Cellular engineering strategies to improve biotherapeutic protein production in mammalian cell culture

The global market for biologics was estimated at ;Apoptosis is the leading form of cell death in bioreactors, additionally; chemicals used to enhance specific productivity such as sodium butyrate (NaBu) are also known to cause apoptosis. As a means to combat the onset of apoptosis, the effect of the overexpression of anti-apoptotic proteins, Bcl-x L, Bcl-xL delta and Bcl-2 delta, is described in batch, fed-batch and perfusion cultures with the addition of a sodium butyrate. While no enhanced benefit was seen with the mutant forms, a three-fold increase in final product yield was achieved using anti-apoptosis engineering in both fed-batch and perfusion cultures.;The use of cell cycle arrest engineering is explored as a means to disconnect cellular growth from division through the inducible expression of p21. To further reduce the metabolic burden, more stable mutant forms of p21 requiring a lower expression level while maintaining similar growth arrest capabilities were compared. Lower effective expression levels were achieved for the mutant p21 proteins but this resulted in only moderate increases in productivity.;Finally these genetic enhancement tools were used in transient gene expression systems to enhance rapid production of a biotherapeutic protein. Transiently expressing Bcl-xL gave added protection from apoptosis during the harsh conditions of transfection, while cell growth arrest alone did not achieve significant gains in productivity. However, when cell cycle arrest was used in combination with Bcl-xL the highest level of enhanced production was found.