Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.
Luca NompariSalvatore Sanna CocconeGian Luca SardoneAlessio CorradoStefania BertiMassimiliano BiaginiMichele RoviniClaudia MagagnoliSimona CianettiSerena OrlandiniSandra FurlanettoRiccardo De RiccoPublished in: ACS pharmacology & translational science (2024)
Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.
Keyphrases
- liquid chromatography
- high throughput
- simultaneous determination
- tandem mass spectrometry
- high resolution mass spectrometry
- mass spectrometry
- quality control
- ultra high performance liquid chromatography
- solid phase extraction
- high performance liquid chromatography
- high resolution
- ms ms
- protein protein
- early stage
- amino acid
- dendritic cells
- gas chromatography
- high speed
- clinical practice
- pseudomonas aeruginosa
- binding protein
- gram negative
- immune response
- multidrug resistant