Presentation on theme: "PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies."— Presentation transcript:
PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies
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]
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
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
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
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.
Tox Study Experimental Design Group No. No. of Animals Test Material ROA Dose (mg/kg) Main Study Recovery M F M F 1 3 3 2 2 Control Article IV and SC * 0 2 3 3 - - ABC-001 SC only 1 3 3 3 2 2 10 4 3 3 2 2 30 5 3 3 2 2 100 6 3 3 - - IV only 1 7 3 3 2 2 30
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 21-724 hours post Day 1 doseX- Day 31-748 hours post Day 1 doseX- Day 41-772 hours post Day 1 doseX- Day 51-796 hours post Day 1 doseX- Day 61-7120 hours post Day 1 doseX- Day 81-7PredoseXX Day 121-796 hours post Day 8 doseX- Day 151-7PredoseXX Day 191-796 hours post Day 15 doseX- Day 221-7PredoseXX Day 221, 6-75 min, 1 hrX- Day 231-724 hours post Day 22 doseX- Day 241-748 hours post Day 22 doseX- Day 251-772 hours post Day 22 doseX- Day 261-796 hours post Day 22 doseX- Day 271-7120 hours post Day 22 doseX- Day 291-7168 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
Toxicokinetic (TK) Assessment Capture protein ABC- Chemiluminescent Substrate Read plate @ 570 nm Monkey-Absorbed Sheep Anti-human IgG (H+L) pAb HRP Dry coated plate Chemiluminescent Substrate Read plate @ 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
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
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.
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 H 2 O
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.
Standard and QC Calibration Standard ABC-001 Concentration, ng/mL 1234567812345678 100 250 500 1250 2500 5000 8000 10000 Working Standard Concentrations Quality Control ABC-001 Concentration, ng/mL QC Low QC Mid QC High 300 1500 7500 QC/Validation Sample Concentrations
Standard and QC performance of TK Sample Analysis Concentration [ng/mL] 100250500125025005000800010000 n 48 50 Overall Mean 101.708249.312487.1961252.2682540.9425164.2368075.7629341.605 S.D. 6.86811.25519.80238.548106.490197.440386.785441.922 %CV 18.104.22.168.22.214.171.124.7 %RE 1.7-0.3-126.96.36.199.30.9-6.6 QC Low (300 ng/mL) Mid (1500 ng/mL) High (7500 ng/mL) Mean309.0281535.6047742.039 S.D.24.371115.454679.087 %CV188.8.131.52 %RE32.43.2 n10099100
Representative TK Graphs Group 5 SC 100 mg/kgGroup 7 IV 30 mg/kg
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.
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
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
Popular ADA assay formats for Mab Drugs ECL / MSDHomogeneous Bridging ELISA
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
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
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 100000 ng/mL Drug Tolerance100-200 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
Drug Tolerance Using H-ELISA ADA at 500ng/mL Drug Concentration µg/mL OD1OD2OD MeanCV 500 0.22020.2054 0.2134.9% 200 0.22270.2144 0.2192.7% 100 0.23820.2274 0.2333.3% 50 0.2360.2409 0.2381.5% 20 0.25090.2454 0.2481.6% 5 0.24060.2485 0.2452.3% 1 0.27350.2606 0.2673.4% 0 0.37030.3595 0.3652.1% Cut-point:0.229 Drug Interference: ( µg/ mL) 100~200 g/mL
ADA sample Analysis Total samples tested for screening assay 350 Samples confirmed positive 60 Titers2-32 Impact of ADA on PK profile No
ADA sample Analysis (QC Performance) Run # OD HPC_1HPC_2LPC_1LPC_2NC Mean%CVMean%CVMean%CVMean%CVMean%CV Run 10.9971.20.9520.90.1102.60.1022.80.0707.5 Run 21.3126.51.2473.40.1234.00.1111.90.07510.3 Run 31.2864.91.1842.10.1135.60.0993.60.0688.6 Run 41.2997.51.3074.10.12111.70.1023.50.0739.5 Run 51.1951.11.1561.20.1231.70.1082.60.0737.5 Run 61.1740.71.1180.40.1201.20.1090.60.0728.1 Run 70.9262.00.8245.50.1064.70.1024.90.0727.0 Run 80.9422.10.8780.20.1042.70.1034.80.0714.4 Run 90.8751.30.9274.00.0996.40.1010.70.0704.1 Overall Mean1.0890.1090.072 Overall SD0.1720.0080.002 Overall CV%184.108.40.206
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