Bioprocess and downstream optimization of recombinant human growth hormone in Pichia pastoris

Saeed Azadi, Seyed Kazem Sadjady, Seyed Alireza Mortazavi, Nasser Naghdi, Arash Mahboubi, Roya Solaimanian

Abstract


The methylotrophic yeast Pichia pastoris is a well-established expression host, which is often used in the production of protein pharmaceuticals. This work aimed to evaluate the effect of various concentrations of ascorbic acid in mixed feeding strategy with sorbitol/methanol on productivity of recombinant human growth hormone (r-hGH). The relevant concentration of ascorbic acid (5, 10, or 20 mmol) and 50 g/L sorbitol were added in batch-wise mode to the medium at the beginning of induction phase. The rate of methanol addition was increased stepwise during the first 12 h of production and then kept constant. Total protein and r-hGH concentrations were analyzed and the results compared with sorbitol/methanol feeding using one-way analysis of variance. Moreover, an effective clarification process using activated carbon was developed to remove process contaminants like pigments and endotoxins. Finally, a three-step chromatographic process was applied to purify the product. According to the obtained results, addition of 10 mmol ascorbic acid to sorbitol/methanol co-feeding could significantly increase cell biomass (1.7 fold), total protein (1.14 fold), and r-hGH concentration (1.43 fold). One percent activated carbon could significantly decrease pigments and endotoxins without any significant changes in r-hGH assay. The result of the study concluded that ascorbic acid in combination with sorbitol could effectively enhance the productivity of r-hGH. This study also demonstrated that activated carbon clarification is a simple method for efficient removal of endotoxin and pigment in production of recombinant protein in the yeast expression system. 


Keywords


Pichia pastoris; ascorbic acid; Activated carbon; Pigment removal.

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Nguyen MT, Koo BK, Vu TTT, Song JA, Chong SH, Jeong B, et al. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase. PLoS One. 2014;9(3):e89038.

Cai Y, Xu M, Yuan M, Liu Z, Yuan W. Developments in human growth hormone preparations: sustained-release, prolonged half-life, novel injection devices, and alternative delivery routes. Int J Nanomedicine. 2014;9:3527-3538.

Aulić S, Bolognesi ML, Legname G. Small-molecule theranostic probes: a promising future in neurodegenerative diseases. Int J Cell Biol. 2013;2013.DOI: 10.1155/2013/150952.

Rudge P, Jaunmuktane Z, Adlard P, Bjurstrom N, Caine D, Lowe J, et al. Iatrogenic CJD due to pituitary-derived growth hormone with genetically determined incubation times of up to 40 years. Brain. 2015;138(11):3386-3399.

Ghavim M, Abnous K, Arasteh F, Taghavi S, Nabavinia MS, Alibolandi M, et al. High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region. Res Pharm Sci. 2017;12(2):168-175.

Ayyar VS. History of growth hormone therapy. Indian J Endocrinol Metab. 2011;15(Suppl3): S162-S165.

Rezaei M, Zarkesh-Esfahani SH. Optimization of production of recombinant human growth hormone in Escherichia coli. J Res Med Sci. 2012;17(7):681-685.

Murasugi A. Secretory expression of human protein in the Yeast Pichia pastoris by controlled fermentor culture. Recent Pat Biotechnol. 2010;4(2):153-166.

Weinacker D, Rabert C, Zepeda AB, Figueroa CA, Pessoa A, Farías JG. Applications of recombinant Pichia pastoris in the healthcare industry. Braz J Microbiol. 2013;44(4):1043-1048.

Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014;98(12):5301-5317.

Rabert C, Weinacker D, Pessoa A, Farías JG. Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system. Braz J Microbiol. 2013;44(2):351-356.

Weidner M, Taupp M, Hallam SJ. Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. J Vis Exp. 2010;36. pii:1862. DOI:10.3791/1862.

Cregg JM, Vedvick TS, Raschke WC. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y). 1993; 1(8):905-910.

Noseda DG, Recúpero MN, Blasco M, Ortiz GE, Galvagno MA. Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter. Protein Expr Purif. 2013;92(2):235-244.

Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol. 2000;54(6):741-750.

Higgins DR, Cregg JM. Introduction to Pichia pastoris. Methods Mol Biol. 1998;103:1-15.

Jozala AF, Geraldes DC, Tundisi LL, Feitosa VA, Breyer CA, Cardoso SL, et al. Biopharmaceuticals from microorganisms: from production to purification. Braz J Microbiol. 2016;47(Suppl. 1):51-63.

