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Daniel Hopkins,
Sujatha Gomathinayagam, Alissa M Rittenhour,
Min Du,
Erik Hoyt,
Khanita Karaveg,
Teresa Mitchell,
Juergen H Nett,
Nathan J Sharkey,
Terrance A Stadheim,
Huijuan Li,
Stephen R Hamilton
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ABSTRACT: The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.
Glycobiology 08/2011; 21(12):1616-26. · 3.58 Impact Factor
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Gavin C Barnard,
Angela R Kull,
Nathan S Sharkey,
Seemab S Shaikh, Alissa M Rittenhour,
Irina Burnina,
Youwei Jiang,
Fang Li,
Heather Lynaugh,
Teresa Mitchell,
Juergen H Nett,
Adam Nylen,
Thomas I Potgieter,
Bianka Prinz,
Sandra E Rios,
Dongxing Zha,
Natarajan Sethuraman,
Terrance A Stadheim,
Piotr Bobrowicz
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ABSTRACT: The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.
Journal of Industrial Microbiology 09/2010; 37(9):961-71. · 1.80 Impact Factor
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Song Lin,
Zheng Shen,
Dongxing Zha,
Nathan Sharkey,
Bianka Prinz,
Stephen Hamilton,
Tej Venkatachalam Pavoor,
Beata Bobrowicz,
Seemab S Shaikh, Alissa M Rittenhour,
Thomas I Potgieter,
Piotr Bobrowicz,
Terrance A Stadheim
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ABSTRACT: A simple cell labeling method for sorting yeast Pichia pastoris antibody expressing strains is described. A small portion of secreted recombinant antibody retained on the cell surface was labeled with fluorescence detection antibody. The signal intensity of the labeled cell was correlated with the cell's antibody productivity. Using this labeling technique to sort a mixture model induced in the same fermenter where the cells of high producing strain were spiked into a population of a low producing strain at the frequency of 1:100,000, one round of sorting achieved a approximately 5000-fold enrichment of the high producing strain. A variety of P.pastoris strains expressing antibody sorted based on the signal intensity on the cell surface yielded titer improvements by 30% to 300%. Our data demonstrate that Pichia cell surface labeling is a simple, effective and reliable method for sorting Pichia antibody expressing strains for productivity improvement.
Journal of immunological methods 03/2010; 358(1-2):66-74. · 2.35 Impact Factor
