Maximizing productivity of CHO cell‐based fed‐batch culture using chemically defined media conditions and typical manufacturing equipment

A highly productive chemically defined fed‐batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell‐based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc‐fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010

[1]  J E Bailey,et al.  Influence of low temperature on productivity, proteome and protein phosphorylation of CHO cells. , 1999, Biotechnology and bioengineering.

[2]  M. Al‐Rubeai,et al.  Apoptosis in cell culture. , 1998, Current opinion in biotechnology.

[3]  A. Nienow.  Reactor Engineering in Large Scale Animal Cell Culture , 2006, Cytotechnology.

[4]  D. Kompala,et al.  Optimization of High Cell Density Perfusion Bioreactors , 2005 .

[5]  Sigma S Mostafa,et al.  Strategies for Improved dCO2 Removal in Large‐Scale Fed‐Batch Cultures , 2003, Biotechnology progress.

[6]  H. Katinger,et al.  Therapeutic potential of neutralizing antibodies in the treatment of HIV-1 infection. , 2003, The Journal of antimicrobial chemotherapy.

[7]  D K Robinson,et al.  Industrial choices for protein production by large-scale cell culture. , 2001, Current opinion in biotechnology.

[8]  P. Hudson,et al.  Recombinant antibodies for cancer diagnosis and therapy , 2003, Expert opinion on biological therapy.

[9]  F. Asselbergs,et al.  Amplification and expression of recombinant genes in serum‐independent Chinese hamster ovary cells , 1995, FEBS letters.

[10]  J. Gray,et al.  Effects of methotrexate on transfected DNA stability in mammalian cells , 1990, Molecular and cellular biology.

[11]  M. Butler,et al.  Effect of Ammonia on the Glycosylation of Human Recombinant Erythropoietin in Culture , 2000, Biotechnology progress.

[12]  D. I. Wang,et al.  Stoichiometric analysis of animal cell growth and its application in medium design , 1994, Biotechnology and bioengineering.

[13]  S. Rosenberg,et al.  Culture of human tumor infiltrating lymphocytes in hollow fiber bioreactors. , 1990, Journal of immunological methods.

[14]  R. Mutharasan,et al.  Cell cycle‐ and growth phase‐dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures , 1990, Biotechnology and bioengineering.

[15]  U von Stockar,et al.  Measurement of volumetric (OUR) and determination of specific (qO2) oxygen uptake rates in animal cell cultures. , 1998, Journal of biotechnology.

[16]  M B Sliwkowski,et al.  Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. , 2000, Biotechnology and bioengineering.

[17]  B. Buckland,et al.  Fed-batch culture of recombinant NS0 myeloma cells with high monoclonal antibody production. , 1997, Biotechnology and bioengineering.

[18]  G. Belfort,et al.  Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors–separators , 1986, Biotechnology and bioengineering.

[19]  Thomas Ryll,et al.  Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues , 2004, Cytotechnology.

[20]  J. Piret,et al.  Dependence on glucose limitation of the pCO2 influences on CHO cell growth, metabolism and IgG production , 2007, Biotechnology and bioengineering.

[21]  J L Cleland,et al.  The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. , 1993, Critical reviews in therapeutic drug carrier systems.

[22]  P. Salmon,et al.  A novel function for selenium in biological system: Selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production , 2006, Biotechnology and bioengineering.

[23]  James M Piret,et al.  Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures. , 2002, Biotechnology and bioengineering.

[24]  F Meuwly,et al.  Conversion of a CHO cell culture process from perfusion to fed-batch technology without altering product quality. , 2006, Journal of biotechnology.

[25]  Suzanne S Farid,et al.  Process economics of industrial monoclonal antibody manufacture. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[26]  S. Gupta,et al.  Effects of Elevated pCO2 and Osmolality on Growth of CHO Cells and Production of Antibody‐Fusion Protein B1: A Case Study , 2008, Biotechnology progress.

[27]  F. Melchers,et al.  Complete replacement of serum by albumin, transferrin, and soybean lipid in cultures of lipopolysaccharide-reactive B lymphocytes , 1978, The Journal of experimental medicine.

[28]  John Thrift,et al.  The "push-to-low" approach for optimization of high-density perfusion cultures of animal cells. , 2006, Advances in biochemical engineering/biotechnology.

[29]  J. Piret,et al.  Decreased pCO2 accumulation by eliminating bicarbonate addition to high cell‐density cultures , 2007, Biotechnology and bioengineering.

[30]  J. Lehmann,et al.  Bulk culture of animal cells for antibody production: A comparison of reactors , 1990 .

[31]  Oxygen limitation is a pitfall during screening for industrial strains , 2006, Applied Microbiology and Biotechnology.

[32]  M. Dalakas,et al.  B cells as therapeutic targets in autoimmune neurological disorders , 2008, Nature Clinical Practice Neurology.

[33]  T Etcheverry,et al.  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. , 2000, Biotechnology and bioengineering.

[34]  M. Betenbaugh,et al.  Anti‐apoptotic genes Aven and E1B‐19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture , 2007, Biotechnology and bioengineering.

[35]  T. Ryll,et al.  A Comparison of Different Methods To Determine the End of Exponential Growth in CHO Cell Cultures for Optimization of Scale‐Up , 2000, Biotechnology progress.

[36]  Alan J Dickson,et al.  Stability of protein production from recombinant mammalian cells , 2003, Biotechnology and bioengineering.

[37]  S. Chamow,et al.  Therapeutic antibody expression technology. , 2001, Current opinion in biotechnology.

[38]  T. Ryll,et al.  Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells. , 1999, Glycobiology.

[39]  J. Chalmers,et al.  Fabrication and use of a transient contractional flow device to quantify the sensitivity of mammalian and insect cells to hydrodynamic forces , 2002, Biotechnology and bioengineering.

[40]  Andrew J Racher,et al.  Antibody production. , 2006, Advanced drug delivery reviews.

[41]  N. Robinson,et al.  Deamidation of human proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  H. Prentice,et al.  Improving Performance of Mammalian Cells in Fed‐Batch Processes through “Bioreactor Evolution” , 2007, Biotechnology progress.

[43]  G. Chiang,et al.  Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells. , 2005, Biotechnology and bioengineering.

[44]  M C Manning,et al.  Controlling deamidation rates in a model peptide: effects of temperature, peptide concentration, and additives. , 2001, Journal of pharmaceutical sciences.

[45]  Raymond Davis,et al.  Identification of novel small molecule enhancers of protein production by cultured mammalian cells , 2008, Biotechnology and bioengineering.

[46]  G. Chiang,et al.  Bcl-xL mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells , 2005 .

[47]  F. Wurm.  Production of recombinant protein therapeutics in cultivated mammalian cells , 2004, Nature Biotechnology.

[48]  J. Goor.  Improvement of Industrial Cell Culture Processes by Caspase-9 Dominant Negative and Other Apoptotic Inhibitors , 2004 .

[49]  Zizhuo Xing,et al.  Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors , 2009, Biotechnology and bioengineering.

[50]  S S Ozturk,et al.  Effects of cell density and temperature on oxygen consumption rate for different mammalian cell lines. , 1999, Biotechnology and bioengineering.

[51]  M. Al‐Rubeai,et al.  Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture , 2004, Cytotechnology.

[52]  Weiwei Hu,et al.  An investigation of small‐molecule surfactants to potentially replace pluronic F‐68 for reducing bubble‐associated cell damage , 2008, Biotechnology and bioengineering.

[53]  T. Ryll,et al.  Biochemistry of growth inhibition by ammonium ions in mammalian cells , 1994, Biotechnology and bioengineering.