Table 1.
An overview of the role of MS technologies in the vaccine development
| Antigen | Study | Technique and instrumentation | Reference |
|---|---|---|---|
| HIV gp120 Recombinant envelope glycoprotein (rgp120) expressed in CHO cells. | First study analyzing the site‐specific N‐glycosylation of gp120. This report describes the structural characterization of the expressed gp120. | Reversed phase HPLC of the tryptic digest. Peptides collected from RP‐HPLC were further identified by amino acid analysis (AAA) or N‐terminal sequencing analysis. | Leonard et al38 |
| HIV‐1 gp120 Expressed in CHO Cells. | Mass spectrometric characterization of the glycosylation pattern. Of HIV‐1 gp120. | MALDI and nanoESI‐LC‐MS/MS using a hybrid quadrupole‐time‐of‐flight tandem mass spectrometer (Q‐TOF) was used to assign glycosylation sites. | Zhu et al111 |
| HIV gp140 JR‐FL and CON‐S Env expressed in CHO and HEK293 cells. | A glycopeptide‐based mass mapping approach was used to characterize the glycosylation of two Env protein vaccine candidates in a glycosylation site‐specific fashion. | Trypsin digested HIV envelope glycoprotein was either subjected to off‐line RP‐HPLC fractionation for MALDI or RP‐HPLC/ESI‐FTICR MS analyses. | Go et al41 |
| HIV gp120/gp41 C.CON and C.97ZA012 Env expressed in HEK 293T cells. | Mass spectrometry‐based glycosylation profiling of two rVV expressed clade C HIV‐1 Envs was performed. | Trypsin digested HIV envelope glycoprotein was either subjected to off‐line RP‐HPLC fractionation for MALDI or RP‐HPLC/ESI‐FTICR MS analyses. | Go et al41 |
| HIV gp120 Expressed in HEK293 cells. | Ion mobility ESI‐MS was used to perform the analysis of N‐glycans from engineered glycoforms of intact, folded HIV gp120. | IMS‐MS was performed on a Synapt G2 mass spectrometers. ESI‐MS/MS was performed with a Waters Q‐TOF in negative ion mode. | Harvey et al57 |
| HIV gp140 B.700010040.C9 and C.1086 Env expressed in HEK293T cells. | Glycopeptide‐based mass mapping approach was used to perform site specific glycosylation analysis of the transmitted/founder Envs. | LC/ESI‐MS was performed using a Thermo Scientific LC‐ESI‐LTQ‐FTICR MS directly coupled to the Dionex UltiMate capillary LC system equipped with a FAMOS well plate autosampler. | Go et al42 |
| HIV gp140 JRFL gp140 ΔCF Env expressed in HEK 293 cells. | LC‐MS/MS based comprehensive analysis of a partially deglycosylated HIV vaccine candidate Env using PNGase F, Endo H and Endo F3 glycosidases. | LC/ESI‐MS was performed using a Thermo Scientific LC‐ESI‐LTQ‐FTICR MS directly coupled to the Dionex UltiMate capillary LC system equipped with a FAMOS well plate autosampler. | Go et al33 |
| HIV gp140 CN54gp140 Env expressed in CHO cells. | Comprehensive mass spectrometric analysis of the site‐specific glycosylation of gp120. | LC‐ESI‐MS was performed using an ion trap as well as a Q‐TOF instrument and standard software for glycopeptide identification. | Pabst et al112 |
| HIV gp120 1086.C Env expressed in CHO and HEK293 cell lines. | Evaluated and compared the host‐cell specific glycosylation pattern of recombinant HIV‐1 gp120 expressed in CHO and 293T cell lines. | An integrated glycopeptide‐based LC‐MS/MS mapping workflow that includes a partial deglycosylation step, fragmentation techniques (ETD and CID) was carried out using a hybrid LC‐ESI‐LTQ‐FTICR MS. | Go et al45 |
| HIV gp120 Expressed in HEK293 and ACH‐2 cell lines. | LC‐MS/MS based site‐specific glycosylation profiling of recombinant gp120 was performed using a novel spectral‐aligning strategy. | MS analysis was performed using a Thermo Q Exactive mass spectrometer via higher energy collisional dissociation and spectral‐aligning strategy. | Yang et al21 |
