Advertisement

Cytotechnology

, Volume 69, Issue 3, pp 511–521 | Cite as

The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside

  • Yasuhiro Takagi
  • Takuya Kikuchi
  • Ryuta Wada
  • Takeshi Omasa
Original Article

Abstract

Recently, with the dramatic increase in demand for therapeutic antibodies, Chinese hamster ovary (CHO) cell culture systems have made significant progress in recombinant antibody production. Over the past two decades, recombinant antibody productivity has been improved by more than 100-fold. Medium optimization has been identified as an important key approach for increasing product concentrations. In this study, we evaluated the effects of deoxyuridine addition to fed-batch cultures of antibody-expressing CHO cell lines. Furthermore, we investigated the effects of combined addition of deoxyuridine, thymidine, and deoxycytidine. Our results suggest that addition of these pyrimidine nucleosides can increase CHO cell growth, with no significant change in the specific production rate. As a result of the increased cell growth, the antibody concentration was elevated and we were able to achieve more than 9 g/L during 16 days of culture. Similar effects of nucleoside addition were observed in fed-batch cultures of a Fab fragment-expressing CHO cell line, and the final Fab fragment concentration was more than 4 g/L. This nucleoside addition strategy could be a powerful platform for efficient antibody production.

Keywords

Chinese hamster ovary (CHO) cell Therapeutic antibody Nucleoside Cell culture medium 

