Robust Cell-Based Assays for Detection of Neutralizing Antibodies to Follow on Biologics like Insulin, GLP1 & Avastin
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1 Robust Cell-Based Assays for Detection of Neutralizing Antibodies to Follow on Biologics like Insulin, GLP1 & Avastin Abhi Saharia, Ph. D. Director of Marketing
2 Bioassays in Biologics Development Research Development Marketing Target ID Lead Discovery & Optimization Preclinical Development Clinical Testing & Development Post Release Marketing Discovery, Screening & Lead Identification Lot Release Testing & Stability Studies Neutralizing Antibody Studies
3 Challenges for NAb Assay Development Challenges Extensive assay development Long, multi-step protocol Complex assay Non-specific assay readout Low assay sensitivity Low drug tolerance Specialized instrument required Negative Impact 3-6 months of time Result in 4-6 days Difficulty with precision & transfer Matrix interference High cost & sensitivity of detection Assay interference & sensitivity High cost
4 Agenda EFC Assay Technology NAb Assays with PathHunter EFC Technology GLP1 NAb Assay Insulin NAb Assay Avastin NAb Assay
5 PathHunter Enzyme Fragment Complementation Technology Objectives of PathHunter NAb assays Create single endpoint assays Target early events for target-specific functional response Develop assay with high matrix tolerance Simplify assay protocol Increase sensitivity of NAb assays Increase assay reproducibility Eliminate cell culture as part of the assay
6 Flexibility of the EFC Technology Monitor protein levels degradation or expression Monitor protein translocation nucleus, cytoplasm, early endosome, golgi, membrane
7 Agenda EFC Assay Technology NAb Assays with PathHunter EFC Technology GLP1 NAb Assay Insulin NAb Assay Avastin NAb Assay
8 GLP1 Receptor Agonists : Drugs & Mechanism of Action Exenatide (Byetta/Bydureon), 2005/2012 Lixisenatide (Lyxumia), EU 2013 Liraglutide (Victoza, Saxenda), 2010 Albiglutide (Tanzeum), 2014 Exendin-4 GLP1 (7-37) Existing GLP1R Assays camp ELISA Radio-immuno assay Calcium flux Insulin release assay
9 GLP1 & Exendin-4 Bioassays 30 min
10 GLP1R Assay Specificity
11 GLP1R Assay Reproducibility 3-7% CV for PathHunter Assays Plate to plate (Intra-lot) reproducibility Day to day reproducibility Operator reproducibility High inter-lot reproducibility GLP1R Day to day and Operator Reproducibility Day 1 Operator 1 Day 2 Operator 2 Day 3 Operator 3 Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Avg %CV EC E E E E E-09 Avg %CV EC E E E E E-09 Avg %CV EC E E E E E-09 Average 2.37nM %RSD 5.53%
12 Agenda EFC Assay Technology NAb Assays with PathHunter EFC Technology GLP1 NAb Assay Insulin NAb Assay Avastin NAb Assay
13 Insulin : Drugs & Mechanism of Action Several insulin drugs on market Rapid Acting Short Acting Intermediate Long Acting Pre-mixed (intermediate & short) Existing assays Glucose uptake Radio Immunoassay Receptor phosphorylation Reporter gene
14 Insulin Bioassay 3 hrs
15 INSR Bioassay Matrix Tolerance Condition EC50 (ng/ml) S:B Ratio 10% NHS % NHS % NHS % NHS
16 INSR NAb Assay Sensitivity Assay conditions 10% human serum Cell seeding = 24 hrs Incubation time = 3 hrs Approximate sensitivity of INSR assay is ng/mL Commercially Developed anti-insulin antibody
17 Agenda EFC Assay Technology NAb Assays with PathHunter EFC Technology GLP1 NAb Assay Insulin NAb Assay Avastin NAb Assay
18 Anti-VEGF Antibody : Drugs & MOA Existing assay HUVEC proliferation assay
19 Bevacizumab (Avastin ) Bioassay
20 R L U R L U Robust & Reproducible Bevacizumab Assay V E G F R 2 D im e r iz a tio n B io a s s a y (H E K ) V E G F R 2 D im e r iz a tio n B io a s s a y (H E K ) P la te A v a s tin P la te 2 P la te V E G F V E G F [g /m L ] A v a s tin [g /m L ] n g /m l V E G F HillSlope EC50 Plate e-009 S /B 4.8 Plate e-009 Plate e HillSlope EC50 Avastin e-008 S /B VEGF e-009
21 R L U Assay Specificity & Matrix Tolerance Assay Specificity Matrix Tolerance K D R / K D R (H E K ) V E G F V E G F V E G F B V E G F C Condition EC50 (ng/ml) S:B Ratio 10% NHS % NHS % NHS L ig a n d [g /m L ] 74% NHS % NHS
22 Anti-Bevacizumab Neutralizing Antibody EC50 of Anti-Avastin Antibody = 470 ng/ml Approximate sensitivity of assay = ng/ml
23 RLU Anti-Bevacizumab Neutralizing Antibody Assay VEGF only 0 Anti-Avastin (ng/ml) 0 A B 0 33 F 100 E 300 D 900 C VEGF Series1 (EC80) Avastin (ND50) KDR/KDR BioAssay VEGF+ Avastin VEGF + Avastin + anti-avastin Samples prepared in 10% pooled normal human serum
24 Summary Objectives for PathHunter NAb assays Create single endpoint assays Target early events for target-specific functional response Develop assay with high matrix tolerance Simplify assay protocol Increase sensitivity of NAb assays Increase assay reproducibility Eliminate cell culture
25 R L U R L U R L U RLU Thaw & Use Bioassay Cells Live Culture Assay Frozen Thaw & Use Assay IL 5 R A / C S F 2 R B A s s a y IL5RA / CSF2RB Assay S/B: 4.6 E C -5 0 : p M S/B: 4.9 EC-50: 342pM Advantages of single use cells Avoid cell culture & associated variability Controlled propagation & harvest Controlled passage number Consistent performance on thaw Requires controlled two-tier banking Save time and money Get more reproducible results IL -5 [g /m L ] Er yth ropoiet i n A ssay S /B : E C -5 0 : p M E ry th ro p o ie tin [M ] E r y th r o p o ie tin A s s a y S /B : 6.1 E C -5 0 : p M IL-5 [M] E ry th ro p o ie tin [M ]
26 Cell Bank Characterization Three-tiered cell banking Controlled cell culture Controlled harvest Cell viability post thaw and pre-thaw Freezing process Sterility and mycoplasma Intra-lot reproducibility
27 Application of EFC Bioassays Biosimilar Bioassay Kit Cell Line Insulin Lispro Bioassay Kit Developed Developed Insulin Glargine Bioassay Kit Developed Developed Insulin Bioassay Kit Developed Developed Exendin-4 Bioassay Kit Developed Developed GLP1 Bioassay Kit Developed Developed GLP1 (7-37) Bioassay Kit Developed Developed Epoetin Alfa Bioassay Kit Developed Developed Darbepoetin Alfa Bioassay Kit Developed Developed Ranibizumab Bioassay Kit Developed Developed Aflibercept Bioasay Kit Developed Developed Bevacizumab Bioassay Kit Developed Developed Ustekinumab Bioassay Kit * Developed Growth Hormone Bioassay Kit * Developed G-CSF Bioassay Kit * Developed Glucagon Bioassay Kit * Developed Denosumab Bioassay Kit * Developed FSH Bioassay Kit * Developed PTH Bioassay Kit * Developed LH/hCG Bioassay Kit * Developed Infliximab Bioassay Kit * Developed Golimumab Bioassay Kit * Developed Etanercept Bioassay Kit * Developed Certolizumab pegol Bioassay Kit * Developed Adalimumab Bioassay Kit * Developed GHRH Bioassay Kit * Developed Anakinra Bioassay Kit * Developed Canakinumab Bioassay Kit * Developed Additional assays under development Tocilizumab Panitumumab Cetuximab 750+ bioassays for novel targets GPCRs Receptor Kinases TGFβ Receptor Superfamily Interleukin & Cytokine Receptors * Thaw and use bioassay cells are under development
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