Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilize d for the separation of recombinant human growth hormone (hGH) variants on a C-18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Thr ee of the obtained peaks were characterized by tryptic mapping and mass spe ctrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14 /Met125 and Met125 sulfoxide) while the third contained a deamidated form ( Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time. (C) 1999 Elsevier Science B.V. A ll rights reserved.