Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography. |
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Authors: | G Karlsson P Gellerfors A Persson B Norén P O Edlund C Sandberg S Birnbaum |
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Institution: | Pharmacia and Upjohn, Stockholm, Sweden. |
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Abstract: | Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 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. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; 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. |
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