Pharmaceutical Research

, Volume 27, Issue 7, pp 1348–1360 | Cite as

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

  • Hirotaka Nishi
  • Makoto Miyajima
  • Hiroaki Nakagami
  • Masanori Noda
  • Susumu Uchiyama
  • Kiichi FukuiEmail author
Research Paper



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


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.


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.


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.


low ionic strength condition monoclonal antibody opalescence phase separation self-association 



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

Supplementary material

11095_2010_125_Fig10_ESM.gif (221 kb)
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)

11095_2010_125_MOESM1_ESM.tif (121 kb)
High Resolution Image (TIFF 120 kb)
11095_2010_125_Fig11_ESM.gif (373 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)

11095_2010_125_MOESM2_ESM.tif (651 kb)
High Resolution Image (TIFF 651 kb)
11095_2010_125_Fig12_ESM.gif (207 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)

11095_2010_125_MOESM3_ESM.tif (103 kb)
High Resolution Image (TIFF 103 kb)
11095_2010_125_Fig13_ESM.gif (193 kb)
Fig. S4

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

11095_2010_125_MOESM4_ESM.tif (89 kb)
High Resolution Image (TIFF 88 kb)


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hirotaka Nishi
    • 1
    • 2
  • Makoto Miyajima
    • 1
  • Hiroaki Nakagami
    • 1
  • Masanori Noda
    • 2
  • Susumu Uchiyama
    • 2
  • Kiichi Fukui
    • 2
    Email author
  1. 1.Formulation Technology Research LaboratoriesDaiichi Sankyo Co., Ltd.HiratsukaJapan
  2. 2.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan

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