Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.
A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.
Schweikart
F
F
Department of Medical Biochemistry at the Medical Centre, Faculty of Medicine, University of Geneva, Switzerland.
Jones
R
R
Jaton
J C
JC
Hughes
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry*; D019149:Bioreactors; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D006031:Glycosylation; D006825:Hybridomas; D007070:Immunoglobulin A / Q000096:biosynthesis / Q000737:chemistry*; D007137:Immunoglobulin alpha-Chains / Q000096:biosynthesis / Q000737:chemistry*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007070:Immunoglobulin A; D007137:Immunoglobulin alpha-Chains; D009844:Oligosaccharides; D011134:Polysaccharides