Next-gen glycoengineering: combining cellular and metabolic engineering to fine tune mAb β1,4-galactosylation
- 1School of Chemical & Bioprocess Engineering, University College Dublin
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 {beta}4-galactosyltransferase overexpression (C /GT+), achieving broad, tuneable control of Fc {beta}4-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(R) 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 {beta}4-galactosylation as the strongest correlate of Fc{gamma}RIIIA (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.
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BibTeX
@misc{tsopanoglou2026nextgen,
title = {{Next-gen glycoengineering: combining cellular and metabolic engineering to fine tune mAb β1,4-galactosylation}},
author = {A. Tsopanoglou and M. Ghahremanzamaneh and I. Gomez Aquino and K. Doyle and B. Glennon and S. Carillo and J. Bones and I. Jimenez del Val},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.26.754714},
url = {https://www.biorxiv.org/content/10.64898/2026.09.26.754714v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Next-gen glycoengineering: combining cellular and metabolic engineering to fine tune mAb β1,4-galactosylation
AU - A. Tsopanoglou
AU - M. Ghahremanzamaneh
AU - I. Gomez Aquino
AU - K. Doyle
AU - B. Glennon
AU - S. Carillo
AU - J. Bones
AU - I. Jimenez del Val
PY - 2026
DA - 2026/09/26
PB - bioRxiv
DO - 10.64898/2026.09.26.754714
UR - https://www.biorxiv.org/content/10.64898/2026.09.26.754714v1
AB - 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 {beta}4-galactosyltransferase overexpression (C /GT+), achieving broad, tuneable control of Fc {beta}4-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(R) 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 {beta}4-galactosylation as the strongest correlate of Fc{gamma}RIIIA (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.
ER -