Applications and analysis of hydrolysates in animal cell culture

Abstract

Animal cells are used in the manufacturing of complex biotherapeutic products since the 1980s. From its initial uses in biological research to its current importance in the biopharmaceutical industry, many types of culture media were developed: from serum-based media to serum-free to protein-free chemically defined media. The cultivation of animal cells economically has become the ultimate goal in the field of biomanufacturing. Serum serves as a source of amino acids, lipids, proteins and most importantly growth factors and hormones, which are essential for many cell types. However, the use of serum is unfavorable due to its high price tag, increased lot-to-lot variations and potential risk of microbial contamination. Efforts are progressively being made to replace serum with recombinant proteins such as growth factors, cytokines and hormones, as well as supplementation with lipids, vitamins, trace elements and hydrolysates. While hydrolysates are more complex, they provide a diverse source of nutrients to animal cells, with potential beneficial effects beyond the nutritional value. In this review, we discuss the use of hydrolysates in animal cell culture and briefly cover the composition of hydrolysates, mode of action and potential contaminants with some perspectives on its potential role in animal cell culture media formulations in the future.

Keywords

Hydrolysates / Animal cells / Cell culture

References

[1] Aguilar-Toalá JE, Deering AJ, . New insights into the antimicrobial properties of hydrolysates and peptide fractions derived from chia seed (Salvia hispanica L.). Probiotics Antimicrob Proteins, 2020, 12(4): 1571-1581.

[2] Alden N, Raju R, . Using metabolomics to identify cell line-independent indicators of growth inhibition for Chinese hamster ovary cell-based bioprocesses. Metabolites, 2020

[3] Al-Mohammadi A-R, Osman A, . Powerful antibacterial peptides from egg albumin hydrolysates. Antibiotics, 2020, 9(12): 901.

[4] Amagliani L, O'Regan J, . The composition, extraction, functionality and applications of rice proteins: a review. Trends Food Sci Technol, 2017, 64: 1-12.

[5] Andreassen RC, Pedersen ME, . Screening of by-products from the food industry as growth promoting agents in serum-free media for skeletal muscle cell culture. Food Funct, 2020, 11: 2477-2488.

[6] Ballez JS, Mols J, . Plant protein hydrolysates support CHO-320 cells proliferation and recombinant IFN-γ production in suspension and inside microcarriers in protein-free media. Cytotechnology, 2004, 44(3): 103-114.

[7] Bertrand JA, Sudduth TQ, . Nutrient content of whole cottonseed. J Dairy Sci, 2005, 88(4): 1470-1477.

[8] Bosques CJ, Collins BE, . Chinese hamster ovary cells can produce galactose-α-1,3-galactose antigens on proteins. Nat Biotechnol, 2010, 28(11): 1153-1156.

[9] Bowie JH, Brinkworth CS, . Collision-induced fragmentations of the (M-H)− parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages. Mass Spectrom Rev, 2002, 21(2): 87-107.

[10] Burteau CC, Verhoeye FR, . Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line. In Vitro Cell Dev Biol, 2003, 9: 291-296.

[11] Carrel A, Burrows MT. An addition to the technique of the cultivation of tissues in vitro. J Exp Med, 1911, 14(3): 244-247.

[12] Carrel A, Burrows MT. Cultivation of tissues in vitro and its technique. J Exp Med, 1911, 13(3): 387-396.

[13] Chabanon G, Chevalot I, . Smith R, . Influence of rapeseed protein hydrolysis conditions on animal cell growth in serum-free media supplemented with hydrolysates BT. Cell technology for cell products, 2007, Dordrecht: Springer, 667-669.

[14] Chalamaiah M, Yu W, . Immunomodulatory and anticancer protein hydrolysates (peptides) from food proteins: a review. Food Chem, 2018, 245: 205-222.

[15] Champagne ET, Wood D, . Champagne ET, . The rice grain and its gross composition. Rice chemistry and technology, 2004, St Paul: AACC, 77-107.

[16] Cheng HN, He Z, . A review of cottonseed protein chemistry and non-food applications. Sustain Chem, 2020, 1(3): 256-274.

[17] Cheong CW, Lee YS, . Chicken feather valorization by thermal alkaline pretreatment followed by enzymatic hydrolysis for protein-rich hydrolysate production. Waste Manag, 2018, 79: 658-666.

[18] Cho MS, Yee H, . Versatile expression system for rapid and stable production of recombinant proteins. Biotechnol Prog, 2003, 19(1): 229-232.

