Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

Abstract

The Global Diabetes Compact was launched by the World Health Organization in April 2021 with one of its important goals to increase the accessibility and affordability of life-saving medicine—insulin. The rising prevalence of diabetes worldwide is bound to escalate the demand for recombinant insulin therapeutics, and currently, the majority of recombinant insulin therapeutics are produced from E. coli inclusion bodies. Here, a comprehensive review of downstream processing of recombinant human insulin/analogue production from E. coli inclusion bodies is presented. All the critical aspects of downstream processing, starting from proinsulin recovery from inclusion bodies, inclusion body washing, inclusion body solubilization and oxidative sulfitolysis, cyanogen bromide cleavage, buffer exchange, purification by chromatography, pH precipitation and zinc crystallization methods, proinsulin refolding, enzymatic cleavage, and formulation, are explained in this review. Pertinent examples are summarized and the practical aspects of integrating every procedure into a multimodal purification scheme are critically discussed. In the face of increasing global demand for insulin product, there is a pressing need to develop a more efficient and economical production process. The information presented would be insightful to all the manufacturers and stakeholders for the production of human insulins, insulin analogues or biosimilars, as they strive to make further progresses in therapeutic recombinant insulin development and production.

Keywords

Recombinant human insulin / Insulin analogues / E. coli inclusion bodies / Downstream processing / Purification

References

[1] Astolfi Filho S, De Lima BD, Thiemann JE, de Sousa HR, Vilela L (2004) Vector for expression of heterologous protein and methods for extracting recombinant protein and for purifying isolated recombinant insulin. United States patent 6,699,692, 2 Mar 2004.

[2] Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Redwan EM. Cell factories for insulin production. Microb Cell Fact, 2014, 13(1): 141.

[3] Bai Q, Kong Y, Geng XD. Studies on renaturation with simultaneous purification of recombinant human proinsulin from E. coli with high performance hydrophobic interaction chromatography. J Liquid Chromatogr Relat Technol, 2003, 26(5): 683-695.

[4] Balcerek J, Sznilik K, Jaros S, Wieczorek M (2018) Method for preparation of a recombinant protein from a precursor. United States patent US 15/532,811, 19 Apr 2018.

[5] Baneyx F. Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol, 1999, 10(5): 411-421.

[6] Batas B, Chaudhuri JB. Protein refolding at high concentration using size-exclusion chromatography. Biotechnol Bioeng, 1996, 50(1): 16-23.

[7] Brange J. Stability of insulin: studies on the physical and chemical stability of insulin in pharmaceutical formulation, 1994, Boston: Kluwer Academic.

[8] Brange J. Galenics of insulin: the physico-chemical and pharmaceutical aspects of insulin and insulin preparations, 2012, Berlin: Springer.

[9] Brange J, Langkjœr L. Insulin structure and stability. Stability and characterization of protein and peptide drugs, 1993, Boston: Springer, 315-350.

[10] Carr D (2002) The handbook of analysis and purification of peptides and proteins by reversed-phase HPLC. Hesperia: Grace Vydac.

[11] Carr D. A guide to the analysis and purification of proteins and peptides by reversed-phase HPLC, 2016, New York: Advanced Chromatography Technologies.

[12] Castellanos-Serra LR, Hardy E, Ubieta R, Vispo NS, Fernandez C, Besada V, Falcon V, Gonzalez M, Santos A, Perez G, Silva A. Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence. FEBS Lett, 1996, 378(2): 171-176.

[13] Chance R, Frank B. Research, development production and safety of biosynthetic human insulin. Diabetes Care, 1993, 16(3): 133-142.

[14] Chance RE, Glazer NB, Wishner KL. Insulin lispro (humalog) biopharmaceuticals, an industrial perspective, 1999, Berlin: Springer, 149-172.

[15] Chang SG, Choi KD, Jang SH, Shin HC. Role of disulfide bonds in the structure and activity of human insulin. Mol Cells, 2003, 16: 3.

[16] Chatani E, Imamura H, Yamamoto N, Kato M. Stepwise organization of the β-structure identifies key regions essential for the propagation and cytotoxicity of insulin amyloid fibrils. J Biol Chem, 2014, 289(15): 10399-10410.

