Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns

Abstract

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.

Figure 1

OMV protein pattern by SDS-PAGE assay for testing and control of the main components: PorA, PorB + FbpA comigration, OmpA, NspA, OpcA, FetA, and YaeT. Protein identification was obtained by in-gel digestion followed by LC-MS/MS of the major Coomassie Blue-stained bands. Batches 1, 2, and 3 are three different preparations of the OMV (batch consistency check).

Figure 2

Best chromatographic profiles of RP-UHPLC scouting. Injection of 5 μL MenB OMV bulk for each run, without sample dilution and treatment. (A) Blue line is the BEH C4 column eluted in an acetonitrile/water/TFA mixture, (B) brown line is the AWP C8 column eluted in an acetonitrile/water/TFA mixture, (C) pink line is the AWP C8 column eluted in a methanol/water/TFA mixture, and (D) black line is the BEH C4 column eluted in a methanol/water/TFA mixture.

Figure 3

Ishikawa diagram for the RP-UHPLC OMV protein pattern. pCMPs already locked (to not be further explored) are identified in bold, pCMPs to be explored are identified in bold and underlined, and all the other parameters are not considered as pCMPs.

Figure 4

D-Optimal DoE_1-graphic effects analysis. The assessment of parameter effect for N, nB, R1, and K′ outputs is represented by a box and whiskers plot. The green bars represent the effects of each tested parameter from level −1 to level +1. The gray bars report the error. If the error contains zero, the parameter does not have a significant effect. On the contrary, the parameter/level has a positive or negative effect on the response under consideration.

Figure 5

Fractional factorial DoE-graphic effects analysis. The assessment of parameters effect for N, nB, R1, and K′ outputs is represented by a box and whiskers plot. The green bars represent the effects of each tested parameter from level −1 to level +1. The gray bars report the error. If the error contains zero, the parameter/level does not have a significant effect. On the contrary, the parameter has a positive or negative effect on the response under consideration.

Figure 6

RP-UHPLC protein pattern profiling of MenB OMV by Acquity BEH C8, 1.7 μm, 2.1 × 150 mm.

Figure 7

MenB OMV protein pattern comparison between RP-UHPLC (gray bars) and SDS-PAGE (blank filled bars) assays. Additional details on RP-UHPLC method performances are reported in Table 2. The SDS-PAGE assay variability is reported in the literature (18).

Figure 8

RP-UHPLC protein pattern profiling of MenB GMMA. The identity of each RP-UHPLC peak was assessed using mass spectrometry (data not shown).