Controlling apoptosis to optimize yields of proteins from mammalian cells.

Apoptosis is the foremost method of cell death in bioreactors and can be caused by nutrient limitation, toxin accumulation, and growth factor withdrawal. By delaying the onset of this form of programmed cell death, one can achieve longer sustained viabilities in culture, thereby increasing product yield. Described here is a genetic-based, step-by-step method to generate an apoptosis-resistant cell line. This cell line, then, can be used as a platform for biotherapeutic protein production. The key steps include antiapoptotic transgene selection and transfection followed by clonal isolation and screening. With the proper screening methods, one can obtain a robust cell line that resists the harsh conditions of late-stage and/or high-density culture.

Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures.

Apoptosis is now recognized as a significant problem in mammalian cell culture. Therefore, in this study, a single gene and multigene approach to inhibit apoptosis has been examined. Stable Chinese hamster ovary (CHO) cell lines were generated to overexpress different single, dual, and triple combinations of three apoptosis inhibitor genes. Two upstream inhibitors involved in the mitochondrial pathway, Bcl-X(L) and Aven, were expressed in addition to a downstream inhibitor of caspases. The caspase inhibitor, a variant of XIAP containing only the caspase inhibitory BIR domains (XIAP-BIRs), has been shown previously to enhance viabilities in mammalian cultures. Stable clonal cell lines were generated and tested for three apoptotic insults: Sindbis virus infection, the chemical reagent etoposide, and spent medium. For all single gene experiments, the Bcl-X(L)-containing cell lines provided superior protection to either the Aven- or XIAP-BIRs-containing cell lines following initial exposure to the insults. However, the cell lines expressing two or more anti-apoptosis proteins were more effective at inhibiting cell death than those expressing just one anti-apoptosis gene. The cell lines overexpressing Bcl-X(L) in combination with XIAP-BIRs were especially effective in delaying cell death for all three apoptotic insults. Expression of all three anti-apoptosis genes in concert was only slightly more effective than using Bcl-X(L) and XIAP-BIRs for some insults. During exposure to spent medium, CHO-BIRS + Aven + BclX(L) was the best inhibitor of apoptosis (IAP) initially, whereas CHO-BIRs + BclX(L) was particularly effective at later times of the experiment. In conclusion, the utilization of a mitochondrial dysfunction inhibitor used in combination with a caspase inhibitor was more effective in thwarting the progression of apoptosis than either inhibitor expressed individually. Thus, the concurrent expression of multiple apoptosis inhibitors may be the most effective strategy to increase survival of mammalian cells in culture.

Study of caspase inhibitors for limiting death in mammalian cell culture.

Apoptosis in mammalian cell culture is associated with decreased bioproduct yields and can be inhibited through altering the intracellular signaling pathways mediating programmed cell death. In this study, we evaluated the capacity to inhibit caspases to maintain high viable cell numbers in CHO and 293 cultures. Two genetic caspase inhibitors, XIAP and CrmA, were examined along with a mutant of each, XIAP-BIR123NC, which contains three BIR domains but lacks the RING finger, and CrmA-DQMD, which has CrmA's pseudosubstrate site replaced with that of another caspase inhibitor, p35. Stable CHO pooled and 293 clonal cell lines expressing each protein were exposed to apoptotic insults, including spent medium, Sindbis virus, and etoposide. For each insult the mutated protein resulted in higher viabilities than its wild-type counterpart. However, the mutants provided different levels of protection, depending on the insult considered. CrmA-DQMD was the preferred inhibitor for spent medium-induced apoptosis, whereas XIAP-BIR123NC conferred better protection for etoposide-induced death. Addition of Z-VAD.fmk to the genetically engineered cells enhanced viabilities in the presence of spent medium or etoposide; however, the largest increases in viability were experienced by the control cells, indicating an overlap in caspase inhibition between the genetic and chemical inhibitors. Finally, parental 293 cells were treated with caspase-8 and -9 inhibitors, Z-IETD.fmk and Z-LEHD.fmk, in concert with spent medium or etoposide exposure. Spent medium-induced death was delayed more readily with the caspase-8 inhibitors, CrmA-DQMD and Z-IETD.fmk, and etoposide-induced death was stalled more so with XIAP-BIR123NC and Z-LEHD.fmk. These results suggest that the apoptosis pathways induced and the level of protection afforded by a particular caspase inhibitor may vary with the insult considered.

Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.