[17] Chen S, Adijanto L, Wang NH. In vitro folding of methionine-arginine human lyspro-proinsulin S-sulfonate—disulfide formation pathways and factors controlling yield. Biotechnol Prog, 2010, 26(5): 1332-1343.

[18] Chen Y, Wang Q, Zhang C, Li X, Gao Q, Dong C, Liu Y, Su Z. Improving the refolding efficiency for proinsulin aspart inclusion body with optimized buffer compositions. Protein Expr Purif, 2016, 122: 1-7.

[19] Chouhan R, Goswami S, Bajpai AK. Recent advancements in oral delivery of insulin: from challenges to solutions. Nanostructures for oral medicine, 2017, New York: Elsevier, 435-465.

[20] Clark ED. Refolding of recombinant proteins. Curr Opin Biotechnol, 1998, 9(2): 157-163.

[21] Coleman MP, Ortigosa AD, Sleevi MC, Kartoa CH (2019) Process for purifying insulin and analogues thereof. United States patent US 10,421,795, 24 Sep 2019.

[22] Cowley DJ, Mackin RB. Expression, purification and characterization of recombinant human proinsulin. FEBS Lett, 1997, 402(2–3): 124-130.

[23] Cytiva (2021) Sephadex G-25 Medium. https://www.cytivalifesciences.com/en/us/shop/chromatography/resins/size-exclusion/sephadex-g-25-medium-p-05608#tech-spec-table. Accessed 14 Apr 2021.

[24] Dickhardt R, Unger B (1997) Chromatographic process for purification of insulin. United States patent US 5,621,073, 15 Apr 1997.

[25] Dickhardt R, Unger B, Hafner L (1993) Process for the purification of insulins by chromatography. United States patent US 5,245,008, 14 Sep 1993.

[26] Evnin LB, Vásquez JR, Craik CS. Substrate specificity of trypsin investigated by using a genetic selection. Proc Natl Acad Sci, 1990, 87(17): 6659-6663.

[27] Fortune Business Insights (2020) Human insulin market size, share & industry analysis, by type (analogue insulin, traditional human insulin), by diabetes type (type 1, type 2), by distribution channel (retail pharmacy, hospital pharmacy, online pharmacy), and regional forecast, 2019–2026. https://www.fortunebusinessinsights.com/industry-reports/human-insulin-market-100395. Accessed 14 Apr 2021.

[28] Frank BH, Pettee JM, Zimmerman RE, Burck PJ (1981) In: Rich D, Gross E, eds. Peptides: proceedings of the Seventh American Peptide Chemistry Symposium. Berlin: Springer, p 729–739.

[29] Freeman JS. Insulin analog therapy: improving the match with physiologic insulin secretion. J Am Osteopath Assoc, 2009, 109(1): 26-36.

[30] GE Healthcare, 28-9966-22, Edition AA (2012) Application note: High-throughput screening and process development for capture of recombinant pro-insulin from E. coli.

[31] GE Healthcare 29-0018-56 (2012) Application note: high-throughput screening, process development, and scale-up of an intermediate purification step for recombinant insulin.

[32] Govender K, Naicker T, Baijnath S, Chuturgoon AA, Abdul NS, Docrat T, Kruger HG, Govender T (2020) Sub/supercritical fluid chromatography employing water-rich modifier enables the purification of biosynthesized human insulin. Journal of Chromatography B 1155:122126.

[33] Gráf L, Jancso A, Szilágyi L, Hegyi G, Pintér K, Náray-Szabó G, Hepp J, Medzihradszky K, Rutter WJ. Electrostatic complementarity within the substrate-binding pocket of trypsin. Proc Natl Acad Sci, 1988, 85(14): 4961-4965.

[34] Guo ZY, Qiao ZS, Feng YM. The in vitro oxidative folding of the insulin superfamily. Antioxid Redox Signal, 2008, 10(1): 127-140.

[35] Harrison RG, Todd P, Rudge SR, Petrides DP. Bioseparations science and engineering, 2015, USA: Oxford University Press

[36] Healthcare GE (2006). Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods. https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=14026. Accessed 14 Apr 2021.

