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Electrophoretic separation of recombinant tissue‐type plasminogen activator glycoforms: Validation issues for capillary isoelectric focusing methods
Author(s) -
Moorhouse Kathryn G.,
Rickel Carina A.,
Chen Anthony B.
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170223
Subject(s) - isoelectric focusing , chromatography , recombinant dna , plasminogen activator , capillary electrophoresis , isoelectric point , chemistry , capillary action , biochemistry , materials science , biology , composite material , gene , enzyme , endocrinology
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 charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue‐type plasminogen activator (rt‐PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The p I of rt‐PA measured with synthetic p I standards was in the range pH 6.5–7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50–1000 μ/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15–30°C, and decreased predictably with increasing voltages (400–600 V/cm) and decreasing N , N , N ′, N ′‐tetramethylethylene diamine (TEMED) concentrations (0.4–1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.