Current Best Practices in Commercial Kit Evaluation and Validation for Biomarker Assays 10th Workshop on Recent Issues in Bioanalysis, 2016 Paul Rhyne, Ph.D. - Director of Immunoassay Services Copyright 2016 Q 2 Solutions. All rights reserved.
Agenda Introduction Commercial kits Validation of kits Case studies Questions and Wrap Up 2
The need for biomarkers Surrogate markers Improved Drug Safety Quantitative measures of biological effects Improved Drug Efficacy 3
Biomarker Assay Considerations Suitable for Intended use Technology Platforms Analytical Validation Interpretable data 4
Biomarker Assays De Novo Commercial kits Clinical Published Commercial tools 5
Agenda Introduction Commercial kits Validation of kits Case studies Questions and Wrap Up 6
Commercial Kit Types Commercial kits RUO Only In Vitro Diagnostic CLIA CE Marked 7
InVitro Diagnostic Assays 3 Categories: Class I, II, and III Multiple FDA approval processes Premarketing Approval 510(k) Must conform to strict quality system regulations (21 CFR 522) Designed and approved for a specific use Closed systems that are not typically open to modification May not provide sensitivity and range of detection beyond approved use 8
Clinical Laboratory Improvement Amendments (CLIA) Established by congress in 1988 Defines quality standards for all non-research laboratory testing on human specimens for providing information for diagnostics, prevention, treatment of disease or impairment of or assessment of health 3 Categories based on complexity: waived, moderate, and high FDA is responsible for categorization of assays CLIA is designed to provide accuracy, reliability and timeliness of patient results regardless of where the test is performed May not guarantee that the test has performance characteristics beyond the approved use 9
Conformité Européenne European Conformity Manditory conformity marking for products placed in the European Economic Area (EEA) CE-Mark signifies that product conforms to the essential requirements of the European Community Legislation The CE-marking is a self certification process by the manufacturer. Certain categories require a more stringent review by 3rd party to obtain a CE-mark CE-marked labeling does not imply or guarantee that the assay will be suitable for measurement of clinical biomarkers 10
Research Use Only assays RUO Exempt from approval by regulatory agencies RUO serves as a warning that the product is not approved for IVD, clinical diagnostic, or patient management use No requirements or guidelines for RUO labels Manufacturing quality, assay characterization, and documentation is up to each manufacturer RUO kits present many challenges for conducting clinical biomarker measurements 11
Commercial Kit Vendors Commercial kits Distributor or manufacturer Flexible supply Technical support & experience Assay characterization Documentation 12
Agenda Introduction Commercial kits Validation of kits Case studies Questions and Wrap Up 13
Biomarker work plan Define Project Needs Intended use of data Engage stakeholders Documentation Assay performance Requirements Validation plan Sample Analysis Sample type Numbers Stability 14
Preclinical Drug Development Phase Early Clinical Late Clinical Method Validation Planning POB Surrogate Safety POC MOA POM Safety Target Proximal FIH doses MOA Safety Exploratory Validation Advanced Validation 15
Tasks Exploratory Method Validation Advanced Method Validation Performance Parameters Calibrators Selectivity May need to use additional calibrators & anchor points 3 analytical runs Perform if not done in feasibility assessment Spike recovery of 10 lots each of matrix from normal and patient populations Dilution Linearity Confirm Confirm Parallelism Confirm Confirm Accuracy and Precision VS or QC samples Typically surrogate matrix Surrogate or biological matrix VS/QC at 5 levels VS/QC at 5 levels MC 2 pools (High and Low) 2 or more pools Accuracy and precision 3 analytical runs, including MC 6 analytical runs including MC Stability Reagent stability Optional Confirm according to validation protocol QC storage stability As needed (usually prepared fresh) As needed (usually prepared fresh) MC storage stability Confirm with MC Confirm with MC Method robustness Lot-to-lot variability As needed Test with MC on 3 lots if available Analyst, instrument, & site variations As needed Multiple analysts, instruments, sites Documentation Validation Plan/Validation report Recommended Required QA audited documentation Intended use dependent Required Bioanalysis (2015) 7(2), 229-242 16
