Analytical Characterization and Predictive Tools for Highly Concentrated Protein Formulations

  • Andrea AllmendingerEmail author
  • Stefan Fischer
  • Robert Mueller
Part of the AAPS Advances in the Pharmaceutical Sciences Series book series (AAPS, volume 38)


This chapter focuses on different aspects of development and characterization of highly concentrated protein formulations. This includes the introduction of predictive tools used during early development, specific challenges for analytical methods, and extended characterization of rheological and structural properties. The application of predictive tools during clinical lead selection and early-stage development helps to assess whether a selected clinical candidate may successfully pass the challenges of a high-concentration product in later development. In particular, parameters like B22 (second virial coefficient) and kD (diffusional interaction parameter) are described and underlying principles, technologies, limitations, and applicability to highly concentrated formulations are elaborated. The specific challenges of high-concentration protein formulations with respect to analytical characterization are discussed in detail and practical recommendation is given. Different common techniques to measure viscosity are described including dependencies on protein concentration, temperature, and shear stress. Structural characterization of highly concentrated protein formulations requires analytical tools capable to measure in the non-diluted regime. Only a limited number of analytical techniques are available for this challenging task like small-angle scattering techniques (SAS) and quartz crystal impedance analysis (QCIA)/microbalance with dissipation monitoring (QCM-D). These techniques are described, and measuring principle, applications in literature as well as limitations are outlined.


High-concentration protein formulation Protein–protein interaction Analytical characterization Non-ideality Second virial coefficient Diffusion coefficient Rheology Viscosity Protein aggregation Opalescence Small-Angle scattering techniques 


  1. 1.
    Jarasch A, et al. Developability assessment during the selection of novel therapeutic antibodies. J Pharm Sci. 2015;104(6):1885–98.PubMedCrossRefGoogle Scholar
  2. 2.
    Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market. MAbs. 2015;7(1):9–14.PubMedCrossRefGoogle Scholar
  3. 3.
    Salinas BA, et al. Understanding and modulating opalescence and viscosity in a monoclonal antibody formulation. J Pharm Sci. 2010;99(1):82–93.PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Curtis RA, Prausnitz JM, Blanch HW. Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes. Biotechnol Bioeng. 1998;57(1):11–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Perchiacca JM, Tessier PM. Engineering aggregation-resistant antibodies. Annu Rev Chem Biomol Eng. 2012;3:263–86.PubMedCrossRefGoogle Scholar
  6. 6.
    Vazquez-Rey M, Lang DA. Aggregates in monoclonal antibody manufacturing processes. Biotechnol Bioeng. 2011;108(7):1494–508.PubMedCrossRefGoogle Scholar
  7. 7.
    Nishi H, et al. Phase separation of an IgG1 antibody solution under a low ionic strength condition. Pharm Res. 2010;27(7):1348–60.PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Cromwell ME, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J. 2006;8(3):E572–9.PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Scherer TM, et al. Intermolecular interactions of IgG1 monoclonal antibodies at high concentrations characterized by light scattering. J Phys Chem B. 2010;114(40):12948–57.PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Some D. Light-scattering-based analysis of biomolecular interactions. Biophys Rev. 2013;5(2):147–58.PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Arzensek D, Kuzman D, Podgornik R. Colloidal interactions between monoclonal antibodies in aqueous solutions. J Colloid Interface Sci. 2012;384(1):207–16.PubMedCrossRefGoogle Scholar
  12. 12.
    Moon YU, et al. Osmotic pressures and second virial coefficients for aqueous saline solutions of lysozyme. Fluid Phase Equilib. 2000;168(2):229–39.CrossRefGoogle Scholar
  13. 13.
    Patro SY, Przybycien TM. Self-interaction chromatography: a tool for the study of protein-protein interactions in bioprocessing environments. Biotechnol Bioeng. 1996;52(2):193–203.PubMedCrossRefGoogle Scholar
  14. 14.
    Hedberg SH, et al. Self-interaction chromatography of mAbs: accurate measurement of dead volumes. Pharm Res. 2015;32(12):3975–85.PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Tessier PM, Lenhoff AM, Sandler SI. Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography. Biophys J. 2002;82(3):1620–31.PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Payne RW, et al. Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography. Biopolymers. 2006;84(5):527–33.PubMedCrossRefGoogle Scholar
  17. 17.
    Saito S, 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. 2012;29(2):397–410.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Connolly BD, et al. Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter. Biophys J. 2012;103(1):69–78.PubMedPubMedCentralCrossRefGoogle Scholar
  19. 19.
    Lehermayr C, et al. Assessment of net charge and protein-protein interactions of different monoclonal antibodies. J Pharm Sci. 2011;100(7):2551–62.PubMedCrossRefGoogle Scholar
  20. 20.
    Yadav S, et al. The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions. Mol Pharm. 2012;9(4):791–802.PubMedCrossRefGoogle Scholar
  21. 21.
