Dynamics of protein uptake within the adsorbent particle during packed bed chromatography

Authors

  • Jürgen Hubbuch,

    1. Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Jölich, Germany; telephone: +49 2461 616 966; fax: +49 2461 612 490
    Search for more papers by this author
  • Thomas Linden,

    1. Merck & Co., Inc., Vaccine Bioprocess R&D, Sumneytown Pike, West Point, Pennsylvania 19486-0004
    Search for more papers by this author
  • Esther Knieps,

    1. Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Jölich, Germany; telephone: +49 2461 616 966; fax: +49 2461 612 490
    Search for more papers by this author
  • Jörg Thömmes,

    1. IDEC Pharmaceuticals Corp., 11011 Torreyana Road, San Diego, California 92121
    Search for more papers by this author
  • Maria-Regina Kula

    Corresponding author
    1. Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Jölich, Germany; telephone: +49 2461 616 966; fax: +49 2461 612 490
    • Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf, 52426 Jölich, Germany; telephone: +49 2461 616 966; fax: +49 2461 612 490
    Search for more papers by this author

Abstract

A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical “shrinking core” behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 359–368, 2002.

Ancillary