Controlled release of the histone deacetylase inhibitor sodium butyrate for increasing recombinant protein production /

Abstract

Improving the yields of biopharmaceutical products from mammalian cell culture is of paramount importance to manufacturing companies; increasing product yield includes enhancing recombinant gene expression in mammalian cells. One avenue for investigation is the use of histone deacetylase inhibitors, such as sodium butyrate, which function by opening up genomic DNA for transcription. However, a limitation to the use of sodium butyrate is its toxicity. This research aimed to examine the controlled release of sodium butyrate onto CHO-K1 cells, to increase recombinant protein production, while minimizing loss of viability. Reused lobster exoskeleton was demineralised and deproteinated and was used as a release vehicle due to its nanoporous biocompatible structure. Biocompatibility of the shell was confirmed by in vitro toxicity assays. Adsorption and release of butyrate was measured by GC-MS and the effect of sodium butyrate on recombinant protein (human insulin-GFP) production in CHO K1-INS/GFP clonal cells was quantified using fluorescent microscopy. Cell viability was quantified using the dyes neutral red, propidium iodide and Hoechst staining. When compared on a per mM basis, controlled release of 100mM sodium butyrate via the lobster shell (7.69 mM actual release) at 24 hours (p=0.0218) and 48 hours (7.47 mM actual release) (p=.0454) resulted in statistically significant increases in protein production compared to 25mM bolus exposure. 250mM controlled release (21.86 actual release) was higher at 48 hours compared to 25mM bolus exposure (p=0.0221). Concurrently, cell viability for controlled release at 48hrs was 99.75% for 100mM and 99.46% for 250mM. For the bolus dose data, the viability at 48hrs was 99.70% for 5mM, 98.36% for 10mM and 93.20% for 25mM, indicating a moderating effect of controlled release on butyrate associated toxicity.

xi

A burst release mechanism for the release of sodium butyrate from the shell is suggested based on release data. Meanwhile, the activation of autophagic prosurvival and anti-apoptotic pathways; which are likely to be active under the conditions of increased protein production by controlled butyrate exposure in comparison to bolus dosing, are proposed as mechanisms for improved cell survival. The study concludes that controlled release of sodium butyrate could be used to significantly improve protein yields in biologics manufacturing, and the use of reused lobster exoskeleton presents a viable vehicle for release.