Non-native capillary isoelectric focusing for the analysis of the microheterogeneity of glycoproteins.
A simple capillary isoelectric focusing method in bare fused-silica capillaries under denaturing conditions was developed for the separation of the glyco-isoforms of recombinant tissue plasminogen activator (rTPA) which have pI values ranging from pH 6 to 8. The glycoprotein was denatured in 8 M urea and separated in the presence of 0.75% carrier ampholytes, 0.4% hydroxypropylmethylcellulose and 4 M urea. Excellent separation of the glyco-isoforms of rTPA was achieved. Good reproducibilities for peak area and migration times were obtained using pressure mobilization of the focused protein zones. The method optimized for rTPA was also successfully applied to other glycoproteins (recombinant human erythropoietin and a monoclonal antibody) with different carbohydrate contents (3-55%).
Kubach
J
J
Analytical Division Europe, Hewlett-Packard, Waldbronn, Germany.
Grimm
R
R
D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D007536:Isomerism; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator