Characterization of Complex Prophylactic Vaccines with Protein and GlycoconjugateComponents

Paul W. Brown, Deanna C. Schuchmann, Nathan A. Lacher, Robert L. Dufield, James A. Carroll and Jason C. Rouse

Mass Spectrometry and Biophysical Characterization GroupAnalytical Research and DevelopmentPfizer, Inc.

12-September-20129th CASSS Symposium

Innovate

<20

kDa

Outline

•Introduction to Vaccine Products

1) Examples of vaccines; heterogeneous products and can comprise proteins, polysaccharides conjugates, adjuvants, etc.

2) Mass spectrometry characterization for vaccine product characterization.

3) Three examples of the use of mass spectrometry to provide enhanced product characterization:

•Detection of an uncommon post-translational modification of a protein constituent in a vaccine

•Detection and identification of a low abundance host cell protein in drug substance by a top-down proteomics approach

•Discovery of an unusual modification in a protein-polysaccharide conjugate induced by conjugation chemistry

2

<20

kDa

Importance of Vaccines

3

“With the exception of safe water, no other modality, not even antibiotics, has had such a major effect on mortality reduction and population growth.(1.)

1. Plotkin S, Orenstein W, and Offit P. Vaccines, 5th ed. Saunders, 2008

Examples of VaccinesGlycoconjugate Vaccine(polysaccharide antigen conjugated to carrier protein)

Virus-Like Particle Vaccine

Recombinant Protein Vaccine

Hapten Conjugate Vaccine(small molecule antigen conjugated to carrier protein)

Killed or Inactivated

4

Strategy for Antibody Characterization usingMass Spectrometry

Measure Molecular Mass

Measure Proteolytic Peptide Masses

1) Confirm amino acid sequence(100% coverage)

2) ID and localize PTMs3) Determine occupancies

Measure Subunit Masses

Intact IgG Subunits

TrypsinDigest

Characterize majorand minor isoforms

1) Confirm AA Sequence2) Localize PTMs3) Determine occupancies4) Confirm disulfides

(non-reduced)

Peptide Level

Reduce

PNGaseDigest/

AB der

Released N-Glycans1) Confirm structures2) Quantitate structures

(LC/fluorescence)

5

Reduce

Type of Vaccine

Typical Mass(kDa)

MSCharacterization Strategy

Special Techniques

Recombinant Protein

25-250 Intact and peptide map

•Confirm disulfide linkages using non-reduced peptide map

Virus-Like Particle

50-3000 Reduce and measure subunit mass, reduced peptide map

•Denature and/or Reduce disulfides to reduce complexity•Measure Intact by SEC/MALLS

HaptenConjugate

30-100 Intact and peptide map

•Need assay to measure drug load•Determine sites of conjugation

Glyco-conjugate

50-200 for polysaccharide,500 to 5000 for conjugate

Peptide map •SEC/MALLS to measure mass of polysaccharide and conjugate•Perform peptide map of conjugate•NMR to confirm structure of polysaccharide

Strategy for Vaccine Characterization usingMass Spectrometry

6

Evaluation only.

Created with Aspose.PowerPoint.

Copyright 2004 Aspose Pty Ltd.

Molecular Mass Distribution of Purified Polysaccharide Determined by SEC/MALLS

Massaldi H, et al. (2010) Features of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14. Biotechnol Appl Biochem. 55:37-43

Molar Mass against Time

Mol

ar M

ass

(g/m

ol)

1.0X107

1.0X105

1.0X105 90,000

50,000

7

Example #1: Detection of an uncommon post-translational modification

<20

kDa

Recombinant Protein Component in Vaccine

Non-glycosylated, single chain protein expressedin E. coli (~55 kDa with no disulfides)

MS approach-intact protein mass analysis-proteolytic digestion

8

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 450.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

3.5e-1

4.0e-1

4.5e-1

5.0e-1

36.68

0 54500 54750 55000 55250 55500 55750 56000 56250 56500 56750

55126.4

55166.4

LC/MS Analysis of Drug Substance

Spectrum of Intact Protein

Intact Protein

+N-Acetylation (+42 Da)

+178 Da modification (~5%)

37.6 min.55126.4

55168.4

55304.0

9

F: FTMS p ESI Full ms [200.00 2000.00]

1650 1700 1750 1800 1850m/z

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

160000

170000

180000

190000

200000

Inte

nsity

1668.70301

1690.68381

1846.750741712.66612

1734.648681650.69406 1805.76224

178.0477 Da

(M + Na)

LC/MS of Lys-C Peptide Map

11.0 11.5 12.0Time (min)

11.47

10.98

11.6711.25 11.8910.80

Unknown (~5%)178 Da modification

K3

K1 2-16

K1

The MS/MS data confirmed modification on N-terminus.Two formulas match within 5 ppm, C6H10O6 (valid) and CH7ON8P (“unreasonable”).

