Glycoengineering design options for IgG1 in CHO cells using precise gene editing

Abstract

Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.

EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity

“…The CRISPR/Cas9 system was used for gene KO in CHO cells as described previously (47). CHOZN GS Ϫ/Ϫ cells with stable expression of human ␣-galactosidase A were used as the parental clone for KO man1a1/1a2/1b1/1c1.…”

Direct quality control of glycoengineered erythropoietin variants

“…To address this need various glycoengineering approaches have been developed 8 – 13 . Attractive emerging strategies include the so-called GlycoDelete approach wherein complex N -glycans are reduced to trisaccharide stems 10 , 11 , and a second approach wherein combinatorial knock out (KO) and knock in (KI) of various glycosyltransferase enzymes in CHO are used to create a cell with custom designed glycosylation capacity to produce a more homogenous glycosylation profile 8 , 14 (Fig. 1a ).…”

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification

“…CHO Cell Culture and Generation of Acp2/5 Knockout-A CRISPR/ Cas9 based approach was used for the gene knockout (KO) in CHO cells as described preciously (19). CHOZN GSϪ/Ϫ cells with stable expression of alpha-galactosidase were used as the parental clone (as below: wild type) for the Acp2/5 KO.…”

“…We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases. Molecular & Cellular Proteomics 18: [16][17][18][19][20][21][22][23][24][25][26][27]2019. DOI: 10.1074/mcp.RA118.000967.…”