Method Adaptation Additional calibrators Curve fitting Matrix controls Reagent substitution Bulk quantities 17
How to select a kit Calibration Range Selectivity Sensitivity Linearity Validation 18
Common Kit Issues LOQ vs LOD Lot to lot consistency Expiration dates Calibrator matrix Analyte stability 19
Agenda Introduction Commercial kits Validation of kits Case studies Questions and Wrap Up 20
Case Study: Making QCs Measure Human Thymidine Kinase 1 in human serum Commercial kit available Human TK1 standards Human Serum based QCs Standard curve works well Kit QCs fall into the lower and middle sections of curve Needed to make an HQC 21
Commercial kit to measure Thymidine Kinase 1 Activity Kit Top Std (4232 ng TK/L) serially diluted 2X with 100% Low endogenous serum down to 66.1 ng TK/L 1:10 MRD into Reaction Buffer 22
Case Study: Making QCs Measure Human Thymidine Kinase 1 in human serum Manufacturer could not provide bulk standards to make HQC Found commercial recombinant TK1 from different vendor Recombinant standard curve very different from kit curve 23
Commercial kit to measure Thymidine Kinase 1 Activity Two different curves Blue: Kit curve Purple: Novus standard 24
Case Study: QCs Few commercial kit manufacturers provide QCs in matrix Material to generate standard curves varies from manufacturer to manufacturer Recombinant standards may not reflect purified forms Spiking matrix to make QCs is not trivial 25
Case Study: Lot to Lot consistency Changes in kit lots over the course of a study Manufacturer may not standardize lot to lot consistency Reagent changes across lots vary Clinical trials may be longer than kit expiration dates 26
Monitoring matrix controls Wang et al., AAPS J. (2009) 11(2), 385-394 Low 0.22 0.20 0.18 0.16 0.14 0.12 0.10 Changed reference lot 0.08 0.80 106 212 318 424 530 636 742 848 954 1060 1166 1272 1378 1484 Sample 0.70 high 0.60 0.50 106 212 318 424 530 636 742 848 954 1060 1166 1272 1378 1484 Sample Each color represents a separate kit lot Wang et al., AAPS J. (2009) 11(2), 385-394 27 27
Changes in kit lots Wu et al., J. Pharm. Biomed. Anal. (2009) 49, 1203-1212 Minimized variations by preparing standards and QC samples from one consistent source of high concentration reference material; included sample controls throughout. 28
Changes in kit lots for multiplexed assays Multiple lot changes alter standard curves 90 80 70 60 50 40 30 20 10 0-10 -20-30 -40-50 -60-70 -80-90 11 pg/ml 22 pg/ml 49 pg/ml 121 pg/ml 277 pg/ml 655 pg/ml 1563 pg/ml Tau Nominal Concentrations Source: www.tecan.com/platform/content/element/9550/2-1rhyne.pdf 29
Case Study: Lot to lot changes Lot-to-lot variability is common with commercially available kits, as vendors change lots of critical reagents as part of normal production cycle Recommended that every effort is made to use a single lot across clinical trials, however, this can be challenging over large clinical trials Longitudinal consistency, evaluated using large lots of endogenous controls, is essential Trend analysis that shows a bias in the results may require a correction factor to compensate for differences, to avoid a negative impact on the interpretation of data 30
Reagent changes Biomarker ELISA In house developed ELISA used to measure a biomarker in response to drug treatment Detection antibody is directly conjugated to HRP Bridging over new lot of antibody during trial 31
Mean OD ELISA standard curve in matrix 2.5 current detection lot, spiked matrix 2 1.5 current detection lot, blank matrix 1 0.5 0 100 10 1 0.1 0.01 Detection Antibody Concentration [ ug/ml] 32
