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Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition

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ABSTRACT

Purpose

Phase separation of monoclonal antibody A (MAb A) solution and its relation to protein self-association are studied.

Methods

A phase diagram of MAb A and its dependence on ionic strength and pH were investigated. The protein self-associations were characterized by dynamic light scattering (DLS), analytical ultracentrifugation analysis (AUC) and viscosity measurement.

Results

MAb A solution with a clear appearance in an isotonic ionic strength condition turned opalescent in a low ionic strength condition, followed by liquid-liquid phase separation (LLPS) into light and heavy phases. The protein concentrations of the two phases were dependent on the ionic strength and pH. The two phases became reversibly miscible when the ionic strength or temperature was increased. DLS and AUC showed that MAb A under a low ionic strength condition self-associates at a protein concentration above the critical concentration of 16.5 mg/mL. The viscosity of the heavy phase was high and dependent on the shear rate. These results indicate that attractive protein-protein interaction in the heavy phase induces LLPS.

Conclusions

LLPS was induced in MAb A solution in a low ionic strength condition due to reversible protein self-association mediated mainly by attractive electrostatic interaction among the MAb A molecules in the heavy phase.

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ACKNOWLEDGEMENTS

We would like to thank Mr. Yuichi Shinozaki and Ms. Tazuko Watanabe (Anton Paar Japan K.K.) for the viscosity measurements.

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Authors and Affiliations

  1. Formulation Technology Research Laboratories, Daiichi Sankyo Co., Ltd., 1-12-1 Shinomiya, Hiratsuka, Kanagawa, 254-0014, Japan

    Hirotaka Nishi, Makoto Miyajima & Hiroaki Nakagami

  2. Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Hirotaka Nishi, Masanori Noda, Susumu Uchiyama & Kiichi Fukui

Authors
  1. Hirotaka Nishi
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  2. Makoto Miyajima
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  3. Hiroaki Nakagami
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  4. Masanori Noda
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  5. Susumu Uchiyama
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  6. Kiichi Fukui
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Corresponding author

Correspondence to Kiichi Fukui.

Electronic supplementary material

Fig. S1

Effect of polysorbate 80 concentation on the protein concentation in the light (●) and heavy (▲) phases at 25°C in low ionic strength buffer of 5 mM sodium phosphate, 10 mM sodium phosphate, 10 mM sodium chloride, 5% sucrose, pH 5.5 N = 3 (GIF 221 kb)

High Resolution Image (TIFF 120 kb)

Fig. S2

Visual appearance of MAb A solution a) at 53 mg/mL and b) at 107 mg/ml in low ionic strength buffer of 5 mM sodium phosphate, 10 mM sodium chloride, 5% sucrose, pH 5.5 at ambient temperature (GIF 372 kb)

High Resolution Image (TIFF 651 kb)

Fig. S3

Autocorrelation function obtained in DLS measurement of MAb A in isotonic ionic strength condition of 10 mM sodium phosphate, 140 mM sodium chrolide, pH 7.2 (GIF 207 kb)

High Resolution Image (TIFF 103 kb)

Fig. S4

SEC chromatogram of MAb A before (red line) and after (Blue line) LLPS (GIF 192 kb)

High Resolution Image (TIFF 88 kb)

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Nishi, H., Miyajima, M., Nakagami, H. et al. Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition. Pharm Res 27, 1348–1360 (2010). https://doi.org/10.1007/s11095-010-0125-7

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  • DOI: https://doi.org/10.1007/s11095-010-0125-7

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