Serrato J Antonio, Hernández Vanessa, Estrada-Mondaca Sandino, Palomares Laura A, Ramírez Octavio T
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, CP, Mexico.
Biotechnol Appl Biochem. 2007 Jun;47(Pt 2):113-24. doi: 10.1042/BA20060216.
SFM (serum-free medium) is preferred to media containing animal-derived components when culturing mammalian cells for the production of therapeutic recombinant proteins and mAbs (monoclonal antibodies). 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 beta-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.
在培养哺乳动物细胞以生产治疗性重组蛋白和单克隆抗体（mAb）时，无血清培养基（SFM）比含有动物源成分的培养基更受青睐。尽管如此，从培养基中去除动物源成分会强烈改变培养性能并改变蛋白质糖基化。在本研究中，将含有10%（v/v）胎牛血清（FBS）的传统培养基[杜尔贝科改良伊格尔培养基（DMEM）/FBS]中的单克隆抗体糖基化谱、细胞外糖苷酶活性、杂交瘤生长和单克隆抗体产量与在SFM或化学限定培养基（CDM）中获得的进行了比较。SFM和CDM支持的细胞和单克隆抗体浓度高于DMEM/FBS；然而，毛细管电泳（CE）分析显示单克隆抗体糖基化模式有重要变化。糖基化模式在所有培养基中均表现出广泛的微异质性，范围从复杂型到高甘露糖型和寡甘露糖型聚糖。在DMEM/FBS中产生的单克隆抗体呈现26种聚糖结构，而在CDM或SFM培养物中发现的聚糖微异质性较低，分别为24种和22种结构。在DMEM/FBS和CDM中，没有末端半乳糖的复杂聚糖（G0）分别占总聚糖的28%和32%，42%和46%分别对应半乳糖基化结构（G1加G2）。相比之下，SFM中的G0聚糖占58%，而只有28%对应G1和G2结构。细胞外β-半乳糖苷酶活性在SFM中增加了约3倍，这可以解释与其他两种培养基中的培养物相比G0含量更高的原因。在SFM和CDM培养基中产生的单克隆抗体中观察到唾液酸化结构有理想的减少，但岩藻糖基化形式有不理想的增加。无论使用何种培养基，约80%的潜在单克隆抗体糖基化位点被占据。
