RAPID ONE-STEP CAPILLARY ISOELECTRIC-FOCUSING METHOD TO MONITOR CHARGED GLYCOFORMS OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN-ACTIVATOR

A rapid (<10 min) one-step capillary isoelectric focusing (cIEF) metho d was developed to monitor charged glycoforms of recombinant human tis sue-type plasminogen activator (rt-PA). Focusing takes place between t he detector and the anode and the electro-osmotic flow (EOF) sweeps th e separated glycoforms past the detector, towards the cathode. The sep aration uses a neutral coated capillary and hydroxypropylmethylcellulo se (HPMC) to reduce the EOF to a constant and reproducible value. The method uses an ampholyte mix with a 50:50 ratio of pH 5-8 and pH 3-10 ampholytes in 4 M urea and 0.1% HPMC to produce maximal resolution whi lst maintaining protein solubility during focusing. The electropherogr ams were compared to isoelectric focusing (IEF) slab gels of samples o f intact rt-PA. In both cases approximately ten charged species could be detected. Data analysis indicated that the intra-assay precision wa s <5% for peak migration times and <10% for normalized peak areas. The number of charged species detected by each of the two methods was con sistent for samples of intact rt-PA, rt-PA types I and II and for neur aminidase-digested rt-PA. Overall the data indicate that the automated cIEF method can be an adjunct to slab-gel IEF in the characterization and routine analysis of recombinant glycoproteins.