An analysis of structure-antibody recognition relationships in nine licenced polysaccharide-tetanus toxoid (TT) conjugate vaccines was performed. The panel of conjugates used included vaccine components to protect against disease caused by Haemophilus influenzae type b, Neisseria meningitidis groups A, C, W and Y and Streptococcus pneumoniae serotype 18C. Conformation and structural analysis included size exclusion chromatography with multi-angle light scattering to determine size, and intrinsic fluorescence spectroscopy and fluorescence quenching to evaluate the protein folding and exposure of Trp residues. A capture ELISA measured the recognition of TT epitopes in the conjugates, using four rat monoclonal antibodies: 2 localised to the HC domain, and 2 of which were holotoxoid conformation-dependent. The conjugates had a wide range of average molecular masses ranging from 1.8×10(6) g/mol to larger than 20×10(6) g/mol. The panel of conjugates were found to be well folded, and did not have spectral features typical of aggregated TT. A partial correlation was found between molecular mass and epitope recognition. Recognition of the epitopes either on the HC domain or the whole toxoid was not necessarily hampered by the size of the molecule. Correlation was also found between the accessibility of Trp side chains and polysaccharide loading, suggesting also that a higher level of conjugated PS does not necessarily interfere with toxoid accessibility. There were different levels of carrier protein Trp side-chain and epitope accessibility that were localised to the HC domain; these were related to the saccharide type, despite the conjugates being independently manufactured. These findings extend our understanding of the molecular basis for carrier protein recognition in TT conjugate vaccines.

Physico-chemical analysis of pneumococcal polysaccharide (PS)-protein conjugate vaccine components used for two commercially licensed vaccines was performed to compare the serotype- and carrier pro...

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.

Objectives: The purpose of this study was to further characterize the ultrafiltration (UF) method for determining free saccharide levels in glycoconjugate vaccines and compare it with other methods used for the determination of free saccharide levels in meningococcal glycoconjugate vaccines. Methods: We performed experiments on both meningococcal glycoconjugates and capsular polysaccharides, and compared UF, deoxycholate (DOC) precipitation, and solid-phase extraction (SPE) methods. Meningococcal capsular polysaccharides from groups A (MenA), C (MenC), and W (MenW) were depolymerized and characterized using SEC-MALS (size-exclusion chromatography with multi-angle laser light scattering) to determine the molecular weight and hydrodynamic size and then subjected to UF. The free saccharide content was quantified using HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection). Results: The characterization of size-reduced group C polysaccharide revealed weight-average molecular mass (Mw) ranging from 22,200 g/mol to 287,300 g/mol and hydrodynamic radii of 3.7 to 19.5 nm. Pore size studies confirmed that polysaccharides with diameters up to 15 nm filtered through the 100 kDa cellulose membrane. The smallest PS fragment tested (22,200 g/mol, 7.4 nm diameter) was partially recovered from the 30 kDa membrane. For MenC-CRM₁₉₇, DOC yielded the lowest free saccharide content (<1%), UF gave moderate results (7–8%), and SPE showed the highest and most variable values (up to 15%). For MenA- and MenW-CRM₁₉₇, UF and DOC consistently provided low free saccharide levels (<2% and 3–11%, respectively). Conclusions: The upper limits on the size of free group C meningococcal polysaccharides that can be ultrafiltered were assessed. Differences in the relative amount of free saccharide were observed between various methods used to control meningococcal conjugate vaccines.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period.