PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies

1. PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies

2. Types of ELISA • Noncompetitive binding assay or Sandwich method • Antigen measuring system [Plate wells coated with antibodies ; Enzyme labelled antibodies] • Antibody measuring system [Plate wells coated with antigens ; Enzyme labelled anti-antibodies] • Competitive binding assay [Plate wells coated with antibodies ; Enzyme labelled antigens]

3. Antibody measuring system Plate wells coated with suitable antigen Add sample containing the antibody Incubate: till antigen antibody reaction is complete Wash remove unbound antibody Add Anti-antibodylabelled with Enzyme Incubate till labelled anti-antibodiesbinds antigen-antibody complex Wash  remove unbound labelled anti-antibody Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour proportional to antibody in patient sample Noncompetitive or Sandwich Assay

4. Antigen measuring system Plate wells coated with suitable antibody Add sample containing the antigen Incubate: till antigen antibody reaction is complete Wash remove unbound antigen Add Antibody labelled with Enzyme Incubate till antigen binds labelled antibody Wash  remove unbound labelled antibody Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour proportional to antigen in patient sample Non-competitive or Sandwich Assay

5. Plate wells coated with antibodies Known quantities of patient sample containing antigen + antigen labelled with enzyme Incubate: till antigen antibody reaction is complete Wash remove unbound antigens Add substrate ; incubate Enzyme + Substrate  Product  measure colour Colour inversely related to antigen in patient sample Competitive Binding assay

6. Tox Study Experimental Design Study title: A 4-Week Intravenous Infusion and Subcutaneous Injection Toxicity Study with ABC-001 in Cynomolgus Monkeys Followed by an 8-Week Recovery Objective: ABC-001 is a humanized IgG1 monoclonal antibody which binds human and cynomolgus monkey 001 cytokines. The objectives of this study are to determine the potential toxicity of ABC-001 when given by either 15-minute intravenous infusion or subcutaneous injection once weekly for 4 consecutive weeks to cynomolgus monkeys, to evaluate the potential reversibility of any adverse findings following an 8-week dose-free recovery period, and to provide additional nonclinical safety data to support the use of ABC-001 in humans. In addition, the toxicokinetic (TK) and immunogenic characteristics of ABC-001 will be determined.

7. Tox Study Experimental Design

8. Tox Study Experimental Design Toxicokinetic (TK) and Monkey Anti-Human Antibody (MAHA) Sample Collection Schedule

9. Read plate @ 570 nm Read plate @ 570 nm Chemiluminescent Chemiluminescent Substrate Substrate Monkey Monkey - Absorbed Sheep Absorbed Sheep Y - Anti Anti - - human IgG (H+L) pAb human IgG (H+L) pAb HRP Y ABC - -001 Dry coated Dry coated plate plate Capture protein Toxicokinetic (TK) Assessment Chemiluminescence-based Immunoassay Using Covalent Binding Plate to Quantify ABC-001 in Monkey Serum

10. Assay Materials and Procedure Equipment • SpectraMax® L Luminescence Microplate Reader • Software: SoftMax Pro Gxp (Version 5.4.4) • Micro-plate Incubator/Shaker, Awareness Technology, Inc. • BioTek Elx405 96-well plate washer (System No. 262) • Precision Pipettes to deliver 10, 100, and 1000 µL • Vortex mixer • Adhesive sealing films for microplates, Excel Scientific, Inc. • Absorbent materials for blotting the strips

11. Assay Materials and Procedure Biological Matrix • Blank monkey serum was purchased from Bioreclamation Inc. The serum lots used in the assay were Lot Nos. CYN105050 and CYN111113. • The pooled serum was used to prepare the standards and QC samples, and for sample dilution. The serum was stored at -70 ºC.

12. Reagents and Materials  • Reference Standard:ABC-001 • Immobilizer amino F96 Black Plate: NUNC, Catalog # 436008 • StabilGuard from SurModics, Catalog # SG01-1000 • Human 001 Protein Carrier-Free, eBioscience, Catalog # 34-8239-85 • Sheep anti-human IgG (H+L) Monkey–Adsorbed–Peroxidase (1:50 dilution in StabilZyme HRP Conjugate Stabilizer), The Binding Site Group Ltd. • Pooled monkey serum, Bioreclamation Inc. • SuperSignal West Pico Chemiluminescent Substrate kit, Thermo Scientific, Catalog # 34080 • 10X DPBS, Mediatech, Inc, Catalog # 20-031-CV • 1X DPBS, Mediatech, Inc, Catalog # 20-031-CM • Tween 20, Sigma, Catalog # P1379 • Sodium Chloride, Sigma, Catalog # P9416 • ProClin300, Supelco, Catalog # 48912-U • SuperBlock, ScyTek, Catalog # AAA999 • Purified H2O

