Separation and Identification of Growth Hormone Variants with High Performance Liquid Chromatography Techniques

Abstract

Liquid chromatography techniques were used to separate and identify human growth hormone (hGH) variants. N-terminal modified forms, such as des-Phe (2-191) and methionyl-hGH (met-1-191), were separated from recombinant human growth hormone (rhGH (1-191] by hydrophobic interaction chromatography (HIC). A proteolytically cleaved ('clip') form of rhGH which has a break in the polypeptide chain between Thr(142) and Tyr(143), also proved to be separable from rhGH by HIC. In addition, a mutated form of rhGH with only two amino acid substitutions, Glu(65) to Val(65) and Glu(66) to Lys(66), on a random coil domain of the molecule, was separated from rhGH by HIC, indicating that these substitutions altered the hydrophobicity of the molecule. Treatment of rhGH with hydrogen peroxide led to sulphoxide formation in two methionine residues Met(14) and Met(125); it was not possible to oxidize Met(170). The oxidized forms of rhGH were readily separated from rhGH(1-191) by reversed-phase chromatography. Analyses of rhGH batches showed very low levels (less than 0.3%) of oxidized rhGH, indicating that rhGH is highly resistant to oxidative reactions. Deamidations were induced in rhGH by heat treatment. The primary deamidation site was found to be Asn(149). Monodesamido rhGH and didesamido rhGH were efficiently separated from rhGH(1-191) by anion-exchange chromatography.