Deconvoluted Spectrumof Unknown and K1

214 nm

K1 N-Acetyl elutes later

1668.7030 K1

1846.75071690.6838

SpeciesObserved Mass (Da)

Theoretical Mass (Da)

Mass Accuracy (ppm)

Gluconoylation of K1 peptide 1846.7507 1846.7552 2.5

Identification of the 178 Modification in Lys-CPeptide Map

10

Anion Exchange Chromatography of Three Lots of Drug Substance Dionex ProPac WAX-10

AU

0.00

0.05

0.10

0.15

0.20

0.25

13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 23.00 2

Batch 03

Batch 01

Batch 02

Peak represents6-Phospho-gluconoylation (+258 Da).All peaks were fraction collected and analyzed byLC/MS.

Deamidation and gluconoylation (+178 Da)

Intactunmodifiedprotein

11(min.)

Gluconoylation •Mass addition 178.0477 Da is consistent with gluconoylation.•MS/MS confirmed modification is localized to N-terminus.•Gluconoylation is a non-enzymatic reaction, so some random gluconoylation of lysine side chains is expected.•Previously detected in Escherichia coli produced proteins (ref below).•6-Phosphogluconoylation (+258 Da) was observed in some lots at lower levels.

+178 Da modification in Delta Mass Database“Spontaneous α-N-6-Phosphogluconoylation of a “His Tag” in Escherichia coli: The Cause of Extra Mass of 258 or 178 Da in Fusion Proteins”, Anal Biochem. 1999 Feb 1;267(1):169-84.

d-Gluconolactone

OO

HO

OHHO

OH

+ H2N-R

OH

HO

OH

OH OHN-R

O

H

Gluconoylamine group(from protein)

12

Example #2: Vaccine Protein Constituent Characterization: Detection of a LowLevel Host Cell Protein

<20

kDa

Recombinant Protein Vaccine

Non-glycosylated, single chain protein expressedin E. coli (~30 kDa)

MS approach-intact protein mass analysis-proteolytic digestion-top-down ID on HCP

13

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35Time (min)

LC/MS Analysis of Drug Substance

Peak 1 has Clip #1 and unknown (0.3%)

Peak 2 (Clip #2) Peak 3

(Clip #3)

20X Zoom

214 nm

21 22 23 24 25 26 27 28 29 30 31 32Time (min)

IntactProtein

14

p [ ]

900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

z=3

z=4

z=11

z=10z=4

z=1z=9z=3

z=1 z=1z=8 z=6z=7

z=6

ESI Mass Spectrum of Peak 1 With Two Components

* ions associated with unknown^ low level clip 1 of vaccine protein

^

** *

* * *

^

p [ ]

1183.4 1183.6 1183.8 1184.0 1184.2 1184.4 1184.6 1184.8m/z

Monoisotopic mass of unknown is 10641.547 Da

Zoom view of 9+

charge state

15

CID of 9+ charge stateprovided highest mascot score

Considerations for MASCOT SearchingImplemented “Didehydro” variable modification to reconcile

intra-molecular disulfide bonds

Submit deconvoluted deisotoped mass spectra list

Utilize Mascot TD due to its higher mass limit

Utilize MS3 for verification and unusual modifications

Signal dilution with higher mass

Switch from TFA to formic acid

Considerations for Top-Down

16

p @ [ ]

600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1253.0899z=7

941.9673z=2

1461.7699z=6

1357.7091z=6820.4065

z=2

1263.2376z=7

770.8724z=2 906.4489

z=2

1412.2451z=6

1523.7625z=2

988.4729z=1

706.8430z=2 1096.5794

z=8 1147.0847z=2598.9656

z=3 1613.8549z=4

526.5635z=?

1010.5061z=?

