A preparation of recombinant birch pollen allergen of Betula verrucosa isoform 1a (Bet v 1a) containing chemically modified (carbamylated) variants has been analyzed by CZE and CIEF. In CZE, employing a 100 mmol/L MES buffer at pH 6.50, with 0.4 mmol/L tetraethylenepentamine (TEPA) added, allowed for the resolution of 17 protein fractions. The CIEF profiling of the allergen preparation required a combination of a wide-pH-range carrier ampholyte (CA) of pH 3-10 with two narrow-range CAs of pH 5-6 and 5-7. For CIEF, 91 mmol/L of glycine at pH 2.12 and 20 mmol/L of CHES at pH 10.00 were applied as anolyte and catholyte, respectively. The generated pH gradient was nonlinear with a flat slope for pH 4-6, thus providing an improved resolution. In CIEF, up to 18 protein fractions were distinguished as well. The pI of the target allergen Bet v 1a was 4.9 as determined by means of two pI marker compounds flanking the allergen. Relative purity of the target allergen within the preparation containing carbamylated variants was in accordance for both separation systems and varied between 40.7 and 42.8%.

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 (mu(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 microg/mL for both methods. Evaluation of robustness for CZE revealed susceptibility of micro(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.