, Volume 63, Issue 3, pp 247–258 | Cite as

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

  • Suwen Hu
  • Lei Deng
  • Huamao Wang
  • Yingping ZhuangEmail author
  • Ju Chu
  • Siliang Zhang
  • Zhonghai Li
  • Meijin GuoEmail author
Original Research


The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 105 cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.


Mouse-human chimeric anti-EGFRvIII antibody Recombinant CHO-C12 cells Balanced amino acid feed Perfusion culture 



This study was supported by the National High Technology Research & Development Program (863 Program) of China (No. 2007AA02Z216), the National Special Fund for State Key Laboratory of Bioreactor Engineering (No.2060204),the National Basic Research Program of China (No.2007CB714303) and the Qianjiang Scholarship grant from Hangzhou Municipal Government, China.


  1. Banik GG (1996) High-density hybridoma perfusion culture. Appl Biochem Biotechnol 61:211–229CrossRefGoogle Scholar
  2. Bibila TA, Robinson DK (1995) In pursuit of the optimal fed-batch process for monoclonal antibody production. Biotechnol Prog 11:1–13CrossRefGoogle Scholar
  3. Bibila TA, Ranucci CS, Glazomitsky K (1994) Monocloal antibody process development using medium concentrates. Biotechnol Prog 10:87–96CrossRefGoogle Scholar
  4. Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58:671–685CrossRefGoogle Scholar
  5. Chen PF, Harcum SW (2005) Effects of amino acid additions on ammonium stressed CHO cells. J Biotechnol 117:277–286CrossRefGoogle Scholar
  6. Chu L, Robinson DK (2001) Industrial choices for protein production by large-scale cell culture. Curr Opin Biotechnol 2:180–187CrossRefGoogle Scholar
  7. Ciardiello F, Tortora G (2001) A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 7:2958–2970Google Scholar
  8. Dai MS, Liu TQ (2002) A study of the metabolism parameters of NSCs. Tissue Eng 12:101–110Google Scholar
  9. Derouazi M, Martinet D, Besuchet SN, Flaction R, Wicht M, Bertschinger M, Hacker DL, Beckmann JS, Wurm FM (2006) Genetic characterization of CHO production host DG44 and derivative recombinant cell lines. Biochem Biophys Res Commun 340:1069–1077CrossRefGoogle Scholar
  10. DeZengotita VM (2000) Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production. Biotechnol Bioeng 69:566–576CrossRefGoogle Scholar
  11. Diedrich U, Lucius J, Baron E, Behnke J, Pabst B, Zoll B (1995) Distribution of epidermal growth factor receptor gene amplifecation in brain tumors and correlation to prognosis. J Neurol 242:683–688CrossRefGoogle Scholar
  12. Gainet M, Guardiola E, Dufresne A, Pivot X (2003) Epidermal growth factor receptors (EGFR): a new target for anticancer therapy. Cancer Radiother 7:195–199Google Scholar
  13. Konstantinov KB, Tsai Y, Moles D, Matanguihan R (1996) Control of long-term perfusion Chinese hamster ovary cell culture by glucose auxostat. Biotechnol Prog 12:100–109CrossRefGoogle Scholar
  14. Kuwae S, Ohda T (2005) Development of a fed-Batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells. J Biosci Bioeng 100:502–510CrossRefGoogle Scholar
  15. Liu L, Backlund LM, Nilsson BR, Grander D, Ichimura K, Goike HM, Collins VP (2005) Clinical significance of EGFR amplification and the aberrant EGFRvIII transcript in conventionally treated astrocytic gliomas. J Mol Med 83:917–926CrossRefGoogle Scholar
  16. Ljunggren J, Haggstrom L (1990) Glutamine limited fed-batch culture reduces ammonium ion production in animal cells. Biotechnol Lett 12:705–710CrossRefGoogle Scholar
  17. Meuwly F, Weber U, Ziegler T et al (2006) Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality. J Biotechnol 123:106–116CrossRefGoogle Scholar
  18. Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirenz G, Gunn G, Zoltick PW, Biegel JA, Hayes RL, Wong AJ (1995) Frequent expression of a utant epidermal growth factor receptor in multiple human tumors. Cancer Res 55:5536–5539Google Scholar
  19. Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, Huang HJ (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. P Natl Aacd Sci USA 91:7727–7731CrossRefGoogle Scholar