Fortis F, Guerrier L, Righetti PG, Antonioli P, Boschetti E. A new approach for the removal of protein impurities from purified biologicals using combinatorial solid-phase ligand libraries. Electrophoresis. 2006;27(15):3018-3027.

Minyasab SA, Dhamane SP, Hazra P, Iyer H. A method of purifying human growth hormone and purified growth hormone thereof. 2010. Pub. No: WO/2010/134084. International Application No: PCT/IN2009/000380. Google Patents.

Whittaker MM, Whittaker JW. Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris. J Biol Inorg Chem. 2002;7(1-2):136-145.

Hao J, Xu L, He H, Du X, Jia L. High-level expression of Staphylococcal Protein A in Pichia pastoris and purification and characterization of the recombinant protein. . Protein Expr Purif. 2013;90(2):178-185.

Azadi S, Mahboubi A, Naghdi N, Solaimanian R, Mortazavi SA. Evaluation of sorbitol-methanol co-feeding strategy on production of recombinant human growth hormone in Pichia pastoris. Iran J Pharm Res. 2017;16 (4):1555-1564.

Huang J, Barent R, Inan M, Gouthro M, Roxas PV, Smith LA, et al. Purification of the N- and C- terminal subdomains of recombinant heavy chain fragment C of botulinum neurotoxin serotype C. Methods Mol Biol. 2007;389:77-98.

Lee CY, Lee SJ, Jung KH, Katoh S, Lee EK. High dissolved oxygen tension enhances heterologous protein expression by recombinant Pichia pastoris. Process Biochem. 2003;38(8):1147-1154.

Invitrogen Co. Pichia Fermentation Process Guidelines, San Diego, CA. 2002.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle protein dye binding. Anal Biochem. 1976;72:248-254.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.

British Pharmacopoeia Commission, British Pharmacopoeia, Monograph on Somatropin, Her Majesty's Stationery Office, London, UK, TSO, 2013.

Wilhelmsen TW, Skibeli V, Arntzen FC. Stability study of somatropin by capillary zone electrophoresis. Procedia Chem. 2010;2(1):34-45.

Kim MJ, Park HS, Seo KH, Yang HJ, Kim SK, Choi JH. Complete solubilization and purification of recombinant human growth hormone produced in Escherichia coli. PLoS One. 2013;8(2):e56168.

Sreekrishna K. Gene Expression in Pichia and Other Methylotroph yeast. In: Flickinger MC, editor. Upstream Industrial Biotechnology. Vol 2. New Jersey: Wiley; 2013. pp. 203.

Ferrer P, Valero F. Coping with Physiological Stress During Recombinant Protein Production by Bioreactor Design and Operation. In: Mandenius CF, editor. Bioreactors: Design, Operation and Novel Applications. Weinheim: Wiley-VCH. 2016; pp. 243.

Vanz AL, Lünsdorf H, Adnan A, Nimtz M, Gurramkonda C, Khanna N, et al. Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes. Microb Cell Fact. 2012;11:103-113.

Xiao A, Zhou X, Zhou L, Zhang Y. Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol. 2006;72(4):837-844.

Çalık P, Bozkurt B, Zerze GH, İnankur B, Bayraktar E, Boy E, et al. Effect of co-substrate sorbitol different feeding strategies on human growth hormone production by recombinant Pichia pastoris. J. Chem. Technol. Biotechnol. 2013;88(9): 1631-1640.

Ramon R, Ferrer P, Valero F. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol. 2007;130:39-46.

Celik E, Calik P, Oliver SG. Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of cosubstrate sorbitol. Yeast. 2009;26(9):473-484.

Wang Z, Wang Y, Zhang D, Li J, Hua Z, Du G, et al. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol cofeeding with methanol in Pichia pastoris fermentation. Bioresour Technol. 2010;101(4):1318-1323.

Zhu T, You L, Gong F, Xie M, Xue Y, Li Y, et al. Combinatorial strategy of sorbitol feeding and low-temperature induction leads to high-level production

of alkaline beta-mannanase in Pichia pastoris. Enzyme Microb Technol. 2011;49(4):407-412.

Gao MJ, Li Z, Yu RS, Wu JR, Zheng ZY, Shi ZP, et al. Methanol/ sorbitol co-feeding induction enhanced porcine interferon-alpha production by P. pastoris associated with energy metabolism shift. Bioprocess Biosyst Eng. 2012;35(7):1125-1136.

Niu H, Jost L, Pirlot N, Sassi H, Daukandt M, Rodriguez C, et al. A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain. Microb Cell Fact. 2013;12:33-40.

Damasceno LM, Pla I, Chang HJ, Cohen L, Ritter G, Old LJ, et al. An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif. 2004;37:18-26.


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