Applications of anti-apoptosis genes to extend cell culture bioprocessing.

摘要

Apoptosis or programmed cell death is a regulated physiological process in which cells respond to particular external and internal stimuli by activating a cascade of caspases. Mammalian cell culture has been the system of choice for therapeutic recombinant protein production due to proper protein folding, assembly, and posttranslational modification. The Sindbis virus expression system produces high levels of recombinant proteins in mammalian cells; however, the expression is limited to transient level due to apoptosis. Modifications in both the virus and the host cells investigated were (1) a single point mutation P726S in the nsP2 domain was employed to generate non-cytopathic Sindbis virus, and (2) the host cells were engineered to express the antiapoptosis Bcl-2 and Bcl-2Delta proteins. The combined effect of the P726S mutation and the Bcl-2Delta gene expression showed persistent infection in CHO cells that produced 5-fold higher of GFP than the control. ER stress induced apoptosis was activated in Sindbis virus infected cells. In the mammalian cell bioreactors, the host cells undergo apoptosis due to nutrient and growth factor limitations, oxygen depletion, toxic byproducts, and shear stress, leading to reduction in viable cells and decrease in protein production. BHK cells secreting recombinant Factor VIII were engineered to express anti-apoptosis genes Aven and E1B-19K. The Aven-E1B19K cell line was examined in the perfusion bioreactor operating at decreasing cell specific perfusion rate (CSPR), decreasing the amount of media available per cell per day. The Aven-E1B19K cell line maintained high viability in the low perfusion rate in which the parental showed a viability decrease. Different metabolism was observed between the Aven-E1B19K and the parental cell line. CHO cells secreting Arylsulfatase B were modified to express Aven and EIB-19K. 3 Aven-E1B19K clones were analyzed in two scale-down perfusion models---pseudoperfusion and push culture. The Aven-EIB19K clones were resistant to apoptosis in both scale-down models, as evidence by higher mitochondria membrane potential, fewer early apoptotic (Annexin-V+, 7-AAD-) cells, and lower caspase-3 activation in the Aven E1B19K clones compared to the parental.