High‐gradient magnetic affinity separation of trypsin from porcine pancreatin

Abstract

We introduce a robust and scale-flexible approach to macromolecule purification employing tailor-made magnetic adsorbents and high-gradient magnetic separation technology adapted from the mineral processing industries. Detailed procedures for the synthesis of large quantities of low-cost defined submicron-sized magnetic supports are presented. These support materials exhibit unique features, which facilitate their large-scale processing using high magnetic field gradients, namely sufficiently high magnetization, a relatively narrow particle size distribution and ideal superparamagnetism. Following systematic optimization with respect to activation chemistry, spacer length and ligand density, conditions for preparation of effective high capacity (Q_max = 120 mg g⁻¹) strongly interacting (K_d < 0.3 μm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies ≈95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of ≈4.1 at the expense of only a 4% loss in α-amylase activity. Efficient recovery of trypsin from the same feedstock was demonstrated at a vastly increased scale using a high-gradient magnetic separation system to capture loaded benzamidine-linked adsorbents following batch adsorption. With the aid of a simple recycle loop over 80% of the initially adsorbed trypsin was recovered in-line with an overall purification factor of ≈3.5. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 301–313, 2002.