Capillary Electrophoresis of r-Hirudin and a Polyethylene Glycol Derivative of r-Hirudin (PEG-Hirudin)

Abstract

Recombinant (r-) hirudins and PEG-hirudin are currently tested for anticoagulant therapy. For their concentration measurement, radioimmunoassay and HPLC methods are available. The separation of r- and PEG-hirudin is currently performed by HPLC. However, the sensitivity of the method is low. Capillary electrophoresis is a rapid, selective technique that requires low sample amounts. Our aim was the development of a capillary electrophoresis method to measure r- and PEG-hirudin. The results are as follows: In a borate solution (0.3% boric acid and 0.4% sodium tetraborate, pH 9.5) r-hirudin was separated from PEG-hirudin in a purified system using a fused silica capillary (50 cm long and 75-μm i.d. and reversed polarity). A neutral capillary with a 20 mM tricine buffer (pH 8.0, field strength 500 V/cm) was also effective in resolving r- from PEG-hirudin. A linear correlation was found between the peak area and the concentration between 20 μg/mL and 10 mg/mL for hirudin (r² = 0.99) and between 1.25 and 10 mg/mL for PEG-hirudin (r² = 0.99). In human plasma mixtures, r- and PEG-hirudin were completely separated. The linear correlation between the peak area and the concentration was r² = 0.99.

Conclusion: In a fused silica capillary, r- and PEG-hirudin are separated in a purified system. Capillary electrophoresis which is performed in a neutral capillary, resolves r- from PEG-hirudin in a purified system, in plasma and in urine. The sensitivities of the methods are comparable. Capillary electrophoresis separates r- from PEG-hirudin and may be applied to biologic systems to measure the concentration and purity of r- and PEG-hirudin.

Keywords:

Thrombin inhibitor - capillary electrophoresis - r-hirudin - PEG-hirudin