| HIV gp120 C97ZA012 gp140 trimeric Env expressed in HEK293T cell lines (transiently & stably expressed). | Mass spectrometry based glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV‐1 gp140 trimers in 293T cells. | High‐ and low‐resolution LC–MS experiments were performed using two different platforms; LC‐ESI‐LTQ‐FTICR MS coupled to a nanoAcquity UPLC system and the second was an LTQ Velos mass spectrometer with ETD (ThermoScientific) coupled to Acquity UPLC system (Waters). | Go et al46 |
| HIV trimers Soluble native SOSIP.664 trimers (92UG037.8 and CZA97.012 clade A and clade C env genes) expressed in HEK293F cells. | Comprehensive mass spectrometric analysis of the native SOSIP.664 trimers. | LC/MS based glycosylation analysis and HDX‐MS was performed using Waters Synapt G2Si MS. LC‐ESI‐MSMS based disulfide mapping was performed using a LTQ Orbitrap Velos Pro (Thermo Scientific). | Ringe et al47 |
| HIV Env trimers HIV‐1 JR‐FL Env Δ712 and Env Δ808 expressed in CHO cells | MS‐based glycosylation analysis was used to evaluate whether Env glycosylation is dependent on the Env form, ie, membrane anchored or soluble. | High‐resolution LC‐MS/MS was performed using an Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific) coupled to an Acquity UPLC system (Waters). | Go et al113 |
| HIV gp120 JRCSFBG505 Env expressed in HEK293T cells. | Env glycosylation analysis by ion mobility mass spectrometry. | Ion mobility experiments were carried out with a Waters Synapt G2 traveling wave ion mobility mass spectrometer fitted with an ESI ion source. | Harvey et al59 |
| HIV gp120 Gp120SU Env expressed in BaL/SUPT1‐R5 cell line [CLN204]. | A study of the site‐specific N‐glycosylation of virion‐derived gp120 by mass spectrometry showing the dominance of oligomannose‐type glycans on almost all N‐glycosylation sites. | MALDI‐MS/MS was performed using Applied Biosystems 4800 MALDI‐TOF/TOF mass spectrometer operated in reflector positive‐ion mode. Nano‐LC‐ES‐MS/MS analysis was performed using HPLC system connected to a Q‐TOF (API Q‐STAR® Pulsar I, Applied Biosystems/MDS Sciex). | Panico et al114 |
| HIV trimers BG505 SOSIP.664 trimers expressed in stable HEK293T cell line. | A parallel mass‐spectrometric‐based approach exploiting two different ionization modes was used to perform quantitative, site‐specific N‐glycosylation analysis to understand the glycan shield of the BG505 SOSIP.664 trimer. | IM‐ESI MS with a Waters Synapt G2Si mass spectrometer and RP‐HPLC separated glycopeptide pools were analyzed using an Autoflex Speed MALDI‐TOF/TOF (Bruker). Enriched glycopeptides were also analyzed on a Q‐Exactive Orbitrap mass spectrometer coupled to a Dionex Ultimate 3000 nanoLC system. | Behrens et al51 |
| HIV gp120 1086.C and TV1.C Env expressed in stable CHO cell lines. | Mass spectrometry based characterization of a bivalent HIV‐1 Subtype C gp120 protein boost for proof‐of‐concept HIV vaccine efficacy trials in Southern Africa. | High‐ and low‐resolution LC–MS experiments were performed using two different platforms; LC‐ESI‐LTQ‐FTICR MS coupled to a nanoAcquity UPLC system and the second was an LTQ Velos mass spectrometer with ETD (ThermoScientific) coupled to Acquity UPLC system (Waters). | Zambonelli et al48 |
| HIV gp120 1086.C and TV1.C Env expressed in stable CHO cell line. | Comprehensive characterization of reference standard lots of HIV‐1 subtype C Gp120 proteins for clinical trials in southern African regions (HVTN 100 Phase I/II clinical trial material and HVTN 702 Phase IIb/III clinical trial material). | Intact molecular weight was measured using Bruker UltrafleXtreme MALDI‐TOF/TOF instrument. Mol. Weight of de‐N‐glycosylated was determined by LC‐MS using a Waters Xevo G2‐S QTOF. N‐Linked glycosylation site mapping was carried out by analyzing the reduced and alkylated tryptic peptides digested by Endo H, Endo F3, or PNGase F by LC‐MS/MS using a Thermo LTQ Orbitrap MS. LC‐MS/MS analysis for the disulfide mapping was carried out using LTQ Orbitrap with both CID and ETD. | Wang et al63 |
