Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research.

Autor: Haryadi R; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Chan KF; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Lin PC; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Tan YL; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Wan C; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Shahreel W; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Tay SJ; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Nguyen-Khuong T; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Walsh I; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore., Song Z; Bioprocessing Technology Institute, A*STAR, 20 Biopolis Way, #06-01, Centros, 138668, Singapore. song_zhiwei@bti.a-star.edu.sg.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2024 Oct 04; Vol. 14 (1), pp. 23068. Date of Electronic Publication: 2024 Oct 04.
DOI: 10.1038/s41598-024-73722-z
Abstrakt: This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody's single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody's interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human β-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.
(© 2024. The Author(s).)
Databáze: MEDLINE
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