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Thermodynamic and Fluorescence Analyses to Determine Mechanisms of IgG1 Stabilization and Destabilization by Arginine

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ABSTRACT

Purpose

To investigate mechanisms governing the stabilization and destabilization of immunoglobulin (IgG1) by arginine (Arg).

Methods

The effects of Arg on the aggregation/degradation, thermodynamic stability, hydrophobicity, and aromatic residues of IgG1 were respectively investigated by size-exclusion chromatography, differential scanning calorimetry, probe fluorescence, and intrinsic fluorescence.

Results

Arg monohydrochloride (Arg–HCl) suppressed IgG1 aggregation at near-neutral pH, but facilitated aggregation and degradation at acidic pH or at high storage temperature. Equimolar mixtures of Arg and aspartic acid (Asp) or glutamic acid (Glu) suppressed aggregation without facilitating degradation even at high temperature. Arg–HCl decreased the thermodynamic stability of IgG1 by enthalpic loss, which was counteracted by using Asp or Glu as a counterion for Arg. The suppression of aggregation by Arg–HCl was well correlated with the decrease in hydrophobicity of IgG1. The intrinsic fluorescence of IgG1 was unaffected by Arg–HCl.

Conclusions

Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

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Abbreviations

Arg–Asp:

Arginine–aspartic acid mixture

Arg–Glu:

Arginine–glutamic acid mixture

Arg–HCl:

Arginine monohydrochloride

Bis-ANS:

4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt

C p :

Molar heat capacity at constant pressure

DSC:

Differential scanning calorimetry

SEC:

Size-exclusion chromatography

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors thank Dr. Kohei Tsumoto (Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo) for thoughtful discussions. The authors also thank many individuals of our departments for their helpful comments.

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Authors and Affiliations

  1. Product Engineering Department, Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, Kita-ku, Tokyo, 115-8543, Japan

    Masakazu Fukuda, Daisuke Kameoka, Masaya Yasutake, Yoshimi Imaeda & Akihiko Mizutani

  2. CMC Development Department, Chugai Pharmaceutical Co., Ltd., 5-5-1 Ukima, Kita-ku, Tokyo, 115-8543, Japan

    Satoshi Saitoh & Akiko Koga

  3. Discovery Research Department, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura City, Kanagawa, 247-8530, Japan

    Takuya Torizawa

Authors
  1. Masakazu Fukuda
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  2. Daisuke Kameoka
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  3. Takuya Torizawa
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  4. Satoshi Saitoh
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  5. Masaya Yasutake
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  6. Yoshimi Imaeda
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  7. Akiko Koga
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  8. Akihiko Mizutani
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Correspondence to Masakazu Fukuda.

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Fukuda, M., Kameoka, D., Torizawa, T. et al. Thermodynamic and Fluorescence Analyses to Determine Mechanisms of IgG1 Stabilization and Destabilization by Arginine. Pharm Res 31, 992–1001 (2014). https://doi.org/10.1007/s11095-013-1221-2

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  • DOI: https://doi.org/10.1007/s11095-013-1221-2

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