2016/09/30

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

Mass Spectrometry: What is it good for?

National Institute of General Medical Sciences

22

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.

Analysis of Biological products by Mass Spectrometry:

Primary sequence

Tertiary structure

Post translational modifications

Degradation products

Host cell proteins

Bioavailability

Potency

Annual Influenza Vaccine

~ 100, 000 A and B sequence entries (GISAID)

Host cell proteins

Influenza proteins -

three or four strains 15 μg hemagglutinin/0.5mL ea

A(H1N1) A(H3N2) B

http://www.itqb.unl.pt/labs/protein-

modelling/activities/haemagglutinin

http://www.rcsb.org/pdb/explore

1 DTLCIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDKHNGK LCKLRGVAPL

_51 HLGKCNIAGW ILGNPECESL STASSWSYIV ETPSSDNGTC YPGDFIDYEE

101 LREQLSSVSS FERFEIFPKT SSWPNHDSDK GVTAACPHAG AKSFYKNLIW

151 LVKKGNSYPK LSKSYINDKG KEVLVLWGIH HPSTSADQQS LYQNADAYVF

201 VGSSRYSKTF KPEIAIRPKV RDREGRMNYY WTLVEPGDKI TFEATGNLVV

251 PRYAFAMERN AGSGIIISDT PVHDCNTTCQ TPKGAINTSL PFQNIHPITI

301 GKCPKYVKST KLRLATGLRN IPSIQSRGLF GAIAGFIEGG WTGMVDGWYG

351 YHHQNEQGSG YAADLKSTQN AIDEITNKVN SVIEKMNTQF TAVGKEFNHL

401 EKRIENLNKK VDDGFLDIWT YNAELLVLLE NERTLDYHDS NVKNLYEKVR

451 SQLKNNAKEI GNGCFEFYHK CDNTCMESVK NGTYDYPKYS EEAKLNREEI

501 DGVKLESTRI YQILAIYSTV ASSLVLVVSL GAISFWMCSN GSLQCRICI

- Increased instrument resolution and sensitivity.

- Increased peptide IDs, ~50% sequence coverage

Ambiguous IDs

Strain ID – Method Development

1 dose vaccine (15 µg HA/500 µl)

Transfer to filter

(10K MWCO)

Reduction, alkylation, quench reaction

Centrifugation wash steps

Deglycoslyation (+3)

Protein digestion - centrifuge enzyme

solution through filter

New collection tube

Dry down flowthrough (=peptides)

Resuspend in injection buffer

trypsin chymotrypsin

DTT

iodoacetamide

DTT

iodoacetamide

PNGaseF in

H2O18

PNGaseF in

H2O18

Fusion : LC-MSMS

Triplicate injections

Peak list processing

Merge 6 LC-MSMS runs search

In-house influenza database

- On-filter digestion, triplicate preps

- Hundreds of peptide IDs, >90% sequence coverage

Routinely Achieve Unambiguous ID

1 DTLCIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDKHNGK LCKLRGVAPL

_51 HLGKCNIAGW ILGNPECESL STASSWSYIV ETPSSDNGTC YPGDFIDYEE

101 LREQLSSVSS FERFEIFPKT SSWPNHDSDK GVTAACPHAG AKSFYKNLIW

151 LVKKGNSYPK LSKSYINDKG KEVLVLWGIH HPSTSADQQS LYQNADAYVF

201 VGSSRYSKTF KPEIAIRPKV RDREGRMNYY WTLVEPGDKI TFEATGNLVV

251 PRYAFAMERN AGSGIIISDT PVHDCNTTCQ TPKGAINTSL PFQNIHPITI

301 GKCPKYVKST KLRLATGLRN IPSIQSRGLF GAIAGFIEGG WTGMVDGWYG

351 YHHQNEQGSG YAADLKSTQN AIDEITNKVN SVIEKMNTQF TAVGKEFNHL

401 EKRIENLNKK VDDGFLDIWT YNAELLVLLE NERTLDYHDS NVKNLYEKVR

451 SQLKNNAKEI GNGCFEFYHK CDNTCMESVK NGTYDYPKYS EEAKLNREEI

501 DGVKLESTRI YQILAIYSTV ASSLVLVVSL GAISFWMCSN GSLQCRICI

Strain ID – Method Development

H1

98%

H3

90%

HB

95%

N1

89%

N2

89%

NB

91%

HCP

Hemagglutinin (HA)

Neuraminidase (NA)

Host cell proteins

But how much is in there?

Digest

LC-MS

Quant

intensity

m/z

Hi3 QconCAT Synthetic Peptides

Influenza Antigen Quantitation

MS Protein Quantitation – Hi3 Method

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.

