Abstract
Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28–34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.
Highlights
Recombinant proteins that require posttranslational modifications, similar to those found in humans, are preferentially produced in mammalian cells [1]
temperature downshifts (TDS) after 48 h of culture affected the growth of Chinese hamster ovary (CHO) TF70R cells, but extended their cultivation time and maintained higher cell viability for up to 7 days longer than those cultures that were maintained at 37°C
This resulted in a 2.3-fold and 1.6-fold increase in recombinant human tissue plasminogen activator (rh-tPA) volumetric and specific productivity, respectively, with a concomitant decrease in glucose consumption and lactate production in TDS cultures as compared to cultures maintained at 37°C
Summary
Recombinant proteins that require posttranslational modifications, similar to those found in humans, are preferentially produced in mammalian cells [1]. There exists a critical need to improve these bioprocesses in order to increase recombinant protein yields while maintaining product stability, activity, and biosafety [1,3,4]. Effects of temperature downshifts (TDS) from 37°C to temperatures ranging from 28°C to 34°C (a procedure known as “biphasic cultures under moderate hypothermia”) have been reported, with the optimal conditions dependent on the product and cell line under investigation [9,10]. TDS usually reduces cell growth but increases cell viability, slows cell metabolism, delays cell death, and prolongs RNA half-life. Such effects have been related to increases in recombinant protein production [2,3,10,11,12,13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
