Glycosylation analysis of Chinese hamster ovary produced glycoproteins

Abstract

Glycosylation is a critical quality attribute of biotherapeutics produced from mammalian cells. Small changes in the glycan structures may have profound effects on the efficacy and functions of the glycoprotein, such as fucosylation of the IgG glycan reducing the ability of the IgG to bind to the FcϒRIIIA receptor. Chinese hamster ovary produced glycoprotein biotherapeutics may contain antigenic glycan structures that must be defined and monitored. Therefore, structural analysis of glycans is a key component in the development and production of glycosylated products. We discuss the current techniques available for glycan, glycopeptide and whole glycoprotein analysis. We emphasize recent analytic developments in high-throughput automated methods, as well as, further refinement of methods for separation and identification of isomers and potentially antigenic structures.

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

“…CHO cells are not known to express Nacetylglucosaminyltransferase III (Gn TIII), the enzyme responsible for producing a bisecting N-acetylglucosamine, which is known to inhibit core fucosylation and ultimately increase FcγRIIIa binding [61]. Additionally, CHO cells have the ability to express both N-acetylneuraminic acid (Neu5Ac) and low levels of the potentially immunogenic Nglycolylneuraminic acid (Neu5Gc) [62]. For these reasons, CHO cells are a suitable choice for exploring SiaNAz expression but require strong quality control in regards to protein glycosylation.…”

“…Two forms of sialylated species were observed: intact and lactonized. Given that sialylation in CHO cells takes place mostly in alpha-2,3 configuration [62], lactonization is possible as a reversible process. Overall, the ratio of SiaNAz to sialic acid increased when the concentration of Ac 4 ManNAz in the cell culture increased.…”

“…With respect to observation of SiaNAz, lower signals are observed upon esterification, suggesting that this reaction affects the azido function. The useful part of this experiment was to distinguish between α2-3 sialylation inherent to CHO cells [62] and α2,6 sialylation due to the plasmid's sialyltransferase [41,42], as indicated in ESM Fig. S5.…”

An integrated approach to analyze EG2-hFc monoclonal antibody N-glycosylation by MALDI-MS

“…21 When antibodies are expressed in Chinese hamster ovary (CHO) cells, Asn297 typically bears complex type biantennary glycans with a high level of core fucosylation and a variable number of galactose residues. 16 Three main glycoforms are present in most IgG molecules: G0F (no galactose and one core fucose), G1F (one galactose and one core fucose), and G2F (two galactose and one core fucose). Many reports describing variations of the IgG Fc glycosylation have demonstrated the importance of glycosylation as a post-translational modification.…”

“…16 Over the past few decades, there have been several studies exploring the structural, biological, and clinical roles of Ig glycosylation. These studies focused mainly on IgG molecules, the predominant type of mAb biotherapeutic.…”