Chemical Degradation Kinetics of Recombinant Hirudin (HVI) in Aqueous Solution: Effect of pH

Abstract

Purpose. To gain information on the chemical stability pattern and the kinetics of the degradation of recombinant hirudin variant HVI (rHir), a thrombin-specific inhibitor protein of 65 amino acids, in aqueous solution as a function of pH.

Methods. Stability of rHir was monitored at 50°C in the framework of a classical pH-stability study in aqueous buffers pH 1−9.5. Two capillary electrophoresis (CE) protocols were used; one for the kinetics of succinimide formation at Asp⁵³-Gly⁵⁴ (C-terminal tail) and Asp³³-Gly³⁴ (loop section), the other for the kinetics of rHir degradation. To check for potential effects of conformational changes by thermal denaturation, circular dichroism (CD) measurements were performed between 25 and 80°C.

Results. Throughout the pH range studied no effect of thermal denaturation on rHir confirmation at 50°C was observed. rHir was most stable at a neutral pH whereas, at slightly acidic pH, an intermediate stability plateau was found. Both, strongly acidic and alkaline conditions led to fast rHir degradation. Depending on the pH of degradation, rHir was found to degrade in various combinations of multiple parallel and sequential degradation patterns. Special focus was on succinimide formation at Asp⁵³-Gly⁵⁴ (C-terminal tail) and Asp³³-Gly³⁴ (loop) and on the potential of isoAsp formation in position 53 and 33.

Conclusions. Chemical rHir stability in the intermediate pH range depends strongly on succinimide formation. At slightly acidic conditions succinimides represent the major degradation product (up to 40%). Around neutral pH succinimides react further, presumably by isoAsp formation, and concentrations remain low. Relative preference of succinimide formation in the C-terminal tail domain versus the loop domain is explained by higher backbone flexibility in the tail.