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

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Presentation on theme: "PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies."— Presentation transcript:

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

2 Types of ELISA 1.Noncompetitive binding assay or Sandwich method 1)Antigen measuring system [Plate wells coated with antibodies ; Enzyme labelled antibodies] 2)Antibody measuring system [Plate wells coated with antigens ; Enzyme labelled anti- antibodies] 2.Competitive binding assay [Plate wells coated with antibodies ; Enzyme labelled antigens]

3 Noncompetitive or Sandwich Assay 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-antibody labelled with Enzyme – Incubate till labelled anti-antibodies binds antigen- antibody complex – Wash  remove unbound labelled anti-antibody – Add substrate ; incubate – Enzyme + Substrate  Product  measure colour – Colour proportional to antibody in patient sample

4 Non-competitive or Sandwich Assay 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

5 Competitive Binding assay 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

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 Group No. No. of Animals Test Material ROA Dose (mg/kg) Main Study Recovery M F M F Control Article IV and SC * ABC-001 SC only IV only

8 Tox Study Experimental Design Study DayGroups No(s). Time points (relative to the End of IV Infusion, EOI or SC injection)TKMAHA Day 11-7PredoseXX Day 11, 6-75 min, 1 hrX- Day hours post Day 1 doseX- Day hours post Day 1 doseX- Day hours post Day 1 doseX- Day hours post Day 1 doseX- Day hours post Day 1 doseX- Day 81-7PredoseXX Day hours post Day 8 doseX- Day 151-7PredoseXX Day hours post Day 15 doseX- Day 221-7PredoseXX Day 221, 6-75 min, 1 hrX- Day hours post Day 22 doseX- Day hours post Day 22 doseX- Day hours post Day 22 doseX- Day hours post Day 22 doseX- Day hours post Day 22 doseX- Day hours* post Day 22 doseXX Day 431, 3-5, 7NAX- Day 571, 3-5, 7NAXX Day 711, 3-5, 7NAX- Day 851, 3-5, 7NAXX Toxicokinetic (TK) and Monkey Anti-Human Antibody (MAHA) Sample Collection Schedule

9 Toxicokinetic (TK) Assessment Capture protein ABC- Chemiluminescent Substrate Read 570 nm Monkey-Absorbed Sheep Anti-human IgG (H+L) pAb HRP Dry coated plate Chemiluminescent Substrate Read 570 nm MonkeyAbsorbed Sheep Anti-human IgG (H+L) pAb - Y Y Dry coated plate 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 Elx 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. CYN and CYN 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 # StabilGuard from SurModics, Catalog # SG Human 001 Protein Carrier-Free, eBioscience, Catalog # 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 # X DPBS, Mediatech, Inc, Catalog # CV 1X DPBS, Mediatech, Inc, Catalog # CM Tween 20, Sigma, Catalog # P1379 Sodium Chloride, Sigma, Catalog # P9416 ProClin300, Supelco, Catalog # U SuperBlock, ScyTek, Catalog # AAA999 Purified H 2 O

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 Calibration Standard ABC-001 Concentration, ng/mL Working Standard Concentrations Quality Control ABC-001 Concentration, ng/mL QC Low QC Mid QC High QC/Validation Sample Concentrations

15 Assay Performance- Standard Curves Run ID STD STD STD STD STD STD STD STD STD ng/mL n Overall Mean (ng/mL) S.D %CV %RE 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 Nominal Concentration LLOQ LQC MQC HQC ULOQ ng/mL Mean Observed Concentration* Intra-Batch Accuracy (%RE) Inter-Batch Accuracy (%RE) Intra-Batch Precision (%CV) Inter-Batch Precision (%CV) %Total Error n Number of Runs %Total Error = ABS(%RE) + %CV of inter-Batch *Calculated via inter-batch statistics (ANOVA) Overview of Precision and Accuracy for QC Samples

17 Assay Performance- Selectivity Selectivity Evaluation Sample NameMS01MS02MS03MS04MS05MS06MS07MS08MS09MS10PMS1PMS2PMS3 Sample Unspiked Observed Conc. (ng/mL) Sample Spiked Conc. (ng/mL)100 Sample Spiked Observed Conc. (ng/mL) %CV %RE

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

19 Dilution Integrity Dilution Linearity of ABC-001 in Monkey Serum Original Concentration = 1.5 mg/mL Sample Name DL1 DL2 DL3 DL4 DL5 Dilution Factor Nominal Conc. (ng/mL) Observed Conc.(ng/mL) OOR %CV of raw data %RE NA Original Concentration = 0.5 mg/mL Sample Name DL1 DL2 DL3 DL4 DL5 Dilution Factor Nominal Conc. (ng/mL) Observed Conc.(ng/mL) OOR %CV of raw data %RE NA Note: OOR = Out of quantitation range (Assay response greater than that of ULOQ) NA = Not applicable

20 Assay Performance - Robustness Tested at the lower limit at each incubation step Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean %RE of Mean -5.4% %CV 7.2% MQC (1500 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean 1524 %RE of Mean 1.6% %CV 8.6% HQC (7500 ng/mL) Replicate * 2.8 Replicate Replicate Replicate * 1.7 Replicate Replicate Mean 8391 %RE of Mean 11.9% %CV 11.6% Note: * Exceeding acceptance criteria of ±20%.

