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Facile development of medium optimization for antibody production: implementation in spinner flask and hollow fiber reactor

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Abstract

Most bio-industrial mammalian cells are cultured in serum-free media to achieve advantages, such as batch consistency, suspended growth, and simplified purification. The successful development of a serum-free medium could contribute to a reduction in the experimental variation, enhance cell productivity, and facilitate biopharmaceuticals production using the cell culture process. Commercial serum-free media are also becoming more and more popular. However, the cell line secrets its own recombinant product and has special nutritional requirements. How can the composition of the proprietary medium be adjusted to support the specific cell’s metabolism and recombinant protein? This article uses statistical strategies to modify the commercial medium. A design of experiments is adopted to optimize the medium composition for the hybridoma cell in a serum-free condition. The supplements of peptone, ferric citrate, and trace elements were chosen to study their impact on hybridoma growth and antibody production using the response surface methodology. The stimulatory effect of the developed formulation on hybridoma growth was confirmed by the steepest ascent path. The optimal medium stimulated the hybridoma growth and antibody production in three diverse systems: a static plate, an agitated spinner flask, and a hollow fiber reactor. The cells in the developed serum-free medium had a better antibody production as compared to that in the commercial medium in the hollow fiber reactor. Our results demonstrated that the facile optimization for medium and antibody production was successfully accomplished in the hybridoma cells.

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References

  • Alcaín FJ, Löw H, Crane FL (1995) Iron at the cell surface controls both DNA synthesis and plasma membrane redox system. Protoplasma 184:233–237

    Article  Google Scholar 

  • Chabanon G, Alves da Costa L, Farges B, Harscoat C, Chenu S, Goergen JL, Marc A, Marc I, Chevalot I (2008) Influence of the rapeseed protein hydrolysis process on CHO cell growth. Biores Technol 99:7143–7151

    Article  CAS  Google Scholar 

  • Chua F, Oh SKW, Yap M, Teo WK (1994) Enhanced IgG production in eRDF media with and without serum, A comparative study. J Immunol Methods 167:109–119

    Article  CAS  Google Scholar 

  • Coombs MRP, Grant T, Greenshields AL, Arsenault DJ, Holbein BE, Hoskin DW (2015) Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells. Exp Mol Pathol 99:262–270

    Article  Google Scholar 

  • Coronel J, Klausing S, Heinrich C, Noll T, Figueredo-Cardero A, Castilho LR (2016) Valeric acid supplementation combined to mild hypothermia increases productivity in CHO cell cultivations. Biochem Eng J 114:101–109

    Article  CAS  Google Scholar 

  • Corrêa AL, Senna JPM, de Sousa ÁPB (2016) Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies. Cytotechnology 68:419–427

    Article  Google Scholar 

  • Davami F, Eghbalpour F, Nematollahi L, Barkhordari F, Mahboudi F (2015) 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 19:194–205

    PubMed  PubMed Central  Google Scholar 

  • Dhanasekaran M, Indumathi S, Lissa RP, Harikrishnan R, Rajkumar JS, Sudarsanam D (2013) A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition. Cytotechnology 65:187–197

    Article  CAS  Google Scholar 

  • Dong J, Mandenius CF, Lübberstedt M, Urbaniak T, Nüssler AKN, Knobeloch D, Gerlach JC, Zeilinger K (2008) Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology. Cytotechnology 57:251–261

    Article  CAS  Google Scholar 

  • Ferreira SL, Santos WND, Quintella CM, Neto BB, Bosque-Sendra JM (2004) Doehlert matrix: a chemometric tool for analytical chemistry—review. Talanta 63:1061–1067

    Article  CAS  Google Scholar 

  • García Münzer DG, Ivarsson M, Usaku C, Habicher T, Soos M, Morbidelli M, Pistikopoulos EN, Mantalaris A (2015) An unstructured model of metabolic and temperature dependent cell cycle arrest in hybridoma batch and fed-batch cultures. Biochem Eng J 93:260–273

    Article  Google Scholar 

  • Knöspel F, Schindler RK, Lübberstedt M, Petzolt S, Gerlach JC, Zeilinger K (2010) Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology. Cytotechnology 62:557–571

    Article  Google Scholar 

  • Kovár J (1988) Hybridoma cultivation in defined serum-free media: growth-supporting substances. V. Trace elements. Folia Biol 34:35–41

    Google Scholar 

  • Lee J, Tscheliessnig A, Chen A, Lee YY, Adduci G, Choo A, Jungbauer A (2009) Adaptation of hybridomas to protein-free media results in a simplified two-step immunoglobulin M purification process. J Chromatogr A 1216:2683–2688

    Article  CAS  Google Scholar 

  • Liu CH, Chang TY (2006) Rational development of serum-free medium for Chinese hamster ovary cells. Process Biochem 41:2314–2319

    Article  CAS  Google Scholar 

  • Liu YF, Tang RH, Zhang QX, Shi JY, Li XM, Liu ZQ, Zhao W (1986) Stimulation of cell growth of Tetrahymena pyriformis and Chlamydomonas reinhardtii by trace elements. Biol Trace Elem Res 9:89–99

