Nutrient Supplementation Strategies for Biopharmaceutical Production, Part 2 Feeding for Optimal Recombinant Protein Production

level: interMediAte Some of the numerous feeding strategies are more appropriate than others for certain types of cell culture production systems. Once a nutrient supplement has been identified as described in Part 1 of this three-part review (1), a supplementation strategy must be chosen. Supplementing at too great a rate may expose log-phase cells to stresses such as increased osmolality and lactate levels that would inhibit biomass expansion. But inadequate supplementation can lead to early apoptosis through rapid depletion of selected important components. For commercially available supplements, guidelines often suggest specific protocols that will usually yield good results and can be further optimized by subsequent experimentation. Additional culture-specific methods are available to laboratories with access to monitoring equipment. As mentioned in Part 1, monitoring one component is an excellent way of approaching relative nutrient homeostasis in the bioreactor, especially with stoichiometrically balanced nutrient supplements (1). They may be added semicontinuously (e.g., daily or twice daily), with each addition providing enough supplement to cover its expected consumption until the next addition. Supplements also can be added continually to match predicted consumption, but this approach is more generally applicable at the research scale than in commercial production. Supplementation of cell cultures generally refers to adding nutrients that replace those consumed, but another type of supplement can be considered. Cell metabolic pathways are important to optimizing protein expression because of the dichotomous pathway of biomass expansion and product synthesis. Cells that are expanding rapidly may not have the highest specific productivity (output per cell), but once maximum cell density is reached, specific productivity can peak and be held at high levels for extended periods of time. Nutrient supplements can be designed to promote both phases. So nutritional supplements (those containing major nutrients such as glucose and specific amino acids that are consumed during the expansion phase) represent one type of supplement. And metabolic supplements (those containing molecules to guide cells toward maximum productivity) represent the other type. Timing their addition may promote superior productivity. A number of molecules have been identified as helpful in the production phase of cell culture. When glucose is replaced by a more slowly metabolized hexose such as galactose or mannose, a slower metabolism results with correspondingly lowered lactic acid production. At the same time, inducers of protein synthesis such as butyrate and pentanoic acid can be used for switching cell machinery from further replication at the plateau phase into protein expression. Numerous small molecules are being researched as protein synthesis stimulators in high-throughput systems.