A Mass Spectrometry based ELISA Assay for Adenovirus Vaccine Potency Testing
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1 A Mass Spectrometry based ELISA Assay for Adenovirus Vaccine Potency Testing Virus capsid session Jonathan Knibbe CASSS MS 22 September 2017 Melinda, Tree of Life Melinda s artwork reflects her journey living with HIV.
2 Content Our HIV vaccine candidate Potency testing MS MS detection on a potency bioassay 2
3 Our HIV Vaccine candidate: 3
4 Our HIV Vaccine candidate: principle Genes responsible for protective immune response Protein responsible for protective immune response (transgene protein) HIV Virus Mode of action = transgene protein production 4
5 Our HIV Vaccine candidate: Tetravalent adenovirus 26 (AdVac ) vector product Ad26-HIV1 Ad26-HIV2 90% identical sequence Ad26-HIV3 Ad26-HIV4 81% identical sequence Barouch, D.H. et al. Nat. Med (2010) 5
6 Our HIV Vaccine candidate: potency testing Lot release requires potency test Ad26-HIV1 Ad26-HIV2 Potency the specific ability or capacity of the product, as indicated by appropriate laboratory tests to effect a given result (21 CFR 600.3(s)) Ad26-HIV3 Ad26-HIV4 given result = transgene protein production Barouch, D.H. et al. Nat. Med (2010) 6
7 Transgene expression-elisa + Suitable for Quality Control (QC) laboratory + Sensitive + Selective multiple dilutions specific antibody Test Standard sample cell model Detection Dose response curves dilution vs. transgene High sequence homology = no specific antibody USP<1032> Design and Development of Biological Assays. 7
8 Can we use Mass Spectrometry to quantify the transgene proteins? Mass Spectrometry Bioassay Can we combine a bioassay with a MS detector? 8
9 TGE-MS: concept and challenges Challenge#2: QC-proof Sample prep Challenge#4: Test design Challenge#1: find proteotypic peptides Challenge#3: QC-proof MS detection Test Reference sample ELISA infection cell model tryptic digestion Selected Reaction Monitoring (SRM) MS on triple quad Potency determination?? 9
10 Challenge#1 find proteotypic peptides Falcon tube 0.6% 10% 0.1% H2O/CH3CN H2O/CH3CN HAc in Transgen Peptide 0.1%FA FA = FA/CH3CN (95/5) (50/50) 80% ref (50/50) CH3CN HIV HIV HIV1 HIV HIV1.3a HIV HIV2 HIV HIV HIV HIV3 HIV HIV ACTIN Act GAPDH G3P VIRAL Hex In-silico digest Proteome-Wide Screen Peptide selection study #of candidates HIV1 36 HIV2 34 HIV3 71 HIV4 71 #of candidates HIV1 7 HIV2 10 HIV3 4 HIV4 5 #of candidates HIV1 1 HIV2 1 HIV3 1 HIV4 1 10
11 Challenge #2: QC-proof sample prep 1. Disruption and solubilization 2. Protein content determination 3.Reduction & alkylation 4. Enzymatic proteolysis 5. MS-compatible peptide recovery Lysis buffer e.g. 8.0M Urea 6.0M Guanidine-HCL + mechanical force e.g. BCA e.g. DTT IAA e.g. Trypsin/Lys-C e.g. SPE columns Lot release test = Quality Control laboratory Simple, robust Dose response curve with replicates High-throughput 11
12 Challenge #2: QC-proof sample prep 1. Disruption and solubilization 2. Protein content determination 3.Reduction & alkylation 4. Enzymatic proteolysis 5. MS-compatible peptide recovery 250 A v. Total P rotein(µ g/m L) Experiment 1 (n=93) Experiment 2 (n=90) Optimized seeding, infection & lysis In-solution digestion in 96-well plate format 96 samples/run Simple, robust 12
13 Challenge#3: QC-proof MS detection Requirements Selective and sensitive peptide quantification in complex matrix Lot release test = Quality Control laboratory Simple, robust, compatible software Dose response curve with replicates High-throughput Triple quadrupole MS must-have instrument 13
14 Challenge#3: QC-proof MS detection A LC-MS/MS method was developed: <15min/run Validated SRM-MS quantification of signature peptides HIV1 HIV2 HIV3 HIV4 Linearity (r 2 ) Precision (total CV) % Accuracy (overall bias) % Chromatograms of HIV peptides spiked at LLOQ level 14
15 TGE-MS: concept and challenges 96 well plate tryptic digestion Challenge#4: test design Single proteotypic peptides selected Validated LC-MS/MS method Test Reference sample ELISA infection cell model tryptic digestion Selected Reaction Monitoring (SRM) MS on triple quad Potency determination?? 15
16 Challenge #4: Test design Example development data HIV3 (N=3) 0.08 pm ol/sam ple ELISA four-parameter logistic curve 0.00 VP/w ell MS Simple linear model (y=α+βx) When using a linear model estimation: Use at least three and preferably four adjacent concentrations; require that the slope of the linear segment is sufficiently steep; and require that the lines fit to Standard and Test samples are straight and that the lines are parallel (USP <1032>) 16
17 Challenge #4: Test design Example development data HIV3 (N=3) 0.08 pm ol/sam ple ELISA four-parameter logistic curve -When using a linear model estimation: - 4 concentrations (dose) -Steep straight lines -Parallel lines 0.00 VP/w ell MS Simple linear model (y=α+βx) 17
18 Challenge #4: Test design 80% -6 dilution levels -6 points / dilution -3 runs Test Reference sample -Parallel lines? -Do we find 80% potency? 18
19 Challenge #4: Test design results Parallelism test F-test: pass (F>0.05) ChiSquare: pass (>0.95) All transgene proteins, all runs individually & combined Potency target=80% Found: HIV1: 76.4% ±7.1% HIV2: 78.6% ±1.0% HIV3: 81.2 ±3.4% HIV4: 81.4% ±5.3% 19
20 Can we use Mass Spectrometry to quantify the transgene proteins? Mass Spectrometry Bioassay Yes we can Can we combine a bioassay with a MS detector? 20
21 Should we combine a bioassay with a MS detector? Mass Spectrometry + Cheap reagents (SILS) + Easy to multiplex (measure multiple targets/sample) + Sensitive + Selective TGE-ELISA Sensitivity Need for antibodies 21
22 The People Janssen Vaccines Leiden Arjen Scholten Jonathan Knibbe Annemiek Verwilligen Hans Kanbier Janssen Biologics Leiden Crina Balog Lars Meijer Janssen Pharmaceuticals Beerse Tom Verhaeghe Lieve Dillen Mark Verhemeldonck 22
23 Bioassay Mass Spectrometry future? CASSS MS 2017 CASSS MS 2020? 23
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