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) method was developed to monitor charged glycoforms of recombinant human tissue-type plasminogen activator (rt-PA). Focusing takes place between the detector and the anode and the electro-osmotic flow (EOF) sweeps the separated glycoforms past the detector, towards the cathode. The separation uses a neutral coated capillary and hydroxypropylmethylcellulose (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 whilst maintaining protein solubility during focusing. The electropherograms were compared to isoelectric focusing (IEF) slab gels of samples of intact rt-PA. In both cases approximately ten charged species could be detected. Data analysis indicated that the intra-assay precision was < 5% for peak migration times and < 10% for normalized peak areas. The number of charged species detected by each of the two methods was consistent for samples of intact rt-PA, rt-PA types I and II and for neuraminidase-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.
Moorhouse
K G
KG
Department of Quality Control Clinical Development, Genentech, S. San Francisco, CA 94080, USA.
Eusebio
C A
CA
Hunt
G
G
Chen
A B
AB
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000737:chemistry*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator