Bioassays without cell culture? Simple, cell-based potency & NAb bioassays for biosimilars. Abhi Saharia, Ph. D. Director of Marketing, Biologics

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1 Bioassays without cell culture? Simple, cell-based potency & NAb bioassays for biosimilars Abhi Saharia, Ph. D. Director of Marketing, Biologics

2 Cell-based potency assays for Biosimilars Biosimilar Bioassay Kit Cell Line Insulin Lispro Developed Developed Insulin Glargine Developed Developed Insulin Developed Developed Exendin-4 Developed Developed GLP1 (7-37) Developed Developed Epoetin Alfa Developed Developed Darbepoetin Alfa Developed Developed Ranibizumab Developed Developed Aflibercept Developed Developed Bevacizumab Developed Developed Ustekinumab Developed Developed Growth Hormone Developed Developed G-CSF Developed Developed Biosimilar Bioassay Kit Cell Line Glucagon * Developed Denosumab * Developed FSH * Developed PTH * Developed LH/hCG * Developed Infliximab * Developed Golimumab * Developed Etanercept * Developed Certolizumab pegol * Developed Adalimumab * Developed GHRH * Developed Anakinra * Developed Canakinumab * Developed Tocilizumab * Developed Additional assays under development Panitumumab Cetuximab 750+ bioassays for novel targets GPCRs Receptor Kinases TGFβ Receptor Superfamily Interleukin & Cytokine Receptors * Thaw and use bioassay cells are under development

3 Bioassays For Development of Biosimilars INNOVATOR DRUG BIOSIMILAR DRUG Research Development Marketing Target ID Lead Discovery & Optimization Preclinical Development Clinical Trials (I & III) Post Release Marketing Discovery, Screening & Lead ID Lot Release Testing & Stability Studies Benchmarking & Combo Studies Neutralizing Antibody Studies

4 Challenges for 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

5 Agenda PathHunter EFC Assay Technology Case Study Potency bioassay for Bevacizumab Comparison to HUVEC proliferation assay Neutralizing antibody bioassay for Bevacizumab Use of cryopreserved ready-to-assay cells Summary Process Quality control of cell banks Advantages

6 Enzyme Fragment Complementation (EFC)

7 Flexibility of the EFC Technology Monitor protein levels degradation or expression Monitor protein translocation nucleus, cytoplasm, early endosome, golgi, membrane

8 Agenda PathHunter EFC Assay Technology Case Study Potency bioassay for Bevacizumab Comparison to HUVEC proliferation assay Neutralizing antibody bioassay for Bevacizumab Use of cryopreserved ready-to-assay cells Summary Process Quality control of cell banks Advantages

9 Anti-VEGF Antibody : Drugs & MOA Existing assay HUVEC proliferation assay

10 Bevacizumab (Avastin ) Bioassay

11 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

12 R L U S/B Ratio Stability of Assay Signal T im e C o u r s e o f D e te c tio n R e a g e n t h r re a d 2 h r re a d 3 h r re a d h r re a d 5 h r re a d 2 4 hr re a d V E G F [g /m l] hr 2hr 3hr 4hr 5hr 24hr Read Time Time EC50 (ng/ml) S:B Ratio 1 hr hrs hrs hrs hrs hrs

13 M e a s u r e d R e la tiv e P o te n c y, % Potency, Linearity, Accuracy and Precision A. Potency Expected Potency Measured Potency Day Mean Potency SD Recovery RSD,% Day 2 150% Day Day Day 2 125% Day Day Day 2 75% Day Day Day 2 50% 45.9 Day Accuracy= 95.9% Precision= 4.1% B. Linearity V E G F R 2 D im e riz a tio n B io A s s a y y = x r 2 = E x p e c te d R e la tiv e P o te n c y, %

14 Comparison to HUVEC Proliferation Assay HUVEC Proliferation Assay PathHunter Bevacizumab Bioassay EC 50 VEGF ng/ml 5 ng/ml EC 50 Bevacizumab ng/ml ng/ml S:B Ratio 2.5 fold >4.3 fold Assay run time 96 hours 16 hours Specificity Low High Cell type Primary cells with donor variability Clonal, frozen ready-to-assay cells Cell Culture? Required No cell culture necessary

15 Immunogenicity Screening Strategy

16 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

17 Anti-Bevacizumab Neutralizing Antibody EC50 of Anti-Avastin Antibody = 470 ng/ml Approximate sensitivity of assay = ng/ml

18 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

19 Agenda PathHunter EFC Assay Technology Case Study Potency bioassay for Bevacizumab Comparison to HUVEC proliferation assay Neutralizing antibody bioassay for Bevacizumab Use of cryopreserved ready-to-assay cells Summary Process Quality control of cell banks Advantages

