Biopharmaceutical Evaluation of Intermolecular Interactions by AUC-SE

  • Shuntaro Saito
  • Susumu UchiyamaEmail author


Analytical ultracentrifugation sedimentation equilibrium (AUC-SE) is a useful technique to investigate the weak reversible intermolecular interactions among proteins in solution. It provides biophysical information such as the average apparent molecular weight, stoichiometry, and association constant of associating proteins. Several studies of intermolecular interaction in biopharmaceuticals by AUC-SE are introduced in this chapter. AUC-SE also provides the second virial coefficient (B2), which represents the type, i.e., repulsive and attractive, and a magnitude of intermolecular interactions. The B2 values obtained from the protein solution at low concentrations showed good correlation with aggregation and viscosity of MAb at high concentrations, indicating that B2 can be an effective indicator of aggregation propensity and viscosity. These findings suggest that AUC-SE provides clues to understand the self-association in biopharmaceuticals and to establish effective manufacturing process, formulation, and administration of biopharmaceuticals.


Analytical ultracentrifugation sedimentation equilibrium Self-association Colloidal stability Aggregation Viscosity Second virial coefficient Antibody 


  1. Alford JR, Kendrick BS, Carpenter JF, Randolph TW (2008) Measurement of the second osmotic virial coefficient for protein solutions exhibiting monomer-dimer equilibrium. Anal Biochem 377:128–133CrossRefPubMedPubMedCentralGoogle Scholar
  2. Attri AK, Minton AP (2005) New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins. Anal Biochem 337:103–110CrossRefPubMedGoogle Scholar
  3. Bajaji H, Sharma VK, Badkar A, Zeng D, Nema S, Kalonia DS (2006) Protein structural conformation and not second virial coefficient relates to long-term irreversible aggregation of a monoclonal antibody and ovalbumin in solution. Pharm Res 23:1382–1394CrossRefGoogle Scholar
  4. Bhambhani A, Kissmann JM, Joshi SB, Vokin DB, Kashi RS, Middaugh CR (2012) Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions. J Pharm Sci 101:1120–1135CrossRefPubMedGoogle Scholar
  5. Brun VL, Friess W, Bassarab S, Mühlau S, Garidel P (2010a) 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 75:16–25CrossRefPubMedGoogle Scholar
  6. Brun VL, Friess W, Schultz-Fademrecht T, Muehlau S, Garidel P (2009) Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: impact for physical protein stabilization. Biotechnol J 4:1305–1319CrossRefPubMedGoogle Scholar
  7. Brun VL, Friess W, Bassarab S, Garidel P (2010b) Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: a case study with lysozyme. Pharm Develop Technol 15:421–430CrossRefGoogle Scholar
  8. Chari R, Jerath K, Badkar AV, Kalonia DS (2009) Long- and short-range electrostatic interactions affect the rheology of highly concentrated antibody solutions. Pharm Res 26:2607–2618CrossRefPubMedGoogle Scholar
  9. Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003a) Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res 20:1325–1336CrossRefPubMedGoogle Scholar
  10. Chi EY, Krishnan S, Kendrick BS, Chang BS, Carpenter JF, Randolph TW (2003b) Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony-stimulating factor. Protein Sci 12:903–913CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chou DK, Krishnamurthy R, Manning MC, Randolph TW, Carpenter JF (2012) Physical stability of albinterferon-α2b in aqueous solution: effects of conformational stability and colloidal stability on aggregation. J Pharm Sci 101:2702–2719CrossRefPubMedGoogle Scholar
  12. Deszczynski M, Harding SE, Winzor DJ (2006) 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 120:106–113CrossRefPubMedGoogle Scholar
  13. Frost RA, Caroline D (1976) Diffusion of polystyrene in a theta mixed solvent (Benzene-2-Propanol) by Photon-correlation spectroscopy. Macromolecules 10:616–618CrossRefGoogle Scholar
  14. Garidel P, Blume A, Wagner M (2013) Prediction of colloidal stability of high protein formulations. Pharm Dev Technol 20(3):367–374CrossRefGoogle Scholar
  15. Goldberg DS, Bishop SM, Shah AU, Satish HA (2011) Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational stability and colloidal stability. J Pharm Sci 100:1306–1315Google Scholar
  16. Jiménez M, Rivas G, Minton AP (2007) Quantitative characterization of weak self-association in concentrated solutions of immunoglobulin G via the measurement of sedimentation equilibrium and osmotic pressure. Biochemistry 46:8373–8378CrossRefPubMedGoogle Scholar
  17. Kumar V, Dixit N, Zhou L, Fraunhofer W (2011) Impact of short range hydrophobic interactions and long range electrostatic forces on the aggregation kinetics of a monoclonal antibody and a dual-variable domain immunoglobulin at low and high concentrations. Int J Pharm 421:82–93CrossRefPubMedGoogle Scholar
  18. Laue T (2012) Proximity energies: a framework for understanding concentrated solutions. J Mol Recognit 25:165–173CrossRefPubMedGoogle Scholar
  19. Laue T, Shah BD, Ridgeway TM, Pelletier SL (1992) Analytical ultracentrifugation in biochemistry and polymer science. Royal Society of Chemistry, London, pp 90–125Google Scholar
