Clonal growth inhibition as a bioassay for thymosin alpha1: inactivation of Talpha1 by trifluroacetic acid.

Thymosin alpha1 (Talpha1) is an immune response modifying peptide isolated from thymus tissue. The synthetic peptide has been evaluated in clinical trials as an adjuvant to cancer chemotherapy, an enhancer of vaccine potency, and an anti-viral for both hepatitis B and C. Among its multiple in vitro activities is the inhibition of the clonal growth of hepatitis B transfected hepatoblastoma cells. This assay was used to define the relationship between bioactivity and immunoactivity of Talpha1. Talpha1 was treated with 50% trifluoroacetic acid (TFA) for 1 hr to inactivate the peptide. Talpha1 heated at 90 degrees C or at room temperature maintained its bioactivity but TFA completely eliminated the activity in the bioassay. The TFA inactivated Talpha1 had a retention time on reverse-phase chromatography identical to bioactive Talpha1 but reduced immunoreactivity. In addition to demonstrating the utility of clonal growth as a bioassay, these studies demonstrate that immunoreactivity rather than retention time on HPLC may be a better predictor of bioactivity of synthetic Talpha1.