ELECTROPHORETIC SEPARATION OF RECOMBINANT TISSUE-TYPE PLASMINOGEN-ACTIVATOR GLYCOFORMS - VALIDATION ISSUES FOR CAPILLARY ISOELECTRIC-FOCUSING METHODS

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such c harge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) o n the basis of their sialic acid content. Nine to ten distinct peaks w ere consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards w as in the range pH 6.5-7.5 with the migration of the standards affecte d by the presence of the protein. The method showed an acceptable reco very of >100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and tot al protein measured by peak areas was linear over a wide range from 50 -1000 mu g/mL. A detailed study showed that when a capillary had been used fbr some time, capillary age affected peak migration times and, t o a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N, N, N', N'-tetrame thylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall th e data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resu lting from capillary age and lot were resolved. Even in its present fo rmat the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.