Cell Engineering For The Production Of Hybrid Type N-glycans In Mammalian Cells

Protein N-glycosylation modification is one of the most common post-translational modifications in eukaryotes,which affects the structure and function of the proteins,including the folding,molecular recognition,stability and immunogenicity of the proteins.However,there are some problems in the production of recombinant proteins in mammalian cells.One of the most important issues is the heterogeneity of N-glycans on recombinant proteins,which will directly affect the stability and function of recombinant proteins,such as antibody-dependent cell-mediated cytotoxicity(ADCC)and its half-life in the body.Recently,with the development of multiple disciplines such as gene editing technology,cell engineering and glycoengineering,the glycosylation sites and glycan structures on the proteins can be modified to reduce or even eliminate the heterogeneity of N-glycans,thereby improving the characteristics of recombinant proteins.Therefore,engineering mammalian cell expression systems to produce homogeneous glycosylated recombinant proteins has become a hot research topic in the biopharmaceutical industry.The main results were as follows:1.Golgi ?-mannosidase-?(MAN2A1/A2)in the N-glycosylation pathway was knocked out in human embryonic kidney cell 293(HEK293)by CRISPR Cas9 system firstly,blocking the synthesis of complex type N-glycans,and M2D-KO cell line was obtained.Subsequently,the ?1,6-fucosyltransferase(FUT8)was further knocked out to remove the core fucose modification,and finally DF-KO cell line was obtained.2.Lectin staining and mass spectrometry were used to identify the N-glycans on cell membrane.The results showed that both of the two knockout cells could not synthesize complex type N-glycans.The N-glycans on cell membrane of M2D-KO were mainly hybrid type N-glycans with core fucose modification,while the N-glycans on the cell membrane of DF-KO are mainly hybrid type N-glycans without core fucose modification.Subsequently,the secreted recombinant protein lysosomal acid lipase A(LIPA)and Fc were expressed in two knockout cell lines.After purification of the two recombinant proteins,the N-glycosylation modification was identified by using glycosidase treatment and mass spectrometry technologies.The results showed that the N-glycans on the recombinant protein expressed by the M2D-KO and DF-KO cell lines were the same as the N-glycans on the cell membrane.3.In order to reduce the level of high-mannose type N-glycans in DF-KO cell line,we overexpressed the genes MsdS and MAN1A2 which code the ?1,2-mannosidases from Aspergillus saitoi and human respectively.The mass spectrometry results showed that overexpression MAN1A2 which codes human ?1,2-mannosidase had reduced the level of high-mannose type N-glycans.Therefore,in this study we successfully constructed cell lines expressing homogeneous hybrid type N-glycans modification,which provides a theoretical basis for the industrial production of pharmaceutical glycoproteins or recombinant proteins.