PRODUCTION OF HUMAN INSULIN PRECURSOR IN A 10 LITER BIOREACTOR USING A METHANOL PULSE FEEDING
Main Article Content
Abstract
ABSTRACT
Indonesia as a country that had 19.5 million diabetic patients in 2021 is still importing raw materials of human insulin. The development of human insulin production process is needed to support the government’s objective to be independent in medicine aspect. Human insulin precursor (HIP) expressed from Pichia pastoris X33/pD902-IP had been developed and optimized in small-scale cultivation. However, the scaling up in bioreactor 10 L has not been studied. Using a 10 L-bioreactor the fermentation of P. pastoris X33/pD902-IP was developed. Fermentation was done in 120 hours using a basal salt medium (half concentration) for the vegetative and induction media. To induce HIP expression, methanol is fed by pulse strategy with a gradient concentration of 1-3% (v/v) for 48 hours. The dry cell weight (DCW) and HIP titers were 72 g/L and 286 mg/L, respectively. This development is the first fermentation of HIP expressed by P. pastoris X33/pD902-IP in a 10 L-bioreactor.
ABSTRAK
Indonesia sebagai negara dengan penderita diabetes 19,5 juta orang pada tahun 2021 masih mengimpor bahan baku insulin manusia. Pengembangan proses produksi insulin manusia diperlukan dalam rangka mendukung tujuan pemerintah untuk mandiri di bidang produksi bahan baku obat. Ekspresi prekursor insulin manusia oleh Pichia pastoris X33/pD902-IP telah dikembangkan dan dioptimalkan dalam skala kultur kocok. Akan tetapi, produksinya pada bioreaktor 10 L belum dilakukan. Untuk itu, fermentasi P. pastoris X33/pD902-IP dipelajari pada bioreaktor 10 L. Fermentasi dilakukan selama 120 jam dengan menggunakan media garam basal (setengah konsentrasi). Untuk menginduksi ekspresi HIP, metanol diberikan dengan gradien konsentrasi 1-3% (v/v) secara berkala selama 48 jam. Berat sel kering dan titer HIP masing-masing adalah 72 g/L dan 286 mg/L. Proses ini merupakan fermentasi pertama HIP yang dihasilkan P. pastoris X33/pD902-IP pada skala bioreaktor 10 L.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
References
Baeshen MN, Bouback TAF, Alzubaidi MA, Bora RS, Alotaibi MAT, Alabbas OTO, Alshahrani SM, Aljohani AAM, Munshi RAA, Al-Hejin A, Ahmed MMM, Redwan EM, Ramadan HAI, Saini KS, Baeshen NA (2016) Expression and purification of c-peptide containing insulin using Pichia pastoris expression system. Biomed Res Int 2016:3423685. doi: 10.1155/2016/3423685
Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed MMM, Ramadan HAI, Saini KS, Redwan EM (2014) Cell factories for insulin production. Microb Cell Fact 13:141. doi: 10.1186/s12934-014-0141-0
Bataille D (2007) Pro-protein convertases in intermediary metabolism: Islet hormones, brain/gut hormones and integrated physiology. J Mol Med 85:673–684. doi: 10.1007/s00109-007-0167-4
Bernauer L, Radkohl A, Lehmayer LGK, Emmerstorfer-Augustin A (2021) Komagataella phaffii as emerging model organism in fundamental research. Front Microbiol 11:607028. doi: 10.3389/fmicb.2020.607028
Bredell H, Smith JJ, Görgens JF, van Zyl WH (2018) Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha. Yeast 35:519–529. doi: 10.1002/yea.3318
Chongchittapiban P, Borg J, Waiprib Y, Pimsamam J, Tongta A (2016) On-line methanol sensor system development for recombinant human serum albumin production by Pichia pastoris. Afr J Biotechnol 15:2374–2383. doi: 10.5897/ajb2015.15122
Crater JS, Lievense JC (2018) Scale-up of industrial microbial processes. FEMS Microbiol Lett 365:fny138. doi: 10.1093/femsle/fny138
Data Bridge (2021) Insulin Market – Global Industry Trends and Forecast to 2027 | Data Bridge Market Research. In: Data Bridge Mark Res Website. https://www.databridgemarketresearch.com/reports/global-insulin-market#. Accessed 14 Oct 2021
De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouzé P, Van De Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566. doi: 10.1038/nbt.1544
Ginovart M, Prats C, Portell X, Silbert M (2011) Exploring the lag phase and growth initiation of a yeast culture by means of an individual-based model. Food Microbiol 28:810–817. doi: 10.1016/j.fm.2010.05.004
IDF (2021) IDF Diabetes Atlas 10th edition. International Diabetes Foundation
Invitrogen Corporation (2002) Pichia Fermentation Process Guidelines. Prog Bot 67:1–11
Jiao L, Zhou Q, Su Z, Xu L, Yan Y (2018) High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins. Protein Expr Purif 147:1–12. doi: 10.1016/j.pep.2018.02.005
