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Institute for International Research, San Francisco, CA
October 6-8, 2008
Main Conference Day One - Tuesday, October 7, 2008| 8:30 | Conference Registration & Morning Coffee | | 9:30 | Quantitative Cell Based Assays for Bioanalytical Use
Cell based bioactivity assays that measure the biological activity of drug or any other analyte of interest in serum samples also hold a lot of promise in answering some important questions during drug development.
- Shalini Gupta, PhD, Associate Director Clinical Immunology, AMGEN | | 10:15 | 30-Morning Networking Break | | 10:45 | A Sensitive and Robust Western Blot Cell Based Assay to Measure the Activity of Botulinum Neurotoxin Serotype A
Cell based assays are critically important for measurements of BoNT/A activity in field and biological samples, in-process samples, and formulated products, to reduce the need for in vivo testing of toxin activity, and to identify inhibitors of BoNT/A action. A well-designed cell based assay should be able to evaluate, indirectly or directly, all three steps in BoNT activity: receptor binding, internalization and translocation, and catalytic activity. Methods: A new 96-well plate cell based assay has been developed using differentiated Neuro-2a cells and a Western Blot read-out with an Allergan custom antibody to SNAP25197 that specifically recognizes the cleaved product of BoNT/A. Accuracy, precision, parallelism, specificity, and robustness of the assay were evaluated. Results: The assay is based on a 7 point dose-response curve fitted with a four parameter logistic model that generated EC50 values of 3.5 nM on average. The assay is routinely run by five operators with Z’ values greater than 0.5 and signal-to-noise values greater than 10 fold. Further optimization of differentiation, BoNT/A treatment, and Western blot conditions resulted in increased sensitivity and EC50 values of 50 to 100 pM. Conclusion: A new specific and robust mediumthroughput screening Western Blot cell based assay for measuring BoNT/A activity has been developed and characterized. Optimization of the assay resulted in increased sensitivity that at the moment is still insufficient for developing a potency assay for product release, but may be useful for evaluation of more concentrated samples.
- Joanne Wang, Senior Professional, ALLERGAN | | 11:30 | Case Study on the Development and Qualification of a Cell Based Neutralization Assay for an Anti-Toxin Antibody
Neutralizing antibody assay development, cell based assay, qualification and drug interference. A case study of the development, optimization and qualification of a cell based neutralization assay for an anti-toxin monoclonal antibody. Topics to be covered include: - Review of immunogenicity and drug neutralization by anti-drug antibodies
- Selection of NAb assay format, single point vs. titer format
- Challenges for bacterial toxin-based cell assays
- Selection of positive control sera
- Assay optimization: selection of toxin and positive control concentrations, explore cell passage and cell density effects
- Determination of assay cutpoint, sensitivity, specificity and assessing matrix-interference and sample stability conditions
- Anti-drug antibody drug interference issues
- Stephen Ullrich, Senior Scientist, HUMAN GENOME SCIENCES | | 12:15 | Networking Luncheon | | 1:30 | Specific Strategies to Overcome Your Cell Based Assay Challenges - New ideas and data to detect antidrug antibodies in the presence of circulating drug
- Improve assay development efficiency and quality
- Establish sufficient assay sensitivity
- Decrease the time required to validate new instruments and assay technologies
- Strategies for quickly developing novel or state-of-the-art cell functional assays
- Correct analysis of biological assay data & parallel line analysis
- Regulatory perspective on assay filing for product release
- Discuss high sensitive assays with good signal to noise ratio
- Double staining of fluorescence on cell-tissue interaction assay
- Design accurate and efficient biochemical and cell based assays
Moderator:
- Shalini Gupta, PhD, Associate Director Clinical Immunology, AMGEN
Panelists:
Stephen Ullrich, Senior Scientist, HUMAN GENOME SCIENCES
Ester Fernandez-Salas, PhD, Principal Scientist, Neurotoxin Research Program, ALLERGAN | | 2:15 | Transfer of Cell Based Assays: Don’t Assume Too Much Logistics - Assay transfer plan and Method – written in a very clear manner
- Reagents – enough available, qualification of new for use at CRO/other sites
Approach to Assay Transfer - Two step with analytical (feasibility) assessment followed by formal assay transfer
Analytical Performance - Training and Troubleshooting (CRO and internal sites – incubation times, cutpoints, criteria)
- Assessment of criteria (need for combined cutpoints or re-evaluation of assay criteria
- Jason Pennucci, Senior Associate Scientist, AMGEN | | 3:00 | 30-Minute Afternoon Networking Break | | 3:30 | Floating Assay Cut Point Approach to Determining NAb Positivity in Immunogenicity Testing
Significant variations between individual donor matrixes could make a statistically derived assay cut point not obtainable. A floating assay cut point approach provides a solution by leveraging on detecting the difference resulted from the presence of NAbs in an individualized way that is less influenced by matrix variation among the donor population. The benefits and limitations of this approach will be discussed with some example.
