On-Column Preparation of High-Performance Affinity Chromatography Columns.

We describe a custom preparation of high-performance affinity columns based on 3.5 μm nonporous polymeric particles covalently modified with streptavidin. The sorbent was packed into a 2.1 × 20 mm column hardware compatible with pressures up to 10,000 psi (68 MPa). Biotinylated affinity ligands were then immobilized on-column using a repetitive injection method until the streptavidin sorbent was saturated. The saturation was observed as a ligand breakthrough signal by ultraviolet detection. Column binding capacity of immobilized affinity ligands was estimated for d-biotin, biotinylated 25nt oligodeoxythymidine, and biotinylated anti-insulin mAb ligands. The prepared columns were used to quantify mRNA and insulin. Affinity column performance was evaluated in terms of binding capacity, calibration linearity range, and target analyte limit of quantitation. The high-performance streptavidin column is a platform for custom-made affinity chromatography device preparation, enabling routine quantitation of analytes of interest.