    Article  CAS  Google Scholar 

  • Manna L, Febo TD, Armillotta G, Luciani M, Ciarelli A, Salini R, Ventura MD (2015) Production of monoclonal antibodies in serum-free media. Monoclon Antib Immunodiagn Immunother 34:278–288

    Article  CAS  Google Scholar 

  • McGillicuddy N, Floris P, Albrecht S, Bones J (2018) Examining the sources of variability in cell culture media used for biopharmaceutical production. Biotechnol Lett 40:5–21

    Article  CAS  Google Scholar 

  • Nagira K, Hara T, Hayashida M, Osada K, Shiga M, Sasamoto K, Kina K, Murakami H (1995) Development of a protein-free medium with iron salts replacing transferrin for a human–human hybridoma. Biosci Biotechnol Biochem 59:743–745

    Article  CAS  Google Scholar 

  • Price PJ (2017) Best practices for media selection for mammalian cells. Vitro Cell Dev Biol Anim 53:673–681

    Article  CAS  Google Scholar 

  • Reimer CB, Phillips DJ, Aloisio CH, Moore DD, Galland GG, Wells TW, Black CM, McDougal JS (1984) Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes. Hybridoma 3:263–275

    Article  CAS  Google Scholar 

  • Sen S, Roychoudhury PK (2013) Development of optimal medium for production of commercially important monoclonal antibody 520C9 by hybridoma cell. Cytotechnology 65:233–252

    Article  CAS  Google Scholar 

  • Shukla AA, Thömmes J (2010) Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol 28:253–261

    Article  CAS  Google Scholar 

  • Souza AS, Santos WNLD, Ferreira SLC (2005) Application of Box–Behnken design in the optimisation of an on-line pre-concentration system using knotted reactor for cadmium determination by flame atomic absorption spectrometry. Spectrochim Acta Part B 60:737–742

    Article  Google Scholar 

  • Spens E, Häggström L (2005) Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors. Biotechnol Prog 21:87–95

    Article  CAS  Google Scholar 

  • Tan KY, Teo KL, Lim JFY, Chen AKL, Reuveny S, Oh SKW (2015) Serum-free media formulations are cell line-specific and require optimization for microcarrier culture. Cytotherapy 17:1152–1165

    Article  CAS  Google Scholar 

  • van der Valk J, Brunner D, De Smet K, Fex Svenningsen Å, Honegger P, Knudsen LE, Lindl T, Noraberg J, Price A, Scarino ML, Gstraunthaler G (2010) Optimization of chemically defined cell culture media—replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro 24:1053–1063

    Article  Google Scholar 

  • Xing Z, Kenty B, Koyrakh I, Borys M, Pan SH, Li ZJ (2011) Optimizing amino acid composition of CHO cell culture media for a fusion protein production. Process Biochem 46:1423–1429

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Yolmeh M, Jafari SM (2017) Applications of response surface methodology in the food industry processes. Food Bioprocess Technol 10:413–433

    Article  CAS  Google Scholar 

  • Zhang Y, Zhou Y, Yu J (1994) Effects of peptone on hybridoma growth and monoclonal antibody formation. Cytotechnology 16:147–150

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We express gratitude to Ministry of Science and Technology (MOST 106-2221-E-182-050), Chang Gung University (BMRP 758) and Chang Gung Memorial Hospital (CMRPD2G0282, 2H0071) for funding and supporting this research. We would also like to thank the valuable suggestion of bioreactor operation from Frank R. H. Wang, United BioPharma Inc., Hsinchu, Taiwan.

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Authors and Affiliations

  1. Department of Chemical and Materials Engineering, Chang Gung University, 259, Wen-Hwa First Road, Kwei-Shan, Taoyuan, 333, Taiwan

    Chi-Hsien Liu

  2. Research Center for Chinese Herbal Medicine and Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, 261, Wen-Hwa First Road, Taoyuan, Taiwan

    Chi-Hsien Liu

  3. Department of Chemical Engineering, Ming Chi University of Technology, 84, Gung-Juan Road, New Taipei City, Taiwan

    Chi-Hsien Liu

  4. Department of Ophthalmology, Chang Gung Memorial Hospital, 5, Fu-Hsing Street, Taoyuan, Taiwan

    Chi-Hsien Liu & Wei-Chi Wu

  5. Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, 259, Wen-Hwa First Road, Kwei-Shan, Taoyuan, 333, Taiwan

    Yi-Xin Liu

  6. College of Medicine, Chang Gung University, 259, Wen-Hwa First Road, Taoyuan, Taiwan

    Wei-Chi Wu

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  1. Chi-Hsien Liu
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Correspondence to Chi-Hsien Liu.

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Liu, CH., Liu, YX. & Wu, WC. Facile development of medium optimization for antibody production: implementation in spinner flask and hollow fiber reactor. Cytotechnology 70, 1631–1642 (2018). https://doi.org/10.1007/s10616-018-0255-z

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