Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Abstract

Small molecule additives to cell culture media (e.g., sodium butyrate) that are capable of enhancing the expression of recombinant proteins have significant utility in the production and manufacture of therapeutic polypeptides. To identify novel small molecule enhancers (SMEs) of recombinant protein expression in Chinese Hamster Ovary (CHO) cells, we screened two separate small molecule libraries for compounds capable of enhancing the expression of either a fluorescent reporter protein or a monoclonal antibody. Several compounds that increased recombinant protein expression were identified, and these compounds fell into three broad classes: (1) aromatic carboxylic acids, (2) hydroxamic acids, and (3) acetamides. We examined the impact of SME addition to CHO cell cultures expressing different classes of recombinant proteins including monoclonal antibodies (MAbs). For CHO cell pools or clones grown in production shake-flasks or bioreactors, recombinant protein titers up to 60% higher than control cultures were observed. Analysis of mRNA levels suggest that transcriptional activation plays a role in the expression enhancement seen for some SMEs, but other mechanisms may be involved for at least one compound. Finally, we tested many of the identified SMEs for their ability to increase MAb production by a hybridoma cell line. Hexanohydroxamic acid increased shake-flask MAb production by 40% relative to a control. Taken together, these data demonstrate the potential utility of the compounds in the production of therapeutically relevant proteins from diverse cell-based production systems.

Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells

“…Histone deacetylase inhibitors (iHDACs) such as VPA, trichostatin A, and sodium butyrate, when applied to mammalian cells in vivo or in vitro, can prevent the transcriptional silencing of genes. Several iHDACs have been shown to enhance recombinant protein yields in transiently and stably transfected mammalian cells (Allen et al, 2008;Backliwal et al, 2008b;Choi et al, 2005;Davie, 2003;Gorman et al, 1983). Although it has been reported that transiently transfected plasmid DNA becomes complexed with histones that undergo post-translational modifications (Riu et al, 2007), it is not clear if the iHDACs mentioned above enhance TGE yields directly through effects on the histones associated with plasmid DNA or indirectly through effects on cellular gene expression.…”

Understanding the intracellular effects of yeast extract on the enhancement of Fc-fusion protein production in Chinese hamster ovary cell culture

“…Nevertheless, it is still challenging to find out the main mechanisms of enhanced gene transcription due to the complexity of both YE components and transcription process itself. Previous studies showed that the addition of small molecules such as hydroxamic acids (Allen et al 2008;Jiang and Sharfstein 2008) and alkanoic acids (Liu et al 2001;Yang et al 2014) could enhance the transcription of heterologous gene by preventing the deacetylation of specific histone lysine residues. These findings encouraged us to speculate that some organic acids in YE should have the similar function.…”

Enhanced production of recombinant proteins by a small molecule protein synthesis enhancer in combination with an antioxidant in recombinant Chinese hamster ovary cells

“…A wide variety of molecules have been used to accomplish this, including quinoline thioethers [12], dimethyl sulfoxide [3,7,[13][14][15], and aurintricarboxylic acid [7]. Additionally, broad classes of small molecules including aromatic carboxylic acids, hydroxamic acids, and acetamides have been studied [16].…”