Validation of a Peptide Map for Recombinant Porcine Growth Hormone and Application to Stability Assessment

摘要: A reverse-phase HPLC method for the analysis of tryptic digests recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4°C a 20-hr period with TPCK-treated trypsin 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, incorporated separation digestion products peak identification carried out by mass spectrometry (MS). The procedure subsequent chromatography were linear in initial concentration range 4.55 – 45.46 µM (100 to 1000 µg/mL) pGH. variability fragment retention times low normalized area less than 5% all but three fragments. utility procedures demonstrated assessing chemical changes pGH induced incubation elevated pH. Upon 0.2 M Tris buffer pH 9 (ionic strength adjusted 0.5 NaCl) 37°C over 400 hr, significant degradation regions corresponding fragments T23–T25 (residues 181–182 linked disulfide bond residues 184–191), T9 96–108), T5–T18 43– 64 158–166) observed. disappearance peaks both displayed apparent first-order kinetics time investigated half-lives 131 154 respectively. complicated poor resolution parent products. These results demonstrate limitations mapping determination reaction