ABSTRACT
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.
Subject(s)
Adhesins, Bacterial/chemistry , Meningococcal Vaccines/chemistry , Neisseria meningitidis, Serogroup B/chemistry , Recombinant Proteins/chemistry , Adhesins, Bacterial/immunology , Amino Acid Sequence , Animals , Circular Dichroism , Female , Meningococcal Vaccines/immunology , Mice , Molecular Sequence Data , Neisseria meningitidis, Serogroup B/immunology , Peptide Mapping , Protein Conformation , Recombinant Proteins/immunology , Spectrometry, Fluorescence , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunologyABSTRACT
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.
Subject(s)
Meningitis, Meningococcal/prevention & control , Meningococcal Vaccines/chemistry , Meningococcal Vaccines/standards , Circular Dichroism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/standardsABSTRACT
The glycoconjugate vaccines against Neisseria meningitidis groups Y and W135 consist of pools of selected oligosaccharides conjugated to the protein carrier (CRM197). Consistent production of these vaccines requires control and thus determination of the average degree of polymerisation of the oligosaccharides used for conjugation. Acid hydrolysis generates group Y and W135 oligosaccharides with N-acetylneuraminic acid at the reducing end. A method, involving NaBH4 reduction and quantification of this terminal N-acetylneuraminic acid by use of high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) following acid hydrolysis (2M TFA), was developed. The average degree of polymerisation is calculated from the ratio of reduced N-acetylneuraminic acid to total N-acetylneuraminic acid. The assay was qualified by application to group C, Y and W135 oligosaccharide standards characterised by liquid chromatography, mass and NMR spectroscopy.
