ANALYSIS OF A RECOMBINANT PROTEIN PREPARATION ON PHYSICAL HOMOGENEITY AND STATE OF AGGREGATION

The homogeneity of a recombinant protein preparation and the date of aggregation were studied by time-resolved polarized fluorometry. From the rotational correlation time theta distribution pattern, the state of aggregation of a protein in solution in deduced. This distribution is determined from a two-dimensional (r, theta) fit using the maximum entropy method of data analysis and where tau values are the fluorescence lifetimes. An analytical procedure is developed that is validated by measurements on a mixture of two proteins having different molecular weights and containing a single tryptophan residue per polypeptidic chain: recombinant human interferon gamma, r-hu IFN gamma (RU42369) of M(r) 17 000 for the monomer and recombinant mutant W201Y lac operon repressor tetramer, M(r) 152 400. By spiking a solution of 1 mg/mL r-hu IFN gamma with the W201Y lac operon repressor, the lower level of detection of higher molecular weight component is found to be 5 % in intrinsic floursecent probe concentration. In this study we found that (1) purified r-hy IFN gamma in solution after lyophilization is a dimeric molecule, without indication of physical heterogeneity and without high molecular weight aggregates; (2) heat treatment of lyophilized r-hu IFN gamma, 14 days at 40-degrees-C, results in the formation of a detectable amount of higher molecular weight material; (3) the dissociation of the dimer r-hu IFN gamma on dilution was not complete after dilution to 0.01 mg/mL (0.57 muM). Taking advantage of the groat sensitivity of a fluorescence technique and of the capabilities of the data analysis MEM, this new procedure can be widely used to detect a high molecular weight protein contaminant (aggregates) in a homogeneous protein solution.