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Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents

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Abstract

Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol %), N-n-octylacrylamide (25 mol %) and N-isopropylacrylamide (40 mol %), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies.

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Notes

  1. Communication with the supplier indicates that the short fragments are not removed readily from the IgG monomer.

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Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada (Collaborative Strategic Program) and by the Agence Nationale pour la Recherche (program Blanc International, grant INT 1501 and program Investissement d’Avenir ANR-11-LABX-0011-01). F.M.W. also acknowledges financial support from the World Premier International Research Center Initiative (MPI) MEXT Japan.

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Authors and Affiliations

  1. Department of Chemistry and Faculty of Pharmacy, Université de Montréal, CP 6128 Succursale Centre Ville, Montréal, H3C 3J7, QC, Canada

    Dewang Ma & Françoise M. Winnik

  2. Ecole Normale Supérieure-PSL Research University, Département de Chimie, Sorbonne Universités - UPMC Univ Paris 06, CNRS UMR 8640 PASTEUR, 24, rue Lhomond, 75005, Paris, France

    Nicolas Martin & Christophe Tribet

  3. WPI International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan

    Françoise M. Winnik

  4. Department of Chemistry and Faculty of Pharmacy, University of Helsinki, 00014, Helsinki, Finland

    Françoise M. Winnik

Authors
  1. Dewang Ma
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  2. Nicolas Martin
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  3. Christophe Tribet
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  4. Françoise M. Winnik
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Correspondence to Françoise M. Winnik.

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Ma, D., Martin, N., Tribet, C. et al. Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents. Anal Bioanal Chem 406, 7539–7547 (2014). https://doi.org/10.1007/s00216-014-8200-2

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  • DOI: https://doi.org/10.1007/s00216-014-8200-2

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