  20. Okamoto I, Kenyon LC, Emlet DR, Moi T, Sasaki J, Hirosako S, Ichikawa Y, Kishi H, Godwin AK, Yoshioka M, Suga M, Matsumoto M, Wong AJ (2003) Expression of constitutively activated EGFRvIII in non-small cell lung cancer. Cancer Sci 94:50–56CrossRefGoogle Scholar
  21. Ou C, Wu FX, Luo Y, Cao J, Zhao YN, Yuan WP, Li Y, Su JJ (2005) Expression and significance of epidermal growth factor receptor variant type III in hepatocellular carcinoma. Chin J Cancer 24:166–169Google Scholar
  22. Ozturk SS, Palsson BO (1992) Effects of ammonia and L-lactate on hybridoma growth, metabolism, and antibody production. Biotechnol Bioeng 39:234–239CrossRefGoogle Scholar
  23. Paredes C, Sanfeliu A, Gardenas F et al (1998) Estimation of the intracellular fluxes for a hybridoma cell line by material ballances. Enzym Microb Tech 22:187–198CrossRefGoogle Scholar
  24. Parkin DM, Stjernsward T, Muir CS (1984) Estimates of the worldwide frequency of twelve major cancers. Bull World Health Organ 62:163–182Google Scholar
  25. Ramnarain DB, Park S, Lee DY, Hatanpaa KJ, Scoggin SO, Otu H, Libermann TA, Raisanen JM, Ashfaq R, Wong ET, Wu J, Elliott R, Habib AA (2006) Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in glioma cells. Cancer Res 66:867–874CrossRefGoogle Scholar
  26. Rastilho CL, Anspach FB, Deckwer WD (2002) Comparison of affinity membranes for the purification of immunoglobulins. Biotechnol 18:776–781Google Scholar
  27. Ryll T, Dutina G, Reyes A, Gunson J, Krummen L, Etcheverry T (2000) Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention: characterization of separation efficiency and impact of perfusion on product quality. Biotechnol Bioeng 69:440–449CrossRefGoogle Scholar
  28. Serag EHB, Andrew C, Mason MD (2001) Epidemiology of hepatocellular carcinoma. Clin Liver Dis 5:87–107CrossRefGoogle Scholar
  29. Sok JC, Coppelli FM, Thomas SM, Lango MN, Xi S, Hunt JL, Freilino ML, Graner MW, Wikstrand CJ, Bigner DD, Gooding WE, Furnari FB, Grandis JR (2006) Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targenting. Clin Cancer Res 12:5064–5973CrossRefGoogle Scholar
  30. Sun XM, Zhang YX (2001) Effects of ammonia on cell metabolismin in the culture of recombinant CHO cells. Chin J Biotechnol 17:304–309Google Scholar
  31. Thomas MB, Zhu AX (2005) Hepatocellular carcinoma: the need for progress. Clin Oncol 23:2892–2899CrossRefGoogle Scholar
  32. Wang HM (2009) Epidermal growth factor receptor vIII antibody development and application in hepatocellular carcinoma therapy. Dissertation, Fudan UniversityGoogle Scholar
  33. Wang HM, Jiang H, Zhou M, Xu ZB, Liu SG, Li ZH (2009) Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma. Cancer Lett 279:30–38CrossRefGoogle Scholar
  34. Xie L, Wang DIC (1996) High cell density and high monoclonal antibody production through medium desigh and rational control in a bioreactor. Biotechnol Bioeng 151:725–729CrossRefGoogle Scholar
  35. Yang JD, Angelillo Y, Chaudhry M, Goldenberg C, Goldenberg DM (2000) Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. Biotechnol Bioeng 69:74–82CrossRefGoogle Scholar
  36. Yoo HY, Patt CH, Geschwind JF, Thuluvath PJ (2003) The outcome of liver transplantation in patients with Hepatocellular carcinoma in the United States between 1988 and 2001: 5-year survival has improved significantly with time. J Clin Oncol 21:4329–4335CrossRefGoogle Scholar
  37. Zeineldin R, Rosenberg M, Ortega D, Buhr C, Chavez MG, Stack MS, Kusewitt DF, Hudson LG (2006) Mesenchymal transformation in epithelial ovarian tumor cells expressing epidermal growth factor receptor variant III. Mol Carcinog 45:851–860CrossRefGoogle Scholar
  38. Zhu AX (2006) Systemic therapy of advanced Hepatocellular carcinoma: how hopeful should we be. Oncol 11:790–800CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Suwen Hu
    • 1
  • Lei Deng
    • 1
  • Huamao Wang
    • 2
  • Yingping Zhuang
    • 1
    Email author
  • Ju Chu
    • 1
  • Siliang Zhang
    • 1
  • Zhonghai Li
    • 2
  • Meijin Guo
    • 1
    Email author
  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiPeople’s Republic of China
  2. 2.State Key Laboratory of Oncogenes and Related GenesShanghai Jiaotong UniversityShanghaiPeople’s Republic of China

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