RESUMEN
Three preparation batches of the recombinant birch pollen allergen Bet v 1a have been analyzed by capillary zone electrophoresis (CZE) using a separation electrolyte consisting of 100 mmol L(-1) phosphate at pH 6.50 with 2.0 mmol L(-1) tetraethylenepentamine (TEPA) added. TEPA improved the resolution by wall shielding and selective attachment to allergens, but reduced migration repeatability at concentrations >2.0 mmol L(-1). Heterogeneity of preparations determined by CZE and electrospray ionization-quadrupole-time-of flight-MS were in accordance and revealed chemically modified (carbamylated) allergens in one of the preparations. The method was validated according to the ICH-guidelines. Repeatability of effective electrophoretic mobility (mu(eff)) was <0.55% R.S.D. (n = 5). Migration time corrected peak areas were used for quantification. Limit of quantification (LOQ) was 25 microg mL(-1) for the major isoform Bet v 1a, based on a signal-to-noise ratio of 10, and detector response was linear between LOQ and 0.90 mg mL(-1). Purity of the different rBet v 1a preparations was determined to be between 40 and 92% depending on the manufacturing protocol.