Mean OD ELISA standard curve Comparison of new HRP-detection antibody vs current lot 2.5 2 1.5 current detection lot, spiked matrix new detection lot, spiked matrix current detection lot, blank matrix new detection lot, blank matrix 1 0.5 0 100 10 1 0.1 0.01 Detection Antibody Concentration [ ug/ml] New lot of antibody performs poorly 33
OD ELISA: New custom lot of HRP detection antibody Newly labeled lot performs better than previous lot 4 3.5 3 2.5 2 1.5 New HRP detection lot Current detection lot New lot background Current lot background 1 0.5 0 100 10 1 0.1 0.01 Detection Ab Concentration [ug/ml] New custom lot has better performance Be careful what you ask for! 34
Impact of reagent selection Example: Bovine Serum Albumin (MSD Assay) Vendor 1 Vendor 2 Vendor 3 Vendor 4 Lesson: Qualify NEW lots before use 35 35
Case Study: Reagent changes Kit and Assay reagents may need to be changed Don t take reagents for granted Reagent qualification is key Pre-qualifying new reagents is critical 36
Biomarker Assays: Adapting assays for clinical use 37
Case study: Interleukin 23 Assay Disulfide-linked heterodimeric protein 2 subunits p40 (shared with IL-12) p19 (the IL-23 alpha subunit) Regulates late state inflammation and autoimmune responses IL-23 a good therapeutic target (Stelera) Goal: To develop, optimize, and validate an ELISA assay for the quantitative determination of Interleukin-23 present in the human plasma. 38 38
Commercial kit for IL-23 Insufficient sensitivity for clinical use ebioscience ELISA Biosource ELISA O.D. 1 Clinical levels of IL-23 0.1 10 0 10 1 10 2 10 3 10 4 10 5 Concentration (pg/ml) Bioanalysis 2(9):1561-1572, 2010 39
Developed in house assay for IL-23 ebioscience ELISA In-House Luminex Biosource ELISA 10 4 10 3 O.D. 1 MFI 10 2 10 1 0.1 10-1 10 0 10 1 10 2 10 3 10 4 10 5 Concentration (pg/ml) Bioanalysis 2(9):1561-1572, 2010 40
Analysis of plasma samples Comparison of unstimulated, LPS or PHA stimulated, or plasma from RA patients Luminex assay had needed sensitivity for clinical samples 41
Case Study: Adapting assays for clinical use Define needs of assay in regards to clinical performance Evaluate kit performance to ensure assay is suitable If a new assay is needed, define the requirements and build to suit 42
Agenda Introduction Commercial kits Validation of kits Case studies Questions and Wrap Up 43
Summary Evaluation of Commercial kits for Biomarker assays Industry needs Biomarker Assays Many sources of Biomarker Assays De Novo IVD Commercial reagents Commercial kits Biomarker plan: define assay requirements and develop a validation plan Evaluate the biomarker assay and consider Manufacturer experience, availability, support Reagent supply and lot to lot consistency Pre-qualify changes in lots and reagents Monitor performance and be prepared! 44
Khan et.al Bioanalysis (2015) 7(2), 229-242 45
Webinar: Adaptation and validation of commercial kits for biomarker quantification in drug development: Recommendations and case studies. AAPS 2015 LBABFG - Biomarker Discussion Group 46
Thank you Contact us Toll free: 1 855.277.9929 Direct: +1 919.998.7000 International: +44 (0) 1506 814000 Website: www.q2labsolutions.com 47
About the Presenter Dr. Paul Rhyne Scientific Director - Immunoassay Services Dr. Rhyne, currently leads a bioanalytical group for Q 2 Solutions, a Quintiles Quest joint venture. Dr. Rhyne has more than 15 years of scientific leadership experience in laboratory operations, clinical biomarkers, large molecule PK analysis, and Immunogenicity assessment. He obtained his Ph.D. at the University of Tennessee Memphis and completed 3 years of post-doctoral studies at St. Jude Children s Research Hospital. Dr. Rhyne was an Associate Director at Bristol-Myers Squibb Company, a Senior Director at Tandem laboratories, and has held many senior leadership positions at Upstate Biotechnology and Amplistar Inc. A noted industry expert in clinical biomarkers and large molecule bioanalytical analysis, and has published many scientific publications in peer reviewed journals. Dr. Rhyne is an active member of the American Association of Pharmaceutical Scientists including serving on programming committees, steering committee for translational biomarker focus group, and chair of the biomarker discussion group. 48