[19] Chou IN, Prezyna C, . Isolation from lactalbumin hydrolysate of a high molecular weight mitogenic factor. J Biol Chem, 1979, 254(21): 10588-10591.

[20] Das D, Jaiswal M, . PlantPepDB: a manually curated plant peptide database. Sci Rep, 2020, 10: 2194.

[21] Davami F, Eghbalpour F, . Effects of peptone supplementation in different culture media on growth, metabolic pathway and productivity of CHO DG44 cells; a new insight into amino acid profiles. Iran Biomed J, 2015, 19(4): 194-205.

[22] Deparis V, Durrieu C, . Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth. Cytotechnology, 2003, 42: 75-85.

[23] Dumont J, Euwart D, . Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit Rev Biotechnol, 2016, 36(6): 1110-1122.

[24] Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol, 2009, 20(6): 700-707.

[25] Earle WR, Schilling EL, . Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J Natl Cancer Inst, 1943, 4(2): 165-212.

[26] Farges B, Tessier B, . Peptide fractions of rapeseed hydrolysate as an alternative to animal proteins in CHO cell culture media. Process Biochem, 2006, 41: 2297-2304.

[27] Farhana M, Tan HL, . Isolation of antimicrobial peptide from food protein hydrolysates: an overview. Key Eng Mater, 2019, 797: 168-176.

[28] Farup J, Rahbek SK, . Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans. SpringerPlus, 2016, 5: 382-382.

[29] Ferreira CB, Sumner RP, . Lentiviral vector production titer is not limited in HEK293T by induced intracellular innate immunity. Mol Ther Methods Clin Dev, 2019, 17: 209-219.

[30] Fogh J, Wright WC, . Absence of HeLa cell contamination in 169 cell lines derived from human tumors2. JNCI, 1977, 58(2): 209-214.

[31] Franěk F, Hohenwarter O, . Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures. Biotechnol Prog, 2000, 16: 688-692.

[32] Galfrè G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol, 1981, 73: 3-46.

[33] Gey GO. Tissue culture studies of the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Res, 1952, 12: 264-265.

[34] Ghaderi D, Zhang M, . Production platforms for biotherapeutic glycoproteins. occurrence, impact, and challenges of non-human sialylation. Biotechnol Genet Eng Rev, 2012, 28: 147-175.

[35] Girón-Calle J, Vioque J, . Chickpea protein hydrolysate as a substitute for serum in cell culture. Cytotechnology, 2008, 57: 263-272.

[36] Gorissen SHM, Crombag JJR, . Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids, 2018, 50(12): 1685-1695.

[37] Goto S, Kishishita S et al. (2008) Cell culture method and utilization of the same Japan, Chugai Pharmaceutical Co Ltd

[38] Graham FL, Smiley J, . Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol, 1977, 36(1): 59-74.

[39] Harnedy PA, Parthsarathy V, . Atlantic salmon (Salmo salar) co-product-derived protein hydrolysates: a source of antidiabetic peptides. Food Res Int, 2018, 106: 598-606.

[40] Harrison RG, Greenman MJ, . Observations of the living developing nerve fiber. Anat Rec, 1907, 1(5): 116-128.

[41] Havenga MJ, Holterman L, . Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity. Biotechnol Bioeng, 2008, 100(2): 273-283.

[42] He R, Girgih AT, . Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions. J Funct Foods, 2013, 5(1): 219-227.

[43] University of Rochester Medical Center (2021) Nutrition facts: Soy protein isolate, potassium type, crude protein basis, 1 oz., Adult and Children’s Health Encyclopedia. https://www.urmc.rochester.edu/encyclopedia/content.aspx?contenttypeid=76&contentid=16423-1. Accesed 30 May 2021

[44] Hernandez R, Brown DT. Growth and maintenance of baby hamster kidney (BHK) cells. Curr Protoc Microbiol, 2010, Chapter 4: Appendix 4H.

[45] Ho SCL, Nian R, . Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells. J Biosci Bioeng, 2016, 122: 499-506.

[46] Hu Q, Noll RJ, . The Orbitrap: a new mass spectrometer. J Mass Spectrom, 2005, 40(4): 430-443.

[47] Hu D, Zhao L, . Physiological responses of Chinese hamster ovary cells to a productivity-enhancing yeast extract. J Biosci Bioeng, 2018, 126: 636-643.

