There are at least 10 MAb enhancing compounds reported in the literature with more to be discovered. There are no common features to the group of compounds other than that they increase MAb production. This work will focus on a method for identifying MAb enhancing agents and then using these agents to probe the MAb production pathway in an effort to identify the limiting step(s) in the pathway. Once a compound has been identified as a MAb enhancer, using a 72 hr 96-well plate MAb titer assay that we have developed in our lab, our investigation moves to how the compound is influencing the MAb production pathway. The investigation is divided into two main branches, the general MAb productivity pathway and the compound specific pathway, which allows us flexibility in choosing our compounds to investigate. We will use rapamycin and sodium butyrate, two MAb enhancing compounds that are reported to act on two different stages of the MAb production pathway. Rapamycin is reported to inhibit the mTOR pathway, which is part of the translational section of the MAb production pathway. The second compound sodium butyrate is a Histone Deacetylase (HDAC) inhibitor in that it prevents the removal of the acetyl groups from the histones, which allows the transcription process to proceed for a longer period of time. Using these two compounds we will investigate whether the mRNA transcript level of the IgG heavy chain has been altered. The translational step will also be explored using Rap and NaBu to determine if the increases in MAb production positively correlate with the intracellular levels of the IgG heavy and light chain polypeptide level.

The second branch of our investigation will examine the specific targets of rapamycin (mTOR) and sodium butyrate (HDAC) to see if they are actively inhibiting their targets. We are currently using a much lower concentration of sodium butyrate than what is seen in the literature to cause the inhibition of HDAC. The concentration of Rap used to enhance MAb production in our cell line is much greater than what has been reported to be necessary for mTOR inhibition. Since we are using concentrations of rapamycin and sodium butyrate that are outside the reported concentrations used for HDAC inhibition and the inhibition of the mTOR pathway it is especially important to investigate them specifically. Western blots will be used to determine whether our concentration of sodium butyrate is high enough to cause the inhibition of HDAC, which influences transcription. Rapamycin's affect on the mTOR pathway will be probed using western blots to determine if the phosphorylation level of p70s6k and 4E-BP1 have changed which would influence translation and possibly cell size.

The two-branch method itself allows one to have great flexibility in choosing ones' compounds to study. Simultaneously the method allows the unknown mechanisms behind MAb production enhancement to be investigated. The pathway analysis and measurement of both pathway-specific and non-specific effects are widely applicable for testing compounds for MAb production enhancement in hybridoma and other cell lines.