Improved methods for the quantification of free and linked spacer in conjugate vaccine process.
In polysaccharide (PS)-protein conjugate vaccine process, indirect coupling via derivatization of the antigenic PS by diamino spacer molecules is widely used. Such a conjugation technology requires the accurate determination of both the degree of PS-amino substitution (linked spacer) and the removal of residual unlinked (free) diamino spacer. We report two methods for the microdetermination of the spacer primary amino groups, based on their fluorescent labelling. In the first developed assay, activated PS is derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and the free spacer is separated and detected as its AQC derivative by RP-HPLC with fluorescence detection (lambda(Ex,Em) 246/396 nm). In the second assay, activated PS is derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and its FQ derivative is separated by capillary zone electrophoresis (CZE) with laser-induced-fluorescence (LIF) detection (lambda(Ex/Em) 488/590 nm). Compared to the traditional gel filtration and colorimetric assays, these two methods show major advances in terms of sensitivity, reduced analysis time and small sample requirements.
Adam
O
O
Aventis Pasteur, Marcy I'Etoile, France.
Moreau
M
M
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D003959:Diamines / Q000737:chemistry; D019075:Electrophoresis, Capillary; D011135:Polysaccharides, Bacterial / Q000032:analysis / Q000737:chemistry*; D018074:Vaccines, Conjugate / Q000032:analysis / Q000737:chemistry*
D003959:Diamines; D011135:Polysaccharides, Bacterial; D018074:Vaccines, Conjugate; C007297:1,6-diaminohexane