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Background and novelty N-glycans constitute an important information carrier in protein-driven signaling networks. Amongst others, N-glycans contribute to protein folding quality, adjust protein turnover and operate as address label for targeting proteins to specific cells and tissues [1]. Hence, the composition of N-glycans attached to recombinant glycoprotein therapeutics is vital for in-vivo therapeutic efficacy and strongly depends on the choice of the expression host [2,3]. Due to absence or silencing of glycosyltransferase genes homologue to human enzymes, biotechnologically used cell lines are limited by their intrinsic glycosylation machinery and produce host specific glycoforms. Cetuximab, a therapeutic chimeric mouse/human monoclonal antibody (IgG1), is N-glycosylated both at the CH2-domain (Asn299) and at the VH-domain (Asn88) (Figure 1A). Sold under the trade name Erbitux, Cetuximab is expressed from a murine myeloma cell line and targets the human EGF receptor [4], which is overexpressed in about 1/3 of all human cancers. The antibody is highly decorated with the aGal-epitope (Gala1-3Galb14GlcNAc) which has been shown to result in fatal allergic/ hypersensitivity response in several patients [5]. The design of new quality-optimized and functionally improved biopharmaceuticals with properties conferred by host cell unrelated N-glycans requires a rational Golgi engineering strategy. Here, we apply GET, a system that enables the positioning of a desired catalytic glycosyltransferase activity into a favorable localization within the intracellular glycosylation machinery, to suspension CHO cells developed to secrete suitable amounts (200 μg/ml) of Cetuximab as a model glycoprotein. The presented Golgi engineering project aims in the extension of the intrinsic glycosylation repertoire enabling CHO cells to produce new human-type glycosylation motifs as indicated in Figure 1A: (i) GalNAcb1,4GlcNAc-R (LacdiNAc, LDN),(ii) GlcNAc in b1,4 linkage to central mannose residue (bisecting GlcNAc, bGN), (iii) Galb1,4(Fuca1,3)GlcNAc-R (Lewis, Le) and (iv) NeuAca2,3Galb1,4 (Fuca1,3)GlcNAc-R (Sialyl-LewisX, sLe). To assemble (ii) and (iv), we coexpress GnT3 and FT7. As shown earlier, the latter enzyme catalyzes fucosylation exclusively of (iv). Therefore, we included in our study a variant of FT6 that is targeted to the early Golgi compartment with the aim to additionally generate structure (iii) [6,7]. The uncommon LDN motif (i) which is e.g. detected on lutropin is assembled by human B4GalNT3 [8,9]. We analyze oligosaccharides released from the products of genetically engineered CHO cells based on the resolution of single glycosylation sites of VHand CH2glycopeptides by quantitative NP-HPLC-FLD and use our comprehensive oligosaccharide standard library to identify novel oligosaccharide motifs.

bmc proceedings

Golgi engineering of CHO cells by targeted integration of glycosyltransferases leads to the expression of novel Asn-linked oligosaccharide structures at secretory glycoproteins