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Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

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An Erratum to this article was published on 01 June 2012

Abstract

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.

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Acknowledgments

The authors would like to thank Nathan Sharkey and Seemab Shaikh for DasGip and Micro24 runs, Nga Rewa Houston-Cummings and Dongxing Zha for the strain YGLY13992, Bianka Prinz for the strain YGLY14401, Daniel Hopkins and Stephan Hamilton for the strain YGLY12900, Alissa Rittenhour for analytical support, and Erin Giaccone, Sujatha Gomathinayagam, and Sandra Rios for protein purification.

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  1. GlycoFi, Biologics Discovery, Merck & Co., Inc, 21 Lafayette St. Suite 200, Lebanon, NH, 03766, USA

    Byung-Kwon Choi, Shannon Warburton, Heping Lin, Rohan Patel, Meehl Meehl, Marc d’Anjou, Terrance A. Stadheim & Natarajan Sethuraman

  2. Department of Pathology and Cell Biology, Columbia University, 630 W. 168th St, New York, NY, 10032, USA

    Istvan Boldogh & Liza Pon

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  1. Byung-Kwon Choi
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Correspondence to Natarajan Sethuraman.

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Choi, BK., Warburton, S., Lin, H. et al. Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris . Appl Microbiol Biotechnol 95, 671–682 (2012). https://doi.org/10.1007/s00253-012-4067-3

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