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

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.

CMC Strategies and Advanced Technologies for Vaccine Development to Boost Acceleration and Pandemic Preparedness.

This review reports on an overview of key enablers of acceleration/pandemic and preparedness, covering CMC strategies as well as technical innovations in vaccine development. Considerations are shared on implementation hurdles and opportunities to drive sustained acceleration for vaccine development and considers learnings from the COVID pandemic and direct experience in addressing unmet medical needs. These reflections focus on (i) the importance of a cross-disciplinary framework of technical expectations ranging from target antigen identification to launch and life-cycle management; (ii) the use of prior platform knowledge across similar or products/vaccine types; (iii) the implementation of innovation and digital tools for fast development and innovative control strategies.

Development of a New Solid-Phase Extraction Base Method for Free Saccharide Content Estimation of Meningococcal Conjugate Vaccines.

GlaxoSmithKline (GSK) is currently developing a fully liquid presentation to ease the administration of the licensed quadrivalent conjugate vaccine (Menveo) against meningococcal serogroup A, C, W, and Y (MenACWY) infections. Herein, we report a new method for determining the free saccharide (FS) content of CRM197-MenACWY conjugated antigens, with the aim of improving accuracy and reproducibility. Mathematical models have been used to support technical knowledge in reducing the need for experimental development. This results in an improved, faster, and platform-based technique for FS separation with one single pretreatment applicable to all antigens of the multivalent meningococcal vaccine.

Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero® vaccine.

Meningococcal disease is a major cause of morbidity and mortality worldwide. Its epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and Y). While effective vaccines already exist for serogroups A, C, W and Y, except for under clonal outbreaks, no vaccine was available against serogroup B. Recently, a four component vaccine, Bexsero(®), designed to prevent infection caused by this serogroup, has been approved in Europe and other Countries for use in individuals from two months of age and older. The active components of this vaccine are three recombinant proteins identified by reverse vaccinology combined with detergent extracted outer membrane vesicles (DOMV) prepared from a New Zealand epidemic strain. Considering their intrinsic complexity, we performed additional characterization of DOMVs on top of the standard quality control testing carried out for batch release. We applied the Hi3 label-free LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV protein content. We first, successfully investigated the robustness and the accuracy of the methodology for the DOMV characterization and we then applied it to compare six DOMV production lots. Around 100 proteins were quantified from each preparation. When classified according to their predicted cellular localization, about 90% of the total protein amount belongs consistently to the outer membrane compartment. Using nonparametric hypothesis testing and complementary log-log linear regression, the quantifications of a subset of 21 proteins common to all lots and including approximately 90% (85-92%) of the total protein amount quantified in any DOMV lot were found consistent across lots. The relevance of these results is two-fold, showing that the Hi3 quantification methodology is robust for a broad range of proteins and indicating that the manufacturing process currently used for the production of the Bexsero(®) DOMV components is highly reproducible and consistent.

Structural characterisation, stability and antibody recognition of chimeric NHBA-GNA1030: an investigational vaccine component against Neisseria meningitidis.

A new generation multi-component vaccine, principally directed against serogroup B Neisseria meningitidis (4CMenB), has recently been developed. One of its components, identified through reverse vaccinology, is the neisserial heparin-binding antigen (NHBA) which is included in the formulation as a novel NHBA-GNA1030 fusion protein (NHBA-FP). We describe here the biophysical characteristics of this vaccine antigen to understand better its structural properties in solution and concurrent immunogenicity prior to formulation. By deliberately stressing the protein to lose its immune responses, we were able to study the protein's structural changes at the molecular level. The unmodified NHBA-FP was found to be mainly monomeric with mass of 67kDa and secondary structure dominated by ß-sheets and turns (57% average). The antigen was very stable in storage buffer. It could be forced to unfold in a low-salt buffer resulting in the exposure of one of its two tryptophan residues at 50°C. Long-term stress studies (10-15 days at 37°C) showed modification in the chromatographic and electrophoretic profiles with progressive degradation and aggregation. Since there was little change in secondary structure (as monitored by circular dichroism and tryptophan fluorescence spectroscopy), the loss of functional immunogenicity of the thermal stressed protein could be mainly attributed to the observed fragmentation and aggregation. We therefore conclude that the maintenance of the intact, non-fragmented state of the NHBA-FP is important to preserve its functional immunogenicity. This may thus be utilised as an assay for the control testing of the protein.

Structural organization of NadADelta(351-405), a recombinant MenB vaccine component, by its physico-chemical characterization at drug substance level.

The physico-chemical characterization of NadADelta(351-405), a recombinant protein discovered by reverse vaccinology, component of a candidate vaccine against Neisseria meningitidis serotype B is presented. Analytical methods like mass spectrometry, electrophoresis, optical spectroscopy and SEC-MALLS have been applied to unveil the structure of NadADelta(351-405), and to evaluate Product-Related Substances. Moreover, analysis of the protein after intentional denaturation has been applied in order to challenge the chosen methods and to determine their appropriateness and specificity. All the obtained results were inserted in a model allowing in-depth understanding of the antigen NadADelta(351-405): it is present in solution as a homo-trimer, retaining a high percentage of alpha-helix secondary structure, and able to reassemble from monomeric subunits after thermal denaturation; this structural organization is consistent with that foreseen for MenB NadA (Neisseria Adhesin A). The analytical data sets produced during process development for clinical phases I-III material confirm product quality and manufacturing consistency.

Physicochemical characterisation of glycoconjugate vaccines for prevention of meningococcal diseases.

Bacterial capsular polysaccharides covalently linked to an appropriate carrier protein represent the best tool to induce a protective immune response against a wide range of bacterial diseases, such as meningococcal infections. We describe here the physico-chemical characterisation of glycoconjugate molecules designed to prepare a vaccine against Neisseria meningitidis serogroups A, C, W135 and Y. The use of a selective conjugation chemistry resulted in well characterised, reproducible and traceable glycoconjugate that can be consistently manufactured at large scale. A pool of physical and spectroscopic methods was used to establish glycosylation ratio, identity, molecular weight profiles, integrity of carrier protein and sites of glycosylation, assuring effective and consistent lots of vaccines.