Mass Spectrometry: What is it good for?

2016/09/30 Mass Spectrometry: What is it good for? Marybeth Creskey, Lisa Walrond and Terry D. Cyr Regulatory Research Division Centre for Biologics...
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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