Scale-up methodology for the preparative purification of peptide M

An octadecapeptide, peptide M, the epitope of a retinal protein that induces experimental autoimmune uveitis, was synthesized and purified by preparative reversed-phase chromatography. The flow-rate and gradient conditions for maximum separation of impurities were determined on a 30 x 0.39 cm I.D. column of Delta Pak (15-microns spherical C18-bonded silica with 300-A pores). The maximum amount of peptide that was resolved under these conditions was then determined experimentally. Using a scale factor dependent on the square of the column diameters, the flow-rate and amount loaded were increased 164 times on a 30 x 5 cm I.D. column of the same packing. The same resolution was achieved. Batches of 200-342 mg were chromatographed with reproducible results, providing a total yield of 394 mg of pure peptide.