2016/09/30
Mass Spectrometry: What is it good for?
Marybeth Creskey, Lisa Walrond and Terry D. Cyr Regulatory Research Division Centre for Biologics Evaluation Biologics and Genetic Therapies Directorate Health Products and Foods Branch Health Canada National Institute of General Medical Sciences
When your only tool is a hammer… Manufacturers have signalled that they would like to increase the use of MS data in product submissions in support of numerous attributes. Q: Which standard methods are readily replaced and what is required to support and validate the conclusions? - USP and to a lesser extent ICH - run analyses in parallel with standard methods Q: What would be an optimal implementation strategy? - Varying comfort levels with MS technology - include access to additional information - Differing views: introduce with an approved product with many lots run in parallel versus inclusion with a new submission as the only option. 2
Analysis of Biological products by Mass Spectrometry:
Primary sequence Tertiary structure Post translational modifications Degradation products Host cell proteins Bioavailability Potency
Annual Influenza Vaccine Influenza proteins three or four strains 15 μg hemagglutinin/0.5mL ea A(H1N1) A(H3N2) B
http://www.itqb.unl.pt/labs/proteinmodelling/activities/haemagglutinin
http://www.rcsb.org/pdb/explore
~ 100, 000 A and B sequence entries (GISAID) Host cell proteins
Strain ID – Method Development - Increased instrument resolution and sensitivity. - Increased peptide IDs, ~50% sequence coverage 1 _51 101 151 201 251 301 351 401 451 501
DTLCIGYHAN HLGKCNIAGW LREQLSSVSS LVKKGNSYPK VGSSRYSKTF PRYAFAMERN GKCPKYVKST YHHQNEQGSG EKRIENLNKK SQLKNNAKEI DGVKLESTRI
NSTDTVDTVL ILGNPECESL FERFEIFPKT LSKSYINDKG KPEIAIRPKV AGSGIIISDT KLRLATGLRN YAADLKSTQN VDDGFLDIWT GNGCFEFYHK YQILAIYSTV
EKNVTVTHSV STASSWSYIV SSWPNHDSDK KEVLVLWGIH RDREGRMNYY PVHDCNTTCQ IPSIQSRGLF AIDEITNKVN YNAELLVLLE CDNTCMESVK ASSLVLVVSL
Ambiguous IDs
NLLEDKHNGK ETPSSDNGTC GVTAACPHAG HPSTSADQQS WTLVEPGDKI TPKGAINTSL GAIAGFIEGG SVIEKMNTQF NERTLDYHDS NGTYDYPKYS GAISFWMCSN
LCKLRGVAPL YPGDFIDYEE AKSFYKNLIW LYQNADAYVF TFEATGNLVV PFQNIHPITI WTGMVDGWYG TAVGKEFNHL NVKNLYEKVR EEAKLNREEI GSLQCRICI
1 dose vaccine (15 µg HA/500 µl)
DTT iodoacetamide
DTT iodoacetamide
Reduction, alkylation, quench reaction
Transfer to filter (10K MWCO) PNGaseF in H2O18
PNGaseF in H2O18
Centrifugation wash steps Deglycoslyation (+3)
New collection tube
trypsin
chymotrypsin
Protein digestion - centrifuge enzyme solution through filter
Dry down flowthrough (=peptides) Resuspend in injection buffer
Fusion : LC-MSMS Triplicate injections
Peak list processing Merge 6 LC-MSMS runs search In-house influenza database
Strain ID – Method Development - On-filter digestion, triplicate preps - Hundreds of peptide IDs, >90% sequence coverage 1 _51 101 151 201 251 301 351 401 451 501
DTLCIGYHAN HLGKCNIAGW LREQLSSVSS LVKKGNSYPK VGSSRYSKTF PRYAFAMERN GKCPKYVKST YHHQNEQGSG EKRIENLNKK SQLKNNAKEI DGVKLESTRI
NSTDTVDTVL ILGNPECESL FERFEIFPKT LSKSYINDKG KPEIAIRPKV AGSGIIISDT KLRLATGLRN YAADLKSTQN VDDGFLDIWT GNGCFEFYHK YQILAIYSTV
EKNVTVTHSV STASSWSYIV SSWPNHDSDK KEVLVLWGIH RDREGRMNYY PVHDCNTTCQ IPSIQSRGLF AIDEITNKVN YNAELLVLLE CDNTCMESVK ASSLVLVVSL
NLLEDKHNGK ETPSSDNGTC GVTAACPHAG HPSTSADQQS WTLVEPGDKI TPKGAINTSL GAIAGFIEGG SVIEKMNTQF NERTLDYHDS NGTYDYPKYS GAISFWMCSN
Routinely Achieve Unambiguous ID
LCKLRGVAPL YPGDFIDYEE AKSFYKNLIW LYQNADAYVF TFEATGNLVV PFQNIHPITI WTGMVDGWYG TAVGKEFNHL NVKNLYEKVR EEAKLNREEI GSLQCRICI
H1 98%
N1 89%
H3 90%
N2 89%
HB 95%
NB 91%
HCP
Hemagglutinin (HA) Neuraminidase (NA) Host cell proteins But how much is in there?
