Abstract
In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28–34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.
Highlights
In vitro animal cell cultivation is currently the fastest growing area of the global pharmaceutical sector, in the market of recombinant therapeutic proteins (r-proteins), with sales of more than $100 billion in 2013 [1]
We investigated the differential effects of dilution rate, culture temperature, and medium glucose concentration on culture performance and cell metabolism of the rh-tPAproducing Chinese hamster ovary (CHO) cell line
Considering all evaluated conditions, our findings suggest that a reduced dilution rate coupled with a high glucose concentration in medium significantly improves r-protein productivity in CHO cells
Summary
In vitro animal cell cultivation is currently the fastest growing area of the global pharmaceutical sector, in the market of recombinant therapeutic proteins (r-proteins), with sales of more than $100 billion in 2013 [1]. Lower production yields, when compared to other expression systems (e.g., bacteria), are one of the industry’s main challenges in coping with increasing biopharmaceutical demand This is why most efforts nowadays are focused on understanding the mechanisms involved in protein synthesis and the development of optimized processes to enhance productivity. Many strategies aiming to enhance recombinant protein production focus on maximizing specific protein productivity while maintaining high viable cell density in culture for long periods. The operational conditions (e.g., temperature or medium composition) play a significant role in culture performance and proper handling of the cultures may enable considerable increases in r-protein production [5,6,7,8,9,10,11,12]
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