Article

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

  1. Min Zhang1,2,*,
  2. Kerry Koskie1,
  3. James S. Ross1,*,
  4. Kevin J. Kayser1,
  5. Matthew V. Caple1

Article first published online: 11 DEC 2009

DOI: 10.1002/bit.22633

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 105, Issue 6, pages 1094–1105, 15 April 2010

Additional Information

How to Cite

Zhang, M., Koskie, K., Ross, J. S., Kayser, K. J. and Caple, M. V. (2010), Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol. Bioeng., 105: 1094–1105. doi: 10.1002/bit.22633

Author Information

  1. 1

    Cell Sciences & Development (CSD), SAFC Biosciences (a member of Sigma–Aldrich Group), 2909 Laclede Avenue, Saint Louis, Missouri 63103; telephone: 314-771-5765 ext 7727; fax: 314-286-7645

  2. 2

    Current address: Molecular Biology/Ferm/Cell Culture, Bioprocess R&D, Eli Lilly and Company, Indianapolis, Indiana 46285; telephone: 317-433-1791; fax: 317-276-3406

*Cell Sciences & Development (CSD), SAFC Biosciences (a member of Sigma–Aldrich Group), 2909 Laclede Avenue, Saint Louis, Missouri 63103; telephone: 314-771-5765 ext 7727; fax: 314-286-7645.

Publication History

  1. Issue published online: 24 FEB 2010
  2. Article first published online: 11 DEC 2009
  3. Accepted manuscript online: 11 DEC 2009 12:00AM EST
  4. Manuscript Accepted: 30 NOV 2009
  5. Manuscript Revised: 30 OCT 2009
  6. Manuscript Received: 16 JUL 2009

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Keywords:

  • Chinese Hamster Ovary (CHO);
  • glycosylation;
  • sialic acid;
  • sialidase;
  • RNA interference (RNAi)

Abstract

Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNγ) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNγ was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology. Biotechnol. Bioeng. 2010;105: 1094–1105. © 2009 Wiley Periodicals, Inc.

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