High-gradient magnetic affinity separation of trypsin from porcine pancreatin.
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 (Kd < 0.3 microm) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies approximately 95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximately 4.1 at the expense of only a 4% loss in alpha-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 approximately 3.5.
Hubbuch
Jürgen J
JJ
Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800, Kgs. Lyngby, Denmark.
Thomas
Owen R T
OR
D000327:Adsorption; D000818:Animals; D001550:Benzamidines / Q000737:chemistry*; D002417:Cattle; D046911:Macromolecular Substances; D008280:Magnetics; D008956:Models, Chemical; D010194:Pancreatin / Q000737:chemistry*; D010865:Pilot Projects; D011786:Quality Control; D012680:Sensitivity and Specificity; D013552:Swine; D014357:Trypsin / Q000302:isolation & purification*
D001550:Benzamidines; D046911:Macromolecular Substances; D010194:Pancreatin; D014357:Trypsin; C032157:benzamidine