| HIV trimers Eleven different trimeric Env expressed in CHO and HEK293 cells. | Mass spectrometry based approach was used to map the complete glycosylation profile at every site in eleven HIV‐1 Env trimers. | High‐resolution LC‐MS/MS was performed using an Orbitrap Velos Pro hybrid mass spectrometer (Thermo Scientific) equipped with ETD and coupled to an Acquity UPLC system (Waters). | Go et al49 |
| HIV gp120 Monomers of the BG505.SOSIP. | Global N‐Glycan site occupancy of HIV‐1 gp120 by metabolic engineering and high‐resolution intact mass spectrometry was performed. | Released N‐glycan analysis was carried out using Synapt G2Si ion mobility mass spectrometer. Native high‐resolution mass spectrometry was performed on Q Exactive hybrid Quadrupole‐ Orbitrap mass spectrometer. | Struwe et al60 |
| HIV trimers JR‐FL SOSIP.664 (subtype B), B41 SOSIP.664 (subtype B), CRF02_AG_250 SOSIP.664 (subtype AG), 327c SOSIP.664 (subtype C), and BG505 SOSIP.664 trimer (subtype A). All of these Env were expressed in HEK293F cells. | Global site‐specific N‐glycosylation analysis of HIV‐1 Env was performed. This study presents a specific endoglycosidases strategy for MS analysis. | Mass spectrometric analysis was performed on Fusion Orbitrap tribrid mass spectrometer (Thermo Fisher Scientific). | Cao et al62 |
| HIV trimers BG505 SOSIP.664 expressed in stable CHO cell line. | Mass spectrometry based testing and N‐glycosylation characterization performed on the cGMP‐quality BG505 SOSIP.664 trimers. | N‐linked glycosylation analysis was carried by HILIC‐UPLC method and the disulfide bonds were determined by LC‐MS/MS analysis using Q Exactive™ hybrid quadrupole‐Orbitrap™ mass spectrometer. | Dey et al21 |
| Influenza A/New Caledonia/20/99 A/Panama/2007/99 A/Wyoming/3/2003 | HA quantification and identification of influenza A&B strains propagated in PER.C6® cells. | RP‐HPLC performed using Waters Alliance 2695 system and a POROS® R1/10 (2.1 mm × 100 mm) column (PerSeptive Biosystems Inc.) maintained at 65 °C and UV detection at 214 nm. | Kapteyn et al115 |
| Influenza A/New Caledonia/20/99 B/Jiangsu/10/2003 | Selective and quantitative detection of influenza virus proteins in commercial vaccines. | 2D‐HPLC (on‐line coupling of size exclusion HPLC to reversed‐phase HPLC). | Garcia‐Canas et al81 |
| Influenza A/Puerto Rico/8/34 (PR8) A/Vietnam/1203/2004/PR8‐RG2 A/Vietnam/1203/2004 | Quantification of influenza virus hemagglutinins in complex mixtures. HA from viral subtypes H1, H3, H5, and B was determined both directly and rapidly. | Isotope dilution tandem mass spectrometry (IDMS) and a multiplexed multiple reaction monitoring (MRM) approach) was performed using a Symmetry300 reverse phase C18 column and Thermo Quantum TSQ mass spectrometer with an electrospray interface. | Williams et al70 |
| Influenza Trivalent bulk vaccines (TBV) from Northern Hemisphere (NH) season 2006/2008; A/Hiroshima/52/2005 A/New Caledonia/20/99 B/Malaysia/2506/2004 Season 2007/2008; A/Wisconsin/67/2005 A/Solomon Islands/3/2006 B/Malaysia/2506/2004 | HA quantification performed for the active or formaldehyde‐inactivated egg‐based and MDCK cell‐based whole virus samples. | RP‐HPLC performed using Waters Alliance 2695 system using a polystyrene POROS® R1/10 (2.1 mm × 100 mm) column (PerSeptive Biosystems Inc.) maintained at 65 °C and UV detection at 214 nm. | Kapteyn et al116 |
| Influenza | Optimization of digestion parameters for protein quantification. | Isotope dilution tandem mass spectrometry (IDMS) and a multiplexed multiple reaction monitoring (MRM) approach) was performed using Thermo Quantum TSQ mass spectrometer. | Norrgran et al71 |
| Influenza | A combination of separation and identification techniques was used to rapidly and reproducibly analyze influenza vaccine constituents. | SEC‐HPLC analysis was performed, peaks were collected and underwent tryptic digest followed by MALDI/MS. | Garcia‐Canas et al82 |
