A number of small-molecule protein production enhancers work through various metabolic pathways to inhibit cell expansion, thus switching cells into a production phase. These molecules tend to be somewhat cytotoxic, supposedly working through stimulation of apoptotic pathways, so their use must be assessed for each cell line. But these enhancers have been known to double productivity.

Several mechanisms of butyrate action have been suggested such as enhanced gene transcription through gene accessibility stimulation (47). In one study, sodium butyrate and sodium propionate (an alkanoic acid) enhanced MAb production sixfold (48). Whereas butyrate showed toxicity, no cell inhibition was seen with propionate. Similar results were reported comparing pentanoic acid (a carboxylic acid) with butyrate (49).

Another study tested valproic acid (which is both FDA approved and much less expensive than butyrate) as an alternative to butyrate (50). Productivity improvements were observed. Seeking new protein-inducing compounds, Allen et al. used a high-throughput model for screening chemical libraries to identify several basic types of molecules as potential possibilities (51). Cell transcriptional activity helped in the team's search and points to use of molecular approaches in future efforts.

In a different mode, adenosine 5′-monophosphate (AMP) significantly increased hepatitis B surface antigen production in CHO cells but dramatically reduced cell number once added to a culture (52). Extracellular nucleotides were shown to enhance protein production because intracellular adenosine 5′-triphosphate (ATP) pools increased with AMP addition. Yet another study showed rapamycin (a G1-phase arresting agent that slows cell cycle progression to delay apoptosis) to reduce hybridoma cell death and enhance MAb production twofold in fed-batch cultures (53). Dimethyl sulfoxide (DMSO) has also been advocated as a protein-inducing enhancer. Ling et al. showed that 0.2% DMSO added to hybridoma cultures at maximum cell density doubled MAb production with no negative effect on bioactivity and glycosylation (54).

The Next Step

As reviewed here, different nutrient supplements and feeding strategies can improve cell performance. Once basic cell culture parameters have been optimized, nutrient supplementation can usually result in a doubling or greater of cell productivity. The conclusion of this three-part review will examine scale-up and scale-down strategies for rapid nutrient supplement prototyping.