    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.PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    He F, et al. High-throughput assessment of thermal and colloidal stability parameters for monoclonal antibody formulations. J Pharm Sci. 2011;100(12):5126–41.PubMedCrossRefGoogle Scholar
  23. 23.
    Warne NW. Development of high concentration protein biopharmaceuticals: the use of platform approaches in formulation development. European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V. 2011;78(2):208–12.PubMedCrossRefGoogle Scholar
  24. 24.
    Raut AS, Kalonia DS. Opalescence in monoclonal antibody solutions and its correlation with intermolecular interactions in dilute and concentrated solutions. J Pharm Sci. 2015;104(4):1263–74.PubMedCrossRefGoogle Scholar
  25. 25.
    Weinbuch D, Jiskoot W, Hawe A. Light obscuration measurements of highly viscous solutions: sample pressurization overcomes underestimation of subvisible particle counts. AAPS J. 2014;16(5):1128–31.PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Werk T, Volkin DB, Mahler HC. Effect of solution properties on the counting and sizing of subvisible particle standards as measured by light obscuration and digital imaging methods. European J Pharm Sci Official J Eur Fed Pharm Sci. 2014;53:95–108.Google Scholar
  27. 27.
    USP, General Chapters: <787> Subvisible particulate matter in therapeutic protein injections, in USP 392016.Google Scholar
  28. 28.
    Manning MC, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res. 1989;6(11):903–18.PubMedCrossRefGoogle Scholar
  29. 29.
    Mahler HC, et al. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98(9):2909–34.PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Morris AM, Watzky MA, Finke RG. Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature. Biochim Biophys Acta. 2009;1794(3):375–97.PubMedCrossRefGoogle Scholar
  31. 31.
    Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004;93(6):1390–402.PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289(1–2):1–30.PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Manning MC, et al. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544–75.PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Cromwell ME, Hilario E, Jacobson F. Protein aggregation and bioprocessing. AAPS J. 2006;08(03):E572–9.CrossRefGoogle Scholar
  35. 35.
    Gabrielson JP, et al. Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. J Pharm Sci. 2007;96(2):268–79.PubMedCrossRefGoogle Scholar
  36. 36.
    Philo JS. A critical review of methods for size characterization of non-particulate protein aggregates. Curr Pharm Biotechnol. 2009;10(4):359–72.PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Li YI, Weiss WF, Roberts CJ. Characterization of high-molecular-weight nonnative aggregates and aggregation kinetics by size exclusion chromatography with inline multi-angle laser light scattering. J Pharm Sci. 2009;98(11):3997–4016.PubMedCrossRefGoogle Scholar
  38. 38.
    Liu J, Shire SJ. Analytical ultracentrifugation in the pharmaceutical industry. J Pharm Sci. 1999;88(12):1237–41.PubMedCrossRefGoogle Scholar
  39. 39.
    Liu J, et al. Reversible self-association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci. 2005;94(9):1928–40.PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Caldwell KD, Wahlund K-G. Field-flow fractionation. In: Jiskoot W, Crommelin FJA, editors. Methods for structural analysis of protein pharmaceuticals. AAPS; 2005. p. 379–412.Google Scholar
  41. 41.
    Zhu R, et al. Studying protein aggregation by programmed flow field-flow fractionation using ceramic hollow fibers. Anal Chem. 2005;77(14):4581–6.PubMedCrossRefGoogle Scholar
  42. 42.
    Litzen A, et al. Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography. Anal Biochem. 1993;212(2):469–80.PubMedCrossRefGoogle Scholar
  43. 43.
    Shire SJ. Formulation and manufacturability of biologics. Curr Opin Biotechnol. 2009;20(6):708–14.PubMedCrossRefGoogle Scholar
  44. 44.
    Burckbuchler V, et al. Rheological and syringeability properties of highly concentrated human polyclonal immunoglobulin solutions. Eur J Pharm Biopharm. 2010;76(3):351–6.PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Tanford C, editor. Physical chemistry of macromolecules. New York: Wiley; 1961.Google Scholar
  46. 46.
    Kanai S, et al. Reversible self-association of a concentrated monoclonal antibody solution mediated by Fab-Fab interaction that impacts solution viscosity. J Pharm Sci. 2008.Google Scholar
  47. 47.
    Chhabra RP, Richardson JF. Non-newtonian flow and applied rheology—engineering applications. 2nd ed. Elsevier.CrossRefGoogle Scholar
  48. 48.
    Allmendinger A, et al. Rheological characterization and injection forces of concentrated protein formulations: an alternative predictive model for non-newtonian solutions. European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V. 2014; 87(2):318–28.PubMedCrossRefGoogle Scholar
  49. 49.
    Allmendinger A, et al. Sterile filtration of highly concentrated protein formulations: impact of protein concentration, formulation composition, and filter material. J Pharm Sci. 2015;104(10):3319–29.PubMedCrossRefGoogle Scholar
  50. 50.