13. Plate Preparation • Plates can be coated and used freshly or stored at 2-8°C in airtight bag with desiccant. Prepare the plate as following: • Dilute Protein-001 Carrier- Free stock in 1X DPBS; make the final concentration 0.5 µg/mL. • Label Nunc immobilizer 96 well plates. • Add 100 µL per well of the diluted protein solution. • Seal the plates and incubate at room temperature for 2-5 hours. • Place plates in a refrigerator (2-8°C) and incubate overnight. • Wash the plate(s) 3 times using a microplate washer. • Add 200 µL per well of StabilGuard to all wells by reverse pipetting using a multichannel pipette and incubate at RT with shaking for 60±10 min. • Dump StabilGuard and blot dry. Coated plates are ready for use. • For plates to be used later days, aspirate plate and dry at room temperature over night. Store dried plate at 2-8°C in airtight bag with desiccant with an expiration date of three month.

14. Standard and QC Working Standard Concentrations QC/Validation Sample Concentrations

15. Assay Performance- Standard Curves Back Calculated Standard Concentrations and Curve Parameters for ABC-001 Assay in Monkey Serum (5-Parameter Logistic Fit with 1/Y Weighting)

16. Precision and Accuracy Overview of Precision and Accuracy for QC Samples

17. Assay Performance- Selectivity Selectivity Evaluation

18. Matrix Interference Matrix Interference for ABC-001 Assay in Monkey Serum

19. Dilution Integrity Dilution Linearity of ABC-001 in Monkey Serum

20. Assay Performance - Robustness Tested at the lower limit at each incubation step

21. Robustness Tested at the upper limit at each incubation step

22. Coated Plate Stability Stability of Coated Plates at 5°C ± 3°C After 27 Days

23. Freeze -Thaw Stability (4 cycles)

24. Bench Top Stability of 20 Hours

25. Long-term Storage Stability

26. Method transfer (P/A Summary)

27. Standard and QC performance of TK Sample Analysis

28. Representative TK Graphs Group 5 SC 100 mg/kg Group 7 IV 30 mg/kg

29. ISR evaluation • An ISR evaluation was performed on the 112 serum samples listed in the ISR Study Plan. • 107 of 112 samples (95.5%) evaluated met the acceptance criteria : at least 2/3 of all the analyzed ISR samples had no more than a ± 30% difference when compared to the original analysis results. General industrial guideline: For studies up to 1000 samples reanalyze 10% of samples and for studies with over 1000 samples reanalyze 10% of the first 1000 samples and 5% of any remaining samples.

30. Immunogenicity Assessment • Basic package: • Anti-drug binding antibody assays (ADA) • Screening • Confirmation • Titration • Neutralizing antibodies (Nab) • Basic package +/- based on: • Risk assessment • Results from pre-clinical and early clinical studies • Regulatory input

31. ADA Assay Platforms • Platforms • ELISA • Bridging • Direct • Indirect • RIA • Biacore • Electrochemiluminescence (ECL) • No “perfect” assay currently exists • In general, assay format for ADA • Protein therapeutics: direct format • mAbs: bridging format

32. Popular ADA assay formats for Mab Drugs ECL / MSD Homogeneous Bridging ELISA

33. ADA Assay Key Parameters for validation • Screening cut point • Specificity/confirmation cut point • Sensitivity • System suitability controls(QCs) acceptance criteria • Selectivity/Interference • Matrix components • Drug tolerance • Precision • Robustness • Stability • Ruggedness

34. ECL vs. H-ELISA H-ELISA Advantages • Antibody-drug interactions take place in solution–High capacity surface contributes to high drug tolerance • Broad dynamic range and good sensitivity • Instrumentation and consumables are available from multiple sources • Limitations-More complicated detection with an additional wash step ECL Advantages • Antibody-Drug interactions take place in solution • High capacity surface contributes to high drug tolerance • Broad dynamic range and good sensitivity •Limitations–Single vendor technology

35. Anti-ABC01 Antibody Assay (Homogeneous Bridging Elisa format)

36. Key Reagents and Materials • PC: Rabbit anti-ABC-001 pAb • Drug: ABC-001 • Biotinylated ABC-001 . • Digoxigenin-conjugated ABC-001 • Anti-Digoxigenin-POD (anti-Dig POD), Fab Fragments • Immobolizer Clear Streptavidin coated plates: Nunc, Cat # 436014. • TMB: KPL • 1N Sulfuric Acid

37. ADA Assay Summary

38. Drug Tolerance Using H-ELISA

39. ADA sample Analysis

40. ADA sample Analysis (QC Performance)

41. Summary and Conclusions •The H-ELISA format is generic and can be easily applied to other immunogenicity assays •Performance characteristics of the H-ELISA meets industry requirement for ADA assays •Frontage remains proactive in exploring new technologies and searching for alternative solutions to analytical problems

42. Acknowledgement Study Sponsor:

43. Thank You Shawn Li, M.D., Ph.D. Director, Biologics Services Frontage Lab. Inc. Shawnli@frontagelab.com Exton, PA Headquarters