In-depth, Quantitative (Host Cell) Protein Fingerprinting of Adenovirus Based Vaccines by Mass Spectrometry
Annemiek Verwilligen, Scientist Analytical Development, 25SEP15
Melinda, Goddess of HealingMelinda’s artwork reflects her journey living with HIV.
2
Outline
• AdVac® Vaccine Platform• Rationale• Approach all-in-one protein fingerprinting
method• Applications• How to proceed
3
AdVac ® platform at Janssen
4
Product Characterization
WHO, EMA, and FDA Biotechnology guidelines require product characterization on:
• Appearance and Description• Identity• Purity and Impurities• Potency• Quantity
5
Protein characterization of AdVac®
What proteins are present?
Adenovirus capsid and core proteins
Per.C6® Host cell proteins
Current Protein Characterization Strategy
6
Adeno virus proteins
Identification:• 1 dimensional SDS-
PAGE + MS band identification
• C4 RP-UPLC protein profiling
Quantification:• C4 RP-UPLC protein
profiling
Host cell proteins
Identification: • 1 dimensional SDS-
PAGE + MS band identification
• C4 RP-UPLC protein profiling + MS fraction identification
Quantification:• HCP ELISA
7
Review current approach
1-D SDS-PAGE profile of Ad26
Pro’s:• Easy visual interpretation• Generally accepted
Con’s:• Not all AVP identified• Dynamic range limits
identification of HCP• Multiple proteins per band• Laborious
KDa
V
III/IIIaIVa2IV52,55KVIVIIVIIIIX
II
C4 RP-UPLC chromatogram Ad26
8
FASP, 5KDa filter.1• 8 M Urea denaturation,• DTT/IAA reduction/alkylation• LysC/Trypsin Digestion
IKey CSH C18 μRP-UPLC 2h gradient
ESI-Q-TOF MSEdata aquisitionand processing
Workflow
AdVac®DS, DP or intermediate
Identified viral and HC proteins
1Wisnieski et al, Nature Methods, 2009
9
Applications
Example:• Process development support• Batch to batch comparrisson of 3
development Ad26 batches • Drug substance and process intermediates
Ad26 DS1, Ad26 PI1 Ad26 DS2, Ad26 PI2 Ad26 DS3, Ad26 PI3
±10μg proteinAd26 Bulk DS/ analysis
10
Adeno virus protein identification
0102030405060708090
100
DS1 DS2 DS3
% o
f tot
al p
rote
in in
tens
ity
AVPHCP
97.1 95.5 96.6
2.9 4.5 3.40
102030405060708090
100
DS1 DS2 DS3
% o
f tot
al p
rote
in in
tens
ity
Total HCP
µ (x)
Fiber (IV)
pTP
IX
protease
VIII
VI
VII
IVa2
V
Penton (III)
IIIa
5255K
Hexon (II)
11
Adenovirus protein quantification
12
Adenovirus protein quantification
1
2
4
8
16
32
64
128
256
512
1024
Hexon(II)
Penton(III)
Fiber(IV)
IIIa VIII VI IX VII V pTP u (x) AVP IVa2 5255K
Prot
ein
copy
num
ber (
Log 2
)
Heck et al. [1]
Theory [2]
Our data
1
2
4
8
16
32
64
128
256
512
1024
Hexon(II)
Penton(III)
Fiber(IV)
IIIa VIII VI IX VII V pTP u (x) AVP IVa2 5255K
Prot
ein
copy
num
ber (
Log 2
)
Heck et al. [1]
Theory [2]
Our data
1
2
4
8
16
32
64
128
256
512
1024
Hexon(II)
Penton(III)
Fiber(IV)
IIIa VIII VI IX VII V pTP u (x) AVP IVa2 5255K
Prot
ein
copy
num
ber (
Log 2
)
Heck et al. [1]
Theory [2]
Our data
[1] Benevento et al, The Journal of Biological Chemistry, 2014[2] Liu et al, Science , 2010
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Host cell protein identification
Batch n AVP n HCP
PI1 14 194
PI2 14 192
PI3 14 188
DS1 14 10
DS2 14 10
DS3 14 9
0
20
40
60
80
100
PI1 PI2 PI3
% o
f tot
al p
rote
in in
tens
ity
AVP
HCP
58.2
0
20
40
60
80
100
DS1 DS2 DS3
% o
f tot
al p
rote
in in
tens
ity
AVP
HCP
97.195.5 96.7
2.9 4.5 3.3
43.6
56.4
41.8 43.3
56.7
HCP Types Identified
Per.C6® household proteins (o.a. Actin, GAPDH)
Proteasome subunits
Histones
Ribosomal proteins
Heat shock proteins
Transgene proteins
14
Host cell protein quantification
HCP ELISA
• AB mixture generated against cell lysate host cell.
