Establishment and optimization of a potency assay for an effector mab using "ADCC Reporter Bioassay

Transcription

1 Establishment and optimization of a potency assay for an effector mab using "ADCC Reporter Bioassay Mike Sadick, Ph.D. Catalent Pharma Solutions Senior Manager Biopharmaceutical Characterization

2 Scenario/Outline Client came to us with a therapeutic mab with effector activity. Existing assay was classical ADCC lytic assay Not a robust assay Difficult to achieve accurate potency quantification Needed to convert assay to more robust, quantitative assay with better dynamic range. Chose ADCC surrogate assay from Promega Based on NFAT signaling pathway used by FcgRIIIa in NK cells during normal ADCC response Runs as luciferase reporter gene assay Presentation will show Underlying strategy of performance and use of assay Initial response of client system in NFAT-Luc reporter gene assay Optimization Final optimized assay

3 Early Characterization of NFAT Signaling Pathway From: Crabtree and Olson, CELL, Volume 109, Issue 2, Supplement 1, 19 April 2002, Pages S67 S79

4 Antibody-Dependent Cellular Cytoxicity FcgIIIa Receptor P NFATc Immune Response Genes Target Cell NK Cell

5 Parental Cell Line: Jurkat T Cell TCR P NFATc Immune Response Genes

6 Engineered Jurkat Reporter Cell Line NFAT-Luciferase TCR FcgIIIa Receptor P NFATc Immune Luciferase Response reporter Genes

7 Promega ADCC Reporter Gene Assay Slide courtesy of Promega

8 Signal Analysis of Bioassay Data y = d + a - d 1 + (conc/c) b d c b a Concentration

9 Signal Analysis of Bioassay Data c } Potency (% of Reference) Concentration

10 Behavior of Promega ADCC Reporter Gene Assay Using Rituximab (anti-cd20) Slide courtesy of Promega

11 Fold of Induction Fold of Induction Characterization of NFAT-Luc assay by Promega Based on Rituxumab and CD-20 expressing target cells Bioassay Characteristics - ICH Guideline Q2 [R1] Validation of Analytical Procedures Accuracy Precision: Repeatability (intra-assay precision) Intermediate precision (day to day, analyst-to analyst) Reproducibility (lab to lab) Specificity Linearity Range Robustness Design: Two analysts Three days Four plates per day 100% vs 50% 100% vs 75% 100% vs 125% 100% vs 150% Relative potency % 100% 150% Log 10 [B1 antibody], g/ml Log [control antibody], g/ml Repeatability plate1 plate2 plate3 plate Log 10 [B1 antibody], g/ml Log [control antibody], g/ml Slide courtesy of Promega Linearity Y=1.026X R2=0.995

12 Optimization of NFAT-Luc assay by Promega Based on Rituxumab and CD-20 expressing target cells Critical Assay Parameters Constant Effector cell number Other parameters tested: Assay buffer: serum concentration, use of low IgG serum Cell numbers per well Pre-plating and incubation time: target cell plating, antibody/target cells incubation Assay plates: White flat, V- or U-bottom plates Slide courtesy of Promega

13 Format suggested by Promega protocol

14 Improved Format suggested by Promega protocol

15 Catalent-desired goals for bioassays and potency quantification All samples run as pseudo-triplicates Three independent plates executed for each sample set QC Control sample run on each plate Alternative source than reference standard Run in same fashion (full curve potency determination) as test samples If QC control fails assay acceptance criteria, whole plate is invalidated and will be repeated 2 different test samples to be tested on each plate If QC control passes assay acceptance criteria, then test sample data is forward-processed If a test sample fails assay acceptance criteria, then that sample run is invalidated and will be repeated Final potency of test sample is based upon the mean of potency values derived from each of the three final independent plates

16 Desired Final Format Ref Std Ukn 1 Ukn 2 Cntl Ref Ukn Ukn Ref Ukn Std 1 2 Cntl Ukn Std 1 2 Cntl

17 Relative Light Units Initial Experiment: Anti-CD20 and WIL2s versus Client mab + 1 cell target 9-point serial dilution + 0 Anti CD20 + WIL2s Therapeutic (Effector) mab Concentration ng/ml Target cells from continuous culture Target cells cultured in well overnight; mab + responder cells added next day E:T ~ 4:1 Stimulation 6-7 hours Client mab + Target cell

18 Relative Light Units Initial Experiment: Anti-CD20 and WIL2s versus Client mab + 1 cell target 9-point serial dilution + 0 Expanded view of Client mab + Target cell Therapeutic (Effector) mab Concentration ng/ml Target cells from continuous culture Target cells cultured in well overnight; mab + responder cells added next day E:T ~ 4:1 Stimulation 6-7 hours

19 Assay Optimization Optimize E:T ratio (~2:1) Freeze target cells with enough cells/vial for 1 assay plate Add diluted target cells from thawed vial on day 1 of assay Next day add serial dilution of mab, then responder Jurkat NFAT-Luc cells Run stimulation x 6-7 hours Equilibrate assay plate to RT x 15 min Develop Glo substrate 5 min Long second day

20 NFAT-Luc assay with client system, after some early optimization. - Target cells as Thaw & Go - Promega Jurkat NFAT-Luc cells, next day, as Thaw & Go Relative Light Units Therapeutic (Effector) mab Concentration ng/ml Target cells from continuous culture Target cells in plate Overnight; mab + responder cells added next day E:T ~ 2:1 Stimulation x 6-7 hours

21 Same assay, but with both Responder Jurkat NFAT-Luc cells and Target cells as same-day Thaw & Go (Thaw/Thaw format) Relative Light Units Target cells from frozen vial Target cells + mab + responder cells added same day E:T ~ 2:1 Stimulation x 6-7 hours Therapeutic (Effector) mab Concentration ng/ml

22 Relative Light Units Relative Light Units Effect of Target Cell Passage Number Target Cell, Passage #2, Triple-layer TC Flask Target Cell, Passage #3, 175cm 2 TC Flask Target Cell, Passage #5, 175cm 2 TC Flask Target Cell, Passage #7, 175cm 2 TC Flask Therapeutic (Effector) mab Concentration ng/ml Therapeutic (Effector) mab Concentration ng/ml Target cells from frozen vial Target cells in plate overnight; mab + responder cells added following day E:T ~ 2:1 Stimulation x 6-7 hours

23 Assay Improvement Stay with simultaneous Thaw & Go for both the target cells and responder cells (Thaw/Thaw) Run stimulation overnight Additional benefits 3-4 hours in the afternoon to set up the assay 3-4 hours next morning to develop substrate, read plates and analyze data No requirement for a hour day Can actually initiate follow-up experiment same day as data analysis (subsequent) Or 4 assay sets in one week

24 Relative Light Units Thaw/Thaw Assay Execution in Vertical Format with Overnight Stimulation 4 Independent Preparations of Reference Standard Target cells from frozen vial Target cells + mab + responder cells added same day E:T ~ 2:1 Stimulation Overnight Therapeutic (Effector) mab Concentration ng/ml

25 Relative Light Units Test of Initial Ref Std and Assay QC Reference Standard Assay QC Target cells from frozen vial Target cells + mab + responder cells added same day E:T ~ 2:1 Stimulation Overnight Therapeutic (Effector) mab Concentration ng/ml

26 Uniformity Test A H Low mab (~EC 20 ) G F E D C B H G F E D C B A Mid mab (~EC 50 ) High mab (~EC 80 ) H G F E D C B A

27 Further Optimization of Assay 1.Reduce pipette size to minimum volume capacity 1. For 25 µl, use a 50 µl capacity variable single- or multichannel pipette, rather than a 100 µl or 200 µl pipette. 2. Minimizes volume variability/error

28 Uniformity for low concentration (~EC 20 ) H G F E D C B A

29 Uniformity for high concentration (~EC 80 ) G F E D C B A H

30 Relative Light Units Reference Standard and New Assay QC Therapeutic (Effector) mab Concentration ng/ml QC potency = 85.6% RS

31 Relative Light Units Maximize points on the Hillslope (determinants of potency) Therapeutic (Effector) mab Concentration ng/ml QC potency = 86.4% RS

32 Final Optimized Assay; Compromise: Maximum # Points on Hillslope and Well Defined (2 Points) Asymptotes. (two independent dilutions of Reference Standard) Relative Light Units Therapeutic (Effector) mab Concentration ng/ml Alt RS potency = 98% RS

33 Relative Light Units Final Optimized Assay (Reference Standard vs. Assay QC) Therapeutic (Effector) mab Concentration ng/ml QC potency = 92.2% RS

34 Pre-Qualification Results QC Control 50% Sample QC Control % Sample - 2

35 Final Optimized (Sample Range) Assay 50% Sample QC Control 200% Sample

36 Summary Successfully adapted Promega NFAT-Luciferase ADCC surrogate bioassay to the client s mab therapeutic and preferred target 1 cell. Able to adapt the system to use both the responder and target cells as Thaw and Go ( Thaw/Thaw ). Optimizing the assay for uniformity, we were able to adapt the assay to use a vertical map layout, with 8 dilution points. Allows the running of a reference standard, an assay QC and up to 2 test samples per plate Eventual complete assay will use 3 independent plates per sample, with sample run as pseudo-triplicates (every 4 th column) across each plate Eight-point serial dilution was optimized to contain at least 3 points on the hillslope (defining potency) and two points defining each asymptote. Distribution of dose/response points define 4-P Fit extremely well. Assay is expected to be robust, accurate and precise in upcoming qualification.

37 Acknowledgements Tess Bassing Pedro Morales Sarah Driskell

38 Contact Information Michael Sadick, Ph.D. Senior Manager Catalent Pharma Solutions, KCM Development & Clinical Services Large Molecule Analysis and Characterization Biopharmaceutical Characterization Phone: