Skip to main content
SpringerLink
Log in

Behavior of Monoclonal Antibodies: Relation Between the Second Virial Coefficient (B 2) at Low Concentrations and Aggregation Propensity and Viscosity at High Concentrations

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

ABSTRACT

Purpose

To investigate relationship between second virial coefficient B 2 and viscosity and aggregation propensity of highly concentrated monoclonal antibody (MAbs) solutions.

Methods

Intermolecular interactions of 3 MAbs solutions with varying pH were characterized according to B 2 estimated by analytical ultracentrifugation sedimentation equilibrium with initial loading concentrations <10 mg/mL. Viscosity measurements and stability assessments of MAb solutions at concentrations higher than 100 mg/mL were conducted.

Results

B 2 of all MAb solutions depended on solution pH and have qualitative correlation with viscosity and aggregation propensity. The more negative the B 2 values, the more viscous the solution, acquiring increased propensity to aggregate. Solutions with B 2 values of ~2 × 10−5 mL·mol/g2 acquire similar viscosity and aggregation propensity regardless of amino acid sequences; for solutions with negative B 2 values, viscosity and aggregation propensity differed depending on sequences. Results suggest attractive intermolecular interactions represented by negative B 2 values are influenced by surface properties of individual MAbs.

Conclusions

B 2 can be used, within certain limitations, as an effective indicator of viscosity and aggregation propensity of highly concentrated MAb solutions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

REFERENCES

  1. Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC. Monoclonal antibody successes in the clinic. Nat Biotechnol. 2005;23(9):1073–8.

    Article  PubMed  CAS  Google Scholar 

  2. Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004;93(6):1390–402.

    Article  PubMed  CAS  Google Scholar 

  3. Treuheit MJ, Kosky AA, Brems DN. Inverse relationship of protein concentration and aggregation. Pharm Res. 2002;19(4):511–6.

    Article  PubMed  CAS  Google Scholar 

  4. Jiménez M, Rivas G, Minton AP. Quantitative characterization of weak self-association in concentrated solutions of immunoglobulin G via the measurement of sedimentation equilibrium and osmotic pressure. Biochemistry. 2007;46:8373–8.

    Article  PubMed  Google Scholar 

  5. Liu J, Nguyen MDH, Andya JD, Shire SJ. Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci. 2005;94(9):1928–40.

    Article  PubMed  CAS  Google Scholar 

  6. Minton AP. The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. J Biol Chem. 2001;276(14):10577–80.

    Article  PubMed  CAS  Google Scholar 

  7. Minton AP. Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations. J Pharm Sci. 2005;94(8):1668–75.

    Article  PubMed  CAS  Google Scholar 

  8. Harn N, Allan C, Oliver C, Middaugh CR. Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability. J Pharm Sci. 2007;96(3):532–46.

    Article  PubMed  CAS  Google Scholar 

  9. Kamerzell TJ, Kanai S, Liu J, Shire SJ, Wang YJ. Increasing IgG concentration modulates the conformational heterogeneity and bonding network that influence solution properties. J Phys Chem. 2009;113:6109–18.

    Article  CAS  Google Scholar 

  10. Yadav S, Liu J, Shire SJ, Kalonia DS. Specific interactions in high concentration antibody solutions resulting in high viscosity. J Pharm Sci. 2010;99(3):1152–68.

    Article  PubMed  CAS  Google Scholar 

  11. Kanai S, Liu J, Patapoff TW, Shire SJ. Reversible self-association of a concentrated monoclonal antibody solution mediated by Fab–Fab interaction that impacts solution viscosity. J Pharm Sci. 2008;97(10):4219–27.

    Article  PubMed  CAS  Google Scholar 

  12. Zhang J, Liu XY. Effect of protein-protein interactions on protein aggregation kinetics. J Chem Phys. 2003;119(20):10972–6.

    Article  CAS  Google Scholar 

  13. Saluja A, Badkar AV, Zeng DL, Kalonia DS. Ultrasonic rheology of a monoclonal antibody (IgG2) solution: implications for physical stability of proteins in high concentration formulations. J Pharm Sci. 2007;96(12):3181–95.

