Presentation on theme: "Noncompetitive binding assay or Sandwich method"— Presentation transcript:
1 PK and Anti-Drug Antibody Immunoassays Using Covalent Binding Plates - Case Studies
2 Noncompetitive binding assay or Sandwich method Types of ELISANoncompetitive binding assay or Sandwich methodAntigen 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 Noncompetitive or Sandwich Assay Antibody measuring systemPlate wells coated with suitable antigenAdd sample containing the antibodyIncubate: till antigen antibody reaction is completeWash remove unbound antibodyAdd Anti-antibody labelled with EnzymeIncubate till labelled anti-antibodies binds antigen-antibody complexWash remove unbound labelled anti-antibodyAdd substrate ; incubateEnzyme + Substrate Product measure colourColour proportional to antibody in patient sample
4 Non-competitive or Sandwich Assay Antigen measuring systemPlate wells coated with suitable antibodyAdd sample containing the antigenIncubate: till antigen antibody reaction is completeWash remove unbound antigenAdd Antibody labelled with EnzymeIncubate till antigen binds labelled antibodyWash remove unbound labelled antibodyAdd substrate ; incubateEnzyme + Substrate Product measure colourColour proportional to antigen in patient sample
5 Competitive Binding assay Plate wells coated with antibodiesKnown quantities of patient sample containing antigen + antigen labelled with enzymeIncubate: till antigen antibody reaction is completeWash remove unbound antigensAdd substrate ; incubateEnzyme + Substrate Product measure colourColour 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 RecoveryObjective: 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 AnimalsTest MaterialROADose (mg/kg)Main StudyRecoveryMF132Control ArticleIV and SC *-ABC-001SC only1043051006IV only7
8 Tox Study Experimental Design Toxicokinetic (TK) and Monkey Anti-Human Antibody (MAHA) Sample Collection ScheduleStudy DayGroups No(s).Time points (relative to the End of IV Infusion, EOI or SC injection)TKMAHADay 11-7PredoseX1, 6-75 min, 1 hr-Day 224 hours post Day 1 doseDay 348 hours post Day 1 doseDay 472 hours post Day 1 doseDay 596 hours post Day 1 doseDay 6120 hours post Day 1 doseDay 8Day 1296 hours post Day 8 doseDay 15Day 1996 hours post Day 15 doseDay 22Day 2324 hours post Day 22 doseDay 2448 hours post Day 22 doseDay 2572 hours post Day 22 doseDay 2696 hours post Day 22 doseDay 27120 hours post Day 22 doseDay 29168 hours* post Day 22 doseDay 431, 3-5, 7NADay 57Day 71Day 85
9 Toxicokinetic (TK) Assessment Chemiluminescence-based Immunoassay Using Covalent Binding Plate to Quantify ABC-001 in Monkey SerumCapture proteinABC-ChemiluminescentSubstrateRead 570 nmMonkeyAbsorbed SheepAntihuman IgG (H+L) pAbHRPDry coatedplate-001Y
10 Assay Materials and Procedure EquipmentSpectraMax® L Luminescence Microplate ReaderSoftware: 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 µLVortex mixerAdhesive sealing films for microplates, Excel Scientific, Inc.Absorbent materials for blotting the strips
11 Assay Materials and Procedure Biological MatrixBlank monkey serum was purchased from Bioreclamation Inc. The serum lots used in the assay were Lot Nos. CYN and CYNThe 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-001Immobilizer amino F96 Black Plate: NUNC, Catalog #StabilGuard from SurModics, Catalog # SGHuman 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 #10X DPBS, Mediatech, Inc, Catalog # CV1X DPBS, Mediatech, Inc, Catalog # CMTween 20, Sigma, Catalog # P1379Sodium Chloride, Sigma, Catalog # P9416ProClin300, Supelco, Catalog # USuperBlock, ScyTek, Catalog # AAA999Purified H2O