CID Mass Spectrum of Unknown 9+ Charge State

CID is performedin ion trap (LTQ)

17

T : F T M S p ES I F u ll m s2 1 1 8 4 .0 0 @ cid 3 5 .0 0 [3 2 5 .0 0 2 0 0 0 .0 0 ]

2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 00

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Rel

ativ

e Ab

unda

nce

8 7 6 0 .5 8 5 4

1 8 8 2 .9 2 9 4

8 1 3 7 .2 2 3 3

3 0 4 5 .5 1 9 57 8 7 6 .1 0 7 61 6 3 9 .8 0 7 9

3 7 6 9 .9 3 0 1 7 3 7 4 .9 0 4 15 7 1 6 .0 6 9 3

Deconvoluted Deisotoped Spectrum of the Unknown

b16

y27

y80

y85y34

Mass error Under 5 ppm for fragment ions

19 b-type ions 41 y-type ions

MASCOT Score: 267

b8

y72

y74

b18

y40y69

H A H L T H Q Y P A A N A Q V T A A P Q

A I T L N F S E G V E T G F S G A K I T

G P K N E N I K T L P A K R N E Q D Q K

Q L I V P L A D S L K P G T Y T V D W H

V V S V D G H K T K G H Y T F S V Ky8

b6 b7

b26

b8 b10 b11 b12 b13 b14 b15 b17 b18 b20

b85

y13 y10y18

y28 y27y34y36 y35

y48y51y58

y58y59y63y64y66y67y68y69y70y71y72y73y74y75y76y77y78

y79y80y81y82y83y84y85

b16

b23 b24

y38

y40

y83

b18

b21

y76

Yoba: Escherichia coli Protein

y81

18

Internal fragy80-b71

Lessons Learned for Top-down Approach

Top-down approach using CID on the Orbitrap was successfully applied to rapidly identify proteins up to 34 kDa

Very sensitive and can be performed on-line

Mass list can be quickly pasted into text file

General observation: Recombinant proteins produced by bacterial systems or non-mAb proteins can contain detectable levels of HCPs as compared to levels found in mAbs which are purified by Protein A and ion exchange processes. Higher titer in mAbs production and the non-lysing of cells also helps.

19

Conjugation Diagram of Conjugate Vaccine

Example #3: Detection of an Unusual Modificationin a Polysaccharide-Protein Conjugate

PolyActivatedAntigen CRM197 (58kDa)

Conjugate

Activation

Purification

Conjugation/Purification

Many combinations

20

21

Strategy for Glycoconjugate Vaccine Characterization

•SEC/MALLS to measure mass of polysaccharide and conjugate•NMR to confirm structure of polysaccharide•MS approach-proteolytic digestion

103.29 129.57

108.11

87.8284.11125.17

112.7896.31

94.51

114.25

137.77135.18120.5191.77

102.03

25 30 35 40 45 50 55 60 65 70 75 80Time (min)

52.63

68.63

78.59

46.50

77.66

60.0575.25

36.1527.21

39.2154.27

65.1144.3355.6740.39

74.60

28.70 49.60 57.67 64.0934.68 43.7133.87

26.496

K23

K1

and

?K

31 a

nd?

K21

K5-

6

K4

K12

K20

K3

K7

K10

& K

11

K34

K6

K8

245-

253

and

?

K2

and

?K

36K

35 a

nd?

K34

254-

264

?

?

?

??

?

??

?

?

? ? Pyro

-Q 2

45-2

53

UV Profile of Polysaccharide CRM197 Conjugate Digested with Lys-C

85 90 95 100 105 110 115 120 125 130 135 140Time (min)

K29

K17

K39

K26

and

?

K15

K14

K27

*

126-

155 K30

Pyro

-QK

26

*

K2-

3

?

??

?

??

??

?

?

???

??

?

?

?

Unknown modified peptides are about 10X lower.

214 nm

22

Peptide ResiduesObserved

Mass(Da)

TheoreticalMass(Da)

SequenceMass Error(ppm)

K9-10 77-90 1430.8151 1430.8133AGGVV(ac)KVTYPGLTK 1.3

K10-11 83-95 1443.8723 1443.8701VTYPGLT(ac)KVLALK 1.5

Detection and Elucidation of Miscleaved Peptides With Single Site of Acetylation at Internal Lys Residues

Acetylated lysine is impervious to proteolytic digestion and results in a miscleavage.

Acetylated peptides matched intact mass within 5 ppm andmost were confirmed by data dependant acquisition of CID mass spectra.37 of the theoretical 39 miscleaved peptides with one acetylation were detected.