[37] Heldin E, Grönlund S, Shanagar J, Hallgren E, Eriksson K, Xavier M, Tunes H, Vilela L. Development of an intermediate chromatography step in an insulin purification process. The use of a high throughput process development approach based on selectivity parameters. J Chromatogr B, 2014, 973: 126-132.

[38] Helmerhorst E, Stokes GB. Self-association of insulin: Its pH dependence and effect of plasma. Diabetes, 1987, 36(3): 261-264.

[39] Hua QX, Jia W, Frank BH, Phillips NF, Weiss MA. A protein caught in a kinetic trap: structures and stabilities of insulin disulfide isomers. Biochemistry, 2002, 41(50): 14700-14715.

[40] Hwang HG, Kim KJ, Lee SH, Kim CK, Min CK, Yun JM, Lee SU, Son YJ. Recombinant glargine insulin production process using Escherichia coli. J Microbiol Biotechnol, 2016, 26(10): 1781-1789.

[41] Jia XY, Guo ZY, Wang Y, Xu Y, Duan SS, Feng YM. Peptide models of four possible insulin folding intermediates with two disulfides. Protein Sci, 2003, 12(11): 2412-2419.

[42] Johansson K, Frederiksen SS, Degerman M, Breil MP, Mollerup JM, Nilsson B. Combined effects of potassium chloride and ethanol as mobile phase modulators on hydrophobic interaction and reversed-phase chromatography of three insulin variants. J Chromatogr A, 2015, 1381: 64-73.

[43] Kim CK, Lee SB, Son YJ. Large-scale refolding and enzyme reaction of human preproinsulin for production of human insulin. J Microbiol Biotechnol, 2015, 25(10): 1742-1750.

[44] Kossiakoff AA. Tertiary structure is a principal determinant to protein deamidation. Science, 1988, 240(4849): 191-194.

[45] Kroeff EP, Owens RA, Campbell EL, Johnson RD, Marks HI. Production scale purification of biosynthetic human insulin by reversed-phase high-performance liquid chromatography. J Chromatogr A, 1989, 461: 45-61.

[46] Kromasil (2021). Kromasil preparative HPLC applications—success stories. https://www.kromasil.com/notes/?NOTEkpmo. Accessed 14 Apr 2021.

[47] Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol, 1982, 157(1): 105-132.

[48] Ladisch MR, Kohlmann KL. Recombinant human insulin. Biotechnol Prog, 1992, 8(6): 469-478.

[49] Laursen BS, Sørensen HP, Mortensen KK, Sperling-Petersen HU. Initiation of protein synthesis in bacteria. Microbiol Mol Biol Rev, 2005, 69(1): 101-123.

[50] Leng C, Li Q, Wu F, Chen L, Su P. Detection of the single-chain precursor in the production and purification process of recombinant human insulin. Monocl Antibodies Immunodiagn Immunother, 2013, 32(4): 255-261.

[51] Mackin RB. Streamlined procedure for the production of normal and altered versions of recombinant human proinsulin. Protein Expr Purif, 1999, 15(3): 308-313.

[52] Mackin RB. Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin. MethodsX, 2014, 1: 108-117.

[53] Mackin RB, Choquette MH. Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin. Protein Expr Purif, 2003, 27(2): 210-219.

[54] Mergulhao FJ, Monteiro GA, Cabral JM, Taipa MA. A quantitative ELISA for monitoring the secretion of ZZ-fusion proteins using SpA domain as immunodetection reporter system. Mol Biotechnol, 2001, 19(3): 239-244.

[55] Mergulhao FJ, Taipa MA, Cabral JM, Monteiro GA. Evaluation of bottlenecks in proinsulin secretion by Escherichia coli. J Biotechnol, 2004, 109(1–2): 31-43.

[56] Merck Millipore (2021) Dialysis or Diafiltration? https://www.drogallega.es/u/uploads/File/1335219226_DialysisorDiafiltration.MM2.pdf. Accessed 14 Apr 2021.

