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 pa thways were elucidated from resultant products. Methods. Degradation a ssay 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 produ cts were estimated by electrophoresis. FAB-MS peptide mapping and amin o acid composition analysis were used to determine these structures. T he 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 a mide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, As p16-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degrad ation products resulting from cleavage at Asp were increased in propor tion to the extent that pH was lowered below 5. As pH was increased ab ove 5, so were products resulting from deamidation of Asn. Correspondi ngly, levels of intact rhPTH were at a peak at pH5. Conclusions. Degra dation of rhPTH under acidic conditions predominantly occurs by cleava ge at Asp, whereas, above pH5, deamidation of Asn is the more prominen t. rhPTH is most stable at pH5.