Moreover, the implementation of global processes, such as global IT and LIMS systems, as well as a global training plan and a solid method life cycle management program, are key elements to ensure successful global transfers. A few case studies will be presented, illustrating the challenges and some successful solutions to support product globalization. Slides were not available at the time of printing. NOTES:

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Development of the 1st International Standard for Pegylated G-CSF (PEG-G-CSF) Meenu Wadhwa National Institute for Biological Standards and Control (NIBSC), South Mimms, United Kingdom WHO International Standards/Reference Reagents are primary standards intended for the calibration, characterization and validation of potency assays. Produced to defined standards which optimize retention of biological activity and ensure stability, WHO standards are calibrated in units or International Units which are often arbitrarily assigned and relate to the ampoule content of the analyte. These standards are established by the WHO Expert Committee on Biological Standardization of WHO following a multi-centre international collaborative study in which the suitability of a particular preparation to serve as a standard is demonstrated. Until recently, WHO standardardisation activities have mainly covered the first wave of successful biotherapeutics, however with patent expiry and global development, standards for in vitro biological activity of modified proteins and monoclonal antibodies are needed. This presentation will cover the developmental aspects of the 1st international standard for a modified protein and focus on the case of human sequence recombinant PEG-G-CSF. The strategy for assigning the unitage of the PEG-G-CSF standard and the relationship of this IS with the 2nd IS for G-CSF will also be discussed. NOTES:

Bioassay Strategy to Support the Transfer of a Therapeutic Protein for Manufacturing and Filing in China Justin Jia WuXi AppTec Inc., Shanghai, China China is the world’s third-biggest pharmaceutical market with an annual growth of over 20% since 2009. Drug registration in China is regulated by China Food and Drug Administration (CFDA) and local offices. It is very critical to understand the drug registration system in China and regulatory requirements for analytical control strategies. Partnership with selected CRO can facilitate the filing and approval procedure. Here, the strategies for selection of CRO, and collaboration on bioassay selection, development, transfer, and qualification were discussed with practical case studies. NOTES:

Strategies to Assist Global Regulatory Acceptance of Assurance of Potency Anthony Mire-Sluis Amgen Inc., Thousand Oaks, CA USA Potency assays often take the form of cell based bioassays. However, during development, one can often start Phase 1 clinical studies using a binding assay if appropriately justified. At some point during product development it is most likely that a cell based bioassay will be required by regulatory agencies. In a global market, it is prudent to plan ahead regarding the lifecycle of the potency assay (binding assay, then cell based and replace the cell based assay with a binding assay for commercial testing) due to the inherent challenges with cell based bioassays. Regulators are concerned with the ability of the assay to mimic the mechanism of action of the product, the variability/reliability of the assay, how it is controlled etc. In addition, there are several regions where either import testing has to occur or the regulators require the assay to be transferred directly into their laboratories. Therefore, designing as robust an assay as possible would avoid the challenges with assay transfers such as bias, lack of appropriate cell growth and performance, overt variability etc. There are many approaches to increase bioassay robustness such as in-depth characterization, selection and control of the cell line, randomization of plate design, use of a potency standard, frozen ready-to-plate cells and automation. The potential to replace a cell based assay with a binding assay, if possible, is optimal since these assays are generally more simple to perform and more robust. With the advent of Quality by Design, a manufacturer can also consider the bioassay within its total control strategy and may be able to remove the testing from either lot release of drug substance or drug product, or from stability studies, dependent upon the product. NOTES:

Bioassay Challenges during Product Globalization Workshop Session

PANEL DISCUSSION – Questions and Answers Katrin Buss, Federal Institute for Drugs and Medical Devices, BfArM, Germany Camille Dycke, F. Hoffmann-La Roche Ltd., Switzerland Chana Fuchs, CDER, FDA, USA Justin Jia, WuXi AppTec, China Anthony Mire-Sluis, Amgen Inc., USA Meenu Wadhwa, National Institute for Biological Standards and Control (NIBSC), United Kingdom The following questions will guide the panel discussion:

• When should bioassay developers consider the potential assay impact of globalization? • What are best practices in the development and validation of bioassays in support of globalization? • What is a good approach for introduction of a bioassay method change in multiple countries? • Any strategies for minimizing the impact of bioassay import testing? • When should a sponsor begin to plan for method transfers to regulatory agency labs? • What are the likely challenges in the development and performance of a bioassay in support of a

global program? • What are the local GMP requirements that may limit the strategic approaches to avoid redundant

testing?

