A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

Kim Ekelund Ottow, Trine Lütken Maury, Timothy John Hobley, Torsten Lund-Olesen, Mikkel Fougt Hansen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.
    Original languageEnglish
    JournalBiotechnology Journal
    Volume6
    Issue number4
    Pages (from-to)396-409
    ISSN1860-6768
    DOIs
    Publication statusPublished - 2011

    Keywords

    • Trypsin
    • Continuous process
    • Protein modification
    • High gradient magnetic fishing

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