Bioprocess and Biosystems Engineering

, Volume 42, Issue 5, pp 711–725 | Cite as

Process intensification for the production of rituximab by an inducible CHO cell line

  • Kahina Mellahi
  • Denis Brochu
  • Michel Gilbert
  • Michel Perrier
  • Sven Ansorge
  • Yves Durocher
  • Olivier HenryEmail author
Research Paper


Mammalian-inducible expression systems are increasingly available and offer an attractive platform for the production of recombinant proteins. In this work, we have conducted process development for a cumate-inducible GS-CHO cell-line-expressing rituximab. To cope with the limitations encountered in batch when inducing at high cell densities, we have explored the use of fed-batch, sequential medium replacements, and continuous perfusion strategies applied during the pre-induction (growth) phase to enhance process performance in terms of product yield and quality. In shake flask, a fed-batch mode and a complete medium exchange at the time of induction were shown to significantly increase the integral of viable cell concentration and antibody titer compared to batch culture. Further enhancement of product yield was achieved by combining bolus concentrated feed additions with sequential medium replacement, but product galactosylation was reduced compared to fed-batch mode, as a result of the extended culture duration. In bioreactor, combining continuous perfusion of the basal medium with bolus daily feeding during the pre-induction period and harvesting earlier during the production phase is shown to provide a good trade-off between antibody titer and product galactosylation. Overall, our results demonstrate the importance of selecting a suitable operating mode and harvest time when carrying out high-cell-density induction to balance between culture productivity and product quality.


CHO cells Antibody Perfusion Fed batch Inducible expression system 



We are thankful to Mr. Louis Bisson from the Human Health Therapeutics Research Center at National Research Council Canada (Montreal) for HPLC analysis. This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-06407). This is NRC publication #NRC_HHT53416.


