Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Cytotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58:671–685. doi:10.1016/j.addr.2005.12.006

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Jayapal KP, Wlaschin KF, Hu WS, Yap MGS (2007) Recombinant protein therapeutics from CHO cells—20 years and counting. Chem Eng Prog 103:40–47

    CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100:3451–3461. doi:10.1007/s00253-016-7388-9

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Lane AN, Fan TW (2015) Regulation of mammalian nucleotide metabolism and biosynthesis. Nucleic Acids Res 43:2466–2485. doi:10.1093/nar/gkv047

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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 

  • 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

    Article  CAS  Google Scholar 

  • 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 40:3719–3722

    CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 91:515–521

    Article  CAS  Google Scholar 

  • Traut TW (1994) Physiological concentrations of purines and pyrimidines. Mol Cell Biochem 140:1–22. doi:10.1007/BF00928361

    Article  CAS  Google Scholar 

  • Walsh G (2014) Biopharmaceutical benchmarks 2014. Nat Biotechnol 32:992–1000. doi:10.1038/nbt.3040

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398. doi:10.1038/nbt1026

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Bioscience and Bioindustry, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima, 770-8506, Japan

    Yasuhiro Takagi & Takeshi Omasa

  2. Biotechnology Laboratories, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki, 300-2698, Japan

    Yasuhiro Takagi, Takuya Kikuchi & Ryuta Wada

  3. Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Takeshi Omasa

Authors
  1. Yasuhiro Takagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takuya Kikuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryuta Wada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takeshi Omasa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takeshi Omasa.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Takagi, Y., Kikuchi, T., Wada, R. et al. The enhancement of antibody concentration and achievement of high cell density CHO cell cultivation by adding nucleoside. Cytotechnology 69, 511–521 (2017). https://doi.org/10.1007/s10616-017-0066-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-017-0066-7

Keywords

Search

Navigation