Controlled release of nutrients to mammalian cells cultured in shake flasks

Abstract

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.

6.2 High Cell Density Cultivation Process

“…However, it is of major importance to be aware that supplementation for the prevention of nutrient depletion needs to be performed in a way that minimizes the accumulation of the main toxic waste products, lactate and ammonia, which are not only the principal cause of culture death but also affect product quality and quantity [32,33]. Careful optimization of glucose/glutamine ratio, application of an optimized regimen to maintain low glucose/glutamine levels as well as their total or partial substitution with slowly metabolized substrates are strategies that have been reported to successfully decrease the level of lactate and ammonia in cell cultures [25,34]. Metabolic flux analysis has been also employed to identify key enzymes in the glucose/glutamine utilization pathway in order to design genetic engineering approaches to produce lower amounts of toxic metabolites [35].…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…Unlike microbial cultures which run for short durations of 24-48 h, animal cells in fed batch are cultured for 7-15 days. Our group is exploring the use of in situ delivery of base and nutrients through hydrogels to enable fed batch culture of animal cells in shake flasks to increase the quality of data from shake flask screening 67,68 . We mainly show that the release rate of base through these hydrogels increases with decreasing pH providing a rudimentary feedback for pH management 68,69 .…”

Effects of glutamine and asparagine on recombinant antibody production using CHO‐GS cell lines

“…Although pH can be maintained within a narrow range using this approach, addition of alkali increases osmolality which can be harmful to cell growth and protein production . As lactate accumulation leads to decrease of pH and toxicity to the cells, much effort has been made to reduce lactate accumulation, such as optimizing feed components and feeding strategies to control glucose at low level, and using genetic engineering techniques to mitigate the conversion of glucose to lactate . In recent years, progress has been reported in medium development to prevent lactate generation and accumulation, such as by replacing primary carbon source with galactose or pyruvate, and by tuning copper concentration in the media .…”