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Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation

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Abstract

Hollow-fiber-flow field-flow fractionation (HF5) separates protein molecules on the basis of the difference in the diffusion coefficient, and can evaluate the aggregation ratio of proteins. However, HF5 is still a minor technique because information on the separation conditions is limited. We examined in detail the effect of different settings, including the main-flow rate, the cross-flow rate, the focus point, the injection amount, and the ionic strength of the mobile phase, on fractographic characteristics. On the basis of the results, we proposed optimized conditions of the HF5 method for quantification of monoclonal antibody in sample solutions. The HF5 method was qualified regarding the precision, accuracy, linearity of the main peak, and quantitation limit. In addition, the HF5 method was applied to non-heated Mab A and heat-induced-antibody-aggregate-containing samples to evaluate the aggregation ratio and the distribution extent. The separation performance was comparable with or better than that of conventional methods including analytical ultracentrifugation–sedimentation velocity and asymmetric-flow field-flow fractionation.

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Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

    Jun Fukuda & Kenji Kano

  2. Bio Process Research and Development Laboratories, Production Division, Kyowa Hakko Kirin Co., Ltd., Hagiwara-machi, Takasaki, Gunma, 370-0013, Japan

    Jun Fukuda, Takafumi Iwura & Shigehiro Yanagihara

Authors
  1. Jun Fukuda
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  2. Takafumi Iwura
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  3. Shigehiro Yanagihara
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  4. Kenji Kano
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Corresponding authors

Correspondence to Jun Fukuda or Kenji Kano.

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Fukuda, J., Iwura, T., Yanagihara, S. et al. Separation and quantification of monoclonal-antibody aggregates by hollow-fiber-flow field-flow fractionation. Anal Bioanal Chem 406, 6257–6264 (2014). https://doi.org/10.1007/s00216-014-8065-4

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  • DOI: https://doi.org/10.1007/s00216-014-8065-4

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