Glycosylation decreases aggregation and immunogenicity of adalimumab Fab secreted from Pichia pastoris
Glycosylation decreases aggregation and immunogenicity of adalimumab Fab secreted from Pichia pastoris
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糖基化降低毕赤酵母分泌的阿达木单抗 Fab 的聚集和免疫原性
DOI：
10.1093/jb/mvaa116
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Ohkuri Takatoshi
中科院分区：
文献类型：
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作者：
Nakamura Hitomi;Kiyoshi Masato;Anraku Makoto;Hashii Noritaka;Oda-Ueda Naoko;Ueda Tadashi;Ohkuri Takatoshi
治疗蛋白的糖工程已被应用于提高几种治疗方法的临床疗效。在这里，我们研究了糖基化对治疗性抗体阿达木单抗Fab特性的影响。通过定点诱变在阿达木单抗Fab的H链常数区178位引入一个n -糖基化位点（H:L178N Fab）， H:L178N Fab在毕赤酵母中产生。表达的突变体Fab含有长聚糖链和短聚糖链（分别为L-glyco Fab和S-glyco Fab）。在pH漂移诱导的胁迫下Fab发生聚集的条件下，L-glyco Fab和S-glyco Fab都不容易发生聚集，且L-glyco Fab比S-glyco Fab更有效地抑制聚集。此外，对小鼠糖基化和野生型fab的抗原性进行比较发现，糖基化导致抗原性受到抑制。对Fab、L-glyco Fab和S-glyco Fab的药代动力学行为分析表明，糖基化Fab在大鼠体内的半衰期短于野生型Fab，其中L-glyco Fab的半衰期短于S-glyco Fab。因此，我们证明了糖链影响Fab的聚集和免疫原性，糖基化降低了体内的消除半衰期。
Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H chain constant region of adalimumab Fab through site-directed mutagenesis (H:L178N Fab), and the H:L178N Fab was produced inPichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behaviour of the Fab, L-glyco Fab and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-lifein vivo.