A Simplified and Efficient Process for Insulin Production in Pichia pastoris

Polez, Sulena; Origi, Domenico; Zahariev, Sotir; Guarnaccia, Corrado; Tisminetzky, Sergio; Skoko, Nataša; Baralle, Marco

doi:10.1371/journal.pone.0167207

Cited by 35 publications

(15 citation statements)

References 29 publications

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“…Their rapid growth, microbiological safety, and amenability to high-density fermentation in simple media render them particularly suited to large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process [15]. In recent years, as one of the most important industrial organisms for heterologous protein production, Komagataella phaffii (K. phaffii) has been used widely for the production of a broad range of recombinant drug products, including antibody fragments [16], growth factors [17], interferon [18], interleukin [19], insulin precursors [20], and staphylococcal kinase [21] etc.…”

Section: Introductionmentioning

confidence: 99%

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

et al. 2020

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Background As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). Results After codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly. Conclusions The combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.

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Section: Introductionmentioning

confidence: 99%

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

et al. 2020

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“…Growing incidence of diabetic mellitus globally demands for large scale production of insulin and its analogues [6,7]. While initial dependencies were on animal origin insulin, advances in recombinant DNA technologies made it possible to bring its rst insulin product in 1980s.…”

Section: Introductionmentioning

confidence: 99%

Large Scale Purification and Characterization of A21 Deamidated Variant-Most Prominent Post Translational Modification (PTM) for Insulins Which Is Widely Observed in Insulins Pharmaceutical Manufacturing

Shukla

Sriva

et al. 2020

Preprint

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BackgroundThe rapid growth of global diabetic population attracts the large scale manufacturing of insulin and analogues to meet the unmet demand. Biopharmaceutical development demands appropriate understanding of product variant, which are formed due to post-translational modification. Deamidation is the most common post‐translational modification occurring in insulins and insulin analogues. Same time, the same variant is most prominent product variant occur during biopharmaceutical manufacturing process or during the storage. Often, one or the other deamidated variant is used as an impurity standard during in-process and final product analysis in the QC system. However, purification of large quantity of highly purified deamidated material is always being challenging due to very similar mass, similar ionic properties, and high structural similarity of the variant compared to the parent productResultsIn the present work authors tried to come up with a novel, easy and large scale process of purification of deamidated impurity with high purity from insulin. Anion exchange resin PolyQuat offers a high ionic capacity for both strong anion exchange and weak anion exchange species. Due to this unique functionality, PolyQuat offers enhanced performance and better selectivity than conventional ion exchange media, which enables to purify closely related A21 desamido variant from insulin. This single chromatography step can enrich the purity of desamido insulin to >96%. The PolyQuat elution pool was concentrated through the preparative RP-HPLC column in the bind elute mode. The RP-HPLC elution pool was precipitated and lyophilized in powder form. Detailed characterization of human insulin variant (+1 Da desamido) was carried out for its primary, secondary and tertiary structure through state-of-the-art mass spectrometry methods, circular dichroism and intrinsic fluorescence and was compared with human insulin. Intact, reduced and Peptide Mass Fingerprinting analysis identified an increase of 1Da mass in the peptide bearing A21 amino acid. We have also compared the extent of IR-B phosphorylation by A21 desamido insulin against human insulin reference standard. As biological functions are governed by protein structure, IR-B phosphorylation assay was performed to compare functional activity of AspA21 human insulin variant and human insulin. The comparative phosphorylation profiles and relative potency clearly suggested no impact of A21 deamidation on the functional activity of recombinant human insulin. ConclusionsThe present work reveals efficient scalable process to manufacture insulin desamido variant in the powder form with more that 60% recovery with 97% purity. This simplified process for manufacturing deamidated variant presented in this study can be handy for the insulin manufacturers to make large quantity variant standard and use for their process development as well as routine manufacturing process. Purified insulin desamido variant allowed detailed characterization of the species as well helps in using as an impurity standard for process monitoring in QC, which aids in robust control for insulin manufacturing.

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“…Furthermore, large scale human insulin production in P. pastoris has been studied. Human insulin precursor is successfully produced using fed-batch fermentation with protein yield to be 2.26-3.8 g/L (Gurramkonda et al, 2010;Polez et al, 2016;Xie et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

Kurniatin¹,

Masduki²,

Nurainy³

et al. 2019

American Journal of Biochemistry and Biotechnology

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A spacer peptide (Glu-Ala repeats) connects N-terminal of a polypeptide precursor to the signal peptide of α-mating factor (α-MF) from Saccharomyces cerevisiae. During the polypeptide precursor secretion, the signal peptide will be first cleaved by signal peptidase in the endoplasmic reticulum, followed by Kex2 endopeptidase and finally the spacer peptide is removed by dipeptidyl aminopeptidase in the Golgi apparatus. In the case of insulin precursor, unfortunately, mixture forms of the insulin precursor containing a spacer peptide and the correct insulin precursor is observed in Pichia pastoris. To overcome this problem, a gene encoding insulin Aspart precursor without the spacer peptide has been constructed and its expression has been investigated in this study. The gene of insulin Aspart precursor has been successfully expressed extracellularly in P. pastoris KM71 with molecular mass of 6306.8 Da as determined by LC-ESI-MS. The expression level of insulin Aspart precursor is affected by aeration, cell density and methanol as an inducer. In shake flask production, the insulin Aspart precursor was optimally produced by 3% methanol induction every 24 h for two days, at initial cell density (OD600) of 60 and aeration (ratio of culture volume to flask volume) of 1:25. Based on Western blot analysis, there is no intracellular insulin Aspart precursor detected. Therefore, the spacer peptide is not essential for insulin Aspart precursor secretion in P. pastoris and removal of spacer resulted in insulin Aspart precursor homogeneity.

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A Simplified and Efficient Process for Insulin Production in Pichia pastoris

Cited by 35 publications

References 29 publications

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

Large Scale Purification and Characterization of A21 Deamidated Variant-Most Prominent Post Translational Modification (PTM) for Insulins Which Is Widely Observed in Insulins Pharmaceutical Manufacturing

The Use of α-MF Signal Peptide Without Spacer for Producing Insulin Aspart Precursor in Pichia pastoris KM71

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