Next-gen glycoengineering: combining cellular and metabolic engineering to fine-tune mAb β1,4-galactosylation
Abstract
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 β4-galactosyltransferase overexpression (C-/GT+), achieving broad, tuneable control of Fc β4-galactosylation. In DP12 C-/GT+ cells, a split-feed regimen spanned a wide galactosylation range (95-46%) but generated substantial aglycosylated and Man5 glycoforms. The same regimen in a second host, VRC01 C-/GT+, gave only a narrow range, indicating 2FG dosing does not transfer directly across cell lines. Based on a proposed mechanism where 2FG is metabolised via the Leloir pathway into inhibitory fluorinated nucleotide-sugar intermediates, we developed a cell-normalised, multi-bolus feeding strategy that substantially broadened control in VRC01 C-/GT+ cells while suppressing aglycosylation and Man5 formation. In a fed-batch Ambr® 250 process, this strategy achieved comparable galactosylation reduction with roughly ten-fold less 2FG, showing that the fraction of mAb secreted after feeding is a key determinant of control strategy efficiency. Across the resulting glycoform range, Bio-Layer Interferometry identified Fc β4-galactosylation as the strongest correlate of FcγRIIIA (CD16a) binding affinity (R2 = 0.92). Together, these findings establish combined metabolic and cellular glycoengineering as a platform for controlling mAb galactosylation across a broad range, with potential to fine-tune downstream pharmacological activity. Highlights Cell-normalised 2FG feeding tunes mAb galactosylation C-/GT+ hypergalactosylation extends the achievable control range Post-feed production timing governs 2FG feeding efficiency Fed-batch bioreactors need ∼10-fold less 2FG for an equivalent control range Galactosylation is the strongest correlate of CD16a binding affinity