23

103.29 129.57

108.11

87.8284.11125.17

112.7896.31

94.51

114.25

137.77135.18120.5191.77

102.03

25 30 35 40 45 50 55 60 65 70 75 80Time (min)

52.63

68.63

78.59

46.50

77.66

60.0575.25

36.1527.21

39.2154.27

65.1144.3355.6740.39

74.60

28.70 49.60 57.67 64.0934.68 43.7133.87

26.496

K23

K1

and

K22

-23A

cK

31 a

ndK

37-3

8 A

cK21

K5-

6

K4

K12

K20

K3

K7

K10

& K

11

K34

K6

K8

245-

253

and

K5-

6 A

c

K2

and

K8-

9 A

c K

36K

35 a

ndK

9-10

Ac

K34

254-

264

K24

-25

Ac

K21

-22A

c

K18

-19

Ac

K5-

6 A

cK

19-2

0 A

c

K3-

4 A

c

K6-

7 A

cK

7-8

Ac

K11

-12

AcK32

-33

Ac

K4-

5 A

cK

23-2

4 A

c an

d K

12-1

3Ac

Pyro

-Q 2

45-2

53

85 90 95 100 105 110 115 120 125 130 135 140Time (min)

K29

K17

K39

K26

and

K2-

3 A

c

K15

K14

K27

*

126-

155 K30

Pyro

-QK

26

*

K2-

3

K35

-36

Ac

K25

-26A

cK

1-2

Ac

K1-

3 A

c

K10

-11

Ac

K17

-18

Ac

K39

-40

Ac

K38

-39

Ac K13

-14

Ac

K30

-31A

c

K26

-27A

c

K29

-30A

cK

13-1

4Ac

K14

-15A

cK

12-1

4

2Ac

K12

-14A

c

HC

P#5

K7-

9

2Ac

Miscleaved acetylated peptides are about 10X lower.

214 nm

UV Profile of Polysaccharide CRM197 Conjugate Digested with Lys-C

24

Extracted Mass Chromatogram of Polysaccharide CRM197 Conjugate Digested with Lys-C EVERY high resolution mass spectrum (100,000 at m/z 400) in the TIC was deconvoluted and deisotoped to the monoisotopic zero charge state using Thermo Xtract software.

The theoretical masses of the miscleaved acetylated peptides of CRM197 were calculated using Excel.

The theoretical masses were copied from Excel and pasted in Thermo Qual Brower to produce overlaid extracted ion chromatogram (+5 ppm).

What is root cause of acetylation?RT: 10.0 - 160.0

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 16Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

120.8

102.3

113.3

88.0

112.5

76.7

91.682.795.378.9 138.7

65.458.0105.980.2

47.4 110.049.956.046.035.3

40.3 127.8

39.3 129.568.9

38.7126.5 135.5

86.1130.0 156.9155.0114.1 139.6 142.0122.096.275.358.654.7 70.515.8

K6-

7 A

cK

7-8

Ac

K11

-12

Ac

K8-

9 A

c K

9-10

Ac

K5-

6 A

c K

19-2

0 A

c

K20

-21

Ac

K37

-38

Ac

K24

-25

Ac

K21

-22A

cK

22-2

3Ac

K18

-19

Ac

K4-

5 A

cK

23-2

4 A

c an

d K

12-1

3Ac

K32

-33

Ac

K3-

4 A

c

K36

-37

Ac

K7-

9

2Ac

K2-

3 A

cK

35-3

6 A

cK

25-2

6Ac

K1-

2 A

c

K10

-11

Ac

K17

-18

Ac

K39

-40

Ac

K38

-39

Ac

K13

-14

Ac K

30-3

1Ac

K29

-30A

cK

12-1

4Ac

K26

-27A

c

K13

-14A

c

K14

-15A

c

25

6 8 10 12 14 16Time (min)

10.63

6.38

16.8515.7914.7713.74

8.97 9.268.107.73

LC-MS Analysis of Reaction Mixture Following Polysaccharide Activation

UnknownPeak of Interest

214 nm

PolyActivatedAntigen

Activation

Purification

26

ActivatedAntigen

Electrospray Mass Spectrum of Unknown

C4H6ON3+ There is one molecular formula within 5 ppm.

0.1 ppm112.05053

27

m/z 113 envelope

N-Acetyl Triazole

Electrospray Mass Spectrum of UnknownSecondary isotopes at m/z 113 give separate signals at an ultra-high resolution of 250,000.

113.04750

113.05386

0.6 ppm

C313CH6ON3

+

0.4 ppm

C4H6ON3+ There is one molecular formula within 5 ppm.

