Engineering improved apoptosis inhibition in mammalian cell culture using modified proteins and multiple inhibitors

The utility of mammalian cell culture for the production of vaccines, monoclonal antibodies, and other biopharmaceuticals is growing rapidly. Unfortunately, to the detriment of biochemical manufacturers, productivity of these biopharmaceuticals is suboptimal due to programmed cell death, commonly referred to as apoptosis. Apoptosis results in an undesirable decrease in the viable cell ‘factories’ that are used in the production of bioproducts and this may in turn lower bioreactor efficiency. Additionally, cells that have died in culture will lyse spilling their cytosolic contents into the culture medium. Released enzymes may alter or degrade the bioproduct and the cellular debris can be problematic for downstream purification processes. Consequently, this work has focused on genetic methods to inhibit apoptosis. In order to maintain the delicate balance between life and death, cells have evolved a complex apoptotic cascade to signal, prevent, or slow the progression of apoptosis. Utilizing the cells' own anti-apoptotic mechanisms, two commercially-relevant mammalian cell lines, Chinese hamster ovary (CHO) and 293 human embryonic kidney, have been genetically engineered to overexpress single and combinations of proteins involved in the inhibition of apoptosis. In addition, with recent advances in biology, a greater understanding of the role each of these proteins exhibits in the prevention of apoptosis is being elucidated.{09}Using this knowledge, mutations of the anti-apoptosis proteins, XIAP and CrmA, increased their efficacy for stalling the progression of apoptosis in culture. In particular, XIAP, a potent inhibitor of the family of proteases responsible for cellular destruction, has been altered through site-directed mutagenesis and deletions of various homology domains. One mutant, in which the C-terminal region has been removed, has shown great promise in extending lifetimes of cultures. Through expression of this protein individually and in combination with two other proteins, Bcl-X _L and Aven, which are responsible for inhibiting apoptosis at points upstream of XIAP, viabilities were extended under various industry-applicable apoptotic insults including Sindbis virus infection and spent medium exposure. In particular, CHO cell lines expressing combinations of the XIAP mutant with Bcl-X_L and the XIAP mutant with Bcl-X_L and Aven were notably effective at extending viabilities in mammalian cell culture.