Here, we show the establishment of a new host cell line, termed BI-HEX-GlymaxXwhich is capable of producing highly active therapeutic antibodies. impacting the antibody structure itself, thereby avoiding negative effects on the PK/PD of the molecule. During the last decade it has been shown that antibodies with a reduced level of glycan fucosylation are much more potent in mediating ADCC, a mode of action particularly relevant for cancer therapeutics. Therefore, defucosylated antibodies are of major interest for biotherapeutics developers. To produce such antibodies, Boehringer Ingelheim has inlicensed the GlymaxXsystem from ProBioGen, Germany. This technology utilises the bacterial protein RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) which, when stably integrated into host cell lines, inhibits fucose de-novo biosynthesis. The enzyme deflects the fucosylation pathway by turning an intermediate (GDP-4-Keto-6-Deoxymannose) into GDP-Rhamnose, a sugar that cannot be metabolised by CHO cells. As a consequence, recombinant antibodies generated by such host cells exhibit reduced glycan fucosylation and 20-100 fold higher ADCC activity. Here, we show the establishment of a new host cell line, termed BI-HEX-GlymaxXwhich is capable of producing highly active therapeutic antibodies. We furthermore present data on the cell line properties FANCH concerning cell culture performance (e.g. titer, growth, transfection efficiency), process robustness and product quality reproducibility. == Methods == The BI-HEXhost cell line was transfected with the bacterial RMD enzyme and stably expressing clones were selected. The presence of RMD was confirmed by Western blotting. The clones were analysed for stability of RMD expression over time in continous culture (>100 days), glycoprofile structure, CD16 binding and ADCC activity of mAbs produced by these clones before selection of the final new BI-HEX-GlymaxXhost cell. Furthermore, we examined the growth and cultivation properties of the modified BI-HEXGlymaxXcells to ensure that the engineered host cell maintained the favourable manufacturability properties of BI-HEXand we tested the reproducibility of key product quality attributes of the generated antibodies. == Results == Up to date seven different antibodies were produced in our new BI-HEX-GlymaxXhost cell line. All molecules showed Resibufogenin a very significant reduction of fucosylation down to 1-3% compared to the control. Correlating with the low fucose levels, antibodies produced in BI-HEX-GlymaxXexhibited a 20-100 increased ADCC activity (Figure1A). This enhancement also correlated well with an increase in CD16 binding. For the routine cell line and process development we investigated the robustness of the defucosylation and its resulting activity enhancement. The results Resibufogenin indicated a high reproducibility between independent production runs. The ADCC level as well as the CD16 binding was robust for all analysed mAbs (Figure1B). Investigating the cell culture behaviour of the BI-HEX-GlymaxXand its parental BI-HEXcell line, we saw comparable results for their transfection efficiencies, doubling times, titer and production run performance. Depletion studies of RMD showed that this enzyme can be efficiently depleted during downstream purification of the mAb. Resibufogenin == Figure 1. == A) Comparison of ADCC activity of Rituximab produced in either BI-HEXor BI-HEX-GlymaxX.B)ADCC activity of 3 different mAbs produced in BI-HEX-GlymaxX. Three independent production runs were performed for each mAb. The mAbs were individually purified by protein A capture before ADCC activity determination. == Conclusions == Our new BI-HEX-GlymaxXcell line is capable of producing >90% defucosylated antibodies which exhibit a 20-100 fold higher ADCC activity compared to a normal CHO production cell line like BI-HEX. This increase in ADCC activity correlated with a stronger CD16 binding in those molecules. Furthermore, the BI-HEX-GlymaxXcells show the same manufacturing properties (transfection efficiency, doubling times, titer, peak cell density) to its originator cell line. For the depletion of RMD we’ve established a sensitive depletion assay and measured a complete reduction of RMD after the first purification step (protein A capture)..