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Poster Abstracts P-01 Novel PD-1 Blockade Bioassay to Assess Therapeutic Antibodies in PD-1 and PD-L1 Immunotherapy Programs Frank Fan Promega Corporation, Madison, WI USA Programmed death receptor-1 (PD-1) and its ligand (PD-L1) are among the few important immunotherapy targets for cancer. Current PD1 assays measure cell proliferation or cytokine production in primary T cells which are tedious, have high assay variation and small assay window. To enable quantitative potency measurement for key anti-PD-1 drugs in the market or in clinical trials such as pembrolizumab and nivolumab, as well as anti-PD-L1 drugs in clinical trials such as MPDL3280A and BMS-936559, here we report the development of a robust bioluminescent cell-based PD1 blockade bioassay. For this, we built a PD-1 effector cells in Jurkat cells which stably express human PD-1 and a NFAT-RE-luciferase reporter, and a PD-L1 positive artificial Antigen Presenting Cells (PD-L1+ aAPC) in CHO-K1 cells which stably express PD-L1 and an engineered TCR activator. Once these two cell types were co-cultivated, transcriptional activation of NFAT pathway in PD-1 effector cells, mediated by binding of TCR complex with TCR activator in PD-L1+ aAPC, is significantly suppressed by PD-1/PD-L1 engagement. This inhibition can then be specifically reversed by co-incubation of PD-1 or PD-L1 blocking antibodies in dose-dependent manner, but not by the antibody for other immune checkpoint receptors such as anti-CTLA4 ipilimumab. We further developed both PD1 effector cells and PD-L1+ aAPC in Thaw-and-Use format so the cells can be plated for assay without the need of cell culture. The resultant PD1 assay using Thaw-and-Use cells brings the benefit of convenience, low day-to-day variation, and easy lab-to-lab assay transfer. We demonstrate the assay is able to measure relative potency for antibody biologics, and also can detect potency changes for stressed antibody samples. In summary, the reporter-based PD1 blockade assay provides a valuable tool for both drug screening and characterization in early drug discovery, and lot release and stability study in drug manufacture for therapeutic antibody drug candidates in PD-1 and PD-L1 immunotherapy programs. NOTES:

P-02 Simple, Reproducible & Robust Bioassays in Cryopreserved Ready-To-Assay Format Scott Gridley; Abhi Saharia; Mong Saetern; Hanako Daino-Laizure; Sangeetha Gunthuri; Rajini Bompeli; Jane Lamerdin DiscoveRx Corporation, Fremont CA USA Cell-based bioassays often pose a hurdle during a rapidly moving biologics development program. High standards for assay accuracy, precision, reproducibility and robustness are additionally put to the test by the use of continuous culture cells that can add to assay variability and increase the cost and complexity of each assay. Advent of new technology platforms can enable rapid development of cell-based bioassays that are simple, accurate, precise and robust. Here, we discuss the development and application of diverse PathHunter® cell-based assays that cover distinct cellular mechanisms either distal from or proximal to the receptor. These quantitative assays are highly specific, scalable, robust and utilize a homogenous mix-and-read protocol, which facilitates rapid and reproducible detection of drug potency. In order to increase the reproducibility and reduce the complexity of the assay by eliminating cell culture, these assays are developed as cryopreserved ready-to-assay cells that can be plated directly from the frozen state onto the assay plate. The cell preparation, bioassay protocol and reagents have been optimized to provide superior bioassay performance with high reproducibility (<10%RSD). Examples discussed here include assays for Bevacizumab, human growth hormone, GLP1 receptor agonists and erythropoietin. NOTES:

P-03 Bioassay Monitoring Data: Use for Statistical Process Control, Qualification of Critical Reagents, Reference Standard Requalification and Determination of Process Capability Index (CpK) Liming Shi1; Alexander Knorre2 1Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN USA; 2BSL BIOSERVICE Scientific Laboratories GmbH, Planegg / Munich, Germany Typically during QC bioassay performance multiple monitoring data are generated. Examples will be shown for using cell culture monitoring data and bioassay monitoring data for Statistical Process Control (SPC) and for qualification of critical reagents for a bioassay. Monitoring data can also be used for housekeeping activities like Reference Standard requalification. In addition, examples of process capability index (CpK) determination of bioassays will be presented. NOTES:

P-04 Bioassay Method Comparison for the Relative Potency Analysis of Erythropoietin Biosimilar Products Rita Stiemke; Manuela Grassi; Joel Galang; Bill Bakewell; Peter Wunderli PPD, Inc. Middleton, WI USA The characterization of the functional activity applied to comparison of of biosimilar and follow-on biological therapeutics to the innovator products requires these methods be sensitive, precise and accurate to allow for detection of potential differences between the drug products. Two cell-based assay platforms were developed and evaluated for their efficacy in comparing the relative potency of two Erythropoietin biosimilar products. A traditional proliferation assay method applied to determine cell growth in the response to Erythropoietin was developed using Luciferase-derived measurement of cell viability; and the method was shown to be linear, accurate, and precise. A second method was developed using a Stat5-responsive Luciferase reporter gene cell line provided by Promega Corporation; and was also demonstrated to be specific, accurate and precise to the treatment of Erythropoietin. Each method was qualified and used to analyze Erythropoietin from two different sources. The reporter gene assay identified markedly different relative potency between the two Erythropoietin biosimilar products, while the proliferation assay did not detect any differences in the relative potency of the two products. This demonstrates that identification of the proper bioassay platform is critical to evaluation of the differences in biological activity that can occur, which may impact both safety and efficacy profiles in these types of products. NOTES:

P-05 FcRn TR-FRET Platform Binding Assay Development and Applications Frank Shi; Carmelata Chitikila; Andrea Sobjak; Nancy Fields; Patrick Niven; Tam Soden Janssen R&D, LLC, Malvern, PA USA The neonatal Fc receptor for IgG (FcRn) performs two critical roles: to extend antibody half-life in the serum and to transfer IgG from a mother to its fetus. For more comprehensive characterization, and for determining lot-to-lot comparability for all biological programs that contain an Fc region, we developed a simpler, more sensitive and more robust FcRn binding assay to replace the existing EIA method. This new homogeneous assay was developed, validated, and implemented using Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology, demonstrating a low invalid rate (<3%) and > 10 fold higher throughput compared to the EIA method. The TR-FRET FcRn binding assay has proven to be reliable, reproducible, robust, and a high throughput Fc binding assay that does not require extensive sample preparation or handling. This poster describes the FcRn TR-FRET assay development and its applications. NOTES:

P-06 A Human FcγRIIa Reporter Bioassay for Antibody-dependent Cellular Phagocytosis (ADCP) Pathway Activation Terry Surowy; Rich Moravec; Aileen Paguio; Denise Garvin; Frank Fan Promega Corporation, Madison, WI USA Antibody-dependent cellular phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies that is effective at killing tumor cells in addition to antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody drug development is beginning to address and realize the potential of this mechanism of action with increasing interest. Traditional ADCP assays are arduous and highly variable. They are FACS-based rather than plate-based, and rely on isolation of primary cells, typically monocytes, that are then differentiated for many days in culture to generate macrophages. Both target cells and differentiated macrophage effector cells must also be labeled differentially for FACS analysis. We sought to develop a plate-based, less arduous, faster and less variable ADCP bioassay. FcγRIIa is the predominant receptor involved in macrophage ADCP. We describe here our development of a human FcγRIIa (H variant)-specific ADCP reporter bioassay for quantifying antibody biological activity in ADCP pathway activation. The assay uses dual-engineered effector cells that express the receptor and NFAT-RE luciferase. Activation of NFAT response upon target cell-bound antibody binding to receptors results in production of luciferase activity. We demonstrate: specificity of the assay, induction being dependent on the presence of FcγRIIa (H variant) and target-cell bound antibody; use of either suspension or adherent target cells; assay robustness; stability indicating ability using heat-stressed rituximab; assay ability to quantify ADCP pathway activation of chimeric, humanized and fully human antibodies and with the expected isotype specificity. The H variant receptor is able to bind all human IgG isotypes and we demonstrate effective ADCP pathway activation with IgG2 antibodies. In a bioassay qualification study, the assay was accurate, precise and demonstrated good linearity in the 50%-200% relative potency range. With thaw-and-use effector cells and an optimized protocol, this assay provides reduced complexity and variability over traditional ADCP bioassays and enables fast and easy assessment of ADCP pathway activation in a 96-well plate-based format. NOTES:

P-07 Biosimilars: The Process is the Product - The Example of Recombinant Streptokinase Craig Thelwell; Colin Longstaff National Institute for Biological Standards and Control (NIBSC), South Mimms, United Kingdom Background: The bacterial plasminogen activator streptokinase was the first licensed thrombolytic drug. Worldwide streptokinase remains the most used thrombolytic agent for the treatment of myocardial infarction, particularly in developing countries. The originator streptokinase product is the native protein purified from culture filtrates of Streptococcus equisimilis. Many biosimilars (or follow-on biologicals) are also produced this way, although increasingly recombinant streptokinase from E. coli is being used as a cheaper alternative to native streptokinase. Potency assignments for recombinant streptokinase, relative to the WHO International Standard (IS), are highly variable and often dependent on the bioassay system used, and this has potentially dangerous consequences. A proportion of some recombinant streptokinase products appear to be incompletely processed, retaining the amino-terminal methionine engineered for intracellular expression, though the significance of this for functional activity is not known. Objectives: To investigate and quantify the impact of an amino-terminal methionine on streptokinase activity. Methods: Mature native streptokinase (rSK) was cloned and a novel variant constructed to include an amino-terminal methionine (rSK-Met) that is not susceptible to processing during expression. Potencies of rSK and rSK-Met were determined relative to the WHO IS using a chromogenic solution (European Pharmacopoeial) assay, and fibrin-based assays. Results: In the chromogenic solution assay there was no measurable difference between rSK and rSK-Met activities. In the fibrin-based methods however potency estimates for rSK-Met were greatly reduced compared to rSK, and compared with the potencies calculated in the chromogenic solution assay, and this was consistent with potency estimates for several different batches of commercial recombinant streptokinase products also tested. This apparent difference in activity and fibrin selectivity means different potencies would be assigned to therapeutic recombinant streptokinase products depending on the degree of amino-terminal methionine processing, and on the pharmacopoeial assay method used, affecting the dosage patients receive. This has serious health implications and provides an example of the danger in the unregulated clinical use of biosimilars. NOTES:

P-08 Development and Applications: An AphaScreen-based FcRn Binding Assay to Characterize Monoclonal Antibodies Qiang Wu; Ho-Young Lee; Guoying Jiang; Pin Yee Wong; Hélène Gazzano-Santoro The neonatal Fc receptor (FcRn) interacts with IgG Fc to play a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody PK and biological characterization study during the early development. Here we developed a homogeneous Alphascreen based FcRn assay to assess the binding of FcRn to IgG antibody. Following three simple steps, the assay can run up to four samples per plate in 2 h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies. NOTES: