A Mass Spectrometry based “ELISA” Assay for AdenovirusVaccine Potency Testing
Jonathan Knibbe| CASSS MS| 22 September 2017
Virus capsid session
Melinda, Tree of LifeMelinda’s artwork reflects her journey living with HIV.
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Content
Our HIV vaccine candidate
Potency testing
MS detection on a potency bioassayMS
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Our HIV Vaccine candidate:
http://www.janssen.com/johnson-johnson-announces-encouraging-first-human-clinical-data-investigational-hiv-preventive
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Our HIV Vaccine candidate: principle
Mode of action = transgene protein production
Protein responsible for protective immune response(transgene protein)
Genes responsible for protective
immune response
HIV Virus
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Our HIV Vaccine candidate:
Tetravalent adenovirus 26 (AdVac®) vector product
Barouch, D.H. et al. Nat. Med. 16 319-23 (2010)
Ad26-HIV1 Ad26-HIV2
Ad26-HIV3 Ad26-HIV4
90% identical sequence
81% identical sequence
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Our HIV Vaccine candidate: potency testing
Lot release requires potency test
Barouch, D.H. et al. Nat. Med. 16 319-23 (2010)
Ad26-HIV1 Ad26-HIV2
Ad26-HIV3 Ad26-HIV4
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))
‘given result’= transgene protein production
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Transgene expression-ELISA
cell modelTestsample
Standard Detection
USP<1032> Design and Development of Biological Assays.
Dose response curvesdilution vs. transgene
+ Suitable for Quality Control (QC) laboratory+ Sensitive+ Selective
multiple dilutionsspecific
antibody
High sequence homology = no specific antibody
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Can we use Mass Spectrometry to quantify the transgene proteins?
Can we combine a bioassay with a MS detector?
Mass Spectrometry
Bioassay
Challenge#2: QC-proof Sample prep
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TGE-MS: concept and challenges
cell modelTestsample
Reference
Selected Reaction
Monitoring (SRM)MS on triple quad
‘ELISA’infection tryptic
digestion
??
Potency determination
Challenge#3: QC-proof MS detection
Challenge#4: Test design
Challenge#1: find proteotypic peptides
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Challenge#1 find proteotypic peptides
In-silico digest
#of candidatesHIV1 36HIV2 34HIV3 71HIV4 71
Proteome-Wide Screen
#of candidatesHIV1 7HIV2 10HIV3 4HIV4 5
Peptide selection study
#of candidatesHIV1 1HIV2 1HIV3 1HIV4 1
Falcon tube
Transgen Peptide 0.1%FA 10% FA = ref
H2O/CH3CN (95/5)
H2O/CH3CN (50/50)
0.1% FA/CH3CN
(50/50)
0.6% HAc in 80%
CH3CN
HIV1
HIV1.1 87.8 102.7 97.5 98.8 106.0 95.9 HIV1.2 2.7 6.1 10.6 117.7 120.0 121.0 HIV1.3 31.4 68.0 81.2 107.6 117.4 109.4 HIV1.3a 35.3 65.5 74.8 106.3 115.9 111.6
HIV2 HIV2.2 39.1 74.9 87.9 108.9 97.0 103.0 HIV2.3 52.3 89.1 97.9 110.7 109.4 113.2 HIV2.4 101.3 100.2 103.0 104.9 95.7 100.2
HIV3 HIV3.3 58.0 87.8 98.6 98.6 115.8 102.7 HIV3.4 98.7 102.6 99.4 102.9 101.9 101.3 HIV3.5 47.1 80.4 95.2 106.2 113.8 111.6
ACTIN Act1 29.7 69.1 81.0 104.3 99.1 106.6 GAPDH G3P2 89.6 100.0 103.5 106.5 100.9 104.9 VIRAL Hex1 94.6 97.3 102.9 107.5 108.7 103.9
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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
e.g.DTTIAA e.g.
Trypsin/Lys-C
Lot release test = Quality Control laboratory Simple, robust
Dose response curve with replicatesHigh-throughput
Lysis buffer e.g.8.0M Urea6.0M Guanidine-HCL + mechanical force
e.g. SPE columns
e.g.BCA
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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
Optimized seeding, infection & lysis
Av
. T
ota
l P
rote
in(µ
g/m
L)
E x p e r ime n t 1
(n= 9 3 )
E x p e r ime n t 2
(n= 9 0 )
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
In-solution digestion in96-well plate format
96 samples/run Simple, robust
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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 replicatesHigh-throughput
Triple quadrupole MS ‘must-have’ instrument
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Challenge#3: QC-proof MS detection
A LC-MS/MS method was developed:<15min/run Validated
HIV1 HIV2 HIV3 HIV4
Chromatograms of HIV peptides spiked at LLOQ level
Linearity (r2) ≥ 0.9991 Precision (total CV)1.6 - 6.6%Accuracy (overall bias)0.9 – 6.2%
SRM-MS quantification of signature peptides
96 well plate tryptic digestion
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TGE-MS: concept and challenges
cell modelTestsample
Reference
Selected Reaction
Monitoring (SRM)MS on triple quad
‘ELISA’infection tryptic
digestion
??
Potency determination
Validated LC-MS/MS method
Challenge#4: test design
Single proteotypicpeptides selected
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Challenge #4: Test design
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>)
ELISAfour-parameter logistic curve
E x a m p le d e v e lo p m e n t d a ta H IV 3 (N = 3 )
V P /w e ll
pm
ol/
sam
ple
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
MSSimple linear model (y=α+βx)
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Challenge #4: Test design
-When using a linear model estimation:-≥4 concentrations (dose)-Steep straight lines-Parallel lines
ELISAfour-parameter logistic curve
E x a m p le d e v e lo p m e n t d a ta H IV 3 (N = 3 )
V P /w e ll
pm
ol/
sam
ple
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
MSSimple linear model (y=α+βx)
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Challenge #4: Test design
-6 dilution levels-6 points / dilution-3 runs
Testsample
Reference
80%
-Parallel lines?-Do we find 80% potency?
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Challenge #4: Test design results
Parallelism testF-test: pass (F>0.05)ChiSquare: pass (>0.95)
All transgene proteins, all runsindividually & 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%
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Can we use Mass Spectrometry to quantify the transgene proteins?
Can we combine a bioassay with a MS detector?
Mass Spectrometry
Bioassay…Yes we can
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Should we combine a bioassay with a MS detector?
Mass Spectrometry
TGE-ELISA
+ Cheap reagents (SILS)+ Easy to multiplex (measure multiple targets/sample)
• Sensitivity
+ Sensitive+ Selective
• Need for antibodies
The People
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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
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Bioassay – Mass Spectrometry future?
CASSS MS 2017 CASSS MS 2020?