EXAMINATION OF CAPILLARY ZONE ELECTROPHORESIS, CAPILLARY ISOELECTRIC-FOCUSING AND SODIUM DODECYL-SULFATE CAPILLARY ELECTROPHORESIS FOR THE ANALYSIS OF RECOMBINANT TISSUE-PLASMINOGEN ACTIVATOR
Jm. Thorne et al., EXAMINATION OF CAPILLARY ZONE ELECTROPHORESIS, CAPILLARY ISOELECTRIC-FOCUSING AND SODIUM DODECYL-SULFATE CAPILLARY ELECTROPHORESIS FOR THE ANALYSIS OF RECOMBINANT TISSUE-PLASMINOGEN ACTIVATOR, Journal of chromatography, 744(1-2), 1996, pp. 155-165
Citations number
34
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
The microscale techniques of CZE, cIEF and SDS capillary electrophores
is have been evaluated for the analysis of a complex glycoprotein, rec
ombinant tissue plasminogen activator (rtPA). A series of omega-amino
acid buffers (pH approximate to 5) was found suitable for the CZE sepa
ration of rtPA on coated capillaries. rtPA could be resolved into a se
ries of major and minor peaks in an epsilon-aminocaproic acid buffer c
ontaining 0.01% (v/v) Tween 80. For cIEF, a two step method with press
ure mobilization was utilized. Using a commercial instrument, either a
polymer solution with a 50 mu m I.D. capillary or narrow bore capilla
ries without a polymer solution (25 mu m I.D.) were employed. rtPA was
resolved into at least eight species within a pi range of 6.4-9.2 usi
ng Ampholine 3.5-10. Migration time precision for the major peaks rang
ed from 0.2% for CZE to less than or equal to 2-3% R.S.D. for cIEF. To
tal recovery of rtPA from the capillary was also demonstrated for both
methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialyl
ated forms resulted in the expected elution profiles. Finally, the pot
ential of SDS capillary electrophoresis using a coated capillary for a
n rtPA Type I/Type II purity assay was shown.