Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.
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.
Ravenscroft
N
N
Chiron Vaccines SpA, Siena, Italy. neil_ravenscroft@cc.chiron.com
Averani
G
G
Bartoloni
A
A
Berti
S
S
Bigio
M
M
Carinci
V
V
Costantino
P
P
D'Ascenzi
S
S
Giannozzi
A
A
Norelli
F
F
Pennatini
C
C
Proietti
D
D
Ceccarini
C
C
Cescutti
P
P
D016667:Bacterial Capsules / Q000737:chemistry* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000378:metabolism* / Q000627:therapeutic use; D002853:Chromatography, Liquid; D003124:Colorimetry; D018073:Haemophilus Vaccines / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D019741:Haemophilus influenzae type b / Q000276:immunology; D006801:Humans; D013058:Mass Spectrometry; D008581:Meningitis / Q000517:prevention & control; D008585:Meningitis, Meningococcal / Q000517:prevention & control; D008970:Molecular Weight; D009345:Neisseria meningitidis / Q000276:immunology; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D011135:Polysaccharides, Bacterial / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D018074:Vaccines, Conjugate / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification
D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D018073:Haemophilus Vaccines; C061964:Haemophilus influenzae-type b polysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine; D009844:Oligosaccharides; D011135:Polysaccharides, Bacterial; D018074:Vaccines, Conjugate