[48] Hwang CF, Chen YA, . Antioxidant and antibacterial activities of peptide fractions from flaxseed protein hydrolysed by protease from Bacillus altitudinis HK02. Int J Food Sci Technol, 2016, 51(3): 681-689.

[49] Jayapal K, Wlaschin KF, . Recombinant protein therapeutics from CHO cells—20 years and counting. Chem Eng Prog, 2007, 103: 40-47.

[50] Jiang C, Scherfner S, . Demonstrating β-glucan and yeast peptide clearance in biopharmaceutical downstream processes. Biotechnol Prog, 2011, 27: 442-450.

[51] Jordan I, Vos A, . An avian cell line designed for production of highly attenuated viruses. Vaccine, 2009, 27(5): 748-756.

[52] Kiewiet MBG, Faas MM, . Immunomodulatory protein hydrolysates and their application. Nutrients, 2018, 10: 904.

[53] Kim JY, Kim YG, . CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol, 2012, 93(3): 917-930.

[54] Kim J-H, Jang H-J, . In vitro antioxidant actions of sulfur-containing amino acids. Arab J Chem, 2020, 13(1): 1678-1684.

[55] KÖHler, G. and C. Milstein,. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256(5517): 495-497.

[56] Kovesdi I, Hedley SJ. Adenoviral producer cells. Viruses, 2010, 2(8): 1681-1703.

[57] Li F, Vijayasankaran N, . Cell culture processes for monoclonal antibody production. MAbs, 2010, 2: 466-479.

[58] Liang Y, Neta P, . Collision-induced dissociation of deprotonated peptides. Relative abundance of side-chain neutral losses, residue-specific product ions, and comparison with protonated peptides. J Am Soc Mass Spectrom, 2018, 29(3): 463-469.

[59] Lobo-Alfonso J, Price P, . Pasupuleti VK, Demain AL, . Benefits and limitations of protein hydrolysates as components of serum-free media for animal cell culture applications. Protein hydrolysates in biotechnology, 2010, Berlin: Springer, 55.

[60] Merten OW. Development of serum-free media for cell growth and production of viruses/viral vaccines–safety issues of animal products used in serum-free media. Dev Biol, 2002, 111: 233-257.

[61] Minkiewicz P, Iwaniak A, . BIOPEP-UWM database of bioactive peptides: current opportunities. Int J Mol Sci, 2019

[62] Mizrahi A. Primatone RL in mammalian cell culture media. Biotechnol Bioeng, 1977, 19(10): 1557-1561.

[63] Mordor Intelligence (2021) Biopharmaceuticals market—growth, trends, COVID-19 impact, and forecasts (2021–2026)

[64] Möller N, Scholz-Ahrens K, . Bioactive peptides and proteins from foods: indication for health effects. Eur J Nutr, 2008, 47: 171-182.

[65] Mols J, Peeters-Joris C, . Origin of rice protein hydrolysates added to protein-free media alters secretion and extracellular proteolysis of recombinant interferon-gamma as well as CHO-320 cell growth. Biotech Lett, 2004, 26: 1043-1046.

[66] Montserrat-de la Paz S, Rodriguez-Martin NM, . Evaluation of anti-inflammatory and atheroprotective properties of wheat gluten protein hydrolysates in primary human monocytes. Foods, 2020

[67] Mosser M, Chevalot I, . Combination of yeast hydrolysates to improve CHO cell growth and IgG production. Cytotechnology, 2013, 65: 629-641.

[68] Nesse KO, Nagalakshmi AP, . Safety evaluation of fish protein hydrolysate supplementation in malnourished children. Regul Toxicol Pharmacol, 2014, 69(1): 1-6.

[69] Ng JY, Chua ML, . Chlorella vulgaris extract as a serum replacement that enhances mammalian cell growth and protein expression. Front Bioeng Biotechnol, 2020, 8: 1068.

[70] Njoya M, Basitere M, . Analysis of the characteristics of poultry slaughterhouse wastewater (PSW) and its treatability. Water Pract Technol, 2019

[71] O’Flaherty R, Bergin A, . Mammalian cell culture for production of recombinant proteins: a review of the critical steps in their biomanufacturing. Biotechnol Adv, 2020, 43.

[72] Obaidi I, Mota LM, . The role of protein hydrolysates in prolonging viability and enhancing antibody production of CHO cells. Appl Microbiol Biotechnol, 2021, 105(8): 3115-3129.

