A High-Throughput Size Exclusion Chromatography Method to Determine the Molecular Size Distribution of Meningococcal Polysaccharide Vaccine (original) (raw)
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Journal of Biochemical and Biophysical Methods, 2003
The molecular size of meningococcal polysaccharides is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to follow changes in the size distribution and therefore in the distribution coefficient (K D ) of the meningococcal polysaccharides of groups A, C, Y and W-135 used to formulate anti-Neisseria meningitidis vaccines. The experimental conditions were also found to be suitable for a rapid monitoring of the quality (no group A polysaccharide depolymerization) of the tetravalent meningococcal polysaccharide vaccine. D
Journal of Immunological Methods, 2019
The multivalent glycoconjugate vaccines against Neisseria meningitidis are extremely high-priced for the developing world. The high cost is due to the manufacturing setup required to produce an effective vaccine and other inflators like complex production steps including the production and purification of the polysaccharide and consequently its conjugation with a protein and finally formulating the finished multivalent product. There is an urgent need for assays which are simple, precise, can be applicable at multiple steps and contribute in reducing the overall manufacturing cost, thereby making the vaccines more equitable to the developing world. WHO recommends serological tests for polysaccharide identification and quantitation at different stages of conjugate vaccine production. We report development of inhibition ELISAs for the identification and quantification of N. meningitidis serogroup A (MenA) and N. meningitidis serogroup X (MenX) polysaccharides (PSs) in samples from stage of cell banking till production of finished product. The method was qualified on various parameters such as specificity, intermediate precision, sensitivity and accuracy. Our results provide a proof of concept for the use of an inhibition ELISA as a common tool for the identification and quantification of PS at various stages of vaccine development and manufacture.
ELECTROPHORESIS, 2013
Neisseria meningitidis group C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates, thereby constituting a serious public health problem. Bio-Manguinhos/Fiocruz is developing a conjugate vaccine by covalent attachment of capsular polysaccharide to hydrazide-activated tetanus toxoid through reductive amination. It is necessary to quantify free components as a quality control process to prevent exacerbated adverse reactions and/or attenuation of vaccine immunogenicity. Thus, this study aimed to develop and validate a quality control method appropriate for the separation and quantification of free polysaccharide present in this conjugate N. meningitidis group C vaccine using CE. CZE was used to remove unbound polysaccharide, and the electrophoretic conditions were varied to optimize resolution. We were able to develop and validate the proposed method, which was linear and showed a matrix effect. Repeatability and partial reproducibility of the method were also evaluated. The robustness results showed that control of temperature is required for reliable results. The validated method will be used to evaluate the conjugate batches submitted for Phase III clinical studies and for routine quality control of the conjugate vaccine.
Infection and Immunity, 1991
Most group C Neisseria meningitidis strains produce an O-acetyl-positive polysaccharide, a homopolymer of alpha-2----9-linked N-acetylneuraminic acid with O-acetyl groups at the C-7 and C-8 of its sialic acid residues. The majority of disease isolates have been reported to contain this polysaccharide. Some strains produce group C polysaccharide lacking O-acetyl groups. The licensed vaccine contains the O-acetyl-positive polysaccharide. We have measured the antibody specificities to the two polysaccharides in sera from asymptomatic group C meningococcal carriers and vaccinated adults by a new enzyme-linked immunosorbent assay (ELISA) procedure using methylated human serum albumin for coating the group C polysaccharide onto microtiter plates. Inhibition of binding of serum antibodies to polysaccharide-coated plates was measured by ELISA after incubation with O-acetyl-positive and O-acetyl-negative group C polysaccharides. Greater inhibition of binding of carrier sera was observed with...
Purification of meningococcal group C polysaccharide by a procedure suitable for scale-up
Journal of Microbiological Methods, 1996
Neisseriu mertingitidis group C capsular polysaccharide is the antigen for the vaccine. An easier method has been developed for the purification of N. meningitidis group C capsular polysaccharide. In this method, two steps of the traditional procedure have been modified: the removal of protein and lipopolysaccharide.
Determination of meningococcal polysaccharides by capillary zone electrophoresis
Analytical Biochemistry, 2005
Meningococcal polysaccharides are medically important molecules and are the active components of vaccines against Neisseria meningiditis serogroups A, C, W135, and Y. This study demonstrates that free solution capillary zone electrophoresis (CZE) using simple phosphate/borate separation buVers is capable of separating intact, native polysaccharides from these four serogroups. Separation appeared to be robust with respect to variations in test conditions and behaved in expected ways with respect to changes in temperature, ionic strength, and addition of an organic modiWer. Serogroups W135 and Y are composed of sialic acid residues alternating with either galactose or glucose, respectively. Separation of these serogroups could be achieved using phosphate buVer and was therefore not dependent on diVerential complexation with borate. Addition of sodium dodecyl sulfate to the separation buVer (i.e., MEKC) resulted in peak splitting for all four serogroups. Changes in polysaccharide size did not aVect migration time for the size range examined, but serogroup C polysaccharide (a sialic acid homopolymer) was separable from sialic acid monosaccharide. CZE quantiWcation of multiple lots of each of the four serogroups was compared to wet chemical determination by phosphorus or sialic acid measurement. Results from CZE determination showed good agreement with the wet chemical methods.
2012
Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using 13 C, 1 H and 31 P NMR spectroscopy and total correlation spectroscopy (TOCSY) and 1 H-13 C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC-SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000 g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule.