The stability and degradation pathway of recombinant human parathyroid hormone: Deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues

Purpose. The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. Methods. Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60 degrees C. The approximate molecular mass and pi values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. Results. At pH2, eight degradation products were found: 1-30rhPTH, 1-74rhPTH, 1-71rhPTH, 1-56rhPTH, 1-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Asp16-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. Conclusions. Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent. rhPTH is most stable at pH5.