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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
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