Influence of culture conditions on the N-glycosylation of a monoclonal antibody specific for recombinant hepatitis B surface antigen.
MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post-translational modifications, e.g. for N-glycosylation. Hybridoma CB.Hep-1, which secretes an IgG2b mAb, was cultured in vivo in ascites and in vitro in static-flask, spinner-flask, dialysis-membrane and perfusion systems using protein-free, low-serum-containing medium (1% foetal-calf serum) and high-serum-containing medium (8% foetal-calf serum). These CB.Hep-1 mAbs were fully characterized, and insignificant differences in the affinity constant were observed. Glycosylation profiling was performed by labelling the N-glycans released by peptide N-glycosidase F with either of the fluorophore tags 8-aminonaphthalene-1,3,6-trisulphonic acid and 4-aminobenzoic acid. The mAb produced in vivo showed two major biantennary-complex-type N-glycans: monogalactosylated, core-fucosylated and agalactosylated, core-fucosylated. The mAbs produced in vitro in static flasks and spinner flasks were not significantly influenced by the serum content in the culture media and showed a higher degree of N-glycan galactosylation compared with those produced in mouse-ascites, hollow-fibre and membrane systems. The monogalactosylated, core-fucosylated structure was the most abundant N-glycan except for those produced in ascites and hollow fibres, where the agalactosylated, core-fucosylated glycoform was the major specie. MAbs produced in high-cellular-yield systems displayed greater galactosylation heterogeneity influenced by changes in culture media.
Cabrera
Gleysin
G
Department of Carbohydrate Chemistry, Center for Genetic Engineering and Biotechnology, Ave 31/158 and 190, P.O. Box 6162, Havana 10600, Cuba.
Cremata
José A
JA
Valdés
Rodolfo
R
García
Rossana
R
González
Yanelys
Y
Montesino
Raquel
R
Gómez
Héctor
H
González
Marcos
M
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D006031:Glycosylation; D006510:Hepatitis B Antibodies / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000502:physiology
D000911:Antibodies, Monoclonal; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens