Biotechnol. Appl. Biochem. (2007) 47, 113–124 (Printed in Great Britain) doi:10.1042/BA20060216 113 Differences in the glycosylation profile of a monoclonal antibody produced by hybridomas cultured in serum-supplemented, serum-free or chemically defined media J. Antonio Serrato, Vanessa Hern´ andez, Sandino Estrada-Mondaca 1 , Laura A. Palomares and Octavio T. Ram´ ırez 2 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnolog´ ıa, Universidad Nacional Aut´ onoma de M´ exico, Apdo. Postal. 510-3, Cuernavaca, Morelos, CP 62250, Mexico SFM (serum-free medium) is preferred to media con- taining animal-derived components when culturing mammalian cells for the production of therapeutic recombinant proteins and mAbs (monoclonal an- tibodies). Nonetheless, eliminating animal-derived components from media can strongly modify culture performance and alter protein glycosylation. In the present study, mAb glycosylation profiles, extracellular exoglycosidase activities, hybridoma growth and mAb production in traditional medium containing 10 % (v/v) FBS (fetal bovine serum) [DMEM (Dulbecco’s modified Eagle’s medium)/FBS] were compared with those obtained in either SFM or CDM (chemically defined medium). SFM and CDM supported higher cell and mAb concentrations than did DMEM/FBS; however, CE (capillary electrophoresis) analyses revealed important changes in mAb glycosylation patterns. Glycosylation patterns showed a broad microheterogeneity in all the media, ranging from complex to high-mannose and paucimannosidic glycans. mAb produced in DMEM/FBS presented 26 glycan structures, whereas a lower glycan microheterogeneity was found for cultures in CDM or SFM, which presented 24 and 22 structures respectively. In DMEM/FBS and CDM, complex glycans without terminal galactose (G0) represented 28 and 32 % of the total glycans respectively and 42 and 46 % corresponded to galactosylated structures (G1 plus G2) respectively. In contrast, G0 glycans in SFM accounted for 58 %, whereas only 28 % corresponded to G1 and G2 structures. Extracellular β -galactosidase activity increased approx. 3-fold in SFM, which can explain the higher G0 content compared with cultures in the other two media. A desirable decrease in sialylated structures, but an undesirable increase in fucosylated forms, was observed in mAb produced in SFM and CDM media. Approxi. 80 % of potential mAb glycosylation sites were occupied, regardless of the culture medium used. Introduction Since the introduction of hybridoma technology, mAbs (monoclonal antibodies) have been used extensively as in vivo and in vitro diagnostic reagents and as ligands for efficient purification of biomolecules. As of 2005, a total of 31 mAb-based products had been accepted for human ther- apy or in vivo diagnostics and many more are now in clinical trials [1,2]. Optimal growth of hybridoma cells is generally achieved in a defined basal medium containing relatively high levels of FBS (fetal bovine serum) (5–10 %, v/v). Nonetheless, a well-defined production process, in which sera and pro- teins of any origin are preferably omitted from the medium formulation, is fundamental to manufacture highly purified mAb of consistent quality. CDM (chemically defined media) offer the advantage of lot-to-lot consistency and reduced dependence on animal or plant components whose supply can be uncertain. Furthermore, CDM facilitate downstream processing and regulatory approval (http://www.fda.gov/ cber/guidelines.htm#97) and can improve product biosafety by reducing the risk of contamination by adventitious agents [3–6]. In the last decade, hybridoma and myeloma cell lines have been successfully adapted to grow in SFM (serum-free media), protein-free media or CDM, with the concomitant selection for improved cell densities and mAb yields [7–11]. Keywords: chemically defined medium, exoglycosidase, glycosylation, metabolism, monoclonal antibody, serum-free medium Abbreviations used: ADCC, antibody-dependent cell cytotoxicity; APTS, 8-aminopyrene-1,3,6-trisulfonic acid; CDM, chemically defined medium; CE, capillary electrophoresis; CHO cells, Chinese-hamster ovary cells; CPA, corrected peak area; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GlcNAc, N-acetylglucosamine; GU, glucose unit; mAb, monoclonal antibody; 4-MU, 4-methylumbelliferyl; SFM, serum-free medium; UNAM, Universidad Nacional Aut ´ onoma de M´ exico 1 Present address: Grupo de Medicina Regenerativa, Unidad de Ingenier´ ıa de Tejidos y Terapia Celular, Instituto Nacional de Rehabilitaci´ on, Secretar´ ıa de Salud, Av. M´ exico-Xochimilco 289, Arenal de Guadalupe, Tlalpan, CP 14389, M´ exico, DF, M´ exico. 2 To whom correspondence should be addressed (email tonatiuh@ibt.unam.mx). C  2007 Portland Press Ltd