High-gradient magnetic affinity separation of trypsin from porcine pancreatin

Jürgen Hubbuch, Owen R. T. Thomas

    Research output: Contribution to journalJournal articleResearchpeer-review


    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 mum) trypsin-binding adsorbents based on immobilized benzamidine were established. In small-scale studies approximate to95% of the endogenous trypsin present in a crude porcine pancreatin feedstock was recovered with a purification factor of approximate to4.1 at the expense of only a 4% loss in a-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 approximate to3.5.
    Original languageEnglish
    JournalBiotechnology and Bioengineering
    Issue number3
    Pages (from-to)301-313
    Publication statusPublished - 2002


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