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?