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.