Individual second virial coefficient determination of monomer and oligomers in heat-stressed protein samples using size-exclusion chromatography-light scattering

Authors

  • Miriam Printz,

    1. Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet Muenchen, D-81377 Munich, Germany
    Search for more papers by this author
  • Devendra S. Kalonia,

    1. Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269
    Search for more papers by this author
  • Wolfgang Friess

    Corresponding author
    1. Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet Muenchen, D-81377 Munich, Germany
    • Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-Universitaet Muenchen, D-81377 Munich, Germany. Telephone: +49-89-2180-77017; Fax: +49-89-2180-77020
    Search for more papers by this author

Abstract

The objective of this work was to determine the second virial coefficient (B22) of monomer and oligomer protein species in heat-stressed samples individually and simultaneously. A high-performance size-exclusion chromatography equipped with a flow-mode detector system enabling measurement of light scattering (LS) and ultraviolet transmission in the same cell was used to separate and analyze different species. The folded/unfolded nature of the protein was analyzed by extrinsic fluorescence spectroscopy using 4,4′-Bis(1-anilinonaphthalene 8-sulfonate). The B22 of each species was calculated from the concentration and LS data as described in an earlier publication. Upon heat exposure, monomer and formed oligomers yielded more negative B22 values, reflecting stronger attractive forces as compared with those for the initial monomer. The increased attractive forces are attributed to partial unfolding of the protein species, and were reflected in the extent of aggregation. On the basis of the B22 values, the monomer appeared to be the most reactive species after heat stress. In summary, the presented B22 determination technique can be used to analyze and follow the nature of intermolecular interactions for all present protein species (monomer and oligomers) and provide further understanding of the mechanism of protein aggregation and growth of aggregates. © 2011 Wiley-Liss, Inc. © 2011 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:363–372, 2012

Ancillary