  20. Lehermayr C, Mahler HC, Mäder K, Fischer S (2011) Assessment of net charge and protein-protein interactions of different monoclonal antibodies. J Pharm Sci 100:2551–2562CrossRefPubMedGoogle Scholar
  21. Liu J, Nguyen MDH, Andya JD, Shire SJ (2005) Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci 94:1928–1940CrossRefPubMedGoogle Scholar
  22. McMillan WG Jr, Mayer JE (1945) The statistical thermodynamics of multicomponent systems. J Chem Phys 13:276–305CrossRefGoogle Scholar
  23. Narayanan J, Liu XY (2003) Protein interactions in undersaturated and supersaturated solutions: a study using light and x-ray scattering. Biophys J 84:523–532CrossRefPubMedPubMedCentralGoogle Scholar
  24. Neal BL, Asthagiri D, Lenhoff AM (1998) Molecular origins of osmotic second virial coefficients of proteins. Biophys J 75:2469–2477. Holde et al., 2006CrossRefPubMedPubMedCentralGoogle Scholar
  25. Neergaard MS, Kalonia DS, Parshad H, Nielsen AD, Møller EH, van de Weert M (2013) Viscosity of high concentration protein formulations of monoclonal antibodies of the IgG1 and IgG4 subclass-prediction of viscosity through protein-protein interaction measurements. Eur J Pharm Biopharm 49:400–410Google Scholar
  26. Reichert JM, Rosensweig CJ, Faden LB, Dewitz MC (2005) Monoclonal antibody successes in the clinic. Nat Biotechnol 23:1073–1078CrossRefPubMedGoogle Scholar
  27. Sahin E, Grillo AO, Perkins MD, Roberts CJ (2010) Comparative effects of pH and ionic strength on protein-protein interactions, unfolding, and aggregation for IgG1 antibodies. J Pharm Sci 99:4830–4848CrossRefPubMedGoogle Scholar
  28. Saito S, Hasegawa J, Kobayashi N, Kishi N, Uchiyama S, Fukui K (2012) 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 29:397–410CrossRefPubMedGoogle Scholar
  29. Saito S, Hasegawa J, Kobayashi N, Tomitsuka T, Uchiyama S, Fukui K (2013) Effects of ionic strength and sugars on the aggregation propensity of monoclonal antibodies: influence of colloidal and conformational stabilities. Pharm Res 30:1263–1280CrossRefPubMedGoogle Scholar
  30. Salinas BA, Sathish HA, Bishop SM, Harn N, Carpenter JF, Randolph TW (2010) Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation. J Pham Sci 99:82–93CrossRefGoogle Scholar
  31. Saluja A, Badkar AV, Zeng DL, Kalonia DS (2007) Ultrasonic rheology of a monoclonal antibody (IgG2) solution: implications for physical stability of proteins in high concentration formulations. J Pharm Sci 96:3181–3195CrossRefPubMedGoogle Scholar
  32. Saluja A, Fesinmeyer M, Hogan S, Brems DN, Gokarn YR (2010) Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation. Biophys J 99:2657–2665CrossRefPubMedPubMedCentralGoogle Scholar
  33. Schmit JD, He F, Mishra S, Ketchem RR, Woods CE, Kerwin BA (2014) Entanglement model of antibody viscosity. J Phys Chem B 118:5044–5049CrossRefPubMedGoogle Scholar
  34. Shire SJ, Shahrokh Z, Liu J (2004) Challenges in the development of high protein concentration formulations. J Pharm Sci 93:1390–1402CrossRefPubMedGoogle Scholar
  35. Singh SN, Yadav S, Shire SJ, Kalonia DS (2014) Dipole-dipole interaction in antibody solutions: correlation with viscosity behavior at high concentration. Pharm Res 31:2549–2558CrossRefPubMedGoogle Scholar
  36. Sule SV, Cheung JK, Antochshuk V, Bhalla AS, Narasimhan C, Blaisdell S, Shameem M, Tessier PM (2012) Solution pH that minimizes self-association of three monoclonal antibodies is strongly dependent on ionic strength. Mol Pharm 9:744–751CrossRefPubMedGoogle Scholar
  37. Tessier PM, Lenhoff AM, Sandler SI (2002) Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography. Biophys J 82:1620–1631CrossRefPubMedPubMedCentralGoogle Scholar
  38. Treuheit MJ, Kosky AA, Brems DN (2002) Inverse relationship of protein concentration and aggregation. Pharm Res 19:511–516CrossRefPubMedGoogle Scholar
  39. van Holde, KE, Johnson C, Ho PS (2006) Physical biochemistry, Pearson Education Inc., New YorkGoogle Scholar
  40. Verwey EJW, Overbeck JTK (1948) Theory of stability of lyophobic colloids. Elsevier, AmsterdamGoogle Scholar
  41. Winzor DJ, Deszczynski M, Harding SE, Wills PR (2007) Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions. Biophys Chem 128:46–55CrossRefPubMedGoogle Scholar
  42. Williams (1972) Ultracentrifugation of macromelecules: modern topics, Academic Press, New YorkGoogle Scholar
  43. Yadav S, Shire SJ, Kalonia DS (2010) Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J Pharm Sci 99:4812–4829CrossRefPubMedGoogle Scholar
  44. Yadav S, Laue TM, Kalonia DS, Singh SN, Shire SJ (2012) The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions. Mol Pharm 9:791–802CrossRefPubMedGoogle Scholar
  45. Yamakawa H (1962) Concentration dependence of the frictional coefficient of polymers in solution. J Chem Phys 36:2995–3001CrossRefGoogle Scholar
  46. Zhang J, Liu XY (2003) Effect of protein-protein interactions on protein aggregation kinetics. J Chem Phys 119:10972–10976CrossRefGoogle Scholar

Copyright information

© Springer Japan 2016

Authors and Affiliations

  1. 1.Biologics Technology Research LaboratoriesDaiichi Sankyo Co., Ltd.Hiratsuka-shiJapan
  2. 2.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan

Personalised recommendations