High-performance capillary electrophoresis of proteins : Sodium dodecyl sulphate—polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins

Abstract

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship ( r>0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10 000–100 000 and 40 000–200 000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 · 10 5–1 · 10 6 per metre are routinely being obtained. Calibrations graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear ( r>0.999) and the proteins may be quantified with an R.S.D. of ca. 3–7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound ( ca. 60 000 dalton) may be differentiated by HPCE with a gel-filled capillary column.