  1. 1.
    Matthews TE, Berry BN, Smelko J, Moretto J, Moore B, Wiltberger K (2016) Closed loop control of lactate concentration in mammalian cell culture by Raman spectroscopy leads to improved cell density, viability, and biopharmaceutical protein production. Biotechnol Bioeng 113:2416–2424CrossRefGoogle Scholar
  2. 2.
    Yusufi FNK, Lakshmanan M, Ho YS, Loo BLW, Ariyaratne P, Yang Y, Ng SK, Tan TRM, Yeo HC, Lim HL (2017) Mammalian systems biotechnology reveals global cellular adaptations in a recombinant CHO cell line. Cell syst 4:530–542. e536CrossRefGoogle Scholar
  3. 3.
    Lalonde M-E, Durocher Y (2017) Therapeutic glycoprotein production in mammalian cells. J Biotechnol 251:128–140CrossRefGoogle Scholar
  4. 4.
    Vallée C, Durocher Y, Henry O (2014) Exploiting the metabolism of PYC expressing HEK293 cells in fed-batch cultures. J Biotechnol 169:63–70CrossRefGoogle Scholar
  5. 5.
    Wlaschin KF, Hu W-S (2006) Fedbatch culture and dynamic nutrient feeding. Cell Culture Engineering SpringerGoogle Scholar
  6. 6.
    Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnol 22:1393–1398CrossRefGoogle Scholar
  7. 7.
    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 26:1400–1410CrossRefGoogle Scholar
  8. 8.
    Zhou W, Rehm J, Hu WS (1995) High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnol Bioeng 46:579–587CrossRefGoogle Scholar
  9. 9.
    Chee Furng Wong D, Tin Kam Wong K, Tang Goh L, Kiat Heng C, Gek Sim Yap M (2005) Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89:164–177CrossRefGoogle Scholar
  10. 10.
    Kuwae S, Ohda T, Tamashima H, Miki H, Kobayashi K (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
  11. 11.
    Sauer PW, Burky JE, Wesson MC, Sternard HD, Qu L (2000) A high-yielding, generic fed-batch cell culture process for production of recombinant antibodies. Biotechnol Bioeng 67:585–597CrossRefGoogle Scholar
  12. 12.
    Zhang L, Shen H, Zhang Y (2004) Fed-batch culture of hybridoma cells in serum-free medium using an optimized feeding strategy. J Chem Technol Biotechnol 79:171–181CrossRefGoogle Scholar
  13. 13.
    Aehle M, Schaepe S, Kuprijanov A, Simutis R, Lübbert A (2011) Simple and efficient control of CHO cell cultures. J Biotechnol 153:56–61CrossRefGoogle Scholar
  14. 14.
    Lu F, Toh PC, Burnett I, Li F, Hudson T, Amanullah A, Li J (2013) Automated dynamic fed-batch process and media optimization for high productivity cell culture process development. Biotechnol Bioeng 110:191–205CrossRefGoogle Scholar
  15. 15.
    Gagnon M, Hiller G, Luan YT, Kittredge A, DeFelice J, Drapeau D (2011) High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO Fed-batch cultures. Biotechnol Bioeng 108:1328–1337CrossRefGoogle Scholar
  16. 16.
    Toussaint C, Henry O, Durocher Y (2016) Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures. J Biotechnol 217:122–131CrossRefGoogle Scholar
  17. 17.
    Lao MS, Toth D (1997) Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell culture. Biotechnol Prog 13:688–691CrossRefGoogle Scholar
  18. 18.
    Altamirano C, Paredes C, Illanes A, Cairo J, Godia F (2004) Strategies for fed-batch cultivation of t-PA producing CHO cells: substitution of glucose and glutamine and rational design of culture medium. J Biotechnol 110:171–179CrossRefGoogle Scholar
  19. 19.
    Henry O, Durocher Y (2011) Enhanced glycoprotein production in HEK-293 cells expressing pyruvate carboxylase. Metabolic Eng 13:499–507CrossRefGoogle Scholar
  20. 20.
    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
  21. 21.
    Banik GG, Heath CA (1997) High-density hybridoma perfusion culture. Appl Biochem Biotechnol 61:211–229CrossRefGoogle Scholar
  22. 22.
    Robinson DK, Distefano DJ, Gould SL, Cuca G, Seamans TC, Benincasa D, Munshi S, Chan CP, Lee DK, Stanfor-Hollis J, Hollis GF, Jain D, Ramasubramanyan K, Mark GE, Silberklang M (1995) Production of engineered antibodies in myeloma and hybridoma cells – enhancements in gene expression and process design. In: Antibody engineering. ACS Symposium Series 604, pp1–14Google Scholar
  23. 23.
    Sheikholeslami Z, Jolicoeur M, Henry O (2014) Elucidating the effects of postinduction glutamine feeding on the growth and productivity of CHO cells. Biotechnol Prog 30:535–546CrossRefGoogle Scholar
  24. 24.
    Poulain A, Perret S, Malenfant F, Mullick A, Massie B, Durocher Y (2017) Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. J Biotechnol 255:16–27CrossRefGoogle Scholar
  25. 25.
    Gaillet B, Gilbert R, Broussau S, Pilotte A, Malenfant F, Mullick A, Garnier A, Massie B (2010) High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch. Biotechnology bioengineering 106:203–215Google Scholar
  26. 26.
    Mullick A, Xu Y, Warren R, Koutroumanis M, Guilbault C, Broussau S, Malenfant F, Bourget L, Lamoureux L, Lo R (2006) The cumate gene-switch: a system for regulated expression in mammalian cells. BMC Biotechnol 6:43CrossRefGoogle Scholar
  27. 27.
    Sheikholeslami Z, Jolicoeur M, Henry O (2013) Probing the metabolism of an inducible mammalian expression system using extracellular isotopomer analysis. J Biotechnol 164:469–478CrossRefGoogle Scholar
  28. 28.
    Sheikholeslami Z, Jolicoeur M, Henry O (2013) The impact of the timing of induction on the metabolism and productivity of CHO cells in culture. Biochem Eng J 79:162–171CrossRefGoogle Scholar
  29. 29.
    Henry O, Kwok E, Piret JM (2008) Simpler noninstrumented batch and semicontinuous cultures provide mammalian cell kinetic data comparable to continuous and perfusion cultures. Biotechnol Prog 24:921–931CrossRefGoogle Scholar
  30. 30.
    Lee S-Y, Kwon Y-B, Cho J-M, Park K-H, Chang S-J, Kim D-I (2012) Effect of process change from perfusion to fed-batch on product comparability for biosimilar monoclonal antibody. Process Biochem 47:1411–1418CrossRefGoogle Scholar
  31. 31.
    Voisard D, Meuwly F, Ruffieux PA, Baer G, Kadouri A (2003) Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells. Biotechnol Bioeng 82:751–765CrossRefGoogle Scholar
  32. 32.
    Meuwly F, Weber U, Ziegler T, Gervais A, Mastrangeli R, Crisci C, Rossi M, Bernard A, von Stockar U, Kadouri A (2006) Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality. J Biotechnol 123:106–116CrossRefGoogle Scholar
  33. 33.
    Yang WC, Minkler DF, Kshirsagar R, Ryll T, Huang YM (2016) Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity. J Biotechnol 217:1–11CrossRefGoogle Scholar
  34. 34.
    Bonham-Carter J, Weegar J, Nieminen A, Shevitz J, Eliezer E (2011) The use of the ATF system to culture chinese hamster ovary cells in a concentrated fed-batch system. Biopharm Int 24:42–42+Google Scholar
  35. 35.
    Feng Q, Mi L, Li L, Liu R, Xie L, Tang H, Chen Z (2006) Application of “oxygen uptake rate-amino acids” associated mode in controlled-fed perfusion culture. J Biotechnol 122:422–430CrossRefGoogle Scholar
  36. 36.
    Hiller GW, Ovalle AM, Gagnon MP, Curran ML, Wang W (2017) Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures. Biotechnol Bioeng 114:1438–1447CrossRefGoogle Scholar
  37. 37.
    Teng X, Yi X, Sun X, Zhang Y (2011) Modeling and application of controlled-fed perfusion culture of cho cells in a bioreactor. Chem Biochem Eng Q 25:385–394Google Scholar
  38. 38.
    Gu MB, Todd P, Kompala DS (1995) Metabolic burden in recombinant CHO cells: effect ofdhfr gene amplification andlacZ expression. Cytotechnology 18:159–166CrossRefGoogle Scholar
  39. 39.
    Jiang Z, Huang Y, Sharfstein ST (2006) Regulation of recombinant monoclonal antibody production in chinese hamster ovary cells: a comparative study of gene copy number, mRNA level, and protein expression. Biotechnol Prog 22:313–318CrossRefGoogle Scholar
  40. 40.
    Zou W, Edros R, Al-Rubeai M (2018) The relationship of metabolic burden to productivity levels in CHO cell lines. Biotechnol Appl Biochem 65:173–180CrossRefGoogle Scholar
  41. 41.
    Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196CrossRefGoogle Scholar
  42. 42.
    Mellahi K, Cambay F, Brochu D, Gilbert M, Perrier M, Ansorge S, Durocher Y, Henry O (2018) Process development for an inducible rituximab-expressing Chinese hamster ovary cell line. Biotechnol Prog. Google Scholar
  43. 43.
    Ozturk SS (1996) Engineering challenges in high density cell culture systems. Cytotechnology 22:3–16CrossRefGoogle Scholar
  44. 44.
    Yang M, Butler M (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol Bioeng 68:370–380CrossRefGoogle Scholar