Jossen V, Eibl-Schindler R, Pörtner R, Kraume M, Eibl D (2016) Stirred bioreactors: Current state and developments, with special emphasis on biopharmaceutical production processes. In: Current Developments in Biotechnology and Bioengineering: Bioprocesses, Bioreactors and Controls. Pp 179–215. Elsevier, Amsterdam. doi: 10.1016/B978-0-444-63663-8.00007-0
Junker BH (2004) Scale-up methodologies for Escherichia coli and yeast fermentation processes. J Biosci Bioeng 97:347–364. doi: 10.1016/s1389-1723(04)70218-2
Karbalaei M, Rezaee SA, Farsiani H (2020) Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol 235:5867–5881. doi: 10.1002/JCP.29583
Kocaefe-Ozsen N, Yilmaz B, Alkim C, Arslan M, Topaloglu A, Kisakesen HLB, Gulsev E, Cakar ZP (2022) Physiological and molekuler characterization of an oxidative stress-resistant Saccharomyces cerevisiae strain obtained by evolutionary engineering. Front Microbiol 13:822864. doi: 10.3389/fmicb.2022.822864
Kurniatin PA, Masduki FF, Nurainy N, Ihsanawati, Natalia D (2019) The use of a-MF signal peptide without spacer for producing insulin aspart precursor in Pichia pastoris KM71. Am J Biochem Biotechnol 15:198-207. doi: 10.3844/ajbbsp.2019.198.207
Liu WC, Gong T, Wang QH, Liang X, Chen JJ, Zhu P (2016) Scaling-up fermentation of Pichia pastoris to demonstration-scale using new methanol-feeding strategy and increased air pressure instead of pure oxygen supplement. Sci Rep 6:18439. doi: 10.1038/srep18439
Nurdiani D, Hariyatun, Kusharyoto W (2018) Secretory expression of human insulin precursor in Pichia pastoris employing truncated a-factor leader sequence and a short C-peptide. Indones J Biotechnol 23:102–108. doi: 10.22146/ijbiotech.38958
Nurdiani D, Hariyatun, Utami N, Putro EW, Kusharyoto W (2020) Selecting Pichia pastoris recombinant clones for higher secretion of human insulin precursor into the culture supernatant. IOP Conf Ser: Earth Environ Sci 439:012017. doi: 10.1088/1755-1315/439/1/012017
Nurdiani D, Hariyatun, Utami N, Putro EW, Kusharyoto W (2022) Enhancement in human insulin precursor secretion by Pichia pastoris through modification of expression conditions. HAYATI J Biosci 29:22–30. doi: 10.4308/hjb.29.1.22-30
Ozcelik AT, Yilmaz S, Inan M (2019) Pichia pastoris promoters. Methods Mol Biol 1923:97–112. doi: 10.1007/978-1-4939-9024-5_3
Permen Kesehatan (2017) Peraturan Menteri Kesehatan No. 17 Tahun 2017 tentang Rencana Aksi Pengembangan Industri Farmasi dan Alat Kesehatan
Putro EW, Nurdiani D, Hariyatun, Utami N, Kusharyoto W (2021) Capture and intermediate purification of human insulin precursor from Pichia pastoris culture using cation exchange chromatography. IOP Conf Ser: Earth Environ Sci 762:012028. doi: 10.1088/1755-1315/762/1/012028
Putro EW, Nurdiani D, Hariyatun, Utami N, Kusharyoto W, Juanssilfero AB, Silvia RA (2022) Evaluating pulses and modified fed-batch feeding of methanol to increase expression level of human insulin precursor in Pichia pastoris high-density cultivation. Int J Adv Sci Eng Inf Technol 12:1001–1007. doi: 10.18517/ijaseit.12.3.15109
Randone BP (2014) Bioprocess Studies of Biomass and Recombinant Protein Production by the Methylotrophic Yeast Pichia pastoris. Thesis, University College of London
Tripathi L, Mani S, Raut R, Poddar A, Tyagi P, Arora U, de Silva A, Swaminathan S, Khanna N (2015) Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies. Front Microbiol 6:1005. doi: 10.3389/fmicb.2015.01005
Vasiljevic J, Torkko JM, Knoch K-P, Solimena M (2020) The making of insulin in health and disease. Diabetologia 63:1981–1989. doi: 10.1007/S00125-020-05192-7
Vlaev SD, Pavlova K, Rusinova-Videva S, Georgieva K, Georgiev D (2016) Agitation effects and kinetic constants of exoglucomannan production by antarctic yeast strain in a stirred tank bioreactor. Chem Biochem Eng Q 30:393-400. doi: 10.15255/CABEQ.2015.2276
Vogl T, Gebbie L, Palfreyman RW, Speight R (2018) Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Appl Environ Microbiol 84:e02712-17. doi: 10.1128/AEM.02712-17
Wu J, Gong G, Han S, Zhang W, Hu Y, Xie L (2019) Expression, purification, and characterization of the Degludec precursor DesB30. Protein Expr Purif 161:28–39. doi: 10.1016/j.pep.2019.04.010
Yang X (2014) Scale-up of microbial fermentation process. Pp. 669–675. Bull AT, Junker B, Katz L, Lynd LR, Masurekar P, Reeves CD, Zhao H (Eds). Manual of Industrial Microbiology and Biotechnology, Third Edition. doi: 10.1128/9781555816827.ch47
Zhu W, Xu R, Gong G, Xu L, Hu Y, Xie L (2021) Medium optimization for high yield production of human serum albumin in Pichia pastoris and its efficient purification. Protein Expr Purif 181:105831. doi: 10.1016/j.pep.2021.105831