Profiling preparations of recombinant birch pollen allergen Bet v 1a with capillary zone electrophoresis in pentamine modified fused-silica capillaries.
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.
Punzet
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M
Department of Molecular Biology, Division of Chemistry, University of Salzburg, Salzburg, Austria.
Ferreira
F
F
Briza
P
P
van Ree
R
R
Malissa
H
H
Stutz
H
H
D000485:Allergens / Q000737:chemistry* / Q000302:isolation & purification; D029662:Betula / Q000737:chemistry*; D001665:Binding Sites; D001881:Borates / Q000737:chemistry; D002021:Buffers; D002073:Butanes / Q000737:chemistry; D004573:Electrolytes / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005029:Ethylenediamines / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010710:Phosphates / Q000737:chemistry; D028321:Plant Preparations / Q000737:chemistry*; D011058:Pollen / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012822:Silicon Dioxide / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D000485:Allergens; D001881:Borates; D002021:Buffers; D002073:Butanes; D004573:Electrolytes; D005029:Ethylenediamines; D010710:Phosphates; D028321:Plant Preparations; D011994:Recombinant Proteins; C046888:butane; D012822:Silicon Dioxide; C034269:tetraethylenepentamine