Effects of reducing sugars on the chemical stability of human relaxin in the lyophilized state.

Sugars and polyols have been used routinely with lyophilized proteins and peptides as bulking agents, cryoprotectants, and lyoprotectants. However, reducing sugars may present a problem as excipients since they are potentially reactive with proteins. In this stability study of recombinant human relaxin (Rix) with various sugars as excipients in lyophilized formulations, we observed rapid covalent modifications of the protein in the presence of glucose. Analysis of the protein by LC/MS and tryptic mapping indicated two major degradation pathways. Covalent adducts of glucose with amino groups on the side chains of the protein (i.e., Lys and Arg) formed via the Maillard reaction. In addition, a significant amount of Ser cleavage from the C-terminal of the B-chain of relaxin was also identified when glucose was used as the excipient. It was observed that the latter reaction occurred to a greater extent in the solid state than in solution. We proposed a mechanism for this reaction involving an initial reaction of the Ser hydroxyl group with glucose followed by subsequent hydrolysis of the Trp-Ser amide bond via a cyclic intermediate. In contrast to glucose, mannitol (polyhydric alcohol) and trehalose (nonreducing sugar) produced stable, lyophilized formulations of Rix.