There are many pathways to improve recombinant protein manufacturing and production, including cell engineering and cell line selection, media and feed strategies, and chemical supplementation. The addition of chemical additives is a straightforward approach to improve protein production since it is a simple chemical that may be added anytime during development or a production process. Sodium butyrate (NaBu) is one of the better-studied additives that supports protein expression in a variety of mammalian cell lines. NaBu is a short-chain fatty acid that inhibits histone deacetylase and enhances transcription factors by making the chromatin area more accessible. However, adverse effects on cell health, including increased apoptosis and alterations to the glycosylation of the produced protein, may limit NaBu’s broad utility in all processes.

The Betenbaugh and Yarema groups at Johns Hopkins University recently revealed another butyrate-containing additive that shared many of NaBu’s strengths without some of its weaknesses. In this study, the team measured the effects of the new NaBu analog called 1,3,4-O-Bu₃ManNAc (3BNAC) on protein productivity and quality and cell health. The supplements were added at varying equivalent butyrate concentrations to Ham’s F12-K medium supplemented with 10% (v/v) fetal bovine serum. These media blends were used in batch cell cultures growing wild-type CHO-K1 cells and CHO-K1 cell lines producing either human erythropoietin (EPO) or an IgG₁ (IgG). During the experiment, protein production, protein sialylation, cell growth characteristics, and cell metabolites were monitored among the different cell lines and wild-type cultures.

Both levels of EPO and IgG increased with supplementation of NaBu and 3BNAC compared to untreated cultures, and these increases followed the relative supplemented concentrations of each additive. For IgG, these increases were observed mostly in the late exponential and stationary phases of the processes, whereas increases in EPO accumulation were noted earlier. The addition of 3BNAC to both EPO and IgG cell lines did not change the cell growth patterns significantly from the untreated control. All three cultures reached a maximum cell density of ~1.5 million cells/mL. These levels were more than 10% higher than the maximum cell density from the NaBu-treated cultures. There were minor differences in glucose consumption and lactate accumulation among all the tested conditions, with the most significant differences coming from only a slightly slower increase in lactate with the NaBu-treated cultures. There were almost no observed differences in caspase-3 protein levels in 3BNAC, an indicator of cell apoptosis, compared to controls, whereas there was an increase with NaBu addition. Qualitative RT-PCR of four apoptotic marker genes (BCL2L11, BAX, BAK, and Cul2) indicated that the addition of NaBu resulted in the highest expression levels of all four genes in the three different cell lines tested. Additionally, lectin blot analysis of the EPO-producing cell line showed that 3BNAC enhanced the sialyation of the recombinant protein, while NaBu decreased it compared to the control.

There are a variety of methods to improve cell productivity to boost protein production in bioprocesses. In this study, the new butyrate analog 3BNAC was shown to be a useful supplement to boost recombinant protein production likely due to the same mechanisms as NaBu (i.e., butyrate’s ability to increase transcription factor accessibility). 3BNAC increased both the levels of both EPO and IgG therapeutic proteins and sialyation of EPO while not significantly effecting culture metabolites, cell health, or apoptotic activity. Although NaBu exhibited higher increases in productivity, its supplementation resulted in higher levels of cytotoxicity and apoptosis with a decrease in sialyation. The differences in results were attributed to the different sizes of the molecules as the smaller NaBu may be able to diffuse into a cell much faster than the larger 3BNAC. Additionally, the need for an esterase to release the butyrate groups from 3BNAC likely slowed the dosage over time. One significant advantage that NaBu has over 3BNAC is its wide commercial availability. Time will tell if 3BNAC becomes a regular off-the-shelf additive that researchers can quickly implement.

By, Glenn A. Harris