ABSTRACT
Four commercial ampholytes: Ampholine and Pharmalyte (Pharmacia Biotech), Bio-Lyte (Bio-Rad) and Servalyt (Serva) were evaluated for their ability to resolve recombinant tissue-type plasminogen activator (rt-PA) glycoforms by isoelectric focusing (IEF) and capillary IEF (cIEF). Each brand of ampholytes focused rt-PA into 3-4 major and 5-6 minor bands on slab gel electrophoresis. Visually, focused bands stained with Coomassie Blue appeared to be similarly resolved by all the ampholytes except for Ampholines, where the bands were closely grouped and more intensely stained. When cIEF was performed, Pharmalytes and Ampholines resolved rt-PA glycoforms consistent with the slab gels. No discernible peaks were detected during cIEF of rt-PA using Servalyts or Bio-Lytes. UV spectrophotometric scans of the components used for cIEF showed that Servalyts absorbed intensely over a range which overlapped the detector bandpass. Bio-Lytes showed absorption over a narrower UV range but still overlapped the detector bandpass, thus preventing the discernment of protein peaks. For this cIEF system the best ampholytes were Ampholines and Pharmalytes.
Subject(s)
Ampholyte Mixtures/chemistry , Tissue Plasminogen Activator/analysis , Amino Acids/chemistry , Buffers , Electrolytes/chemistry , Electrophoresis, Polyacrylamide Gel/methods , Hydrogen-Ion Concentration , Indicators and Reagents/chemistry , Isoelectric Focusing/methods , Polyamines , Polymers/chemistry , Recombinant Proteins/analysis , Spectrophotometry, UltravioletABSTRACT
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 pI of rt-PA measured with synthetic pI 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 micrograms/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 degrees 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.