    Article  PubMed  CAS  Google Scholar 

  14. Alford JR, Kwok SC, Roberts JN, Wuttke DS, Kendrick BS, Carpenter JF, et al. High concentration formulations of recombinant human Interleukin-1 receptor antagonist: I. Physical characterization. J Pharm Sci. 2008;97(8):3035–50.

    Article  PubMed  CAS  Google Scholar 

  15. Chari R, Jerath K, Badkar AV, Kalonia DS. Long- and short-range electrostatic interactions affect the rheology of highly concentrated antibody solutions. Pharm Res. 2009;26(12):2607–18.

    Article  PubMed  CAS  Google Scholar 

  16. Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98(9):2909–34.

    Article  PubMed  CAS  Google Scholar 

  17. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003;20(9):1325–36.

    Article  PubMed  CAS  Google Scholar 

  18. Gokarn YR, Fesinmeyer RM, Saluja A, Cao S, Dankberg J, Goetze A, et al. Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein. Protein Sci. 2009;18(1):169–79.

    PubMed  CAS  Google Scholar 

  19. Nishi H, Miyajima M, Nakagami H, Noda M, Uchiyama S, Fukui K. Phase separation of an IgG1 antibody solution under a low ionic strength condition. Pharm Res. 2010;27(7):1348–60.

    Article  PubMed  CAS  Google Scholar 

  20. Salinas BA, Sathish HA, Bishop SM, Harn N, Carpenter JF, Randolph TW. Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation. J Pharm Sci. 2010;99(1):82–93.

    Article  PubMed  CAS  Google Scholar 

  21. Wu SJ, Luo J, O’Neil KT, Kang J, Lacy ER, Canziani G, et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng Des Sel. 2010;23(8):643–51.

    Article  PubMed  CAS  Google Scholar 

  22. Nezlin R. Interactions between immunoglobulin G molecules. Immunol Lett. 2010;132(1–2):1–5.

    Article  PubMed  CAS  Google Scholar 

  23. Saluja A, Badkar AV, Zeng DL, Nema S, Kalonia DS. Ultrasonic storage modules as a novel parameter for analyzing protein-protein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements. Biophys J. 2007;92:234–44.

    Article  PubMed  CAS  Google Scholar 

  24. Holde KE, Johnson C, Ho PS. Principles of physical biochemistry. Upper Saddle River: Pearson Education; 2006.

    Google Scholar 

  25. Neal BL, Asthagiri D, Lenhoff AM. Molecular origins of osmotic second virial coefficients of proteins. Biophys J. 1998;75:2469–77.

    Article  PubMed  CAS  Google Scholar 

  26. Attri AK, Minton AP. New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins. Anal Biochem. 2005;337:103–10.

    Article  PubMed  CAS  Google Scholar 

  27. Alford JR, Kendrick BS, Carpenter JF, Randolph TW. Measurement of the second osmotic virial coefficient for protein solutions exhibiting monomer-dimer equilibrium. Anal Biochem. 2008;377:128–33.

    Article  PubMed  CAS  Google Scholar 

  28. Narayanan J, Liu XY. Protein interactions in undersaturated and supersaturated solutions: a study using light and X-ray scattering. Biophys J. 2003;84:523–32.

    Article  PubMed  CAS  Google Scholar 

  29. Brun VL, Friess W, Bassarab S, Mühlau S, Garidel P. A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein-protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody. Eur J Pharm Biopharm. 2010;75(1):16–25.

    Article  PubMed  Google Scholar 

  30. Brun VL, Friess W, Bassarab S, Garidel P. Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme. Pharm Dev Technol. 2010;15(4):421–30.

    Article  PubMed  Google Scholar 

  31. Harding SE, Rowe AJ, Horton JC. Analytical ultracentrifugation in biochemistry and polymer science. London: Royal Society of Chemistry; 1992. p. 90–125.

    Google Scholar 

  32. McGown EL, Hafeman DG. Multichannel pipettor performance verified by measuring pathlength of reagent dispensed into a microplate. Anal Biochem. 1998;258:155–7.

    Article  PubMed  CAS  Google Scholar 

  33. Sahin E, Grillo AO, Perkins MD, Roberts CJ. Comparative effects of pH and inonic strength on protein-protein intaractions, unfolding, and aggregation for IgG1 antibodies. J Pharm Sci. 2010;99(12):4830–48.

    Article  PubMed  CAS  Google Scholar 

  34. Engelsman J, Garidel P, Smulders R, Koll H, Smith B, Bassarab S, et al. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development. Pharm Res. 2011;28(4):920–33.

    Article  Google Scholar 

  35. Yadav S, Shire SJ, Kalonia DS. Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J Pharm Sci. 2010;99(12):4812–29.

    Article  PubMed  CAS  Google Scholar 

  36. Frost RA, Caroline D. Diffusion of polystyrene in a theta mixed solvent (Benzene-2-Propanol) by Photon-correlation spectroscopy. Macromolecules. 1976;10(3):616–8.

    Article  Google Scholar 

  37. Yamakawa H. Concentration dependence of the frictional coefficient of polymers in solution. 1962;36(11):2995–3001.

  38. Lehermayr C, Mahler HC, Mäder K, Fischer S. Assessment of net charge and protein–protein interactions of different monoclonal antibodies. J Pharm Sci. 2011;100(7):2551–62.

    Article  PubMed  CAS  Google Scholar 

  39. Winzor DJ, Deszczynski M, Harding SE, Wills PR. Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions. Biophys Chem. 2007;128:46–55.

    Article  PubMed  CAS  Google Scholar 

  40. Deszczynski M, Harding SE, Winzor DJ. Negative second virial coefficients as predictors of protein crystal growth: evidence from sedimentation equilibrium studies that refutes the designation of those light scattering parameters as osmotic virial coefficients. Biophys Chem. 2006;120:106–13.

    Article  PubMed  CAS  Google Scholar 

  41. Chi EY, Krishnan S, Kendrick BS, Chang BS, Carpenter JF, Randolph TW. Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Sci. 2003;12:903–13.

    Article  PubMed  CAS  Google Scholar 

  42. Hawe A, Kasper JC, Friess W, Jiskoot W. Structural properties of monoclonal antibody aggregates induced by freeze-thawing and thermal stress. Eur J Pharm Sci. 2009;38:79–87.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS & DISCLOSURES

The authors would like to thank Daisuke Ama, Kei Kubota, Yuki Araki, Mami Mitsui and Misako Sawakuri (Daiichi Sankyo Co., Ltd., Tokyo, Japan) for their skillful technical support.

Author information

Authors and Affiliations

  1. Analytical & Quality Evaluation Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo, 134-8630, Japan

    Shuntaro Saito, Jun Hasegawa, Naoki Kobayashi & Naoyuki Kishi

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

    Shuntaro Saito, Susumu Uchiyama & Kiichi Fukui

Authors
  1. Shuntaro Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jun Hasegawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naoki Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Naoyuki Kishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Susumu Uchiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kiichi Fukui
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kiichi Fukui.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material Fig. 1

Residual plots generated from least-squares fitting of the concentration dependence of 1/M W,app for MAb-A (A), MAb-B(B), and MAb-C (C) in 10 mM citrate buffer containing 140 mM NaCl (pH 6). The data obtained for the entire concentration range 1 to 12 mg/mL were used for MAb-A and MAb-C. Data obtained at lower concentrations 1 to 3 mg/mL (□) and higher concentrations 4 to 12 mg/m (■) were used for MAb-B. (JPEG 33 kb)

High Resolution Image (TIFF 3577 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saito, S., Hasegawa, J., Kobayashi, N. et al. Behavior of Monoclonal Antibodies: Relation Between the Second Virial Coefficient (B 2) at Low Concentrations and Aggregation Propensity and Viscosity at High Concentrations. Pharm Res 29, 397–410 (2012). https://doi.org/10.1007/s11095-011-0563-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-011-0563-x

KEY WORDS

Search

Navigation