The Production of Glycoproteins by Transient Expression in Mammalian Cells

Recent advances in the production of proteins in insect and mammalian cells for structural biology

“…5D). As this glycan-type is high-mannose, the oligosaccharide chains can be trimmed to leave one GlcNAc using endo H. Kifunensine has been used in large-scale transient production of glycoproteins in HEK 293T cells for crystallization studies (Nettleship et al, 2009) mainly with endo H treatment (Bishop et al, 2009;Bowden et al, 2008a,b;Carafoli et al, 2009) but also without .…”

“…In general, this is done by small scale transient transfection of mammalian cells which allows screening and analysis to be completed in approximately 4 days (Lee et al, 2009;Nettleship et al, 2009). Expression screening can be performed with either attached (Lee et al, 2009;Nettleship et al, 2009) or suspension (Chapple et al, 2006;Davies et al, 2005) (Chapple et al, 2006). Evaluation of expression levels was by dot blot and was shown to correlate with expression levels subsequently obtained in 50 ml cultures (Chapple et al, 2006).…”

The impact of glycosylation on monoclonal antibody conformation and stability

“…These glycoforms may be targeted by glycosylation engineering, but may also be affected by cell culture conditions. [10][11][12] In this report, we focus on deglycosylated antibodies. Changes in a product's glycosylation pattern may significantly alter its intrinsic properties and stability, [13][14][15][16][17][18][19] thereby adding challenges for downstream process development.…”

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

“…The quantity of plasmid DNA generated with miniprep kits ($25 lg) is sufficient to transfect up to 25 mL of cell culture, which is enough for small-scale affinity purification and crystallization trials [13]. For that purpose, the addition of the glycosyltransferase inhibitor kifunensine can be used to generate glycoproteins with high mannose-type glycosylation that are more susceptible to endoglycosidases treatment and to crystallization [38,39]. Preliminary results have shown that this transfection method can also be applied to suspension CHO cells grown in serum-free medium (data not shown).…”