[57] Mikiewicz D, Bierczyńska-Krzysik A, Sobolewska A, Stadnik D, Bogiel M, Pawłowska M, Wójtowicz-Krawiec A, Baran PA, Łukasiewicz N, Romanik-Chruścielewska A, Sokołowska I. Soluble insulin analogs combining rapid-and long-acting hypoglycemic properties–from an efficient E. coli expression system to a pharmaceutical formulation. PLoS ONE, 2017, 12(3): e0172600.

[58] Min CK, Son YJ, Kim CK, Park SJ, Lee JW. Increased expression, folding and enzyme reaction rate of recombinant human insulin by selecting appropriate leader peptide. J Biotechnol, 2011, 151(4): 350-356.

[59] Mollerup I, Jensen SW, Larsen P, Schou O, Snel L. Insulin purification. Encyclop Ind Biotechnol, 2010, 4: 1-20.

[60] Mollerup JM, Frederiksen SS (2016) Purification of insulin. United States patent US 9,447,163, 20 Sep 2016.

[61] Muzaffar M, Ahmad A. The mechanism of enhanced insulin amyloid fibril formation by NaCl is better explained by a conformational change model. PLoS ONE, 2011, 6(11): e27906.

[62] Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, Vyas S, Uversky VN, Fink AL. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry, 2001, 40(20): 6036-6046.

[63] Nilsson J, Jonasson P, Samuelsson E, Stahl S, Uhlén M. Integrated production of human insulin and its C-peptide. J Biotechnol, 1996, 48(3): 241-250.

[64] Ohno Y, Seki T, Kojima Y, Miki R, Egawa Y, Hosoya O, Kasono K, Seki T. Investigation of factors that cause insulin precipitation and/or amyloid formation in insulin formulations. J Pharm Health Care Sci, 2019, 5(1): 1-1.

[65] Palmer I, Wingfield PT (2012) Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli, Chap 6 (Unit 6.3). In: Current protocols in protein science

[66] Patrick JS, Lagu AL. Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC. Anal Chem, 1992, 64(5): 507-511.

[67] Petrides D, Sapidou E, Calandranis J. Computer-aided process analysis and economic evaluation for biosynthetic human insulin production—A case study. Biotechnol Bioeng, 1995, 48(5): 529-541.

[68] Pharmacia Biotech (2021) Sephadex® G-25 media and pre-packed columns. http://www.chembio.uoguelph.ca/educmat/chm357/g25.pdf. Accessed 14 Apr 2021

[69] Qiao ZS, Min CY, Hua QX, Weiss MA, Feng YM. In vitro refolding of human proinsulin: kinetic intermediates, putative disulfide-forming pathway, folding initiation site, and potential role of c-peptide in folding process. J Biol Chem, 2003, 278(20): 17800-17809.

[70] Qiao ZS, Guo ZY, Feng YM (2006) In vitro folding/unfolding of insulin/single-chain insulin. Protein & Peptide Lett 13(5):423–429

[71] Redwan ER, Matar SM, El-Aziz GA, Serour EA. Synthesis of the human insulin gene: protein expression, scaling up and bioactivity. Prep Biochem Biotechnol, 2007, 38(1): 24-39.

[72] Ritchie C (2012) Protein purification. Mater Methods 2:134. https://doi.org/10.13070/mm.en.2.134.

[73] Schoner RG, Ellis LF, Schoner BE. Isolation and purification of protein granules from Escherichia coli cells overproducing bovine growth hormone. Bio/technology, 1985, 3(2): 151-154.

[74] Sigma (2021) INSULIN, HUMAN, RECOMBINANT EXPRESSED IN E. coli (Product information sheet). https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/i2767pis.pdf. Accessed 14 Apr 2021.

[75] Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact, 2015, 14(1): 41.

[76] Singhvi P, Saneja A, Srichandan S, Panda AK. Bacterial inclusion bodies: a treasure trove of bioactive proteins. Trends Biotechnol, 2020, 38(5): 474-486.

[77] Son YJ, Kim CK, Choi BT, Park YC, Seo JH. Effects of β-mercaptoethanol and hydrogen peroxide on enzymatic conversion of human proinsulin to insulin. J Microbiol Biotechnol, 2008, 18(5): 983-989.