Influenza Antigen Quantitation Hi3
QconCAT
Digest
Quant
intensity
LC-MS
m/z
Synthetic Peptides
MS Protein Quantitation – Hi3 Method Signal intensity from tryptic peptides from three equimolar proteins
Average signal intensity from the three most intense peptides ~ protein amount (± 15% for proteins of similar mass)
Silva JC, Gorenstein MV, Li GZ, Vissers JP, Geromanos SJ. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition. MCP 2006 5:144–56.
MS Protein Quantitation Hi3 standards
Quantification of antigens can be made by comparing to a spiked reference standard
Average Hi3 Peptide Intensity
µG NA /dose
Hi3 versus Western blot
Vaccine sample
Stable isotope labeled peptides
Measurement accuracy – –
Method range –
Efficiency of proteolysis Differential losses during fractionation What is required
Method precision – – –
Multiple labels Multiple lab staff Multiple instruments
Relative Vaccine Antigen Quantitation
Triplicate samples of vaccines and reference antigens spiked equal amounts of labelled tryptic peptides R/K Response ratios between native and labelled peptides measured for each target protein 2-4 target peptides used for each HA subtype [except the Victoria B strain - one peptide] Vaccine antigen quantity calculated relative to corresponding reference antigen
Neuraminidase Quantitation
Absolute quantity determined for each NA subtype Quantitation based on one peptide Quadrivalent vaccines have neuraminidase from both B strains –
This experiment does not distinguish neuraminidase in the two B strains
QconCAT designs Iteration
Design Strategy
QconCAT 1
- 4 peptides - Direct concatenation - N-terminal polyhistidine tag
QconCAT 2
- 3 peptides - include 3 flanking peptides - include Arg between each flanking set - N-terminal polyhistidine tag
QconCAT 3
- 4 peptides - link peptides with a spacer (ASGK) - N-terminal polyhistidine tag
QconCAT 4
- 4 peptides - link peptides with a spacer (ASGK) - C-terminal polyhistidine tag
QconCAT 5
- 4 peptides - link peptides with a spacer (ASGK) - Peptide set are dispersed - C-terminal polyhistidine tag
PolyQuant
Ratio of Average Hi3 Value from all proteins in QconCAT 2 1.8 1.6
1.4 1.2 1 0.8 0.6 0.4 0.2 0 QconCat1
QconCat2
QconCat3
QconCat4
QconCat5
QconCAT Sequence Protein
Peptides
BSA – Bovine Serum Albumin
1- LGEYGFQNALIVR, 2- LVNELTEFAK, 3- DAFLGSFLYEYSR, 4- HLVDEPQNLIK 1- VVGLSTLPEIYEK, 2- LPLVGGHEGAGVVVGMGENVK, 3- SISIVGSYVGNR, 4- ANELLINVK 38 1- EVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSR, 2- STQNAIDEITNK 3- MNYYWTLVEPGDK, 4- MNTQFTAVGK 1- TFFLTQGALLNDK, 2- YNGIITDTIK, 3- YGNGVWIGR, 4- GDVFVIR 29 1- IDLWSYNAELLVALENQHTIDLTDSEMNK, 2- STQAAIDQINGK, 3- SQQAVIPNIGFRPR, 4- LNWLTHLNFK 1- TLLMNELGVPFHLGTK, 2- LVDSVVSWSK, 3- SGYSGIFSVEGK, 4- GWAFDDGNDVWMGR 1- LSGAMDELHNEILELDEK, 233 FTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGR, 32 3- NLNSLSELEVK, 4- ADTISSQIELAVLLSNEGIINSEDEHLLALER 1- GVTLLLPEPEWTYPR, 2- LNVETDTAEIR, 3- YGEAYTDTYHSYANK, 4- GNSAPLIIR 1- GGLEPINFQTAADQAR, 2- ISQAVHAAHAEINEAGR, 3- LTEWTSSNVMEER, 4- NVLQPSSVDSQTAMVLVNAIVFK