[73] O'Neill EN, Cosenza ZA, . Considerations for the development of cost-effective cell culture media for cultivated meat production. Compr Rev Food Sci Food Saf, 2021, 20: 686-709.

[74] Pasupuleti VK, Braun S. Pasupuleti VK, Demain AL. State of the art manufacturing of protein hydrolysates. Protein hydrolysates in biotechnology, 2010, Dordrecht: Springer, 11-32.

[75] Patnaik SK, Stanley P. Lectin-resistant CHO glycosylation mutants. Methods Enzymol, 2006, 416: 159-182.

[76] Petrova I, Tolstorebrov I, . Production of fish protein hydrolysates step by step: technological aspects, equipment used, major energy costs and methods of their minimizing. Int Aquat Res, 2018, 10(3): 223-241.

[77] Pham PL, Perret S, . Transient gene expression in HEK293 cells : peptone addition posttransfection improves recombinant protein synthesis. Biotechnol Bio, 2005, 90: 332-344.

[78] Pintoe Silva MEM, Paton I, . Mineral and vitamin content of beef, chicken, and turkey hydrolysates—mineral and vitamin content of protein hydrolysates. Quim Nova, 2008

[79] Podpora B, Swiderski F, . Spent brewer’s yeast extracts as a new component of functional food. Czech J Food Sci, 2016, 34: 554-563.

[80] Ponten J, Saksela E. Two established in vitro cell lines from human mesenchymal tumours. Int J Cancer, 1967, 2(5): 434-447.

[81] Rasheed S, Nelson-Rees WA, . Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer, 1974, 33(4): 1027-1033.

[82] Richardson J, Shah B, . Metabolomics analysis of soy hydrolysates for the identification of productivity markers of mammalian cells for manufacturing therapeutic proteins. Biotechnol Prog, 2015, 31: 522-531.

[83] Rodríguez-Hernandez C, Torres-García S, . Cell culture: history, development and prospects. Int J Curr Res Acad Rev, 2014, 2: 188-200.

[84] Rungruangsaphakun J, Keawsompong S. Optimization of hydrolysis conditions for the mannooligosaccharides copra meal hydrolysate production. 3 Biotech, 2018

[85] Rutherfurd SM. Methodology for determining degree of hydrolysis of proteins in hydrolysates: a review. J AOAC Int, 2019, 93(5): 1515-1522.

[86] Schlaeger E-J. The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties. J Immunol Methods, 1996, 194: 191-199.

[87] Schneider U, Schwenk H-U, . Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer, 1977, 19(5): 621-626.

[88] Sha S, Agarabi C, . N-glycosylation design and control of therapeutic monoclonal antibodies. Trends Biotechnol, 2016, 34(10): 835-846.

[89] Si D, Shang T, . Production and characterization of functional wheat bran hydrolysate rich in reducing sugars, xylooligosaccharides and phenolic acids. Biotechnol Rep, 2020, 27.

[90] Siemensma A, Babcock J, . Pasupuleti VK, Demain AL, . Towards an understanding of how protein hydrolysates stimulate more efficient biosynthesis in cultured cells. Protein hydrolysates in biotechnology, 2010, Berlin: Springer, 33.

[91] Silvestre MPC. Review of methods for the analysis of protein hydrolysates. Food Chem, 1997, 60(2): 263-271.

[92] Singh BP, Vij S, . Functional significance of bioactive peptides derived from soybean. Peptides, 2014, 54: 171-179.

[93] Song W, Kong X, . Antioxidant and antibacterial activity and in vitro digestion stability of cottonseed protein hydrolysates. LWT, 2020, 118.

[94] Soule HD, Vazguez J, . A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst, 1973, 51(5): 1409-1416.

[95] Spearman M, Lodewyks C, . The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells. Biotechnol Prog, 2014, 30(3): 584-593.

[96] Steinfeld H, Gerber P, . Livestock's long shadow, 2006, Rome: Food and Agriculture Organization of the United Nations.

[97] Sung YH, Lim SW, . Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol, 2004, 63: 527-536.

[98] The Business Research Company (2021). Monoclonal antibodies (MAbS) global market report 2021: COVID-19 impact and recovery to 2030. T. B. R. company. Global. p 200

[99] Thomas T, Thomas TJ. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell Mol Life Sci, 2001, 58: 244-258.

[100] Thompson S, Chesher D. Lot-to-lot variation. Clin Biochem Rev, 2018, 39(2): 51-60.

