BI-HEX®-GlymaxX® cells enable efficient production of next generation biomolecules with enhanced ADCC activity

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

“…Expression of RMD in IgG-producing cells has already been shown to be an effective way of producing afucosylated IgG. 42 , 43 In an effort to streamline the cell line generation for the production of afucosylated IgGs, we constructed a set of plasmids for the co-expression of IgG and RMD. To evaluate the best expression strategy, three plasmids were generated ( Fig.…”

“… 42 Co-expression of RMD and IgG can be achieved in several ways, including introducing the RMD expression cassette into an IgG-expressing cell line 42 or engineering a CHO host to stably express RMD that would act as the starting point for the introduction of an IgG expression vector. 43 Sequential rounds of cell line engineering are time-consuming, so in a third scenario, the RMD and the IgG genes could be expressed simultaneously by introducing independent expression plasmids utilizing different selection markers. However, all three approaches require at least two different selection markers and selection steps, which increase the risk of long-term expression instability upon withdrawal of the selection markers during scale up for manufacturing or increased cost of production by introducing multiple selection reagents during manufacturing.…”

“…RMD functions as a deflecting enzyme to block the fucosylation pathway by enzymatic conversion of GDP-4-keto-6-deoxymannose, a metabolic intermediate of the pathway, to GDP-D-Rhamnose, a dead-end metabolite and a sugar that cannot be metabolized by CHO cells. 42 , 43 In previously published work, an existing mAb-producing cell line was engineered to express RMD or an RMD-expressing CHO cell line was engineered to express a mAb. 43 Both approaches involved two rounds of transfection, selection and screening.…”

“… 42 , 43 In previously published work, an existing mAb-producing cell line was engineered to express RMD or an RMD-expressing CHO cell line was engineered to express a mAb. 43 Both approaches involved two rounds of transfection, selection and screening. Here, we report the development of a simplified, single-step method for the rapid generation of CHO cell lines producing afucosylated mAbs using RMD co-expression.…”

In planta expression of active bacterial GDP‐6‐deoxy‐d‐lyxo‐4‐hexulose reductase for glycan modulation

“…Notably, our data suggest, that suppressed availability of GDP‐ l ‐fucose allows the core α1,3‐fucosyltransferase to use the structurally related UDP‐ l ‐galactose as donor substrate, in line with other results that report the transfer of l ‐galactose to N‐glycans in case of GDP‐ l ‐fucose shortage (Ohashi et al ., 2017; Rayon et al ., 1999). This should be considered by RMD‐based glycan engineering for commercial purposes (Puklowski et al ., 2013). In summary, transient co‐delivery of bacterial RMD into WT plants provides a straightforward alternative for the removal of core fucose compared to laborious genome editing approaches (Jansing et al ., 2019).…”