Oxidation of human insulin-like growth factor I in formulation studies: kinetics of methionine oxidation in aqueous solution and in solid state.

PURPOSE: The aim of this work was to study the kinetics of oxidation of methionine in human Insulin-like Growth Factor I (hIGF-I)1 in aqueous solution and in the solid state by the aid of quantification of oxygen. METHODS: The oxidized from of hIGF-I was characterized by tryptic peptide analysis, RP-HPLC and FAB-MS and quantified by RP-HPLC. The oxygen content was quantified polarographically by a Clark-type electrode. RESULTS: Second-order kinetics with respect to amount of protein and dissolved oxygen was found to be appropriate for the oxidation of methionine in hIGF-I. The rate constants ranged from 1 to 280 M-1 month-1 and had an activation energy of 95 (+/-4) kl/mole. Light exposure, storage temperature and oxygen content were found to have a considerable impact on the oxidation rates. No significant difference in reaction rates was found for the oxidation of hIGF-I in aqueous solution or in the solid state. A method for decreasing the oxygen content in aqueous solution without purging is described. CONCLUSIONS: Polarographic quantification of dissolved oxygen makes it possible to establish the kinetics for oxidation of proteins. The oxidation of methionine in hIGF-I appears to follow second-order kinetics.

Separation and identification of growth hormone variants with high performance liquid chromatography techniques.

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.

Rapid purification of monoclonal antibodies by high-performance liquid chromatography.

Three different high-performance liquid chromatographic (HPLC) techniques, i.e., ion-exchange, hydrophobic interaction and hydroxyapatite chromatography, have been used to purify monoclonal antibodies from ascites fluid. The monoclonal antibodies were raised against coagulation factor VIII. Precipitation of the antibodies by ammonium sulphate prior to HPLC made it possible to purify the antibody in one chromatographic step. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis this highly pure preparation revealed only one extra polypeptide besides the light- and heavy-chain immunoglobulin polypeptides. Attempts were also made to purify the antibody without prior ammonium sulphate precipitation. A combination of ion-exchange and hydrophobic interaction chromatography resulted in an antibody preparation comparable in purity to the one obtained from ammonium sulphate-precipitated immunoglobulin. The rapid HPLC techniques were found to be very useful for purification of monoclonal antibodies on a preparative scale, where sample loadings of up to 25 mg of ascites protein were fully resolved in satisfactory yields.