(Bos Taurus)
ADH – Alcohol Dehydrogenase (Saccharomyces cerevisiae)
H1 – Hemagglutinin A/California (H1N1)
N1 – Neuraminidase A/California (H1N1)
H3 – Hemagglutinin A/Victoria (H3N2)
N2 – Neuraminidase A/Victoria (H3N2)
HB – Hemagglutinin B/Brisbane
NB – Neuraminidase B/Brisbane
OV – Ovalbumin (Gallus gallus)
QconCAT Final Sequence: MAGR ~ BSA-1 ~ ADH-1 ~ H1-1 ~ H3-1 ~ HB-1 ~ N1-1 ~ N2-1 ~ NB-1 ~ OV-1 ~ OV-2~ NB-2 ~ N2-2 ~ N1-2 ~ HB-2 ~ H3-2 ~ H1-2 ~ ADH-2 ~ BSA-2 ~ HB-3 ~ N1-3 ~ N2-3 ~ NB-3 ~ OV-3 ~ BSA-3 ~ ADH-3 ~ H1-3 ~ H3-3 ~ H3-4 ~ H1-4 ~ ADH-4 ~ BSA-4 ~ OV-4 ~ NB-4 ~ N2-4 ~ N1-4 ~ HB-4 ~ LAAALEHHHHHH
Tagged peptides SpikeTides
Low cost commercial peptides from JPT Peptide Technologies Custom synthesized peptides Peptide quantified via a coupled chromophore Chromophore tag removed by trypsin
Hemagglutinin – H1 µg H1 / mL, relative to Reference Antigens 80
E
70
B
60 50
C
D
A
40
30 20 10 0 1A 2A 3A 4A 5B 6B 7B 8B 9B M1 M2 10C11C12C13D14D15E 16F 17F 18F
H1-Rel to M1
H1 Rel to M2
Reference antigens M1 and M2 contain 46 and 35 mg H1/mL, respectively
Neuraminidase – N1 µg N1 / mL vaccine 25
B
F
20
C
D
15
A 10
5
0 1A
2A
3A
4A
5B
6B
7B
8B
9B
M1
M2
10C 11C 12C 13D 14D 15E 16F 17F 18F
Hemagglutinin – H3 F
80
µg H3 / mL, relative to Reference Antigen M3 70
60
A
50
C
B
40
30
20
10
0 1A
2A
3A
4A
5B
6B
7B
8B
9B
M3
10C
11C
12C
13D
14D
15E
H3 rel to M3
Reference antigen M3 stated to contain 55 mg H3 / mL
16F
17F
18F
Neuraminidase – N2 µg N2 / mL vaccine F
8.0
7.0
6.0
5.0
C
B
A
4.0
3.0
2.0
1.0
0.0
1A
2A
3A
4A
5B
6B
7B
8B
9B
M3
10C 11C 12C 13D 14D 15E 16F 17F 18F
Hemagglutinin - B mg HB / mL vaccine 80
70
60
50
40
30
20
10
0 1A
2A
3A
4A
5B
6B
7B
8B
9B
M4
HB - Rel to M4
M5
10C 11C 12C 13D 14D 15E 16F 17F 18F
HB - Rel to M5
Reference antigen M4 and M5 stated to contain 32 and 42 mg HB / mL, respectively.
Neuraminidase - B mg NA (B) / mL vaccine 30
F 25
20
C
D
15
B A 10
5
0
* Quadrivalent vaccines have NB from both B strains.
Overall Comparison of Methods
Comparison of method attributes** Hi 3
Speed
Cost Accuracy Dynamic Range
Precision
** using problematic peptides
QconCAT
SpikeTide
Thank you for your attention HC Lab Colleagues
HC Review Colleagues
- Mass spectrometry - Marybeth Creskey - Lisa Walrond - Daryl Smith - Yi-Min She
Vaccines/Hormones and Enzymes/Cytokines/Monoclonal Antibodies
- Virology - Sean Li - Aaron Farnsworth - Caroline Gravel - NMR - Yves Aubin - Genevieve Gingras
-
Evangelos Bakopanos Sherri Boucher Chantal Depatie Nathalie Fortin Nancy Green Richard Isbrucker Jeremy Kunkel Richard Siggers Jeffrey Skene Dean Smith Tong Wu
