Submission preparation what to watch out for

Transcription

1 Submission preparation what to watch out for EBF 2017 Boris Gorovits AAPS BIOTEC section Pfizer June 2017

2 Analytes Commonly Assessed for ADC PK Unconjugated Drug analyte Total Antibody analyte Conjugated Antibody analyte Conjugated Drug analyte

3 Unique LBA Challenges Posed by ADC Lower dose - high desired assay sensitivity Accumulation of naked Ab impact on ADC detection? Impact of endogenous binding protein on assay performance Plasma vs serum as preferred matrix for ADC bioanalysis Impact of bioanalytical platform transitions Impact of changes of the regulatory landscape Impact of immunogenicity development As promised 3

4 Impact of Assay Platform Change on ADC Assay MSD platform may yield higher sensitivity and relatively broader dynamic range

5 Total Ab to ADC accumulation and impact Typically half life of total antibody is expected to be longer vs. that for half life of conjugated antibody (ADC). Related questions: Was there an appropriate assessment done of the impact of accumulated unconjugated or low DAR ADC fraction on the performance of the ADC assay(s)? Was evaluation of total Ab vs. ADC PK done for all disease indications during compound development? Anjali Advani et al J Clin Onc VOL 28 N

6 Case Study: Specificity Evaluation for Conjugated Ab Assay Potential Issue: Interference from naked Ab due to target protein as capture reagent Spike recovery (%RE) in presence of naked mab Assay can tolerate < 5 µg/ml naked mab 6

7 Case Study: Impact of Reagents on Total Ab Assay Conventional Cysteine side chain based conjugation Detector Anti-hIgG Fc (commercial) Polyclonal antisera against naked Ab Target Summary Unacceptable accuracy (> 20%) Robustness Issues (Inter-Analyst) Unacceptable precision (>20%) High background Extended dynamic range Improved inter-analyst robustness DAR independence 7

8 Case Study: Endogenous Protein Interference Issue: Endogenous protein interference in Species B Species A ROQ (100% serum): ng/ml Freeze-thaw stability: 5 Cycles Bench-top stability: 19 hrs RT Species B Loss in assay sensitivity (10 ng/ml LLOQ not acceptable in both pooled and individual serum lots) Significant under recovery at low-end of ROQ (LLOQ and LQC) after 1 freezethaw cycle 8

9 Case Study: Endogenous Protein Interference Mitigation Strategies: Simultaneous format Increase LLOQ for Species B Prevent interference by adding antibodies against interfering protein in either neat serum or in assay buffer Solution: Addition of antibodies against interfering protein in assay buffer substantially improved recovery at lower end Increased LLOQ for species B 9

10 Assay performance in Plasma vs Serum Why? LCMS assays preferably conducted using plasma. LBA prefer serum Additional assessment: Relative performance of standards Transition from existing method (e.g. in serum) to another matrix? Vs. proactive evaluation and planning Stability Freeze / Thaw Bench top (RT) Selectivity Remember that there is one serum and many types of plasma!

11 Case Study: Assay Performance in Plasma vs Serum Matrix Assay QC Performance Matrix QC samples %RE LQC -15 Plasma MQC -15 HQC -12 Serum LQC 14 MQC 4.0 HQC 17 Similar Standard curve and QC performance in plasma vs serum matrices 11

12 Case study: Assay Performance in Plasma vs Serum Matrix No selectivity issues observed based on assessment of 10 individual lots of serum and plasma No F/T and BT stability issues observed in plasma

13 Multiple analytes. Example B: Anti-Lewis Y mab ADC Hussain et al Bioanalysis 2014 Non-clinical regulated study PK measurements of: Total Antibody Total Drug (Conjugated + Unconjugated) Conjugated Antibody Unconjugated drug Anti-ADC antibody (immune response) Significant onset of anti-adc antibodies observed impacting PK in many but not all ADA+ animals Unconjugated Drug was essentially not detectable Deconjugation of the drug from the ADC was inferred by a comparative analysis of Total Drug, Total Antibody and Conjugated Antibody analytes Inferred change in DAR over time 13

14 Analytical Platform Transition Potential Platform Change or other Assay Life Cycle considerations: Transitions between LBA and LC/MS or HRMS platforms (Clinical assay sensitivity / selectivity) Within a given assay platform A change in reagent lot or reagent type Method modifications with the goal to improve assay performance or characteristics (e.g. sensitivity or specificity) DAR-sensitive to DAR-insensitive (Validated nonclinical LBA to clinical LBA) Improved reagents in clinical assay At the transition from animal assays to human Due to selectivity issues in clinical assay modify develop apply validate

