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Institute for International Research, San Francisco, CA
October 6-8, 2008
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| 8:00 | | Conference Registration and Morning Coffee | | 8:45 | Chairpersons Welcome and Opening Remarks
- EricWakshull, PhD, Senior Scientist/Group Leader, BioAnalytical Research & Development, GENENTECH | |
| 9:00 | Challenges in Generating Information on Molecular Interactions to Support Biotherapeutic Development
Information on molecular interactions has been widely used to support biotherapeutic development. However, accurate information on these interactions such as binding affinities and kinetics are sometimes hard to obtain due to the complexity of large molecules such as monoclonal antibodies. In addition, interpretation of the information could be challenging due to complex biological systems. The talk will use case studies to discuss challenges and strategies to help generate meaningful information on molecular interactions to support biotherapeutic development.
- Jihong Yang, PhD, Scientist, BioAnalytical Research and Development, GENENTECH | | 9:30 | Binding Parameters Of Antibodies Binding To The Cell Surface
As antibodies (Abs) are increasingly used for therapy, it is essential to have reliable methods to compare the binding properties of standard Abs and Ab derivatives. It has become accepted practice to measure the functional affinity (equilibrium association constant). However, because of multivalent binding, such measurements do not adequately describe the interactions that occur. Ab binding is predominantly irreversible, but it is important to determine the level of dissociation that occurs (usually < 30%), and the rate of internalization.
- Jules Mattes, PhD, CENTER FOR MOLECULAR MEDICINE & IMMUNOLOGY | | 10:15 | | 30-Minute Morning Networking Break | | 10:45 | Accelerating Identification of High Potential Therapeutic Antibody Candidates Using Molecular Interaction Analyses
The ability to quickly identify promising therapeutic candidates allows for increased throughput and deep interrogation of multiple antibody phage display libraries in parallel, as well as rapid evaluation of hybridoma-generated antibodies. Biosensor technologies are powerful tools for analyzing antibodies or fragments in complex media such as bacterial extracts and hybridoma culture supernatants, as well as purified proteins. XOMA accelerates the identification of high potential therapeutic candidates through customized development of target-specific biosensor-based assays in order to evaluate clones as early as possible. Case studies will be presented to demonstrate various screening strategies for therapeutic antibody discovery and development including appropriate consideration of the affinity, epitope, function, and potency.
- Marina K. Roell, PhD, Associate Director, Molecular Interactions and Biophysics Group, Preclinical Research and Development, XOMA | | 11:15 | Label-Free Screening Of Bio-Molecular Interactions
The potential of label-free approaches to complement or even displace other detection technologies has never been higher. This presentation will cover the latest and emerging developments in label-free detection systems, their underlying technology principles and end-user case studies that reveal the power and limitations of label-free in areas of drug discovery and diagnosis. Unlike label- and reporter-based technologies that simply confirm the presence of the detector molecule, label-free techniques can provide direct information on analyte binding to target molecules typically in the form of mass or stress addition or depletion from the surface of a sensor substrate. However, these technologies have failed to gain widespread acceptance due to technical constraints, low throughput, high user expertise requirements, and cost. Whilst they can be powerful tools in the hands of a skilled user evaluating purified samples, they are not readily adapted to every day lab use where simple to understand results on high numbers of samples are the norm. This talk seeks to address some of the issues surrounding the unmet needs in the market place, and the difficulties faced by technology developers in meeting these needs with innovative products. It will also outline recent trends from newer technology developers who are in the race to release products for the general lab user.
- Matthew A. Cooper, PhD, Managing Director, CAMBRIDGE MEDICAL INNOVATIONS | |
| 11:45 | Strategies for Applying Molecular Interaction Information to Aid Drug Discovery & Development
Molecular interactions, affinity, in vitro vs. in vivo, alternative technologies…these topics are not often discussed at open forums and this is a great time to initiate such discussions within a broad scientific community that ultimately generates and interprets such information to support Research & Development. This unique panel provides a rare opportunity to openly discuss molecular interaction challenges and allow you to ask experts your most pressing questions!
