title: Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.
abstract: The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.
authors.affiliation: a Vaccine Bioprocess Research and Development; Merck Research Labs; Merck & Co., Inc. ; Whitehouse Station , NJ , USA.
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Dick
authors.forename: John T
authors.initials: JT
authors.lastname: Mehl
authors.forename: John W
authors.initials: JW
authors.lastname: Loughney
authors.forename: Anna
authors.initials: A
authors.lastname: Mach
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
authors.forename: Lan
authors.initials: L
authors.lastname: Zhang
authors.forename: Craig T
authors.initials: CT
authors.lastname: Przysiecki
authors.forename: Lance
authors.initials: L
authors.lastname: Dieter
authors.forename: Van M
authors.initials: VM
authors.lastname: Hoang
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D055808:Drug Discovery / Q000379:methods; D006801:Humans; D022401:Meningococcal Vaccines / Q000737:chemistry*; D038541:Neisseria meningitidis, Serogroup B / Q000276:immunology; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D022401:Meningococcal Vaccines
keywords: BCA, bicinchoninic acid; ELISA, enzyme-linked immunosorbent assay; HPLC –high performance liquid chromatography; LC-MS/MS, liquid chromatography, tandem mass spectrometry; MRM, multiple reaction monitoring; MS, mass spectrometry; MenB, Meningococcal serogroup B; NadA, Neisseria adhesin A; OMPC, outer membrane protein complex; OMV, outer membrane vesicle; PorA, major outer membrane protein (class I outer membrane protein); QC, quality control; RSD, relative standard deviation; SDS-PAGE, sodium dodecyl sulfate, polyacrylamide gel electrophoresis; WRAIR, Walter Reed Army Institute of Research; fHbp, factor H binding protein; mass spectrometry; membrane proteins; meningococcal group B; multiple reaction monitoring; outer membrane vesicle; protein quantification; vaccine