Semi-preparative high-performance liquid chromatographic purification of a 28-amino acid synthetic parathyroid hormone antagonist.

Structure-activity studies of parathyroid hormone (PTH) over the last decade have led to the design and synthesis of a peptide hormone inhibitor of PTH action in vivo, [Tyr-34]bPTH-(7-34)amide. To evaluate the biological properties of this 28-amino acid hormone analogue, sufficient amounts of the peptide needed to be prepared in a high state of purity to permit continuous infusion in groups of animals for periods of several hours. For this purpose, gel chromatography followed by semi-preparative, reversed-phase high-performance liquid chromatography (HPLC) using a lyophilizable solvent gradient system of acetonitrile in 0.1% trifluoroacetic acid was utilized. In a parallel purification scheme, gel filtration followed by ion-exchange chromatography was employed. Comparison of yields and product purity were determined by a battery of chemical analytical techniques. Semi-preparative HPLC generated a highly purified product; substantial heterogeneity was detected in the ion-exchange-purified material. The HPLC-purified product retains the biological activities of binding to PTH receptors in vitro and inhibiting PTH action in vivo.