| Influenza Monovalent bulks: A/Brisbane/59/2007 H1N1 A/Brisbane/59/2007 H1N1 A/California/07/2009 H1N1 A/Uruguay/716/2007 B/Florida/4/2006 | Reversed‐phase HPLC method was developed and optimized for the quantitative determination of HA in influenza vaccine preparations. | RP‐HPLC was performed using a Waters Alliance 2695 coupled to fluorescence detector (λex 280 nm and λem335 nm) and a UV–vis photodiode array detector. MICRA® HPLC NPS‐ODSI, 4.6 mm × 33 mm, column was used and chromatographic separations were carried out at 60 °C. | Lorbetskie et al117 |
| Influenza Hemagglutinins (HA) in trivalent influenza vaccines (TIV). | Quantification of immunoreactive viral influenza proteins using an immunocapture isotope dilution mass spectrometry (IC‐IDMS) method. | The captured proteins were digested, and evolutionarily conserved tryptic peptides were quantified using IDMS‐MRM approach performed using Thermo Quantum TSQ mass spectrometer with an electrospray interface. | Pierce et al75 |
| Trivalent influenza vaccine (Protein Sciences Corp., Meriden, CT). A/Brisbane/59/2007 (H1N1), A/Brisbane/16/2007 (H3N2) B/Florida/4/2006 | LC‐MS was performed for simultaneous identification of HA proteins and process‐related impurities in a trivalent influenza candidate vaccine, comprised of purified recombinant HA (rHA). | LC‐MSE measurements were performed on a Waters Synapt HDMS system coupled with a Waters ACQUITY UPLC for online RP LC separation. The detected site‐specific glycoforms were further confirmed and quantified by HILIC‐ MRM assays. | Xi et al118 |
| Influenza Commercial 2007/2008 vaccine; A/Solomon Islands/3/2006 A/Wisconsin/67/2005 B/Malaysia/2506/2004‐like strains Commercial 2009/2010 vaccine; A/Brisbane/59/2007 A/Uruguay/716/2007 B/Brisbane/60/2008‐like strains. Commercial 2010 vaccine; A/California/7/2009 A/Perth/16/2009 B/Brisbane/60/2008‐like strains. | LC/MS/MS method was performed for the absolute quantification of viral proteins in a complex mixture. | IDMS and MRM approach was performed using a Symmetry300 reverse phase C18 column and Thermo Quantum TSQ mass spectrometer with an electrospray interface. | Williams et al72 |
| Influenza Commercial 2007/2008 vaccine; A/Solomon Islands/3/2006, A/Wisconsin/67/2005 B/Malaysia/2506/2004‐like strains Commercial 2009/2010 vaccine; A/Brisbane/59/2007 A/Brisbane/10/2007 B/Brisbane/60/2008‐like strains. Commercial 2010 vaccine; A/California/7/2009, A/Victoria/210/2009, and B/Brisbane/60/2008‐like strains. | Simultaneous quantification of the viral antigens HA and NA in influenza vaccines. The central premise of MSE quantification is that the “top 3” most intense peptide ions in an LC–MS chromatogram are approximately equal for all proteins at equimolar concentrations. | LC–MSE was performed using nanoAcquity UPLC BEH130 C18 reverse‐phase analytical column (100 μm × 100 mm). The eluting peptides were analyzed with a Waters Synapt HDMS system operating in MSE mode. | Creskey et al83 |
| Influenza A/Brisbane/59/2007 A/Solomon Islands/03/2006 A/New Caledonia/20/1999 A/Vietnam/1203/2004 B/Brisbane/60/2008 monovalent 2009 H1N1 vaccine | Quantification of recombinant HA and NA in influenza virus using a label‐free mass spectrometry (MS) based method that enables simultaneous identification and quantification of HA, NA, and other viral proteins. | Samples were analyzed by LC/MSE using a nanoAcquity UPLC and Synapt G2 mass spectrometer equipped with a NanoLockSpray ion source (Waters). | Getie‐Kebtie et al78 |
| Influenza A/Vietnam/1203/2004 (H5N1) expressed in three different insect cell lines. A/Vietnam/1203/2004 (H5N1) virus cultivated in hen eggs A/bar‐headed goose/Qinghai/14/2008 (H5N1) produced in HEK293 | A method using nanoLC/MSE and quantitative MALDI‐TOF MS permethylation was developed to monitor the glycosylation of HA's from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five™, expresSF + ® and glycoengineered expresSF +), and a human cell line (HEK293). | Mass spectrometry analysis was performed using a BEH C18 column on Waters Synapt G2 HDMS system. MALDI‐TOF analysis of permethylated N‐glycans was performed using a Perseptive Biosystems Voyager DE RF MALDI‐TOF mass spectrometer. Samples were analyzed in positive ion reflectron mode in the 800–5500 m/z range. | An et al119 |