References

  1. Austin WR, Armijo AL, Campbell DO, Singh AS, Hsieh T, Nathanson D, Herschman HR, Phelps ME, Witte ON, Czernin J, Radu CG (2012) Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress. J Exp Med 209:2215–2228. doi: 10.1084/jem.20121061 CrossRefGoogle Scholar
  2. Backliwal G, Hildinger M, Kuettel I, Delegrange F, Hacker DL, Wurm FM (2008) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol Bioeng 101:182–189. doi: 10.1002/bit.21882 CrossRefGoogle Scholar
  3. Barnes LM, Bentley CM, Dickson AJ (2000) Advances in animal cell recombinant protein production: GS-NS0 expression system. Cytotechnology 32:109–123. doi: 10.1023/A:1008170710003 CrossRefGoogle Scholar
  4. Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnol (NY) 10:169–175. doi: 10.1038/nbt0292-169 CrossRefGoogle Scholar
  5. Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58:671–685. doi: 10.1016/j.addr.2005.12.006 CrossRefGoogle Scholar
  6. Carinhas N, Duarte TM, Barreiro LC, Carrondo MJ, Alves PM, Teixeira AP (2013) Metabolic signatures of GS-CHO cell clones associated with butyrate treatment and culture phase transition. Biotechnol Bioeng 110:3244–3257. doi: 10.1002/bit.24983 CrossRefGoogle Scholar
  7. Carvalhal AV, Santos SS, Calado J, Haury M, Carrondo MJ (2003) Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins. Biotechnol Prog 19:69–83. doi: 10.1021/bp0255917 CrossRefGoogle Scholar
  8. Carvalhal AV, Santos SS, Carrondo MJ (2011) Extracellular purine and pyrimidine catabolism in cell culture. Biotechnol Prog 27:1373–1382. doi: 10.1002/btpr.656 CrossRefGoogle Scholar
  9. Chaderjian WB, Chin ET, Harris RJ, Etcheverry TM (2005) Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed. Biotechnol Prog 21:550–553. doi: 10.1021/bp0497029 CrossRefGoogle Scholar
  10. Chen F, Fan L, Wang J, Zhou Y, Ye Z, Zhao L, Tan WS (2012a) Insight into the roles of hypoxanthine and thymidine on cultivating antibody-producing CHO cells: cell growth, antibody production and long-term stability. Appl Microbiol Biotechnol 93:169–178. doi: 10.1007/s00253-011-3484-z CrossRefGoogle Scholar
  11. Chen F, Ye Z, Zhao L, Liu X, Fan L, Tan WS (2012b) Biphasic addition strategy of hypoxanthine and thymidine for improving monoclonal antibody production. J Biosci Bioeng 114:347–352. doi: 10.1016/j.jbiosc.2012.04.015 CrossRefGoogle Scholar
  12. Clincke MF, Molleryd C, Samani PK, Lindskog E, Faldt E, Walsh K, Chotteau V (2013) Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE Bioreactor-part II: applications for antibody production and cryopreservation. Biotechnol Prog 29:768–777. doi: 10.1002/btpr.1703 CrossRefGoogle Scholar
  13. De Leon Gatti M, Wlaschin KF, Nissom PM, Yap M, Hu WS (2007) Comparative transcriptional analysis of mouse hybridoma and recombinant Chinese hamster ovary cells undergoing butyrate treatment. J Biosci Bioeng 103:82–91. doi: 10.1263/jbb.103.82 CrossRefGoogle Scholar
  14. Dietmair S, Hodson MP, Quek LE, Timmins NE, Chrysanthopoulos P, Jacob SS, Gray P, Nielsen LK (2012) Metabolite profiling of CHO cells with different growth characteristics. Biotechnol Bioeng 109:1404–1414. doi: 10.1002/bit.24496 CrossRefGoogle Scholar
  15. Golabgir A, Gutierrez JM, Hefzi H, Li S, Palsson BO, Herwig C, Lewis NE (2016) Quantitative feature extraction from the Chinese hamster ovary bioprocess bibliome using a novel meta-analysis workflow. Biotechnol Adv 34:621–633. doi: 10.1016/j.biotechadv.2016.02.011 CrossRefGoogle Scholar
  16. Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P, van den Bremer ET, Gerritsen J, van Berkel PH (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108:1591–1602. doi: 10.1002/bit.23075 CrossRefGoogle Scholar
  17. Huang YM, Hu W, Rustandi E, Chang K, Yusuf-Makagiansar H, Ryll T (2010) Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment. Biotechnol Prog 26:1400–1410. doi: 10.1002/btpr.436 CrossRefGoogle Scholar
  18. Inoue Y, Fujisawa M, Shoji M, Hashizume S, Katakura Y, Shirahata S (2000) Enhanced antibody production of human-human hybridomas by retinoic acid. Cytotechnology 33:83–88. doi: 10.1023/A:1008155609072 CrossRefGoogle Scholar
  19. Jayapal KP, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics from CHO cells—20 years and counting. Chem Eng Prog 103:40–47Google Scholar
  20. Kim DY, Lee JC, Chang HN, Oh DJ (2005) Effects of supplementation of various medium components on chinese hamster ovary cell cultures producing recombinant antibody. Cytotechnology 47:37–49. doi: 10.1007/s10616-005-3775-2 CrossRefGoogle Scholar
  21. Kishishita S, Kodaira K, Takagi Y, Matsuda H, Okamoto H, Takuma S, Hirashima C, Aoyagi H (2015) Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells. J Biosci Bioeng 120:78–84. doi: 10.1016/j.jbiosc.2014.11.022 CrossRefGoogle Scholar
  22. Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100:3451–3461. doi: 10.1007/s00253-016-7388-9 CrossRefGoogle Scholar
  23. Kyriakopoulos S, Polizzi KM, Kontoravdi C (2013) Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity. J Biotechnol 168:543–551. doi: 10.1016/j.jbiotec.2013.09.007 CrossRefGoogle Scholar