[78] Son YJ, Kim CK, Kim YB, Kweon DH, Park YC, Seo JH. Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B. Biotechnol Prog, 2009, 25(4): 1064-1070.

[79] Sung WL, Yao FL, Zahab DM, Narang SA. Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli. Proc Natl Acad Sci, 1986, 83(3): 561-565.

[80] Thurow H, Blumenstock H, Havenith C (2010) Method of purifying preproinsulin. United States patent US 7,803,763, 28 Sep 2010.

[81] Tiiman A, Noormägi A, Friedemann M, Krishtal J, Palumaa P, Tõugu V. Effect of agitation on the peptide fibrillization: Alzheimer's amyloid-β peptide 1–42 but not amylin and insulin fibrils can grow under quiescent conditions. J Pept Sci, 2013, 19(6): 386-391.

[82] Tikhonov RV, Pechenov SE, Belacheu IA, Yakimov SA, Klyushnichenko VE, Boldireva EF, Korobko VG, Tunes H, Thiemann JE, Vilela L, Wulfson AN (2001) Recombinant Human Insulin: VIII. Isolation of Fusion Protein–S-Sulfonate, Biotechnological Precursor of Human Insulin, from the Biomass of Transformed Escherichia coli Cells. Protein expression and purification 21(1):176–82.

[83] Verma S (2021). Reversed-phase HPLC Buffers. Sigma-aldrich. Reporter US Volume 27.4. https://www.sigmaaldrich.com/technical-documents/articles/reporter-us/reversed-phase-hplc.html#:~:text=The%20typical%20pH%20range%20for,is%20pH%202%20to%208. Accessed 14 Apr 2021.

[84] Waller DG, Sampson T. Medical pharmacology and therapeutics E-Book, 2017, New York: Elsevier.

[85] Walsh G. Therapeutic insulins and their large-scale manufacture. Appl Microbiol Biotechnol, 2005, 67(2): 151-159.

[86] Wang NH, Xie Y, Mun S (2003) Insulin purification using simulated moving bed technology. United States patent US 10/276,948, 20 Nov 2003.

[87] Watson DS, Ortigosa AD, Rauscher MA, Story KM (2018) Chromatography process for purification of insulin and insulin analogs. United States patent US 10,155,799, 18 Dec 2018.

[88] Whittingham JL, Scott DJ, Chance K, Wilson A, Finch J, Brange J, Dodson GG. Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation. J Mol Biol, 2002, 318(2): 479-490.

[89] Winter J, Lilie H, Rudolph R. Recombinant expression and in vitro folding of proinsulin are stimulated by the synthetic dithiol Vectrase-P. FEMS Microbiol Lett, 2002, 213(2): 225-230.

[90] Winter J, Lilie H, Rudolph R. Renaturation of human proinsulin—a study on refolding and conversion to insulin. Anal Biochem, 2002, 310(2): 148-155.

[91] Wintersteiner O, Abramson HA. The isoelectric point of insulin electrical properties of adsorbed and crystalline insulin. J Biol Chem, 1933, 99(3): 741-753.

[92] World Health Organization, WHO (2021) The WHO Global Diabetes Compact. https://www.who.int/docs/default-source/world-diabetes-day/global-diabetes-compact-final.pdf. Accessed 16 Apr 2021.

[93] Yuan J, Zhou H, Yang Y, Li W, Wan Y, Wang L. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns. Biomed Chromatogr, 2015, 29(5): 777-782.

[94] Zhang K, Liu X. Mixed-mode chromatography in pharmaceutical and biopharmaceutical applications. J Pharm Biomed Anal, 2016, 128: 73-88.

[95] Zieliński M, Romanik-Chruścielewska A, Mikiewicz D, Łukasiewicz N, Sokołowska I, Antosik J, Sobolewska-Ruta A, Bierczyńska-Krzysik A, Zaleski P, Płucienniczak A. Expression and purification of recombinant human insulin from E. coli 20 strain. Protein Expr Purif, 2019, 157: 63-69.

[96] Zimmerman RE, Stokell DJ (2010) Insulin production methods and pro-insulin constructs. United States patent US 7

Funding

Agency for Science, Technology and Research, Singapore