High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.
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 greater than 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 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 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.
Tsuji
K
K
Pharmaceutical Product Control Division, Upjohn Company, Kalamazoo, MI 49001.
D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis; D002417:Cattle; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli; D008970:Molecular Weight; D009113:Muramidase / Q000032:analysis; D009218:Myosins / Q000032:analysis; D010047:Ovalbumin / Q000032:analysis; D010763:Phosphorylase b / Q000032:analysis; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D014361:Trypsin Inhibitors / Q000032:analysis; D001616:beta-Galactosidase / Q000032:analysis
D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D010047:Ovalbumin; D010763:Phosphorylase b; D009113:Muramidase; D001616:beta-Galactosidase; D009218:Myosins; D002256:Carbonic Anhydrases