Isolation of a low-molecular-weight growth inhibitory factor from hybridoma cell cultures

A low-molecular-weight growth inhibitory factor was produced by hybridoma cells. The number of viable cells in hybridoma cell cultures reached a maximum of about 5 x 10(5) cells/ml when the inhibitory factor had accumulated to a critical level, after which the number of viable cells declined with a concomitant increase in the number of dead cells. The growth inhibitory factor was purified to apparent homogeneity by ultrafiltration, reverse-phase chromatography, passage through cation exchangers, and gel filtration. Analysis by reverse-phase chromatography and micellar electrokinetic chromatography using a capillary electrophoresis system indicated that the final inhibitory fraction was pure. The factor had a molecular weight of 500 or less, as judged by ultrafiltration, and its behavior upon ion-exchange chromatography indicated that it was uncharged. Its absorbance maximum at 263 nm indicated that it was not a peptide, but that it may have a conjugated system of carbon-carbon double bonds.