| Influenza A/Netherlands/219/2003 (H7N7) A/Shanghai/2/2013 (H7N9) | An accurate and precise IDMS method was used to quantify the HA and the NA proteins in a purified virus preparation of A/Netherlands/219/2003 (H7N7) in the same analytical run. | IDMS was performed using Agilent Technologies 1200 HPLC using a Symmetry300 C18 Waters column. The eluent was introduced into a Thermo Scientific Vantage TSQ triple quadrupole tandem mass spectrometer with an electrospray interface. | Santana et al73 |
| Influenza A/Hong Kong/1/68 (H3N2: HK68) | HA N‐Glycosylation analysis of engineered H3N2 virus strain was carried out by mass spectrometry based approaches. | Nano‐LC‐MSE was used for glycopeptide composition, sequence and site occupancy analysis, and MALDI‐TOF MS permethylation profiling carried out for characterization of released HA N‐glycans. | An et al120 |
| Influenza A/Puerto Rico/8/1934 (H1N1) A/Aichi/2/1968 (H3N2) A/Wilson‐Smith/1933 (H1N1) A/Hong Kong/8/1968 (H3N2) and B/Lee/1940 | IEX‐HPLC method for the quantification of viral influenza particles is presented. | IEX‐HPLC was performed using the Alliance HPLC system equipped with a 2695 separations module, 2475 fluorescence detector and Empower™ software. A monolithic column (5.2mm × 5.0 mm) was used to separate the virus. | Transfiguracion, et al121 |
| Influenza Trivalent influenza vaccines (2011–2012 and 2014–2015) H1N1 H3N2 | Quantification of the antigens HA and NA in influenza vaccines has been reported using an antibody‐free LC–MS based method known as MSE “Hi3”. | MSE Hi3 was performed using a Waters nanoAcquity UPLC with a BEH130 C18 reverse‐phase column (100 µm × 100 mm) and peptide analysis was carried out by a Waters Synapt HDMS system operating in data independent analysis mode. | Smith et al122 |
| Influenza A/California/7/2009 (H1N1 A/California/7/2009 (H1N1) | Evaluation of HA content by RP‐HPLC to generate pandemic influenza vaccine. | Waters Alliance 2695 system using a polystyrene POROS R1/10 (2.1 mm × 100 mm) column and Waters 2996 PDA detector. A UV detector at 214 nm was used. | Kang et al123 |
| Influenza A/Shanghai/2/2013 | IC‐IDMS was performed to evaluate the suitability of the underlying monoclonal and polyclonal antibodies for their capacity to isolate the H7 hemagglutinin in the vaccine for quantification by IDMS. | IDMS was performed using Agilent Technologies 1200 HPLC using a Symmetry300 reverse phase C18 Waters column. The eluent was introduced into a Thermo Scientific Vantage TSQ triple quadrupole tandem mass spectrometer with an electrospray interface. | Pierce et al76 |
| Influenza A/California/7/2009 | Topological N‐glycosylation and site‐specific N‐glycan sulfation of influenza proteins in the highly expressed H1N1 candidate vaccines. | High‐resolution LTQ‐FT and Orbitrap mass spectrometer was used to identify the N‐glycan structure of intact glycopeptides of the protein digests by low‐energy collision‐induced dissociation followed by multi‐stage tandem mass spectrometry (MS3). | She et al124 |
| Influenza A/Mallard/Denmark/64650/03 (H5N7). | Glycosylation patterns of four recombinant H5 hemagglutinins derived from H5N7 were characterized using mass spectrometry based methods. | Two semi quantitative analyses were performed; MALDI‐TOF MS permethylation analysis for the released glycans and LC–MSE analysis for glycosylation profiling and site occupancy. | Parsons et al125 |
