Glycosylation decreases aggregation and immunogenicity of adalimumab Fab secreted from Pichia pastoris

Hitomi Nakamura, Masato Kiyoshi, Makoto Anraku, Noritaka Hashii, Naoko Oda-Ueda, Tadashi Ueda, Takatoshi Ohkuri

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


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 in Pichia 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-life in vivo.

Original languageEnglish
Pages (from-to)435-443
Number of pages9
JournalJournal of biochemistry
Issue number4
Publication statusPublished - Apr 1 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology


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