Signal intensity from tryptic peptides from

three equimolar proteins

MS Protein Quantitation Hi3 standards

Quantification of antigens can be made by comparing to a

spiked reference standard

Average Hi3 Peptide Intensity

Hi3 versus Western blot

Vaccine sample

µG

NA

/dose

Stable isotope labeled peptides

Measurement accuracy

– Efficiency of proteolysis

– Differential losses during fractionation

Method range

– 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

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 5PolyQuant

- 4 peptides

- link peptides with a spacer (ASGK)

- Peptide set are dispersed

- C-terminal polyhistidine tag

QconCAT designs

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

QconCat1 QconCat2 QconCat3 QconCat4 QconCat5

Ratio of Average Hi3 Value

from all proteins in QconCAT

Protein PeptidesBSA – Bovine Serum Albumin (Bos Taurus)

1- LGEYGFQNALIVR, 2- LVNELTEFAK,

3- DAFLGSFLYEYSR, 4- HLVDEPQNLIK

ADH – Alcohol Dehydrogenase (Saccharomyces cerevisiae)

1- VVGLSTLPEIYEK, 2- LPLVGGHEGAGVVVGMGENVK,

3- SISIVGSYVGNR, 4- ANELLINVK

H1 – Hemagglutinin A/California (H1N1)

1- EVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSR, 2- STQNAIDEITNK

3- MNYYWTLVEPGDK, 4- MNTQFTAVGK

N1 – Neuraminidase A/California (H1N1)

1- TFFLTQGALLNDK, 2- YNGIITDTIK,

3- YGNGVWIGR, 4- GDVFVIR

H3 – Hemagglutinin A/Victoria (H3N2)

1- IDLWSYNAELLVALENQHTIDLTDSEMNK, 2- STQAAIDQINGK,

3- SQQAVIPNIGFRPR, 4- LNWLTHLNFK

N2 – Neuraminidase A/Victoria (H3N2)

1- TLLMNELGVPFHLGTK, 2- LVDSVVSWSK,

3- SGYSGIFSVEGK, 4- GWAFDDGNDVWMGR

HB – HemagglutininB/Brisbane

1- LSGAMDELHNEILELDEK, 2-

FTSSANGVTTHYVSQIGGFPDQTEDGGLPQSGR,

3- NLNSLSELEVK, 4- ADTISSQIELAVLLSNEGIINSEDEHLLALER

NB – NeuraminidaseB/Brisbane

1- GVTLLLPEPEWTYPR, 2- LNVETDTAEIR,

3- YGEAYTDTYHSYANK, 4- GNSAPLIIR

OV – Ovalbumin (Gallus gallus)

1- GGLEPINFQTAADQAR, 2- ISQAVHAAHAEINEAGR,

3- LTEWTSSNVMEER, 4- NVLQPSSVDSQTAMVLVNAIVFK

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

QconCAT Sequence

38

29

3332

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

0

10

20

30

40

50

60

70

80

1A 2A 3A 4A 5B 6B 7B 8B 9B M1 M2 10C11C12C13D14D15E16F 17F 18F

µg H1 / mL, relative to Reference Antigens

H1-Rel to M1 H1 Rel to M2

Reference antigens M1 and M2 contain 46 and 35 mg H1/mL, respectively

A

E

DCB

Hemagglutinin – H1

0

5

10

15

20

25

1A 2A 3A 4A 5B 6B 7B 8B 9B M1 M2 10C 11C 12C 13D 14D 15E 16F 17F 18F

µg N1 / mL vaccine

A

C D

FB

Neuraminidase – N1

0

10

20

30

40

50

60

70

80

1A 2A 3A 4A 5B 6B 7B 8B 9B M3 10C 11C 12C 13D 14D 15E 16F 17F 18F

µg H3 / mL, relative to Reference Antigen M3

H3 rel to M3

Reference antigen M3 stated to contain 55 mg H3 / mL

A

F

CB

Hemagglutinin – H3

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

1A 2A 3A 4A 5B 6B 7B 8B 9B M3 10C 11C 12C 13D 14D 15E 16F 17F 18F

µg N2 / mL vaccine

AC

F

B

Neuraminidase – N2

0

10

20

30

40

50

60

70

80

1A 2A 3A 4A 5B 6B 7B 8B 9B M4 M5 10C 11C 12C 13D 14D 15E 16F 17F 18F

mg HB / mL vaccine

HB - Rel to M4 HB - Rel to M5

Reference antigen M4 and M5 stated to contain 32 and 42 mg HB / mL, respectively.

Hemagglutinin - B

* Quadrivalent vaccines have NB from both B strains.

0

5

10

15

20

25

30

mg NA (B) / mL vaccine

A

C D

F

B

Neuraminidase - B

Overall Comparison of Methods

Hi 3 QconCAT SpikeTide

Speed

Cost

Accuracy

Dynamic

Range

Precision

Comparison of method attributes**

** using problematic peptides

Thank you for your attention

HC Lab Colleagues- Mass spectrometry

- Marybeth Creskey

- Lisa Walrond

- Daryl Smith

- Yi-Min She

- Virology

- Sean Li

- Aaron Farnsworth

- Caroline Gravel

- NMR

- Yves Aubin

- Genevieve Gingras

HC Review ColleaguesVaccines/Hormones and

Enzymes/Cytokines/Monoclonal

Antibodies

- Evangelos Bakopanos

- Sherri Boucher

- Chantal Depatie

- Nathalie Fortin

- Nancy Green

- Richard Isbrucker

- Jeremy Kunkel

- Richard Siggers

- Jeffrey Skene

- Dean Smith

- Tong Wu