Purification of a synthetic myristylated peptide by counter-current chromatography.

A preparative purification of myristyl-Gly-Asn-Ile-Phe-Ala-Asn-Leu-Phe-Lys-Gly-Leu-Phe-Gly-Lys-Lys-Glu -NH2 was accomplished using the multi-coil counter-current chromatograph. A partition coefficient was determined in the n-butanol-acetic acid-water (4:1:5) system. Chromatographic runs were made in this system and one modified with ethyl acetate. The peptide material showed anomalous elution behavior due to its surfactant properties. It was found that by loading the sample exclusively in the stationary phase, satisfactory retention of the compound occurred. Finally, conditions utilizing the upper phase as the mobile phase successfully separated the impurities.

Purification of synthetic peptides on a high-resolution preparative reversed-phase column.

A 2-in. I.D. column filled with 10-microns spherical C18 bonded silica with 120 A pores was used for the preparative purification of various synthetic peptides in one step. The small-sized silica packing afforded high resolution and the spherical shape helped maintain a relatively low back-pressure during the chromatography. Conditions for performing the separations were derived from the analytical chromatography of samples on a column of similar 5-microns material. The same amount of organic modifier, but with the gradient duration increased, achieved very similar separations on the preparative column.

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.