13 Plate PreparationPlates 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 Calibration StandardABC-001Concentration, ng/mL12345678100250500125025005000800010000QC/Validation Sample ConcentrationsQuality ControlABC-001Concentration, ng/mLQC LowQC MidQC High30015007500
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)Run IDSTDSTDSTDSTDSTDSTDSTDSTD1STDng/mL198.11263.5501.212382357545990318615507562100.4249.8487.71278252447898443978149957399.03258.3494.31295230853328670911150417493.19275.7514.112032459509794508602509185101.0239.5521.71237246981369736501406101.6238.2515.31268244949168792935250169nOverall Mean (ng/mL)98.89254.2505.71253242851158754920050393S.D.3.07214.5413.3633.4579.67457.9520.7377.4%CV188.8.131.52.184.108.40.206.7%RE-220.127.116.11.2-18.104.22.168-8.00.8
16 Precision and Accuracy Overview of Precision and Accuracy for QC SamplesNominal ConcentrationLLOQLQCMQCHQCULOQng/mL100.0300.01500750010000Mean Observed Concentration*98.35293.51458816010822Intra-Batch Accuracy (%RE)-1.7-2.2-22.214.171.124Inter-Batch Accuracy (%RE)8.8Intra-Batch Precision (%CV)126.96.36.199.016.9Inter-Batch Precision (%CV)10.8%Total Error12.513.011.215.825.1n1817Number of Runs6%Total Error = ABS(%RE) + %CV of inter-Batch*Calculated via inter-batch statistics (ANOVA)
18 Matrix InterferenceMatrix Interference for ABC-001 Assay in Monkey Serum
19 Dilution Integrity Dilution Linearity of ABC-001 in Monkey Serum Original Concentration = 1.5 mg/mLSample NameDL1DL2DL3DL4DL5Dilution Factor1520050015003000Nominal Conc. (ng/mL)10000075001000Observed Conc.(ng/mL)OOR79853109964.8457.0%CV of raw data1.72.01.96.61.2%RENA6.53.6-3.5-8.6Original Concentration = 0.5 mg/mL566.7166.7500.07832333811415188.8.131.52.04.411.314.12.4Note: 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 stepSample NameObserved Concentration (ng/mL)%RE%CVLQC (300.0 ng/mL)Replicate 1301.80.63.4Replicate 2309.73.23.6Replicate 3255.6-14.86.6Replicate 4289.5-3.52.6Replicate 5276.9-7.71.0Replicate 6269.3-10.23.0Mean283.8%RE of Mean-5.4%7.2%MQC (1500 ng/mL)168412.20.115211.42.11470-2.00.916117.41304-13.12.515511.615241.6%8.6%HQC (7500 ng/mL)922823.0*2.87087-5.52.3867015.61.8933924.5*1.77285-2.94.5873816.5839111.9%11.6%Note: * Exceeding acceptance criteria of ±20%.
21 Robustness Tested at the upper limit at each incubation step Sample NameObserved Concentration (ng/mL)%RE%CVLQC (300.0 ng/mL)Replicate 1294.2-1.96.0Replicate 2307.52.53.6Replicate 3248.4-17.212.2Replicate 4295.3-1.69.0Replicate 5283.9-5.42.2Replicate 6250.2-16.60.6Mean279.9%RE of Mean-6.7%8.9%MQC (1500 ng/mL)15946.32.315201.30.21424-5.12.7166210.81345-10.315271.815120.8%7.5%HQC (7500 ng/mL)960528.1*4.26804-9.33.0876116.83.5987031.6*6995-6.7888918.51.4848713.2%15.3%Note: * Exceeding acceptance criteria of ±20%.
22 Coated Plate Stability Stability of Coated Plates at 5°C ± 3°C After 27 DaysSample NameObserved Concentration (ng/mL)%RE%CVLQC (300.0 ng/mL)Replicate 1290.3-3.25.5Replicate 2286.8-4.44.7Replicate 3249.6-16.88.3Replicate 4283.1-5.62.0Replicate 5290.42.2Replicate 6267.9-10.74.2Mean278.0%RE of Mean-7.3%5.8%MQC (1500 ng/mL)15832.115432.90.61414-5.70.115503.40.81346-10.215050.46.51490-0.7%6.1%HQC (7500 ng/mL)838311.87398-1.478152.6880317.44.57015-6.50.079726.378985.3%8.2%
23 Freeze -Thaw Stability (4 cycles) Sample NameObserved Concentration (ng/mL)%RE%CVLQC (300.0 ng/mL)260.4-13.24.4235.6-21.5*13.9265.8-11.46.7265.3-11.63.4Mean256.8%RE of Mean-14.4%5.6%HQC (7500 ng/mL)940225.4*0.475400.51.781698.93.0862215.02.0843312.4%9.3%Note: * Value exceeded acceptance criteria of within ±20%.
