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细胞培养过程对单克隆抗体糖基化修饰的影响和调控
({{custom_author.role_cn}}), {{javascript:window.custom_author_cn_index++;}}The Glycosylation Design and Control of Monoclonal Antibody by Cell Culture
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}在单克隆抗体药生产过程中,其糖基化修饰可能受到多种工艺参数的影响,因而容易产生异质性,并且抗体糖基化和抗体半衰期、免疫源性、ADCC、CDC等密切相关,所以单克隆抗体的糖基化修饰是重要的质量属性,需要在生物药尤其是生物类似药开发过程中重点关注,并加以调控。通过概述培养过程中的细胞株、培养工艺,以及培养基对糖型的影响,讨论如何在工艺开发过程开展研究,确保产品糖基化的一致性,从而保证单抗药物的疗效及安全性。
In monoclonal antibody drug production process, its glycosylation modification may be affected by a variety of process parameters, therefore come into being heterogeneity easily, and Antibody glycosylation is closely related to antibody half-life, immunogenicity, ADCC, CDC, and so on, so glycosylation is an important quality index for monoclonal antibody drugs, It is necessary to pay attention to and regulate the development of biological drugs, especially in the development of biosimilar. This paper strengthens the understanding of stable and consistent glycosylation by discussing the influence of various important control indicators in the culture process, such as pH, DO, osmolarity and temperature, to guide the efficacy and safety of monoclonal antibody drugs.
单克隆抗体 / 糖基化修饰 / 培养工艺 {{custom_keyword}} /
Monoclonal antibody / Glycosylation / Culture process {{custom_keyword}} /
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Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.
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The effect of ammonia on Chinese hamster ovary (CHO) cell growth and galactosylation of recombinant immunoglobulin (rIgG) was investigated using shaking flasks with serum free media containing 0–15 mM NH<sub>4</sub>Cl. The elevated ammonia inhibited cell growth and negatively affected the galactosylation of rIgG. At 15 mM NH<sub>4</sub>Cl, the proportions of monogalactosylated glycan with fucosex (monogalactosylated glycan with fucose) and digalactosylated glycan with fucose (G2F) were 23.9% and 6.3% lower than those at 0 mM NH<sub>4</sub>Cl, respectively. To reduce ammonia formation by cells, glutamate was examined as a substitute for glutamine. The use of glutamate reduced the accumulation of ammonia and enhanced the production of rIgG while depressing cell growth. At 6 mM glutamate, ammonia level did not exceed 2 mM, which is only one third of that at 6 mM glutamine. Also, a 1.7-fold increase in the titer of rIgG and specific rIgG productivity, <i>q</i> <sub>rIgG</sub>, was achieved at 6 mM glutamate. The galactosylation of rIgG was favorable at 6 mM glutamate. The proportion of galactosylated glycans, G1F and G2F, at 6 mM glutamate was 59.8%, but it was 50.4% at 6 mM glutamine. The use of glutamate also increased complement-dependent cytotoxicity activity, one of the effector functions of rIgG. Taken together, substitution of glutamine by glutamate can be considered relevant for the production of rIgG in CHO cells since glutamate not only enhances <i>q</i> <sub>rIgG</sub> but also generates a higher galactosylation essential for the effector function of rIgG.
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