Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

Abstract

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio-better” protein therapeutics and cell culture vaccine production.

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

“…Most of the presently available glycoengineering techniques address and alter the processing machinery by knocking down, inhibiting, or overexpressing glycosylation enzymes (22,25,30,32,55,56) and thus are designed to modify simultaneously all the glycans carried by a given protein. Combining site-directed glycoprotein mutagenesis and classic glycoengineering techniques toward the limiting enzymes, as determined by flux analysis, can yield specific and targeted distributions of glycan on a single site.…”

“…Altering glycostructures in production processes is mostly done by variation of production conditions or by changing the activity level of specific glycosyltransferases and glycosyl hydrolases and has primarily been studied on glycoproteins with a single N-glycosylation site (monoclonal antibody) (22,(29)(30)(31)(32)(33). The analysis of site-specific microheterogeneity on a protein with multiple N-glycosylation sites in cell or tissue-derived extracts requires more elaborate analytical methods that have become available only recently (16,17,(34)(35)(36).…”

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

“…Thus, it will be of great value to integrate our model with models of other metabolic pathways. Such coupled models would enable predictive glycoengineering using both genetic means (Lin et al, 2015; Meuris et al, 2014; Wu and Chan, 2014), and media alterations, such as alternative sugars and other media additives. Furthermore, such a coupled model could help identify the mechanisms underlying media effects on glycosylation.…”

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

“…Fluorescently labeled N-glycans were separated by HILIC on an Acquity UPLC instrument (http://glycobase.nibrt.ie/glycobase/browse_glycans.action ) was used to confirm that a suggested N-glycan elutes in a distinct chromatographic peak using glucose units as well as knowledge of the biosynthetic pathway of N-glycans with an emphasis on specificity of N-16 glycosylation in CHO cell line (31,32). Finding of informative fragments after MS/MS analysis to confirm a structure was performed using GlycoWorkbench (33).…”