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Effect of Ions on Agitation- and Temperature-Induced Aggregation Reactions of Antibodies

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Abstract

Purpose

The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies.

Methods

Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG2 mAbs. The CH2 domain melting temperature (T m1) and reduced valence (z*) of the mAbs were measured.

Results

Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F− < Cl− < Br− < I− < SCN− ~ ClO4 −) and correlated with a decrease in T m1 and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li+, Na+, K+, Rb+, or Cs+) had no effect on T m1, z* or aggregation upon agitation.

Conclusions

The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.

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Acknowledgements

The authors would like to thank Prof. Thomas M. Laue and Ms. Susan Chase of the University of New Hampshire for their help with the reduced valence measurement method.

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Authors and Affiliations

  1. Department of Process and Product Development, AW2/D-3152, Amgen Court West, Seattle, Washington, 98119, USA

    R. Matthew Fesinmeyer, Sabine Hogan, Atul Saluja, Eva Kras & Yatin R. Gokarn

  2. Department of Process and Product Development, Thousand Oaks, California, 91320, USA

    Stephen R. Brych, Linda O. Narhi & David N. Brems

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  1. R. Matthew Fesinmeyer
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  2. Sabine Hogan
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Correspondence to Yatin R. Gokarn.

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R. Matthew Fesinmeyer and Sabine Hogan have contributed equally to this work.

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Fesinmeyer, R.M., Hogan, S., Saluja, A. et al. Effect of Ions on Agitation- and Temperature-Induced Aggregation Reactions of Antibodies. Pharm Res 26, 903–913 (2009). https://doi.org/10.1007/s11095-008-9792-z

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  • DOI: https://doi.org/10.1007/s11095-008-9792-z

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