Panelists:
- Jihong Yang, PhD, Scientist, BioAnalytical Research and Development, GENENTECH
- Jules Mattes, PhD, CENTER FOR MOLECULAR MEDICINE & IMMUNOLOGY
- Marina K. Roell, PhD, Associate Director, Molecular Interactions and Biophysics Group, Preclinical Research and Development, XOMA
- Matthew A. Cooper, PhD,Managing Director, CAMBRIDGE MEDICAL INNOVATIONS | | 12:15 | | Networking Lunch | |
| 1:30 | PK Assay Strategies for Biotherapeutics
Although there are white paper recommendations describing best practices for ligand binding assay development and validation, there is no clear guidance as to whether methods are needed to quantitate and/or differentiate between unbound therapeutic and therapeutic complexed with soluble forms of a target. However, the format and reagents chosen for a given PK assay may impact which drug species are detected and therefore affect the resulting observed PK parameters. A case study will be presented where different assay formats were compared and circulating target appears to have differentially impacted observed PK profiles. In addition, various factors which should be considered when developing a PK assay strategy for a biotherapeutic will be discussed.
- Alyssa Morimoto, PhD, Scientist, BioAnalytical Research and Development, GENENTECH | | 2:15 | Characterizing The In Vivo Disposition And Clearance of Biotherapeutics: Influence of Bioanalytical Assay Format
Interaction with soluble target ligands can alter the disposition of a therapeutic monoclonal antibody. These interactions can also produce interference and artifact in bioanalytical methods. As a result, the choice of assay format is critical to the appropriate characterization of the pharmacokinetics and disposition of this class of biotherapeutic agents. Examples in which differences or limitations in analytical approach influenced the interpretation of preclinical PK/PD studies will be discussed.
- Victor J.Wroblewski, PhD, Research Fellow, Drug Disposition, Global PK/PD/TS, ELI LILLY | | 3:00 | | 30-Minute Afternoon Networking Break | | 3:30 | PK Assay Formats for Antibody Therapeutics
PK assay formats for antibody therapeutics may differentially impact the accuracy of drug measurement, depending on the nature of the target and the dosing schedule. Two case studies in which distinct PK assay formats were compared will be discussed. Specifically, comparisons between an antigen capture vs. an anti-idiotype capture formats, the effect of circulating target levels and the nature of the target (multimeric targets: trimeric vs heptameric molecules) will be covered. Finally, the impact of the PK assay formats on drug measurement in the presence of anti-drug antibodies, will also be discussed.
- Thi Migone, PhD, Senior Director, Clinical Immunology, HUMAN GENOME SCIENCES | | 4:00 | Assay Format Selection During Method Development – First Step
Assay format selection is the crucial step for developing a high quality method for the quantification of macro molecule therapeutics. This presentation will focus on a case study to reveal the principles and technical approaches during assay development. A monoclonal antibody (drug) was developed to its receptor (A) to reach the therapeutic effects. Three types of reagent were available: the recombinant receptor A, anti-human IgG Fc antibody, and antiidiotypic drug antibodies. To support preclinical and clinical studies, two ELISAs were developed; a total assay to measure the bound and free drug and a “free” assay to only measure the “free” drug. Experimental results led the project team to make the decision to use the total assay as the validated assay to support regulated work. The “free” assay was used for research and exploratory purposes only.
- Mark Ma, PhD, Principal Scientist, Pharmacokinetics and Drug Metabolism Department, AMGEN | |
| 4:45 | Analytical Strategies for Measuring Protein Therapeutics— Pharmacokinetic Assays
Robust and specific methodologies are critical for measurement of monoclonal antibody therapeutics to enable thorough safety and pharmacokinetic evaluations. Currently, there is no clear guidance nor is it widely understood what the differences between unbound therapeutic and therapeutic complexed with soluble target mean from a pharmacokinetic, safey toxicology, or clinical viewpoint. Join this panel discussion for a meaningful cross-functional (PK, toxicology, bioanalytical) discussion to enable a definition of best practices.
Panelists:
- Alyssa Morimoto, PhD, Scientist, BioAnalytical Research and Development, GENENTECH
- Victor J.Wroblewski, PhD, Research Fellow, Drug Disposition, Global PK/PD/TS, ELI LILLY
- Thi Migone, PhD, Senior Director, Clinical Immunology, HUMAN GENOME SCIENCES
- Mark Ma, PhD, Principal Scientist, Pharmacokinetics and Drug Metabolism Department, AMGEN | | 5:15 - | Day One Concludes |
| 8:00 | Chairperson's Recap of Day One
- Valerie Quarmby, Principal Scientist/Associate Director, Bioanalytical Research & Development, GENENTECH | |
| 8:15 | Regulatory Perspectives on Bioassays for Antibody-Drug Conjugates
Antibody-based conjugates are designed to deliver a therapeutic agent (e.g. cytotoxic drug) through a targeting moiety. Ideally, an antibodydrug conjugate (ADC) remains non-toxic during circulation in vivo until it reaches its specific target site, where the drug is only activated once it is internalized to the target cell. Although such ADCs are simple in principle, significant chemistry, manufacturing, and control (CMC)- related challenges have presented themselves during regulatory review and approval of these complicated products. The role of bioassays in the characterization of this class of products and some critical CMC issues will be discussed.
