Electrophoretic separation of recombinant tissue‐type plasminogen activator glycoforms: Validation issues for capillary isoelectric focusing methods

Abstract

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.

Validation of capillary zone electrophoresis and capillary isoelectric focusing separations optimized for the characterization of two recombinant products of the birch pollen allergen Bet v 1a

“…In addition, CE provides product information orthogonal to HPLC [40,50]. To date, validation of CE separations of (recombinant) proteins is mainly focused on CZE and CGE-SDS [38,[51][52][53][54], whereas CIEF validation is scarcely reported [41,45,55,56]. Despite the availability of several CE separations for the characterization of (recombinant) allergens [57][58][59][60][61][62][63], only a single validated CZE method has been published so far [57].…”

Capillary Electrophoresis in the Development of Recombinant Protein Biopharmaceuticals

“…Protein recovery from the coated capillary was observed to be > 90%, the limit of detection was determined, and the precision of the area percent shown to be acceptable. However, due to the variability of residual EOF in different lots of the coated capillary, resulting in highly variable migration patterns and migration times, these capillaries were deemed unacceptable [55]. Hunt el al.…”

“…Therefore, these methods could be used to determine concentration (strength) of the active component in the final dosage form. The validation of a one-step clEF method for demon stating consistency of rt-PA glycoforms was attempted by Moorhouse et al [55]. Protein recovery from the coated capillary was observed to be > 90%, the limit of detection was determined, and the precision of the area percent shown to be acceptable.…”

Capillary electrophoretic analysis of proteins and peptides of biomedical and pharmacological interest

“…In addition to zone electrophoretic methods, Cifuentes et al [99] have reported the analysis by cIEF of rEPO: all the isoforms were resolved in the pH 2.5-5.0 interval. Isoelectric focusing techniques were also used for studying the heterogeneity of tissue plasminogen activator, as reported by Thorne et al [100] and by Moorhouse et al [101].…”