Enterokinase monolithic bioreactor as an efficient tool for biopharmaceuticals preparation: on-line cleavage of fusion proteins and analytical characterization of released products

As enzyme immobilization increases stability and reusability of the biocatalyst while reducing operating costs and sample contamination, in this work we report the covalent immobilization of recombinant EK (rEK) on monolithic chromatographic supports with different binding chemistries for the development of a rEK-chromatographic-bioreactor. An on-line assay for the determination of the activity of the immobilized rEK was set up using a synthetic substrate (Gly-Asp4-Lys-β-naphthylamide, GD4K-NA). The assay was used to study the improvement of the operational conditions (temperature and flow rate) on hydrolytic activity of the bioreactor. The immobilization yields, as well as the cleavage activity of immobilized rEK on GD4K-NA, were highly satisfactory when the immobilized enzyme reactor was used in recirculation. The ability of the immobilized rEK to cleave fusion proteins was tested by recirculation of thioredoxin (Trx)-TB10.4 and Trx-Ag85B His-tagged proteins yielding the mature antigens TB10.4 and Ag85B, to be used in the preparation of potential novel glycovaccines against tuberculosis. The prepared rEK-based immobilized enzyme reactors proved to efficiently cleave the considered fusion proteins even if the cleavage specificity at the canonical site was not fully achieved. The immobilized rEK showed very good stability and reusability.