- Jun T. Park, PhD, Division of Monoclonal Antibodies, OBP/CDER/FDA | | 9:00 | Bioanalytical Strategies for Antibody-Drug Conjugates
Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs, via a linker, and represent an exciting new biotherapeutic platform for the treatment of cancer. ADCs can deliver the cytotoxic drug specifically inside target tumor cells, minimizing the exposure to non-target cells and the subsequent side effects. Although the idea of antibody-drug conjugates was conceived over twenty years ago, these are complex drugs to develop and promising clinical data have only recently emerged. The complex structural nature of ADCs requires novel bioanalytical strategies involving the combination of approaches used for protein therapeutics and for small molecule drugs. Due to the possibility that some of the cytotoxic drug may be lost from the conjugate over time in vivo, our strategy includes multiple pharmacokinetic assays using ELISA and mass spectrometric methods, instead of a single ELISA assay typical for the bioanalysis of monoclonal antibody therapeutics. Clearly, ADCs represent a new bioanalytical paradigm and there is a need to develop novel methods to understand the fate of ADCs in vivo. For example, it is valuable to develop methods capable of structural characterization of intact ADCs in vivo for monitoring drug to antibody ratio (DAR) changes. However, the complexity and dynamic range of the background plasma proteome present significant analytical challenges. We have developed a novel affinity mass spectrometric method to successfully characterize intact ADCs in serum and plasma and measured changes in DAR distributions during in vitro plasma stability studies and nonclinical in vivo studies. This presentation will highlight examples of immunoassay and mass spectrometric-based ADC bioanalytical strategies and data used to assess pharmacokinetics and changes in DAR ratios of molecules in preclinical and clinical Development.
- Surinder Kaur, Senior Scientist/Group Leader, GENENTECH | | 9:30 | Key Considerations of an ELISA PK Assay for the Quantification of ADC
In developing a pharmacokinetic (PK) assay to quantify the amount of an Antibody-Drug Conjugate (ADC), it is critical to generate a reagent that specifically recognizes the drug. Currently, the use of a monoclonal antibody (mAb) preparation seems to be the common approach. In this study, the goal was to measure the conjugated antibody independent of its drug load in support of the evaluation of PK parameters. The assay format chosen was an ELISA using an anti-drug mAb as a coating agent to capture the ADC and an anti-idiotypic mAb as detection antibody. Anti-drug mAbs were screened and one particular mAb was selected for its ability to bind to the ADC in a manner that is non-specific as to the drug load and position(s) of conjugation. The coating concentration of the anti-drug mAb must also be optimized to eliminate the drug load discriminating effect. The key aspects of the assay development and validation will be presented.
- Mary Hu, Associate Director, Bioanalytical Development, SEATTLE GENETICS | | 10:15 | Characterizing the In Vitro And In Vivo Stability Of Antibody- Maytansinoid Conjugates
Targeted delivery of immunoconjugates in general, and antibodymaytansinoid conjugates in particular, have been generating significant interest recently based on several ongoing clinical studies. The presentation describes the stability of antibody-maytansinoid conjugates with specific focus on the in vitro and in vivo stability of the linkage between the antibody and the cytotoxic maytansinoid.
- Rajesh Krishnamurthy, Director, Analytical & Pharmaceutical Sciences, IMMUNOGEN | | 10:45 | | 30-minute Morning Networking Break | | 11:15 | A Novel LC/MS/MS Method for the Quantitation of Total N-acetylgamma Calicheamicin Derived from Monoclonal Antibody-Calicheamicin Conjugates in Animal Serum
Monoclonal antibody-calicheamicin conjugates (mAb-cali conjugates) are a group of drugs where monoclonal antibodies are covalently linked to calicheamicin derivatives. In order to use LC/MS/MS to quantify calicheamicin derivative concentrations in biological specimens, calicheamicin moiety need to be dissociated from the protein moiety. Reducing the disulfide bond will quantitatively release calicheamicin derivatives. However, the enediyne moiety in calicheamicin rearranges to form a diradical when the disulfide bond is reduced. The Formation of the diradical makes the calicheamicin derivative quantitation very challenging since free radicals are very reactive. Because of the complexity, calicheamicin derivatives were previously measured using an immunoassay (ELISA) approach. By using dithiothreitol and isopropyl alcohol as disulfide bond reduction agent and free radical capturer, a new sample processing procedure was developed. The processed samples were analyzed using LC/MS/MS to quantify calicheamicin derivative concentrations in animal serum.
- Richard Xue, Principal Research Scientist II, WYETH | |
| 11:45 | Bioanalytical Strategies for Antibody-Drug Conjugates
Antibody-drug conjugates are monoclonal antibodies with covalently bound cytotoxic drugs and represent an exciting new biotherapeutics platform. ADCs can deliver the cytotoxic drug specifically inside target tumor cells, minimizing the exposure to non-target cells and the subsequent side effects. Join us to review best practices for bioanalysis across some of the ADC molecules that are currently in development. Take advantage of our expert panel to gain new insights to this up and coming biologic!
Panelists:
- Jun T. Park, PhD, Division of Monoclonal Antibodies, OBP/CDER/FDA
- Surinder Kaur, Senior Scientist/Group Leader, GENENTECH
- Mary Hu, Associate Director, Bioanalytical Development, SEATTLE GENETICS
- Rajesh Krishnamurthy, Director Analytical & Pharmaceutical Sciences, IMMUNOGEN
- Richard Xue, Principal Research Scientist II, WYETH | | 12:15 | | Networking Lunch | |
| 1:15 | Structural and Functional Comparisons of IgGs and FcyRs Between Man, Mouse and Non-human Primates - Human and primate IgG & FcyR isotypes and polymorphisms
- Glycosylation of IgG-Fab
- Gal alpha 1 – 3 gal and hypersensitivity
- Roy Jefferis, Professor of Molecular Immunology, The Division of Immunity & Infection, UNIVERSITY OF BIRMINGHAM | | 1:45 | Development And Validation Of Cell-Based Assays For Assessment Of Antibody-Dependent Cellular Cytotoxicity (ADCC) Of Therapeutic Antibodies
Characterization of ADCC functionality of therapeutic antibodies has aided tremendously in the understanding of antibody biology and mechanism-of-action of antibody-based drugs. However, implementation of in-vitro systems measuring ADCC activity of therapeutic antibodies has been difficult within controlled environments of the pharmaceutical and biotech industry, due to inherent variability.
To overcome these limitations, we have developed and validated robust cell-based ADCC potency assays using engineered cell lines. Case studies will be presented, demonstrating that these assays are reproducible, precise, accurate, and amenable to validation under GxP.
- Marcel S. Zocher, PhD, Group Leader Bioassay Development Senior Scientist, R&D Biotech Products, F. HOFFMANN-LA ROCHE | | 2:15 | Effects Of Fucose Depletion on Fc Gamma Receptor Binding Of Humanized IgG1 Antibodies
It has been shown that the level of non-fucosylated glycans of an antibody affects its binding affinity toward Fc gamma receptors (FcyRs) and hence the ADCC activity. In the present study, we evaluated different in vitro assays to measure FcyR binding affinities of humanized IgG1 antibodies with varying levels of non-fucoylated glycans. Binding affinities toward human FcyR IIIa F-158 and V-158 allotypes were measured with bioanalytical methods based on ELISA, MSD, or BioCore techniques. Correlations between relative FcyR IIIa binding affinities derived from different assay formats and relative activity from in vitro ADCC assays will be discussed.
- Shan Chung, PhD, Scientist, BioAnalytical Research and Development, GENENTECH | | 2:45 | Comparison Of Biological Activity Among Therapeutic IgG1s With Different Fc Oligosaccharides
The structure of N-linked oligosaccharides attached to the antibody Fc region of human IgG1 has been shown to affect the pharmacokinetics and antibody effector functions of ADCC and CDC. Here, we succeeded in generating core fucose-lacking human IgG1s with three different Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and directly compared their activities. The present study provides basic information on the effects of core fucose-lacking Fc oligosaccharides on antibody biological activities.