15 Analytical Platform Transition Recommended transition procedures: - Transitions between LBA and LC/MS: Conduct assays comparison during new method development stage Verify assay performance using incurred study samples based on pre-defined assessment criteria. May include analysis of available non-clinical tox. study samples. May require conduct of additional non-clin. PK study At minimum, a correlation between methods is expected Potential Risk: results may not agree. Evaluation of the cause for disagreement and impact assessment will be required modify develop apply validate

16 Analytical Platform Transition - Method Change for a given analyte. Includes Reagent change Standard method re-qualification or / and re-validation strategy applies Perform formal cross-validation including clinical incurred samples testing Must pass cross-validation pre-defined assessments acceptance criteria modify develop apply validate

17 Critical Assay Life Cycle Changes Type of Change Entity Effected Driver for Change Timing Continuity Strategy Potential Risk LBA to LC/MS LC/MS back to LBA Critical Regents (LBA) Method (LC/MS) Total and Conjugated Antibody Payload (free or conjugated) Selectivity/ Sensitivity Speed and Cost effectiveness Assay performance based, to improved selectivity, specificity, sensitivity Improved sensitivity and throughput (speed & cost) Prior to or after FIP Most typically after FIP Prior or after FIP After FIP Assay performance verified during development. May include use of incurred samples Data obtained using prior and updated methods may not directly match. Evaluation of the cause and impact is expected. Evaluation needs to include incurred sample test evaluation

18 Sample stability changing regulatory landscape Challenge Samples may be stored for an extended period of time during assay investigations or platform to platform evaluation Platform change may require additional stability assessment Stability testing expectations changed during compound development (3 aliquots are requested vs. 1 previously acceptable) Mitigation strategy Appropriate storage stability needs to be established Same Additional evaluation may be needed. May be able to use totality of data argument

19 Unconjugated payload ADC Building an Exposure Response relationship can be challenging but particularly challenging for unconjugated payload analyte due to low / non-existant PK profiles and MMAE scarce measurable concentrations

20 Changes in Regulatory Expectations or Industry Practice Specificity tests 6 or 10 samples analyzed? Specificity test done at LQC / LLOQ / HQC? Stability covers conditions required? F/T number, frozen stability coverage Stability tests how many aliquots per condition? 1 vs. 3 Dilutional linearity test highest concentration tested on par with Cmax? Immunogenicity assay cutpoints statistically defined? Number of individual samples used for cutpoint assessment? (50?)

21 Continuity of knowledge Personnel that worked on earlier assays may have rotated out of the company, institutional knowledge may be limited or lost Need to be able to explain reasons for applying specific type of assays particularly during early support Transition between methods should be justified Impact of observed ADAs / NAbs on various PK may be requested

22 Immunogenicity evaluation Standards applied for immunogenicity assessment may have significantly evolved during compound development Cutpoint assessment Expected ADA / NAB assay format Expected ADA / NAB assay sensitivity Various methods may have been applied to improve assay concordance with regulatory expectations ELISA MSD for ADA Non-cell to cell based NAb

23 Summary Initial ADC reference material is heterogeneous mixtures of various drug-to-antibody ratio (DAR) species ADC heterogeneity continues to evolve in vivo Due to our limited current understanding on which DAR species provides better correlation for exposure-response relationship, it is important that the bioanalytical LBAs are able to accurately quantitate all DAR species found in circulation Evaluate assay sensitivity to various DAR species DAR sensitivity may impact the observed PK profile and calculation of PK parameters such as clearance and drug exposure 23

24 Summary (contd ) There is no single bioanalytical strategy that fits all Select appropriate assay format, assay condition, capture/detection reagents that are minimally impacted by ADC heterogeneity It is possible that none of the formats will deliver best conditions consider using an alternative analyte (e.g. Conjugated Drug) A fit-for-purpose assay used in discovery may not translate to a robust and reliable assay for GLP and clinical support Differences in observed PK profile between discovery (ETS) and GLP studies Evaluate plasma vs serum matrix for regulated assays early on during assay development 24

25 Acknowledgements PDM Regulated BA Nicole Duriga Timothy Taylor Elena Seletskaia Qiang Qu Carina Carter Terri Caiazzo Seema Kumar L-Wyeth colleagues PDM Non-regulated BA Jo-Ann Wentland Tracey Clark Lindsay King Wenlian Wang PDM PR Frank Barletta Mauricio Leal Eugenia Kraynov

26 Thank you!