Next-gen glycoengineering: combining cellular and metabolic engineering to fine tune mAb β1,4-galactosylation
N-linked Fc galactosylation drives variability in therapeutic mAb quality, affecting complement-dependent cytotoxicity and ADCC. Current control strategies suffer from narrow ranges, productivity loss, or unwanted glycoforms. Here, we combine metabolic and cellular glycoengineering by feeding 2-deoxy-2-fluorogalactose (2FG) to CHO cells engineered for hypergalactosylation via COSMC knockout and…
Therapeutic monoclonal antibodies (mAbs) with N-linked Fc galactosylation exhibit variability in quality, impacting complement-dependent cytotoxicity and ADCC. Current control methods have limited ranges, causing productivity losses and unwanted glycoforms. Researchers have now combined metabolic and cellular glycoengineering techniques.
By feeding 2-deoxy-2-fluorogalactose (2FG) to CHO cells engineered for hypergalactosylation via COSMC knockout and {beta}4-galactosyltransferase overexpression, they achieved broad, tunable control over Fc {beta}4-galactosylation. This method was tested in different cell lines, revealing that 2FG dosing does not translate across all lines.
A new cell-normalised, multi-bolus feeding strategy was developed, which expanded control in certain cell lines while reducing aglycosylation and Man5 formation. This strategy, applied in a fed-batch Ambr(R) 250 process, reduced the amount of 2FG needed, showing its efficiency. Ultimately, Bio-Layer Interferometry identified Fc {beta}4-galactosylation as the strongest predictor of Fc{gamma}RIIIA (CD16a) binding affinity.
These advancements pave the way for controlling mAb galactosylation across a wide range, potentially fine-tuning downstream pharmacological activity.
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