<scp> <i>N</i> </scp> ‐acetyl‐ <scp>D</scp> ‐mannosamine, a novel additive, effectively reducing high mannose glycosylation of monoclonal antibody without affecting other quality attributes

N-linked glycosylation stands as a pivotal quality attribute for monoclonal antibodies (mAbs), particularly the high mannose (Man5) variant, which significantly influences the pharmacokinetics of mAbs. Traditional approaches to modulate Man5 have frequently resulted in suboptimal outcomes. In this investigation, we introduced a novel additive, N-acetyl-d-mannosamine (ManNAc), which selectively targeted and reduced Man5 without compromising other product quality attributes (PQAs). The study further examined optimal concentrations and timing for the incorporation of ManNAc in the mAbs expression process utilizing CHO-K1 cells within a fed-batch shaker flask culture mode. In the ManNAc titration experiments, we established groups at concentrations of 5, 10, 15, 20, 40, 60, 80, and 100 mM. The findings revealed a concentration-dependent decrease in Man5, with reductions reaching as low as 2.9% from an initial 8.9%. Importantly, cellular growth, metabolism, and other PQAs remained unaffected. Regarding the timing of ManNAc addition, groups were set for days N-1, 0, 5, and 11. The results indicated that ManNAc addition on Day 11 did not affect Man5 levels, whereas earlier additions proved effective. A full factorial design was employed to assess the interplay between ManNAc concentration and addition timing, revealing no significant interaction. Consequently, it is recommended to administer 20-40 mM ManNAc prior to Day 4. The strategy of introducing 20 mM ManNAc on Day 0 has been successfully implemented across 12 clones, achieving an average Man5 reduction of 46%. Collectively, these findings delineate a novel and efficacious strategy for the Man5 modulation, promising enhanced control over this critical quality attribute in mAbs production.