CHO starter cell lines for manufacturing of proteins with pre-defined glycoprofiles.

Backround Glycosylation of protein therapeutics is influenced by a multifaceted mix of product intrinsic properties, host cell genetics and upstream process parameters. Industrial CHO cell lines may have several deficits in their glycosylation pattern for some applications, like high fucose content (corresponding to a low ADCC profile) and low galactosylation and sialylation levels (proposed to decrease activity and/ or pharmacokinetics). We have successfully applied the GlymaxX technology [1] abolishing fucose synthesis in well-established CHO DG44 and K1 platforms and pre-existing producer cell lines (glycan modulator GM1). Here we extend this strategy by other engineering approaches to enable production of protein therapeutics with desired glycosylation features. Through stable integration of other genes for glycosylation enzymes we are able to tune galactosylation (glycan modulator GM2) and sialylation (glycan modulator GM3). These glycan modulators can specifically be combined to address certain desired oligosaccharide patterns. We postulate that modulating effects of GM2 and GM3 require a specific expression level. In this case the combination of high level target protein expression and defined levels of glycan modulators becomes extremely rare. Therefore, the characterization of clones with individual stable levels of glycanmodulator expression is a prerequisite for industrial application.