Inhibition of succinimide formation in aqueous Zn-rHirudin suspensions.
OBJECTIVE
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 degrees C) and a 4 degrees 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 degrees C and 292 versus 147 days at 4 degrees 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.
Gietz
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Department of Pharmacy, Swiss Federal Institute of Technology, Zürich (ETH).
Arvinte
T
T
Alder
R
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Merkle
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HP
D000925:Anticoagulants / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019075:Electrophoresis, Capillary; D006629:Hirudins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*; D013535:Suspensions; D015032:Zinc / Q000737:chemistry
D000925:Anticoagulants; D006629:Hirudins; D011994:Recombinant Proteins; D013388:Succinimides; D013535:Suspensions; D015032:Zinc