0.1 ppm112.05053

Zoom of m/z 113 to show fine structure

C4H6ON215N+

0.6 ppm

Ultra-high resolution MS/MS was also used to confirm structure N-Acetyl Triazole 28

Root Cause of Acetylated CRM197(Carrier Protein) is NAT

N-Acetyl Triazole (NAT) acetylates the epsilon-amino group of thelysine residues and N-terminus on carrier protein.

Identity of NAT confirmed with synthetic standard.

N

N

NO CH

CH2CH2CH2CH2NH2

HN

O

+ +CHCH2CH2CH2CH2

HN

HN

O

N

N

NH

O

NAT lysine on carrier protein acetylated lysine

29

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

Comparison of Two Individual Conjugate Samples

K6-

7 A

cK

7-8

Ac

K11

-12

Ac

K8-

9 A

c K

9-10

Ac

K5-

6 A

c K

19-2

0 A

c

K20

-21

Ac

K37

-38

Ac

K24

-25

Ac

K21

-22A

cK

22-2

3Ac

K18

-19

Ac

K4-

5 A

cK

23-2

4 A

c an

d K

12-1

3Ac

K32

-33

Ac

K3-

4 A

c

K36

-37

Ac

K2-

3 A

cK

35-3

6 A

cK

25-2

6Ac

K1-

2 A

c

K10

-11

Ac

K17

-18

Ac

K39

-40

Ac

K38

-39

Ac

K13

-14

Ac

K30

-31A

c

K29

-30A

c

K26

-27A

c

K13

-14A

c K14

-15A

c

Sample #2

Sample #1

3.30E6/ 2.54E7 = 13%

Ratio 13/12 = 1.11.8E6/ 1.47E7 =12%

30

The acetylation levels are consistent

RT: 0.0 160.5

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

87.3

112.5120.1

78.2

101.5

91.075.979.5

82.155.3 111.734.7 46.8 64.746.2

127.547.245.338.7 94.7109.268.1 135.1

138.48.6 153.695.5 142.59.4 69.659.5

87.4 120.0101.678.2 112.655.3 91.1 154.764.846.939.8 127.434.8 135.2 153.368.2

New Process:Acetylated Peptides are reduced 35X

Original Process

N-Acetyl Triazole is Minimized in the New Conjugation Process

31

Extracted mass chromatograms facilitate the comparisons

Conclusions

•Vaccines can be multi-component products where the development effort applied is equivalent to several mAb projects, so mass spectrometry is essential to provide rapid, efficient, comprehensive characterization.

•For vaccine glycoconjugates, the 100kDa to 3MDa molecular masses, in conjunction with high polysaccharide polydispersity and micro-heterogeneity, exceeds the technical capabilities of modern ionization techniques/mass spectrometers to detect and measure these intact species.

•Vaccines require special investigations in order to characterize unique and unexpected posttranslational modifications, as well as low level host cell proteins, as compared to standard mAbs.

•The on-line top-down approach for the ID of low-level HCPs is faster and more efficient than conventional proteomic methods.

•Mass spectrometry support of the conjugation process can expedite vaccine process development and production, ensuring side reactions pertaining to the conjugation chemistry are minimized or eliminated.

+5 ppm allows for tremendous specificity

Software collapses multiple charge states and multiple isotopic distributions

Facilitates the comparison of multiple species and their levels

RT: 10.0 - 150.0

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

96.2

117.3

83.1

105.2

56.8

108.6

140.594.2 117.9101.545.2 124.020.5 72.1 108.876.0 78.624.5

NL: 1.78E4m/z= 762.3874-762.3950+1843.9529-1843.9713+1904.9385-1904.9575+2204.1691-2204.1911+3518.7461-3518.7813+4815.4552-4815.5034 MS 20110928PWB-01_xtract

Message: Xtract is Powerful Tool for ScreeningMultiple Theoretical Masses Simultaneously

Quickly search for oxidation, linker location, “haptens”, HCP, sequence variants, and clips

Six tryptic peptides were detected and associated with this HCP.There were only two peptides that had CID mass spectra during DDA.The response is about 1000 times lower than therapeutic protein..

33

AcknowledgmentsJames CarrollJason RouseDeanna SchuchmannNathan LacherRobert Dufield Tracy ScottJohn BuckleySteve KolodziejMatt ThompsonOlga FrieseJustin SperryKeith JohnsonJacky SmithQin Zou Marta Czupryn

This week: Would enjoy talking to you about how your lab is performing top-down, using Xtract, and vaccine characterization.

34