Process intensification for the production of rituximab by an inducible CHO cell line
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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.
KeywordsCHO 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.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
- 5.Wlaschin KF, Hu W-S (2006) Fedbatch culture and dynamic nutrient feeding. Cell Culture Engineering SpringerGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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