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Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

ABSTRACT: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.

SUBMITTER: Tran AM

PROVIDER: S-EPMC5379694 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

Tran Anh-Minh AM Nguyen Thanh-Thao TT Nguyen Cong-Thuan CT Huynh-Thi Xuan-Mai XM Nguyen Cao-Tri CT Trinh Minh-Thuong MT Tran Linh-Thuoc LT Cartwright Stephanie P SP Bill Roslyn M RM Tran-Van Hieu H

BMC research notes 20170404 1

<h4>Background</h4>Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation ...[more]

PMID: 28376863

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Project description:Despite recent advances in critical care and ventilator management, acute lung injury and acute respiratory distress syndrome continue to cause significant morbidity and mortality. Granulocyte-macrophage colony stimulating factor may be beneficial for patients with acute respiratory distress syndrome.To determine whether intravenous infusion of granulocyte-macrophage colony stimulating factor would improve clinical outcomes for patients with acute lung injury/acute respiratory distress syndrome.A randomized, double-blind, placebo-controlled clinical trial of human recombinant granulocyte-macrophage colony stimulating factor vs. placebo. The primary outcome was days alive and breathing without mechanical ventilatory support within the first 28 days after randomization. Secondary outcomes included mortality and organ failure-free days.Medical and surgical intensive care units at three academic medical centers.One hundred thirty individuals with acute lung injury of at least 3 days duration were enrolled, out of a planned cohort of 200 subjects.Patients were randomized to receive human recombinant granulocyte-macrophage colony stimulating factor (64 subjects, 250 ?g/M) or placebo (66 subjects) by intravenous infusion daily for 14 days. Patients received mechanical ventilation using a lung-protective protocol.There was no difference in ventilator-free days between groups (10.7 ± 10.3 days placebo vs. 10.8 ± 10.5 days granulocyte-macrophage colony stimulating factor, p = .82). Differences in 28-day mortality (23% in placebo vs. 17% in patients receiving granulocyte-macrophage colony stimulating factor (p = .31) and organ failure-free days (12.8 ± 11.3 days placebo vs. 15.7 ± 11.9 days granulocyte-macrophage colony stimulating factor, p = .16) were not statistically significant. There were similar numbers of serious adverse events in each group.In a randomized phase II trial, granulocyte-macrophage colony stimulating factor treatment did not increase the number of ventilator-free days in patients with acute lung injury/acute respiratory distress syndrome. A larger trial would be required to determine whether treatment with granulocyte-macrophage colony stimulating factor might alter important clinical outcomes, such as mortality or multiorgan failure. (ClinicalTrials.gov number, NCT00201409 [ClinicalTrials.gov]).

Dataset Information

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

Publications

Pichia pastoris versus Saccharomyces cerevisiae: a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor.

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