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

  • MAbs. 2025 Dec;17(1):2561823. doi: 10.1080/19420862.2025.2561823.
Ruiqiang Sun  1 ,  Miaomiao Chai  1 ,  Jiahao Man  2 ,  Guiju Yang  2 ,  Hai Shu  1 ,  Qiancheng Wang  1 ,  Cong Tian  1 ,  Linlin Wang  1 ,  Shanhui Liao  3 ,  Yifeng Zhang  4 ,  Yanyan Cao  3 ,  Lisha Xia  2 ,  Haili Yang  2 ,  Ting Zhang  2 ,  Luoyan Ma  2 ,  Shenwang Cao  2 ,  Qiao Gao  5 ,  Yichen Le  5 ,  Mingli Shi  5 ,  Quanxue Li  5 ,  Yinmao Fan  1 ,  Hang Zhou  6
Affiliations
  • 1. Cell Culture Process Development (CCPD), WuXi Biologics INC, Shanghai, China.
  • 2. Analytical Development (AS), WuXi Biologics INC., Shanghai, China.
  • 3. Industrial Microbial Development (IMD), WuXi Biologics INC., Shanghai, China.
  • 4. Cell Line Development (CLD), WuXi Biologics INC, Wuxi, China.
  • 5. Cell Line Development (CLD), WuXi Biologics INC., Shanghai, China.
  • 6. Bioprocess Research and Development (BRD), WuXi Biologics INC., Shanghai, China.
PMID: 40963238 DOI: 10.1080/19420862.2025.2561823
Abstract

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.

Keywords
Cytidine-5’-monophospho-N-acetylneuraminic acid; N-acetyl-D-mannosamine; glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine enzyme; high mannose; monoclonal antibodies; uridine-diphosphate-N-acetylglucosamine.