  24. Lane AN, Fan TW (2015) Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res 43:2466–2485. doi: 10.1093/nar/gkv047 CrossRefGoogle Scholar
  25. Li F, Vijayasankaran N, Shen A, Kiss R, Amanullah A (2014) Cell culture processes for monoclonal antibody production. mAbs 2:466–479. doi: 10.4161/mabs.2.5.12720 CrossRefGoogle Scholar
  26. Luo Y, Chen G (2007) Combined approach of NMR and chemometrics for screening peptones used in the cell culture medium for the production of a recombinant therapeutic protein. Biotechnol Bioeng 97:1654–1659. doi: 10.1002/bit.21365 CrossRefGoogle Scholar
  27. Nakamura T, Omasa T (2015) Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system. J Biosci Bioeng 120:323–329. doi: 10.1016/j.jbiosc.2015.01.002 CrossRefGoogle Scholar
  28. Omasa T (2002) Gene amplification and its application in cell and tissue engineering. J Biosci Bioeng 94:600–605. doi: 10.1016/S1389-1723(02)80201-8 CrossRefGoogle Scholar
  29. Omasa T, Higashiyama K, Shioya S, Suga K (1992) Effects of lactate concentration on hybridoma culture in lactate-controlled fed-batch operation. Biotechnol Bioeng 39:556–564. doi: 10.1002/bit.260390511 CrossRefGoogle Scholar
  30. Omasa T, Takami T, Ohya T, Kiyama E, Hayashi T, Nishii H, Miki H, Kobayashi K, Honda K, Ohtake H (2008) Overexpression of GADD34 enhances production of recombinant human antithrombin III in Chinese hamster ovary cells. J Biosci Bioeng 106:568–573. doi: 10.1263/jbb.106.568 CrossRefGoogle Scholar
  31. Omasa T, Onitsuka M, Kim WD (2010) Cell engineering and cultivation of chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol 11:233–240. doi: 10.2174/138920110791111960 CrossRefGoogle Scholar
  32. Porter AJ, Dickson AJ, Racher AJ (2010) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: realizing the potential in bioreactors. Biotechnol Prog 26:1446–1454. doi: 10.1002/btpr.442 CrossRefGoogle Scholar
  33. Rajendra Y, Peery RB, Barnard GC (2016) Generation of stable Chinese hamster ovary pools yielding antibody titers of up to 7.6 g/L using the piggyBac transposon system. Biotechnol Prog 32:1301–1307. doi: 10.1002/btpr.2307 Google Scholar
  34. Reinhart D, Damjanovic L, Kaisermayer C, Kunert R (2015) Benchmarking of commercially available CHO cell culture media for antibody production. Appl Microbiol Biotechnol 99:4645–4657. doi: 10.1007/s00253-015-6514-4 CrossRefGoogle Scholar
  35. Russell GR, Partick EJ (1980) Effects of variations in nucleoside pool sizes on comparisons of the incorporation of [3H]thymidine into isolated rat liver cells. Cancer Res 40:3719–3722Google Scholar
  36. Sellick CA, Croxford AS, Maqsood AR, Stephens G, Westerhoff HV, Goodacre R, Dickson AJ (2011) Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production. Biotechnol Bioeng 108:3025–3031. doi: 10.1002/bit.23269 CrossRefGoogle Scholar
  37. Staub M, Spasokukotskaja T, Benczur M, Antoni F (1988) DNA synthesis and nucleoside metabolism in human tonsillar lymphocyte subpopulations. Acta Otolaryngol 105:118–124. doi: 10.3109/00016488809125014 CrossRefGoogle Scholar
  38. Takagi M, Hia HC, Jang JH, Yoshida T (2001) Effects of high concentrations of energy sources and metabolites on suspension culture of Chinese hamster ovary cells producing tissue plasminogen activator. J Biosci Bioeng 91:515–521CrossRefGoogle Scholar
  39. Traut TW (1994) Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140:1–22. doi: 10.1007/BF00928361 CrossRefGoogle Scholar
  40. Walsh G (2014) Biopharmaceutical benchmarks 2014. Nat Biotechnol 32:992–1000. doi: 10.1038/nbt.3040 CrossRefGoogle Scholar
  41. Wong NS, Wati L, Nissom PM, Feng HT, Lee MM, Yap MG (2010) An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107:321–336. doi: 10.1002/bit.22812 CrossRefGoogle Scholar
  42. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398. doi: 10.1038/nbt1026 CrossRefGoogle Scholar
  43. Yamaoka T, Kondo M, Honda S, Iwahana H, Moritani M, Ii S, Yoshimoto K, Itakura M (1997) Amidophosphoribosyltransferase limits the rate of cell growth-linked de novo purine biosynthesis in the presence of constant capacity of salvage purine biosynthesis. J Biol Chem 272:17719–17725. doi: 10.1074/jbc.272.28.17719 CrossRefGoogle Scholar
  44. Yu M, Hu Z, Pacis E, Vijayasankaran N, Shen A, Li F (2011) Understanding the intracellular effect of enhanced nutrient feeding toward high titer antibody production process. Biotechnol Bioeng 108:1078–1088. doi: 10.1002/bit.23031 CrossRefGoogle Scholar
  45. Zhang H, Wang H, Liu M, Zhang T, Zhang J, Wang X, Xiang W (2013) Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody. Cytotechnology 65:363–378. doi: 10.1007/s10616-012-9488-4 CrossRefGoogle Scholar
  46. Zhang J, Reddy J, Buckland B, Greasham R (2003) Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process. Biotechnol Bioeng 82:640–652. doi: 10.1002/bit.10608 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Institute of Bioscience and BioindustryTokushima UniversityTokushimaJapan
  2. 2.Biotechnology LaboratoriesAstellas Pharma Inc.TsukubaJapan
  3. 3.Graduate School of EngineeringOsaka UniversitySuitaJapan

Personalised recommendations