| Influenza | Fast and highly selective determination of hemagglutinin content in quadrivalent influenza vaccine by RP‐HPLC method. | HPLC analysis was performed using a Waters Alliance2695 chromatograph coupled to a 2475 Fluorescence and a 2996 UV–vis photodiode array detector. Chromatographic separation of HA in QIV formulations was achieved by using a MICRA®HPLC NPS‐ODSI, 33 mm × 4.6 mm, column at 55 °C by using 0.04% (v/v) aqueous TFA as eluent A and 0.03% (v/v) TFA in 25% ACN and 75% 2‐propanol as eluent B. | Lorbetskie et al85 |
| Influenza A/Victoria/361/2011 (H3N2) | N‐linked glycosylation analysis was performed for the recombinant influenza virus haemagglutinin (HA) from H3N2 strain produced in HEK 293 F cells. | Glycosylation site occupancy of H3N2 strain was performed using a Fusion Orbitrap tribrid mass spectrometer (Thermo Fisher Scientific). A reversed phase BEH C18 column was used to perform chromatographic separation. | Cao et al62 |
| Meningococcal vaccine CRM197 and meningococcal serogroups A (MenA), C (MenC), W135 (MenW) and Y (MenY) glycoconjugates | Mass spectrometry based characterization of glycoconjugate molecules designed to prepare a vaccine against Neisseria meningitides serogroups A, C, W135 and Y was performed. | SEC–MS analysis was performed using a Superdex Peptide PC 3.2/30, and LC‐MS/MS analysis was performed using Vydac C4 column on Q‐TOF (Micromass). | Bardotti et al97 |
| Meningococcal vaccine | Semi quantitative LC‐MS/MS analysis carried out to define conjugation of glycans to the lysines of Cross‐Reactive‐Material‐197 (CRM197). Conjugate vaccines use CRM197 as carrier protein. | NanoLC‐ESI‐MS analysis was carried out on an Ultimate 3000 RSLC‐nano system (Dionex/Thermo Scientific) coupled to an amaZon speed ETD ion trap (Bruker). LC‐MALDI‐TOF‐MS experiments were performed on an ultrafleXtreme MALDI‐TOF/TOF (Bruker). | Crotti et al98 |
| Meningococcal group B Vaccine (Bexsero(®) | BEXSERO is a FDA approved Neisseria meningitidis serogroup B (MenB) vaccine licensed for active immunization to prevent invasive disease caused by MenB. Quantitative proteomics for the vaccine was performed. | Quantification of outer membrane vesicle proteins of the Bexsero® vaccine was performed using 1.7 µm BEH130 C18 analytical column (75 µm × 250 mm, Waters) and nanoAcquity UPLC coupled to a Synapt G2 mass spectrometer. Hi3 methodology was used to perform quantitative proteomics. | Tani et al100 |
| Meningococcal group B vaccine | Label‐free quantitative LC‐MS/MS analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine was carried out. | MRM‐based LC‐MS/MS was carried out using a Waters BEH C18 column and 4000 Q‐Trap mass spectrometer. | Dick et al96 |
| Meningococcal group B vaccine Trumenba (bivalent rLP2086) | Mass spectrometry based characterization of the bivalent rLP2086 vaccine (Trumenba®), a Neisseria meningitidis serogroup B (MenB) vaccine was carried out. | GC/MS was used to analyze the composition of fatty acids released from rLP2086‐A05 and rLP2086‐B01. LC‐MS/MS analysis was carried out a Waters BEH C4 column at 60 °C using UHPLC/UV interfaced to an ultrahigh‐resolution Bruker Daltonics maXis ESI‐QTOF to characterize the primary sequence and PTMs. | Luo et al101 |
| Meningococcal vaccine | Glycoconjugates vaccines consisting of CRM197 and synthetic oligosaccharide epitopes was characterized using mass spectrometry techniques. | The primary structure was assessed by combining intact protein MALDI‐TOF‐MS, LC‐MALDI‐TOF‐MS middle‐down and LC‐ESI‐MS bottom‐up approaches. | Möginger et al126 |
| Multicomponent meningococcal serogroup B vaccine (4CMenB; Bexsero®) | The active components of the Bexsero vaccine; Neisseria heparin binding antigen, factor H binding protein, Neisseria adhesion A, and outer membrane vesicles were separated and analyzed. | A fast, selective and sensitive UHPLC method for the determination of the Bexsero antigens in the vaccine supernatant is presented in this study. | Nompari et al127 |