24 Bench Top Stability of 20 Hours Sample NameObserved Concentration (ng/mL)%RE%CVLQC (300.0 ng/mL)265.9-11.44.5240.8-19.75.4260.7-13.14.7271.3-9.66.9Mean259.7%RE of Mean-13.4%5.1%HQC (7500 ng/mL)829010.53.37191-4.14.8834611.34.3862815.02.681148.2%7.8%
27 Standard and QC performance of TK Sample Analysis Concentration [ng/mL]100250500125025005000800010000n4850Overall MeanS.D.6.86811.25519.80238.548%CV184.108.40.206.220.127.116.11.7%RE1.7-0.3-18.104.22.168.30.9-6.6QCLow (300 ng/mL)Mid (1500 ng/mL)High (7500 ng/mL)MeanS.D.24.371%CV22.214.171.124%RE32.43.2n10099
28 Representative TK Graphs Group 5 SC 100 mg/kgGroup 7 IV 30 mg/kg
29 ISR evaluationAn 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)ScreeningConfirmationTitrationNeutralizing antibodies (Nab)Basic package +/- based on:Risk assessmentResults from pre-clinical and early clinical studiesRegulatory input
31 ADA Assay Platforms Platforms ELISA Bridging Direct Indirect RIA BiacoreElectrochemiluminescence (ECL)No “perfect” assay currently existsIn general, assay format for ADAProtein therapeutics: direct formatmAbs: bridging format
32 Popular ADA assay formats for Mab Drugs ECL / MSDHomogeneous BridgingELISA
33 ADA Assay Key Parameters for validation Screening cut point Specificity/confirmation cut pointSensitivitySystem suitability controls(QCs) acceptance criteriaSelectivity/InterferenceMatrix componentsDrug tolerancePrecisionRobustnessStabilityRuggedness
34 ECL vs. H-ELISA ECL Advantages H-ELISA 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 technologyH-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
37 ADA Assay Summary Screening Assay Cut Point 0.159 Normalization Factor 1.44Confirmation Cut-Point14.79%High Positive ControlIntra-Assay %CV: ≤ 12.6% , Inter-Assay %CV: %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/mLDrug Toleranceµg/mL at 500 ng/mL of PCInterference by Hemolysis and LipemiaNo Interference observedImmuno-depleted Control (LPC)Average: 37.20%Immuno-depleted Control (HPC)Average: 92.88%Relative Sensitivity100 ng/mLMethod Selectivity90% of the spiked and unspiked samples were within 75%-125% of the respective controls.RobustnessPlates with longest incubation times at each step meet acceptance criteriaBench Top StabilityStable up to 24 hrsRefrigerated StabilityStable up to 3 daysFreeze Thaw StabilityStable up to 6 cycles
38 Drug Tolerance Using H-ELISA ADA at 500ng/mLDrug Concentration µg/mLOD1OD2OD MeanCV5000.22020.20540.2134.9%2000.22270.21440.2192.7%1000.23820.22740.2333.3%500.2360.24090.2381.5%200.25090.24540.2481.6%50.24060.24850.2452.3%10.27350.26060.2673.4%0.37030.35950.3652.1%Cut-point:0.229Drug Interference: (µg/mL)100~200 mg/mL
39 ADA sample Analysis Total samples tested for screening assay 350 Samples confirmed positive60Titers2-32Impact of ADA on PK profileNo
40 ADA sample Analysis (QC Performance) Run #ODHPC_1HPC_2LPC_1LPC_2NCMean%CVRun 10.9971.20.9520.90.1102.60.1022.80.0707.5Run 21.3126.51.2473.40.1234.00.1111.90.07510.3Run 31.2864.91.1842.10.1135.60.0993.60.0688.6Run 41.2991.3074.10.12111.73.50.0739.5Run 51.1951.11.1561.70.108Run 61.1740.71.1180.40.1200.1090.60.0728.1Run 70.9262.00.8245.50.1064.77.0Run 80.9420.8780.20.1042.70.1034.80.0714.4Run 90.8751.30.9276.40.101Overall Mean1.089Overall SD0.1720.0080.002Overall CV%126.96.36.199
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