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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
Author(s) -
Kronsteiner Barbara,
Dullnig Verena,
Stutz Hanno
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700892
Subject(s) - chromatography , chemistry , capillary electrophoresis , repeatability , isoelectric focusing , analytical chemistry (journal) , selectivity , biochemistry , enzyme , catalysis
CZE and CIEF separation systems, both developed previously for a quality control of two recombinant products of the major birch pollen allergen Bet v 1a of Betula verrucosa , were validated including aspects of the International Conference on Harmonization. One product contained carbamylated variants as impurities. Linearity of response was confirmed by Mandel's fitting test between 0.028 and 1.90 mg/mL for CZE and between 0.016 and 0.26 mg/mL for CIEF. Repeatability and intermediate precision were evaluated for the effective mobility ( μ eff ) in CZE, for relative mobilization time in CIEF and the peak area ratio of Bet v 1a. LOQ for Bet v 1a was between 10 and 23 μg/mL for both methods. Evaluation of robustness for CZE revealed susceptibility of μ eff of Bet v 1a to alterations in of buffer pH and separation temperature. Selectivity was impaired by an increase in temperature, pH, and buffer concentration. In addition, pH variations influenced the separation profile of impurities. For CIEF, the ratio of narrow pH range carrier ampholytes is the critical parameter to retain robustness. Results demonstrate the suitability of both separation systems to discriminate between nonmodified Bet v 1a and carbamylated variants in the selected recombinant allergen products.