• Result: μg HCP/ml.• LOD: ± 1 ng/ml.• No HCP ID information
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
P04406 Q92841 Q7Z5M8 P10809 P14618 P62805 O60814 Q497I4 Q32P51 P51993
% o
f Tot
al P
rote
in
Uniprot protein number
0
10
20
30
40
50
60
70
80
90
100
AVP HCP
% o
f Tot
al P
rote
in
15
Host cell protein quantification
MS HCP quantification DS1
• Result: % of total protein
• LOD: ± 1 ng/ml per HCP
• Protein specific information
97.1
2.9
A-fucosyltransferase
16
HCP cell protein quantification
Unknown HCP observed in 1 D SDS-PAGE profile process development DS batches
1. 2. 3.
1D SDS-PAGE profile contingency batches
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Host cell protein quantification
05
101520253035404550
1 2 3
Aver
age
Nor
mal
ized
Abun
danc
e
Batch number
GAPDH
0123456789
10
1 2 3
Aver
age
Nor
mal
ized
Abun
danc
e
Batch number
Histone 2B
Extra HCP identified as Glyceraldehyde-3-Phosphate Dehydrogenese (GAPDH)
0
100
200
300
400
500
600
1 2 3
Aver
age
Nor
mal
ized
Abun
danc
e
Batch number
Adenovirus protein II
18
Comparisson current and new approach
SDS-PAGE, C4 RP-UPLC, HCP ELISA
Proteinfingerprinting by MS
Identify all adenovirus proteins
Quantify all adenovirus proteins
Identify HCPs
Quantify total amount of HCPs
Quantify specific HCPs
AVP maturation intermediates
Results within 24 hours
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Conclusion
Because of the short excecution time and the deep coverage this assay exemplifies the importance of mass spectrometry in the characterization of complex (adeno)virus based vaccines.
20
Outlook
• Introduce spiking reference peptides for absolute label free quantification.
• Qualification and implementation of the method for batch release purposes.
• Test the method on more process intermediates.
• Implement method for process development support: batch-to-batch comparison.
21
The Team
Dayar van de SteegJonathan KnibbeArjen ScholtenTatev Haruntunyan
and Analytical Assays team at Janssen IDV Leiden, The Netherlands
25Sep15
Thank you
Melinda, Goddess of HealingMelinda’s artwork reflects her journey living with HIV.
In-depth, Quantitative �(Host Cell) Protein Fingerprinting of Adenovirus Based Vaccines by Mass SpectrometryOutlineAdVac ® platform at JanssenProduct CharacterizationProtein characterization of AdVac® Current Protein Characterization StrategyReview current approachWorkflowApplicationsAdeno virus protein identificationAdenovirus protein quantificationAdenovirus protein quantificationHost cell protein identificationHost cell protein quantificationHost cell protein quantificationHCP cell protein quantificationHost cell protein quantificationComparisson current and new approachConclusionOutlookThe TeamSlide Number 22Slide Number 23
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