Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production

Qiong Wang, Cheng Yu Chung, Weiming Yang, Ganglong Yang, Sandra Chough, Yiqun Chen, Bojiao Yin, Rahul Bhattacharya, Yingwei Hu, Christopher T. Saeui, Kevin J Yarema, Michael J. Betenbaugh, Hui Zhang

Research output: Contribution to journalArticlepeer-review

Abstract

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu3ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu3ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β-1,4- and β-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation. Supplementation of 1,3,4-O-Bu3ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild-type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4-O-Bu3ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild-type CHO cells. Moreover, a detailed mass spectrometric ESI-LC-MS/MS characterization of glycans at each of the three N-glycosylation sites of EPO showed that the 1st N-site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4-O-Bu3ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu3ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.

Original languageEnglish (US)
Article number1800186
JournalBiotechnology Journal
DOIs
StateAccepted/In press - Jan 1 2018

Keywords

  • butyrated ManNAc
  • EPO
  • glycoengineering
  • intact glycopeptide analysis, sialylation

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Molecular Medicine

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