Characterization of Complex Prophylactic Vaccines with Protein and Glycoconjugate Components Paul W. Brown, Deanna C. Schuchmann, Nathan A. Lacher, Robert L. Dufield, James A. Carroll and Jason C. Rouse Mass Spectrometry and Biophysical Characterization Group Analytical Research and Development Pfizer, Inc. 12-September-2012 9 th CASSS Symposium Innovate
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Characterization of Complex Prophylactic Vaccines with ...€¦ · Characterization of Complex Prophylactic Vaccines with Protein and Glycoconjugate Components Paul W. Brown, Deanna
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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
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<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)
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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
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Evaluation only.
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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
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)
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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
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.
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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
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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
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UV Profile of Polysaccharide CRM197 Conjugate Digested with Lys-C
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.
Miscleaved acetylated peptides are about 10X lower.
214 nm
UV Profile of Polysaccharide CRM197 Conjugate Digested with Lys-C
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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
N-Acetyl Triazole is Minimized in the New Conjugation Process
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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
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..
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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.