ABSTRACT
We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.
Subject(s)
Bacterial Capsules/chemistry , Bacterial Vaccines/metabolism , Oligosaccharides/isolation & purification , Bacterial Capsules/immunology , Bacterial Outer Membrane Proteins/analysis , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Vaccines/therapeutic use , Chromatography, Liquid , Colorimetry , Haemophilus Vaccines/analysis , Haemophilus Vaccines/immunology , Haemophilus Vaccines/isolation & purification , Haemophilus influenzae type b/immunology , Humans , Mass Spectrometry , Meningitis/prevention & control , Meningitis, Meningococcal/prevention & control , Molecular Weight , Neisseria meningitidis/immunology , Nuclear Magnetic Resonance, Biomolecular , Oligosaccharides/analysis , Oligosaccharides/immunology , Polysaccharides, Bacterial/analysis , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/isolation & purification , Vaccines, Conjugate/analysis , Vaccines, Conjugate/immunology , Vaccines, Conjugate/isolation & purificationABSTRACT
We have developed a chromatographic method suitable for the fractionation of polysaccharides having a negatively charged group. The method permits the removal of all those polysaccharide fragments having a short sequence and which are likely unsuitable for conjugate vaccine construction. The selected polysaccharide fragments can be used to produce glycoconjugate vaccines containing a restricted saccharide polydispersion. We have applied this chromatographic method to three different antigens, Haemophilus influenzae type b and Neisseria meningitidis group A and group C polysaccharides. The method is easily adapted for manufacturing purposes.
Subject(s)
Antigens, Bacterial/chemistry , Bacterial Capsules/chemistry , Haemophilus Vaccines/chemistry , Polysaccharides, Bacterial/chemistry , Vaccines, Conjugate/chemistry , Chromatography, Ion Exchange , Enzyme-Linked Immunosorbent Assay , Hydrolysis , Mass Spectrometry , Neisseria meningitidis , UltrafiltrationABSTRACT
A simple two-step procedure for purifying F(ab)2 fragments of horse immunoglobulins is described. In the first step, the horse plasma is diluted, made up to 12% (w/v) with ammonium sulphate and digested with pepsin. In the second step, the previously dialyzed solution is chromatographed. Instead of a normal ion-exchange resin, a DEAE-cellulose, covalently linked to a synthetic vinyl polymer, was used (DEAE-Zeta-Prep). With this assembly it is possible to perform chromatography at a high flow-rate without the problems related to the use of large columns. The yield and purity of the final product are satisfactory. This method has been scaled up for industrial application.
