Alteration of Hybridoma Viability and Antibody Secretion in Transfectomas with Inducible Overexpression of Protein Disulfide Isomerase

Abstract

Monoclonal antibody (mAb)-secreting transfectomas with dexamethasone inducible expression of the mammalian endoplasmic reticulum foldase and chaperone protein disulfide isomerase (PDI, ERp59) were generated from the murine 9.2.27 hybridoma in order to obtain in vivo evidence of whether alteration of the level of PDI, believed to be involved in immunoglobulin (Ig) assembly, results in alteration of mAb secretion kinetics. Using an RNase refolding assay, the specific activity of endogenous PDI in the 9.2.27 hybridoma was found to be constant during batch growth. An expression vector for glucocorticoid-inducible overexpression of PDI, pMMTVPDI, was constructed from pMAMneo using a rat PDI cDNA. Cell lysates of stable transfectomas contained 2-4-fold higher levels of PDI mRNA and increased levels of PDI protein, detected by immunoblotting, following induction with 0.1 microM dexamethasone. Monoclonal antibody secretion kinetics were evaluated in 12.5 mL shake flasks, a 100 mL spinner, and a 1 L aerated batch reactor. A transfectoma was found with altered mAb secretion kinetics during cell growth following dexamethasone induction of PDI overexpression. Specific mAb secretion rate was not significantly increased following dexamethasone induction; however, hybridoma viability was sustained longer during the stationary phase of cell growth and hence total antibody yield was increased in comparison to the parent 9.2.27 hybridoma.

In vitro and in vivo RNA interference mediated suppression of Tn‐caspase‐1 for improved recombinant protein production in High FiveTMcell culture with the baculovirus expression vector system

“…Traditionally, this has been accomplished either through the overexpression of specific targets directly involved in the pathway (Chen et al, 2001;Irani et al, 1999) or alteration of the overall protein synthesis machinery, such as the expression of chaperone proteins (Borth et al, 2005;Davis et al, 2000;Higgins et al, 2003;Kitchin and Flickinger, 1995;Yokoyama et al, 2000) or transcriptional factors (Tigges and Fussenegger, 2006) involved in the secretory pathway. However, as illustrated in Figure 1A, a similar strategy can be implemented using RNA interference (RNAi) (Hebert et al, 2008).…”

Effects of overexpression of X‐box binding protein 1 on recombinant protein production in Chinese hamster ovary and NS0 myeloma cells

“…While such a strategy increases heterologous protein production in yeast (Shusta et al, 1998;Smith et al, 2004) and insect cells (Ailor and Betenbaugh, 1998;Hsu and Betenbaugh, 1997), it has not consistently achieved the same effects in mammalian cells (Borth et al, 2005;Davis et al, 2000;Kitchin and Flickinger, 1995). Surprisingly, the reduction of the level of endogenous BiP, rather than its overexpression, was found to improve secretion of tissue plasminogen activator in CHO cells (Dorner et al, 1988).…”

Engineering of chaperone systems and of the unfolded protein response

“…Secretion of some heterologous proteins was increased (Kato et al 2005;Lambert and Merten 1997), but secretion of an equal number of heterologous proteins was not affected (Kitchin and Flickinger 1995;Mohan et al 2007). PDI may enhance secretion via increased rates of disulfide bond formation and isomerization, or because of its chaperone function.…”