[101] Tjio JH, Puck TT. Genetics of somatic mammalian cells. II. Chromosomal constitution of cells in tissue culture. J Exp Med, 1958, 108(2): 259-268.

[102] Todaro GJ, Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol, 1963, 17(2): 299-313.

[103] Tovar-Jimenez X, Muro C, . Hydrolysate antimicrobial activity released from bovine whey protein concentration by the appartyl protease Eap1 of Sporisoriun reilianum. Rev Mex Ing Quim, 2017, 16: 11-18.

[104] Tsuchiya S, Yamabe M, . Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer, 1980, 26(2): 171-176.

[105] Tuomisto HL, Teixeira de Mattos MJ. Environmental impacts of cultured meat production. Environ Sci Technol, 2011, 45: 6117-6123.

[106] Vink T, Oudshoorn-Dickmann M, . A simple, robust and highly efficient transient expression system for producing antibodies. Methods, 2014, 65(1): 5-10.

[107] Wang R, Han Z, . Antibacterial activity of trypsin-hydrolyzed camel and cow whey and their fractions. Animals, 2020

[108] Wasserman AE. Amino acid and vitamin composition of saccharomyces fragilis grown in whey. J Dairy Sci, 1961, 44(3): 379-386.

[109] Wölfel J, Essers R, . CAP-T cell expression system: a novel rapid and versatile human cell expression system for fast and high yield transient protein expression. BMC Proc, 2011, 8(Suppl 8): 133.

[110] Wu P-T, Lau Y-Q, . Ability of chicken protein hydrolysate to lower serum cholesterol through its bile acid binding activity. CyTA, 2020, 18(1): 493-499.

[111] Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol, 2004, 22(11): 1393-1398.

[112] Wurm FM, Wurm MJ. Cloning of CHO cells, productivity and genetic stability—a discussion. Processes, 2017, 5(2): 20.

[113] Xu X, Nagarajan H, . The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol, 2011, 29(8): 735-741.

[114] Xu Q, Bai F, . Utilization of acid hydrolysate of recovered bacterial cell as a novel organic nitrogen source for l-tryptophan fermentation. Bioengineered, 2019, 10(1): 23-32.

[115] Xu J, Xu X, . Biomanufacturing evolution from conventional to intensified processes for productivity improvement: a case study. MAbs, 2020, 12: 1770669.

[116] Yamaguchi K, Itoh K, . Engineered long terminal repeats of retroviral vectors enhance transgene expression in hepatocytes in vitro and in vivo. Mol Ther, 2003, 8(5): 796-803.

[117] Yao T, Asayama Y. Animal-cell culture media: history, characteristics, and current issues. Reprod Med Biol, 2017, 16(2): 99-117.

[118] Yasumura Y, Kawakita Y. Studies on SV40 in tissue culture-preliminary step for cancer research in vitro. Nihon Rinsho, 1963, 21: 1201-1215.

[119] Zang L, Frenkel R, . Metabolomics profiling of cell culture media leading to the identification of riboflavin photosensitized degradation of tryptophan causing slow growth in cell culture. Anal Chem, 2011, 83: 5422-5430.

[120] Zhang XX, Li W, Wang R, Luo XH, Li Y, Chen ZX. Protective effects of rice dreg protein hydrolysates against hydrogen 2 peroxide-induced oxidative stress in HepG-2 cells. Food Funct, 2016

[121] Zhang Y, Tu D, . Fish scale valorization by hydrothermal pretreatment followed by enzymatic hydrolysis for gelatin hydrolysate production. Molecules, 2019

[122] Zhang M, Cao T-T, . Silk sericin hydrolysate is a potential candidate as a serum-substitute in the culture of Chinese hamster ovary and henrietta lacks cells. J Insect Sci, 2019, 19: 10.

[123] Zhang P, Chan W, . Revisiting fragmentation reactions of protonated α-amino acids by high-resolution electrospray ionization tandem mass spectrometry with collision-induced dissociation. Sci Rep, 2019, 9(1): 6453.

[124] Zheng X, Baker H, . Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture. Part IV. Application of proteomics to the manufacture of biological drugs. Biotechnol Prog, 2006, 22(5): 1294-1300.

[125] Zhou K, Canning C, . Effects of rice protein hydrolysates prepared by microbial proteases and ultrafiltration on free radicals and meat lipid oxidation. LWT Food Sci Technol, 2013, 50(1): 331-335.

Funding

National Research Foundation, Singapore, and the Agency for Science, Technology and Research (A*STAR)(H20H8a0003)