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

Autor: Wang Q; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Chung CY; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Yang W; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA., Yang G; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA., Chough S; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Chen Y; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Yin B; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Bhattacharya R; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21231, USA., Hu Y; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA., Saeui CT; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21231, USA., Yarema KJ; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21231, USA., Betenbaugh MJ; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA., Zhang H; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Jazyk: angličtina
Zdroj: Biotechnology journal [Biotechnol J] 2019 Apr; Vol. 14 (4), pp. e1800186. Date of Electronic Publication: 2018 Oct 08.
DOI: 10.1002/biot.201800186
Abstrakt: 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-Bu 3 ManNAc 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-Bu 3 ManNAc 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-Bu 3 ManNAc 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-Bu 3 ManNAc 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-Bu 3 ManNAc treatments mainly affects the 2nd and 3rd N-glycan sites of EPO protein. In summary, these results demonstrate 1,3,4-O-Bu 3 ManNAc 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.
(© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Databáze: MEDLINE
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