20 What are Cryopreserved Ready-to-assay cells? Remove the frozen cells from LN 2 Do not thaw cells in water bath Place on dry ice Thaw in hood with media Plate cells directly No cell culture necessary No need to count cells You are ready to run the assay

21 R L U R L U R L U RLU Advantages of Cryopreserved Ready-to-assay 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 ]

22 Long Term Support with a 2-Tiered Cell Bank Master Cell Bank (MCB) Tested with certificate of analysis (CofA) Kept aside for client/customer Tested annually to ensure long term stability (>5 yrs) Working Cell Bank (WCB) Custom generated with vials from the MCB Tested with CofA Kept on site and shipped on demand Lots produced for vials

23 Cell Bank Characterization Two-tiered cell banking Controlled cell culture Controlled harvest Cell viability post thaw and pre-thaw Freezing process Sterility and mycoplasma Intra-lot & inter-lot reproducibility

24 Bioassay WCB Certificate of Analysis Pass Criteria for Internal QC Custom Lot >90% >90% >100% Negative Negative <=30 %CV <=30 %CV

25 Bioassay Kits Certificate of Analysis Reagents Part # Lot # Contents Quantity Expiration Date PathHunter XXX Cells 10 Vials PathHunter BioAssay Detection Kit Kit Detection Reagent Bottle Detection Reagent Bottle Ligand XXX Ligand XXX Reconstitution Buffer Cell Plating Reagent XX AbHunter Antibody Dilution Buffer AbHunter Antibody Dilution Buffer 1 Kit 1 Vial 1 Vial 2 Bottles M 1 Set Bottles 96-well Tissue Culture Plates Plates

26 R L U R L U Highly Reproducible Assay 3-7% CV for PathHunter Assays Plate to plate (Intra-lot) reproducibility Day to day reproducibility Operator reproducibility High inter-lot reproducibility Bottom Top LogEC50 HillSlope EC G L P 1 R - In tr a -L o t R e p r o d u c ib ility Plate e-009 E x e n d in -4 [M ] Plate e-009 Plate e-009 Plate e-009 Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate e-009 R elative P otency % 94.5 % 93.3 % % % G L P 1 R - In te r -L o t R e p r o d u c ib ility L o t 1 GLP1R Day to day and Operator Reproducibility L o t 2 L o t 3 Plate 1 Plate 2 Plate 3 Plate 4 Plate Day 1 Operator 1 Avg %CV S:B EC E E E E E Bottom Top LogEC50 HillSlope EC50 E x e n d in -4 [M ] Lot e-009 Lot e-009 Lot e-009 S :B Day 2 Operator 2 Day 3 Operator 3 Avg %CV S:B EC E E E E E-09 Avg %CV S:B EC E E E E E-09

27 R L U >6 Years of Stability & Inter-lot reproducibility G L P 1 R A s s a y S ta b ility G L P 1 (7-3 6 ) [M ] Year EC 50 (nm) S/B Slope

28 Agenda PathHunter EFC Assay Technology Case Study Potency bioassay for Bevacizumab Comparison to HUVEC proliferation assay Neutralizing antibody bioassay for Bevacizumab Use of cryopreserved ready-to-assay cells Summary Process Quality control of cell banks Advantages

29 Cell-based potency assays for Biosimilars Biosimilar Bioassay Kit Cell Line Repeatability (%CV) Accuracy (%) Precision (%RSD) Linearity (R 2 ) Ranibizumab Developed Developed Aflibercept Developed Developed Bevacizumab Developed Developed Epoetin Alfa Developed Developed Darbepoetin Alfa Developed Developed Exendin-4 Developed Developed GLP1 (7-37) Developed Developed Growth Hormone Developed Developed G-CSF Developed Developed Insulin Lispro Developed Developed n.d Insulin Glargine Developed Developed n.d Insulin Developed Developed n.d Ustekinumab Developed Developed In process Biosimilar Bioassay Kit Cell Line Glucagon * Developed Denosumab * Developed FSH * Developed PTH * Developed LH/hCG * Developed Infliximab * Developed Golimumab * Developed Etanercept * Developed Certolizumab pegol * Developed Adalimumab * Developed GHRH * Developed Anakinra * Developed Canakinumab * Developed Tocilizumab * Developed Additional assays under development Panitumumab Cetuximab Rituximab Trastuzumab 750+ bioassays for novel targets GPCRs Receptor Kinases TGFβ Receptor Superfamily Interleukin & Cytokine Receptors * Thaw and use bioassay cells are under development