| Gonorrhea Vaccine | A comprehensive proteomic platform − isobaric tagging for absolute quantification coupled with 2D‐LC‐MS was used to characterize potential gonococcal vaccine antigens. | 2D LC‐MS/MS was performed after the cell envelope‐associated proteins were precipitated, trypsinzed and labeled with iTRAQ reagents. Desalted SCX fractions were analyzed by LC/ESI MS/MS with a ThermoScientific nano HPLC coupled to a hybrid Orbitrap Elite ETD mass spectrometer. | Zielke et al128 |
| Dengue vaccine Sanofi Pasteur tetravalent (CYD). Four chimeric viruses produced in mammalian Vero cells | Mass spectrometry based site‐specific characterization of envelope protein N‐glycosylation was performed. First report assessing the specific N‐glycosylation pattern of the E‐protein using mass spectrometry. | N‐glycosylation profiling of the E‐protein was performed using MALDI‐TOF (Ultraflextreme MALDI‐TOF‐MS in reflectron positive mode for neutral glycans and in negative mode for acidic glycans). The characterization of sitespecific N‐glycosylation was assessed by nanoLC–ESI‐MS/MS using a Bruker Maxis Q‐TOF mass spectrometer or an ion trap HCT. | Dubayle et al87 |
| Ebolavirus glycoprotein Recombinant Ebola glycoprotein expressed in HEK 293 cells | Comprehensive characterization of glycosylation was done for EBOV Yambuku GP1. | Identification of N‐glycans from Ebola virus glycoproteins by matrix‐assisted laser desorption/ionisation time‐of‐flight and negative ion electrospray tandem mass spectrometry. | Ritchie et al89 |
| Ebolavirus‐like particles (EBOV VLPs, eVLPs) Recombinant Ebola glycoprotein expressed in HEK 293 cells | GP1 concentration is a critical quality attribute of EBOV vaccines and LC‐HRMS was used to perform quantitation of GP1 in eVLP vaccine preparations. | AQUA Ultimate (heavy and light) peptides were used to perform LC‐MS/MS analysis using ultimate 3000 HPLC and Orbitrap Elite mass spectrometer with a HESI‐2 ion source (Thermo Fisher Scientific). | Cazares et al91 |
| Ebolavirus glycoprotein Recombinant Ebola glycoprotein expressed in HEK 293 cells | The study describes and compares the N‐linked and O‐linked glycosylation patterns for GP1,2 of five pathogenic ebolaviruses (BDBV, SUDV, TAFV, and two EBOV variants). | N‐ and O‐glycan profiling of permethylated glycans by MALDI TOF was performed. Positive ion reflectron MALDI‐TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker Daltonics). | Collar et al90 |
| Ebolavirus glycoprotein Recombinant Ebola glycoprotein expressed in in insect (Sf9) and HEK 293 cells | The study describe and compare the N‐linked and O‐linked glycosylation patterns for GP1,2 expressed in HEK293 and insect (Sf9) cells. | Glycan compositions and N‐glycan site occupancy was determined after MALDI‐TOF‐MS analysis using 2,5‐dihydroxybenzoic acid as matrix. Positive ion reflectron MALDI‐TOF/TOF mass spectra were acquired using an Autoflex III mass spectrometer (Bruker Daltonics). | Clarke et al129 |
| Chikungunya (CHIKV) virus‐like particle (VLP) vaccine.Recombinantly expressed in HEK293 and SfBasic cells | A sensitive RP‐ HPLC method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution was developed to characterize and quantitate CHIKV VLP components. | HPLC separation was performed using XBridge BEH300 C4 column held at 60 °C. MALDI‐TOF was performed to characterize the separated proteins using Bruker Autoflex III operated in reflector mode. The PTMs on the viral glycoproteins E1 and E2 were further identified by LC‐MS using a Synapt G2 mass spectrometer. | Shytuhina et al92 |
| Chikungunya (CHIKV) virus‐like particle (VLP) vaccine.Recombinantly expressed in HEK293 and SfBasic cells | The glycosylation patterns of CHIKV virus‐like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells were characterized. | HILIC with fluorescence and mass spectrometry (MS) based characterization of N‐glycosylation profiles from mammalian and insect cell derived chikungunya VLP was performed. | Lancaster et al93 |