ISSN:
1573-904X
Keywords:
recombinant hirudin
;
protein formulation
;
protein stability
;
capillary electrophoresis
;
cyclic imide
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Purpose. The formation of succinimide intermediates at Asp-Gly sites and their hydrolysis products, e.g., isoAsp isomers, represents a common source of microheterogeneity in therapeutic proteins. Here we report on the stabilization effect of a zinc chloride induced precipitation of recombinant hirudin HV1 (rHir), an anticoagulant protein. Methods. rHir was precipitated by zinc chloride at neutral pH to form a Zn-rHir suspension. An Arrhenius-type study (at 50, 40, 30, and 25°C) and a 4°C stability study were performed. Monitoring of rHir, rHir succinimides at Asp33-Gly34 (Q5) and Asp53-Gly54 (Q4), and further side products was by capillary electrophoresis (CE). Results. The activation energies of rHir degradation in both aqueous rHir solution and Zn-rHir suspension were similar, i.e. 104.5 and 110.3 kJ/mol, respectively. Zn-rHir suspension demonstrated improved shelf-life stability (t90%, 95% confidence limit) versus rHir solution, i.e., 23 versus 3 days at 25°C and 292 versus 147 days at 4°C, respectively. In rHir solution, Q4 (Asp53-Gly54 succinimide) levels were slightly above Q5 (Asp33-Gly34 succinimide) levels. In Zn-rHir suspension, however, Q4 succinimide levels dropped markedly whereas Q5 levels were not affected. Correspondingly, in Zn-rHir isoAsp53-rHir levels were reduced but not isoAsp33-rHir levels. Conclusions. In Zn-rHir suspensions, interactions of zinc and rHir show site-specific inhibition of succinimide formation only at Asp53-Gly54 (Q4), located in the highly flexible C-terminal tail of rHir. In contrast, succinimide formation at Asp33-Gly34 (Q5), located in a less flexible loop domain is not affected, reflecting steric hindrance.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1018925209080
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