反应详情

A solution of a mature rVWF (35 U VWF:Ag/ml) in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose was prepared according to Example 23. Then branched mPEG succinimidyl glutarate (PE-SG/chain length: 20 kD) supplied by NOF corporation (NOF Europe, Grobbendonk, Belgium) was added to this solution under gentle stirring (5 mg PEG-SG/mg protein) and the pH value was adjusted to 7.4 by drop wise addition of 0.5 M NaOH. Than the PEGylation was carried out under gentle stirring for 1 h at room temperature. Subsequently the reaction mixture was applied onto an equilibrated ion-exchange chromatography resin (Fractogel EMD TMAE 650M) in 20 mM HEPES buffer, 150 mM NaCl, pH 7.4, containing 0.5% sucrose. Then the column was washed with 20 CV equilibration buffer to remove excess reagent and the PEGylated ANVF was eluted with elution buffer (20 mM HEPES, 0.5 M NaCl, 0.5% sucrose, pH 7.4). The eluate was concentrated by ultrafiltration/diafiltration with a membrane consisting of regenerated cellulose and with a molecular weight cut-off of 100 kD using a buffer system consisting of 20 mM HEPES, 150 mM NaCl, 0.5% sucrose, pH 7.4. The PEGylated derivative obtained showed a slightly diminished VWF:RCo/VWF:Ag ratio of 0.79 as compared with the rVWF starting material (VWF:Rco/VWF:Ag ratio: 0.89). In addition, the PEGylated rVWF starting material had a FVIII binding capacity of 83% as measured by the ECA test (Example 16).

1. customwas prepared
2. additionwas added to this solution
3. stirringunder gentle stirring for 1 h at room temperature
4. washThen the column was washed with 20 CV equilibration buffer
5. customto remove excess reagent
6. washthe PEGylated ANVF was eluted with elution buffer (20 mM HEPES, 0.5 M NaCl, 0.5% sucrose, pH 7.4)
7. concentrationThe eluate was concentrated by ultrafiltration/diafiltration with a membrane
8. customThe PEGylated derivative obtained