Kg. Moorhouse et al., ELECTROPHORETIC SEPARATION OF RECOMBINANT TISSUE-TYPE PLASMINOGEN-ACTIVATOR GLYCOFORMS - VALIDATION ISSUES FOR CAPILLARY ISOELECTRIC-FOCUSING METHODS, Electrophoresis, 17(2), 1996, pp. 423-430
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.