Uncommon Peptide Bond Cleavage of Glucagon from a Specific Vendor under near Neutral to Basic Conditions.

Abstract

The main purposes of this manuscript are to report a surprising and interesting degradation reaction of glucagon from a specific vendor in which glucagon underwent cleavage among several peptide bonds quickly under near neutral to basic conditions, and to propose the root cause of mechanism for the degradation reaction. The degradation reaction was monitored by HPLC and the fragment structures were confirmed by LC-MS. Possible impurities responsible for the degradation were either confirmed or excluded by a variety of techniques such as addition of chelator EDTA and transitional metal ions or separation by ultrafiltration. This type of degradation was rarely reported in literature, especially considering its extreme cleavage efficiency. Contamination by a thermostable high molecular impurity (such as a peptidase with molecular weight between 10 and 30 KDa) during the manufacturing process was the main reason for this interesting phenomenon. The degradation phenomenon described here could be used as an excellent example showing that products ordered from vendors meeting the rudimentary quality standards might contain impurities which could cause significant degradation. We suggest that a simple solution, i.e. additional tests of stability under real or accelerated conditions by manufacturers and inclusion of the "accelerated stability criteria" in the Certificate of Analysis (CoAs), especially for sensitive biological reagents prone to faster degradation, would be very helpful for avoiding losses for both vendors and users.