ManNAc attenuates Man5 glycoform abundance through GNE-mediated metabolic channeling of UDP-GlcNAc to N-glycosylation modifications via CMP-Neu5Ac biosynthesis

N-glycosylation, a critical quality attribute of monoclonal antibodies, plays a pivotal role in regulating pharmacokinetics and pharmacodynamics through high-mannose (Man5) glycoform modulation. While our previous work demonstrated that N-acetyl-D-mannosamine (ManNAc) supplementation effectively reduces Man5 levels without compromising antibody yield or other critical quality attributes, the mechanistic basis remained unclear. This study systematically investigates ManNAc’s regulatory mechanism through a multi-parametric analysis. Cellular uptake studies revealed a 3-day latency period preceding Man5 reduction post-ManNAc administration. Subsequent transcriptional profiling showed no significant alterations in Man5-associated enzyme expression (Mgat1, Mgat2, Man2a1, SLC35A3), while metabolomic analysis demonstrated marked elevation of intracellular ManNAc, uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), and cytidine-5’-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) levels. Mechanistic studies revealed two critical findings: (1) Chinese hamster ovary cells exhibit minimal endogenous N-acetyl-D-glucosamine-2-epimerase expression, and (2) CMP-Neu5Ac exerts potent inhibition on glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) activity in vitro, despite ManNAc’s lack of transcriptional regulation on GNE. We propose a metabolic flux redirection model, where ManNAc-derived CMP-Neu5Ac accumulation inhibits GNE activity, thereby shunting UDP-GlcNAc from sialic acid biosynthesis toward N-glycosylation pathways to reduce Man5 levels. This work not only identifies UDP-GlcNAc substrate limitation as a key constraint in antibody glycosylation but also establishes exogenous monosaccharide supplementation as a novel metabolic engineering strategy for Man5 optimization. These findings provide critical mechanistic insights for precision glycoengineering of therapeutic antibodies.