Reaktion #162269

Reaktionsgleichung

NaOH

NaCl

sucrose

NaCl

sucrose

HEPES

HEPES

TMAE

→

Lysine

Reaktionsbedingungen

Aufarbeitung

1. 1
   Sonstigewas prepared
2. 2
   workup.ADDITIONwas added to this solution
3. 3
   workup.STIRRINGunder gentle stirring for 1 h at room temperature
4. 4
   WaschenThen the column was washed with 20 CV equilibration buffer
5. 5
   Sonstigeto remove excess reagent
6. 6
   Waschenthe PEGylated ANVF was eluted with elution buffer (20 mM HEPES, 0.5 M NaCl, 0.5% sucrose, pH 7.4)
7. 7
   EinengenThe eluate was concentrated by ultrafiltration/diafiltration with a membrane
8. 8
   SonstigeThe PEGylated derivative obtained

Vorschrift

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).

Quelle