ABSTRACT
Mixed-mode chromatography was investigated for the purification of the recombinant allergen rBet v 1a expressed in Escherichia coli (E. coli) and used as an active principle for specific immunotherapy (SIT) treatment against birch pollen allergy. The screening of micro-volumes of three mixed-mode sorbents established that rBet v 1a could be captured without any pre-treatment of the crude feedstock on HEA or PPA HyperCel sorbents equilibrated in "physiological-like" conditions. On a mini-column pre-packed with PPA HyperCel sorbent, rBet v 1a was recovered at pH 4, partially separated from a major methionine Bet v 1 contaminant and purified approximately 9-fold in a single step (85% purity).
Subject(s)
Allergens/isolation & purification , Betula/chemistry , Chromatography, Liquid/methods , Pollen/chemistry , Resins, Synthetic/chemistry , Allergens/chemistry , Allergens/genetics , Chromatography, Liquid/instrumentation , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Rhinitis, Allergic, Seasonal/immunologyABSTRACT
Pre-packed MediaScout MiniChrom columns of 2.5, 5 and 10 mL were investigated for screening three mixed-mode chromatography sorbents (HEA, PPA and MEP HyperCel). Packing performance was of good quality and the three sorbents displayed higher capacity than traditional HIC sorbents in physiological-like conditions. Each sorbent offered a unique selectivity. Bovine beta-lactoglobulin was partially purified after loading milk whey directly on HEA HyperCel sorbent. The combination of small pre-packed columns and SELDI-MS appeared to be a valuable strategy for high-throughput screening of chromatography sorbents and for enabling rapid process development and optimization.