- Yao Zhuang, PhD, Senior Scientist, AMGEN | | 4:15 | Development, Validation, & Transfer Strategies for Cell Based Assays
Assay Development - Design accurate and efficient biochemical and cell based assays
- Develop cell to cell interaction assay
- Develop cytotoxicity and ADCC (antibody dependent cell cytotoxicity)
Assay Validation - Validate cell based assays using the most up to date guidelines
- Differentiate qualification vs. validation requirements
- Interpretation of guidance documents for bioassays
Assay Transfer - Interface with development for assay transferring
- Transfer criteria for cell based NAb assays to CROs
- How to improve the monitoring bioassays
- Measure anti-drug antibodies in the presence of drug
- Use of primary cell line in a lot release bioassay
- Applications for cell line toxicology
- Yao Zhuang, PhD, Senior Scientist, AMGEN
- Wei Zhang, Scientist II, BIOGEN IDEC
- Jason Pennucci, Senior Associate Scientist, AMGEN | | 5:00 | End of Day One |
Main Conference Day Two - Wednesday, October 8, 2008| 8:30 | Morning Coffee | | 9:00 | Chairpersons’ Recap of Day One
- Shalini Gupta, PhD, Associate Director Clinical Immunology, AMGEN | | 9:15 | Statistical Process Control for Improved Assay Development Production and Transfer
The most important aspect of how we develop, validate, maintain, and possibly transfer assays is rooted in the data. It is the data that inform us as to how we should act, where we should go, and if our processes are capable of performing at some required level. Data analysis is the key to this and statistical techniques are tools that we use. Discusses types of charts that are used, how these charts can tell us if our processes are in control, and where we can use these charts in the development, production, and transfer processes.
- Martin Kane, MS CRE, Senior Manager, Process Statistics, HUMAN GENOME SCIENCES | | 10:00 | Optimization and Analysis of Sources of Variation in theWestern Blot Neuro-2a cell based Assay Via Application of the Weighted Four-Parameter Logistic Model
Purpose. The Neuro-2a cell based assay is a potency assay that uses 96-well plates and differentiated Neuro-2a cells in a Western Blot read-out. It has been designed to measure all three steps in BoNT/A activity: receptor binding, internalization and translocation, and catalytic activity. Mathematical modeling of the Neuro-2a dose-response curve via the four-parameter logistic model [4PL] has been used not only (1) to improve this assay but also (2) to characterize it after finalization. Methods: (1) The 4PL was applied to actual data and then used to simulate the likely results for three different dose regimens of interest. The 4PL was then applied to the simulated data and the resulting model parameters and convergence were monitored. The optimal dose regimen was defined as the one yielding estimated parameters as close to nominal as possible while also causing the fewest convergence problems. (2) After optimization, routine testing was initiated, yielding a historical database spanning nine months. Weighted four-parameter logistic models were separately fitted to the individual response curve data in this historical database. The fitted asymptotes, EC50, HillSlope, and weighting parameters values were then regressed on variables measuring the relevant extrinsic variability sources (such as operator id and cell passage numbers). Results: (1) The nature of the collaborative relationship, and its benefits, between the Depts. of Biostatistics and Biological Sciences will be discussed. Appropriate dose optimization was a direct result of this collaborative effort. (2) Statistically significant sources of extrinsic assay variability will be noted and quantified. The results will then be used to indicate how a unified mixed-effects weighted four-parameter logistic model might be built. The benefits of unified mixedeffects model-building will be noted.
- David Paul, PhD, Manager, Department of Biostatistics, ALLERGAN | | 10:45 | Morning Networking Break | | 11:15 | Statistical Analysis of Cell Based Assay Data - Setting assay acceptance criteria
- Statistical approaches and strategies for determination of robust cut points in immunogenicity assays
- Find one software package that one can easily use to trend data, perform anova, and test for parallelism all in one swoop
- The multitude of projects/multi-tasking and staying on top of new technologies and their applications to existing programs FDA/USP recommended non-linear curve fit data analysis
- Setting minimum requirements
- Identifying testing parameters
- Comparison with preclinical data
- Setting specification limits on bio-assays
- Match sensitivity of cell based assay to ELISA like methods controlling sensitivity drift - Martin Kane, MS CRE, Senior Manager, Process Statistics, HUMAN GENOME SCIENCES
- David Paul, Manager Biostatistician, Biostatistics, ALLERGAN
| | 12:00 | Networking Luncheon | | 1:15 | Evaluation of Fluorometric and Electrochemiluminescence Assay Platforms for the Detection of Blocking Antibodies in Human Serum
The performance of a membrane based Delfia® fluorometric assay and a cell based ECL assay using MSD platform were evaluated for the detection of blocking antibodies in normal human serum and in autoimmune disease serum. In this particular case, the cell based ECL assay provided a more sensitive, drug tolerant platform for the detection of blocking antibodies in autoimmune disease serum. Overall this work highlights that during the course of clinical development; careful consideration of study population, dose levels and anticipated levels of circulating drug is required prior to the selection of assay configuration. Membrane based Delfia® assay that provides ease of use may potentially be used for the detection of antibodies in a healthy volunteer study but in studies with autoimmune disease population, cell based ECL assay format was more appropriate.
- Darshana Jani, Senior Supervisor, Laboratory Management Clinical Science and Technology Department, BIOGEN IDEC | | 2:00 | New Technologies Applied to Cell Based Assay Development - Homogeneous methods - add and read type cell based assays
- Assays using primary cells, frozen, division-arrested cells, 3-D cultures.
- Nanotechnology and its application in cell based assays
- Automated potency assays with better precision and accuracy
- 3D co-culture tube formation assay
- Tumor cell coculture with endothelial cell assay
- Bioluminescence Resonance Energy Transfer or BRET
- Appropriate liquid handling instrumentation for complete automation of cell based assays
- cell based assays used for high throughput screening in drug discovery
- Molecular techniques used in cell based assays especially for lot release potency assays (example: mRNA, signaling pathways)
- New Technology discussions
- PCR-based assays
- MSD
- Luminex
- FACS-based assays
- Shalini Gupta, PhD, Associate Director Clinical Immunology, AMGEN
- Darshana Jani, Senior Supervisor, Laboratory Management Clinical Science and Technology Department, BIOGEN IDEC
- Stephen Ullrich, Senior Scientist, HUMAN GENOME SCIENCES | | 3:00 | 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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- Ester Fernandez-Salas, PhD, Principal Scientist, Neurotoxin Research Program, ALLERGAN
- Jaya Goyal, PhD, Associate Director, Clinical Science and Technology, BIOGEN IDEC
- Shalini Gupta, PhD, Associate Director Clinical Immunology, AMGEN
- Velvizhi Heine, Scientist II, BIOGEN IDEC
- Darshana Jani, Senior Supervisor, Laboratory Management Clinical Science & Technology Department, BIOGEN IDEC
- Xu-Rong Jiang, PhD, MD, Associate Director, Analytical Biochemistry, MEDIMMUNE
- Martin Kane, MS CRE, Senior Manager, Process Statistics, HUMAN GENOME SCIENCES
- David Paul, PhD, Manager, Department of Biostatistics, ALLERGAN
- Jason Pennucci, Senior Associate Scientist, AMGEN
- Susan Pederson, Associate Director, PreClinical Development, ZYMOGENETICS
- An Song, Senior/Group Leader, Development Sciences, GENENTECH
- Stephen Ullrich, Senior Scientist, HUMAN GENOME SCIENCES
- Joanne Wang, Senior Professional, ALLERGAN
- Wei Zhang, Scientist II, BIOGEN IDEC
Yao Zhuang, PhD, Senior Scientist, AMGEN Linda Zuckerman, PhD, Director, Portfolio and Program Managment, ZYMOGENETICS
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Deadline for Abstracts:
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Email vbernardino@iirusa.com for more information
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Registration:
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Please visit www.iirusa.com/cba for more information on registration or call (888) 670-8200 U.S. or (941) 951-7885 Intl. You can also email vbernardino@iirusa.com for more information.
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E-mail:
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vbernardino@iirusa.com
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