- Yutaka Kanda, PhD, Senior Scientist, Antibody Research Laboratories, KYOWA HAKKO KOGYO | |
| 3:15 | Assessment of Fc Functionality
There are many possible in vitro ways to assess Fc functionality, and best practices have yet to be defined. We will discuss how in vitro data are typically generated and how they might relate to in vivo activity, efficacy and safety. Academic and industry experts come together to discuss the Fc biology, and strategies, challenges and methodologies for assessing Fc effector functions. Our experts open up communication on how data from in vitro analyses relates to efficacy or toxicity in vivo and best practices to assess Fc functionality.
Panelists:
- Roy Jeffries, Professor of Molecular Immunology, The Division of Immunity & Infection, UNIVERSITY OF BIRMINGHAM
- Marcel S. Zocher, PhD, Group Leader Bioassay Development Senior Scientist, R&D Biotech Products, F. HOFFMANN-LA ROCHE
- Shan Chung, PhD, Scientist, BioAnalytical Research and Development, GENENTECH
- Yutaka Kanda, PhD, Senior Scientist, Antibody Research Laboratorie, KYOWA HAKKO KOGYO | | 3:45 | | Conference Concludes |
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Organized by:
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Institute for International Research |
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Invited Speakers:
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- Shan Chung, PhD, Scientist, BioAnalytical Research & Development, GENENTECH
Matthew A. Cooper, PhD, Managing Director, CAMBRIDGE MEDICAL INNOVATIONS Jaya Goyal, PhD, Associate Director, Clinical Science & Technology, BIOGEN IDEC Velvizhi Heine, Scientist II, BIOGEN IDEC Mary Hu, Associate Director, Bioanalytical Development, SEATTLE GENETICS Roy Jefferis, Professor of Molecular Immunology, The Division of Immunity & Infection, UNIVERSITY OF BIRMINGHAM Xu-Rong Jiang, PhD, MC, Associate Director, Analytical Biochemistry, MEDIMMUNE Yutaka Kanda, PhD, Senior Scientist, Antibody Research Laboratories, KYOWA HAKKO KOGYO Surinder Kaur, Senior Scientist/Group Leader, GENENTECH Rajesh Krishnamurthy, Director, Analytical & Pharmaceutical Sciences, IMMUNOGEN Jian-Feng Lu, PhD, Principal Scientist, Pharmacokinetics, AMGEN Mark Ma, PhD, Principal Scientist, Pharmacokinetics & Drug Metabolism Department, AMGEN Jules Mattes, PhD, CENTER FOR MOLECULAR MEDICINE & IMMUNOLOGY Thi Migone, PhD, Senior Director, Clinical Immunology, HUMAN GENOME SCIENCES Alyssa Morimoto, PhD, Scientist, BioAnalytical Research & Development, GENENTECH Jun T. Park, PhD, Division of Monoclonal Antibodies, OBP/CDER/FDA Susan Pederson, Associate Director, PreClinical Development, ZYMOGENETICS Valerie Quarmby, Principal Scientist/Associate Director, Bioanalytical Research & Development, GENENTECH Marina K. Roell, PhD, Associate Director, Molecular Interactions & Biophysics Group, Preclinical Research & Development, XOMA An Song, PhD, Senior Scientist/Group Leader, BioAnalytical Research & Development, GENENTECH Eric Wakshull, PhD, Senior Scientist/Group leader, BioAnalytical Research & Development, GENENTECH Victor J. Wroblewski, PhD, Research Fellow, Drug Disposition, Global PK/PD/TS, ELI LILLY Richard Xue, Principal Research Scientist II, WYETH Jihong Yang, PhD, Scientist, BioAnalytical Research & Development, GENENTECH Wei Zhang, Scientist II, BIOGEN IDEC Marcel S. Zocher, PhD, Group leader Bioassay Development Senior Scientist, R&D Biotech Products, F. HOFFMAN-LA ROCHE Linda Zuckerman, PhD, Director, Portfolio & Program Management, ZYMOGENETICS
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Deadline for Abstracts:
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Email rgalbraith@iirusa.com for more information
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Registration:
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Please visit www.iirusa.com/bamd for more information on registration or call (888) 670-8200 U.S. or (941) 951-7885 Intl. You can also email rgalbraith@iirusa.com for more information.
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E-mail:
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rgalbraith@iirusa.com
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