High‐gradient magnetic affinity separation of trypsin from porcine pancreatin

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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 (Qmax = 120 mg g−1) strongly interacting (Kd < 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.