21 Robustness Tested at the upper limit at each incubation step Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean %RE of Mean -6.7% %CV 8.9% MQC (1500 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean 1512 %RE of Mean 0.8% %CV 7.5% HQC (7500 ng/mL) Replicate * 4.2 Replicate Replicate Replicate * 1.3 Replicate Replicate Mean 8487 %RE of Mean 13.2% %CV 15.3% Note: * Exceeding acceptance criteria of ±20%.

22 Coated Plate Stability Stability of Coated Plates at 5°C ± 3°C After 27 Days Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean %RE of Mean -7.3% %CV 5.8% MQC (1500 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean 1490 %RE of Mean -0.7% %CV 6.1% HQC (7500 ng/mL) Replicate Replicate Replicate Replicate Replicate Replicate Mean 7898 %RE of Mean 5.3% %CV 8.2%

23 Freeze -Thaw Stability (4 cycles) Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) * Mean %RE of Mean -14.4% %CV 5.6% HQC (7500 ng/mL) * Mean 8433 %RE of Mean 12.4% %CV 9.3% Note: * Value exceeded acceptance criteria of within ±20%.

24 Bench Top Stability of 20 Hours Sample Name Observed Concentration (ng/mL) %RE %CV LQC (300.0 ng/mL) Mean %RE of Mean -13.4% %CV 5.1% HQC (7500 ng/mL) Mean 8114 %RE of Mean 8.2% %CV 7.8%

25 Long-term Storage Stability Nominal Concentration LQC, 300 ng/mLHQC, 7500 ng/mL 6-Month Stability (188 Days) Mean ng/mL %RE %CV n33 12-Month Stability (364 Days) Mean ng/mL %RE %CV611.5 n33

26 Method transfer (P/A Summary) Sample Batch /plate Rep 1Rep 2Rep 3 Intrabatch (within-run) StatisticsAbsolute n Mean (ng/mL) Stdv.%CV%RE ∑%CV+ %RE LLOQ1/ / ng/mL2/ Interbatch (Between-run) Statistics: LQC1/ / ng/mL2/ Interbatch (Between-run) Statistics: MQC1/ / ng/mL2/ Interbatch (Between-run) Statistics: HQC1/ / ng/mL2/ Interbatch (Between-run) Statistics: ULOQ1/ / ng/mL2/ Interbatch (Between-run) Statistics:

27 Standard and QC performance of TK Sample Analysis Concentration [ng/mL] n Overall Mean S.D %CV %RE QC Low (300 ng/mL) Mid (1500 ng/mL) High (7500 ng/mL) Mean S.D %CV %RE n

28 Representative TK Graphs Group 5 SC 100 mg/kgGroup 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 / MSDHomogeneous 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 # – TMB: KPL – 1N Sulfuric Acid

37 ADA Assay Summary Screening Assay Cut Point0.159 Normalization Factor1.44 Confirmation Cut-Point14.79% High Positive ControlIntra-Assay %CV: ≤ 12.6%, Inter-Assay %CV: 13.8 % Low Positive ControlIntra-Assay %CV: ≤ 14.5%, Inter-Assay %CV: 17.4% Negative ControlIntra-Assay %CV: ≤ 16.5%, Inter-Assay %CV: 21.1% Hook EffectNo Hook effect up to ng/mL Drug Tolerance µg/mL at 500 ng/mL of PC Interference by Hemolysis and Lipemia No Interference observed Immuno-depleted Control (LPC)Average: 37.20% Immuno-depleted Control (HPC)Average: 92.88% Relative Sensitivity100 ng/mL Method Selectivity 90% of the spiked and unspiked samples were within 75%-125% of the respective controls. Robustness Plates with longest incubation times at each step meet acceptance criteria Bench Top Stability Stable up to 24 hrs Refrigerated Stability Stable up to 3 days Freeze Thaw Stability Stable up to 6 cycles

38 Drug Tolerance Using H-ELISA ADA at 500ng/mL Drug Concentration µg/mL OD1OD2OD MeanCV % % % % % % % % Cut-point:0.229 Drug Interference: ( µg/ mL) 100~200  g/mL

39 ADA sample Analysis Total samples tested for screening assay 350 Samples confirmed positive 60 Titers2-32 Impact of ADA on PK profile No

40 ADA sample Analysis (QC Performance) Run # OD HPC_1HPC_2LPC_1LPC_2NC Mean%CVMean%CVMean%CVMean%CVMean%CV Run Run Run Run Run Run Run Run Run Overall Mean Overall SD Overall CV%

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 Shawn Li, M.D., Ph.D. Director, Biologics Services Frontage Lab. Inc. Exton, PA Headquarters Thank You


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