  45. 45.
    Pereira S, Kildegaard HF, Andersen MR (2018) Impact of CHO metabolism on cell growth and protein production: an overview of toxic and inhibiting metabolites and nutrients. Biotechnol J 13:1700499CrossRefGoogle Scholar
  46. 46.
    Noh SM, Shin S, Lee GM (2018) Comprehensive characterization of glutamine synthetase-mediated selection for the establishment of recombinant CHO cells producing monoclonal antibodies. Sci Rep 8:5361CrossRefGoogle Scholar
  47. 47.
    Yoon SK, Kim SH, Lee GM (2003) Effect of low culture temperature on foreign protein production in recombinant CHO Cells. In: Yagasaki K, Miura Y, Hatori M, Nomura Y (eds) Animal cell technology: Basic & applied aspects. Springer, Dordrecht, pp 163–167CrossRefGoogle Scholar
  48. 48.
    Kou T-C, Fan L, Zhou Y, Ye Z-Y, Zhao L, Tan W-S (2011) Increasing the productivity of TNFR-Fc in GS-CHO cells at reduced culture temperatures. Biotechnol Bioprocess Eng 16:136–143CrossRefGoogle Scholar
  49. 49.
    Bollati-Fogolín M, Forno G, Nimtz M, Conradt HS, Etcheverrigaray M, Kratje R (2005) Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality. Biotechnol Prog 21:17–21CrossRefGoogle Scholar
  50. 50.
    Kumar N, Gammell P, Clynes M (2007) Proliferation control strategies to improve productivity and survival during CHO based production culture. Cytotechnology 53:33–46CrossRefGoogle Scholar
  51. 51.
    Hossler P, Khattak SF, Li ZJ (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19:936–949CrossRefGoogle Scholar
  52. 52.
    Karengera E, Robotham A, Kelly J, Durocher Y, De Crescenzo G, Henry O (2017) Altering the central carbon metabolism of HEK293 cells: impact on recombinant glycoprotein quality. J Biotechnol 242:73–82CrossRefGoogle Scholar
  53. 53.
    Butler M (2006) Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems. Cytotechnology 50:57CrossRefGoogle Scholar
  54. 54.
    Ha TK, Lee GM (2014) Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture. J Biotechnol 180:23–29CrossRefGoogle Scholar
  55. 55.
    Chen P, Harcum SW (2006) Effects of elevated ammonium on glycosylation gene expression in CHO cells. Metab Eng 8:123–132CrossRefGoogle Scholar
  56. 56.
    Costa AR, Rodrigues ME, Henriques M, Oliveira R, Azeredo J (2014) Glycosylation: impact, control and improvement during therapeutic protein production. Crit Rev Biotechnol 34:281–299CrossRefGoogle Scholar
  57. 57.
    Lipscomb ML, Mowry MC, Kompala DS (2004) Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor. Biotechnology Prog 20:1402–1407CrossRefGoogle Scholar
  58. 58.
    Jefferis R (2005) Glycosylation of recombinant antibody therapeutics. Biotechnol Prog 21:11–16CrossRefGoogle Scholar
  59. 59.
    Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20:471–478CrossRefGoogle Scholar
  60. 60.
    Walther J, Lu J, Hollenbach M, Yu M, Hwang C, McLarty J, Brower K (2018) Perfusion cell culture decreases process and product heterogeneity in a head-to-head comparison with fed-batch. Biotechnol J 2018 e1700733Google Scholar
  61. 61.
    Park JH, Jin JH, Lim MS, An HJ, Kim JW, Lee GM (2017) Proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing CHO cells. Sci Rep UK 7:44264CrossRefGoogle Scholar
  62. 62.
    Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P, van den Bremer ETJ, Gerritsen J, van Berkel PHC (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108:1591–1602CrossRefGoogle Scholar
  63. 63.
    Fan Y, Jimenez Del Val I, Muller C, Lund AM, Sen JW, Rasmussen SK, Kontoravdi C, Baycin-Hizal D, Betenbaugh MJ, Weilguny D, Andersen MR (2015) A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture. Biotechnol Bioeng 112:2172–2184CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Kahina Mellahi
    • 1
  • Denis Brochu
    • 2
  • Michel Gilbert
    • 2
  • Michel Perrier
    • 1
  • Sven Ansorge
    • 3
  • Yves Durocher
    • 3
  • Olivier Henry
    • 1
    Email author
  1. 1.Department of Chemical EngineeringÉcole Polytechnique de MontréalMontrealCanada
  2. 2.Human Health Therapeutics Research CentreNational Research Council CanadaOttawaCanada
  3. 3.Human Health Therapeutics Research CentreNational Research Council CanadaMontrealCanada

Personalised recommendations