Abstract
Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.
Highlights
Over the years, the demand for recombinant proteins as biopharmaceuticals has increased dramatically, attaching a special relevance to monoclonal antibody production [1]
We aimed to evaluate the effect of mild hypothermia on the culture performance of two highly productive Chinese hamster ovary (CHO) cell lines producing anti-TNFα, the impact of culture temperature on myc and xbp1s expression, and their relationship with cell metabolism and anti-TNFα productivity
The impact of mild hypothermia on cell growth, metabolism, recombinant protein production and expression of myc and xbp1s were successfully assessed in both anti-TNFα producing CHO cell lines
Summary
The demand for recombinant proteins as biopharmaceuticals has increased dramatically, attaching a special relevance to monoclonal antibody production [1]. Proof of that are their positive clinical results and increased approval of therapeutic antibody drugs for clinical uses by international organisations in the United States and Europe [1] Such scenario of increased demand for these therapeutic agents places considerable pressure on the development of highly efficient production processes to develop less expensive drugs [6,7]. To this date, Chinese hamster ovary (CHO) cells are the main platform for the production of a great number of recombinant therapeutic antibodies [8] due to their easy gene manipulation, adaptation to suspension cultures and capacity to properly perform post-translational modification, glycosylations [9,10]. Since production of a recombinant protein is directly related to specific productivity and the integral of viable cell (IVC), efforts to maximize production are directed towards a synergistic combination of both approaches selecting highly productive cell lines and optimizing environmental culture condition
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