    Rathore N, et al. Characterization of protein rheology and delivery forces for combination products. J Pharm Sci. 2012;101(12):4472–80.PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Fischer I, et al. Calculation of injection forces for highly concentrated protein solutions. Int J Pharm. 2015;493(1–2):70–4.PubMedCrossRefGoogle Scholar
  52. 52.
    Osswald T, Rudolph N. Polymer rheology—fundamentals and applications. Hanser Publishers.Google Scholar
  53. 53.
    Ostwald W. Ueber die Geschwindigkeitsfunktion der Viskositaet disperser Systeme. Kolloid-Z. 1925;36(99).Google Scholar
  54. 54.
    Malkin AY, editor. Rheology fundamentals. ChemTec Publishing; 1994.Google Scholar
  55. 55.
    Bird RB, Carreau PJ. A nonlinear viscoelastic model for polymer solutions and melts. Chem Eng Sci. 1968;23:427–34.CrossRefGoogle Scholar
  56. 56.
    Yasuda K, Armstrong RC, Cohen RE. Shear flow properties of concentrated solutions of linear and star branched polystyrenes. Rheol Acta. 1981;20:163–78.CrossRefGoogle Scholar
  57. 57.
    Saluja A, Kalonia DS. Nature and consequences of protein-protein interactions in high protein concentration solutions. Int J Pharm. 2008;358(1–2):1–15.PubMedPubMedCentralCrossRefGoogle Scholar
  58. 58.
    Jezek J, et al. Viscosity of concentrated therapeutic protein compositions. Adv Drug Deliv Rev. 2011;63(13):1107–17.PubMedCrossRefGoogle Scholar
  59. 59.
    Teraoka I, editor. Polymer solutions: an introduction to physical properties. New York: Wiley; 2002.Google Scholar
  60. 60.
    Goodwin JW, Hughes RW, editors. Rheology for chemists—an introduction. Cambridge: Royal Society of Chemistry; 2000.Google Scholar
  61. 61.
    Ross PD, Minton AP. Hard quasispherical model for the viscosity of hemoglobin solutions. Biochem Biophys Res Commun. 1977;76(4):971–6.PubMedCrossRefGoogle Scholar
  62. 62.
    Mooney M. The viscosity of a concentrated suspension of spherical particles. J Colloid Sci. 1951;6(2):162–70.CrossRefGoogle Scholar
  63. 63.
    Mehl JW, Oncley JL, Simha R. Viscosity and the shape of protein molecules. Science. 1940;92(2380):132–3.PubMedCrossRefGoogle Scholar
  64. 64.
    Metzger T. The rheology handbook. 4th ed. Hanover: Vincentz Network; 2014.Google Scholar
  65. 65.
    USP, General Chapters: <913> Viscosity—rolling ball method, in USP 392016.Google Scholar
  66. 66.
    Malkin AY, Isayev AI. Rheology—concepts, methods, and applications. 2nd ed. ChemTec Publishing.Google Scholar
  67. 67.
    USP, General Chapter <911> Viscosity—capillary methods, in USP 392016.Google Scholar
  68. 68.
    USP, General Chapter <912> Viscosity—rotational methods, in USP 392016.Google Scholar
  69. 69.
    Patapoff TW, Esue O. Polysorbate 20 prevents the precipitation of a monoclonal antibody during shear. Pharm Dev Technol. 2009;14(6):659–64.PubMedCrossRefGoogle Scholar
  70. 70.
    Bee JS, et al. Response of a concentrated monoclonal antibody formulation to high shear. Biotechnol Bioeng. 2009;103(5):936–43.PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Dharmaraj VL, et al. Rheology of clustering protein solutions. Biomicrofluidics. 2016;10(4):043509.PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    He F, et al. High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions. Anal Biochem. 2010;399(1):141–3.PubMedCrossRefGoogle Scholar
  73. 73.
    Parmar AS, Muschol M. Lysozyme as diffusion tracer for measuring aqueous solution viscosity. J Colloid Interface Sci. 2009;339(1):243–8.PubMedCrossRefGoogle Scholar
  74. 74.
    Allmendinger A, et al. High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation. J Pharm Biomed Anal. 2014;99:51–8.PubMedCrossRefGoogle Scholar
  75. 75.
    Chari R, et al. Long- and short-range electrostatic interactions affect the rheology of highly concentrated antibody solutions. Pharm Res. 2009;26(12):2607–18.PubMedCrossRefGoogle Scholar
  76. 76.
    Yadav S, Shire SJ, Kalonia DS. Viscosity behavior of high-concentration monoclonal antibody solutions: correlation with interaction parameter and electroviscous effects. J Pharm Sci. 2012;101(3):998–1011.PubMedPubMedCentralCrossRefGoogle Scholar
  77. 77.
    Lilyestrom WG, et al. Monoclonal antibody self-association, cluster formation, and rheology at high concentrations. J Phys Chem B. 2013;117(21):6373–84.PubMedPubMedCentralCrossRefGoogle Scholar
  78. 78.
    Godfrin PD, et al. Effect of hierarchical cluster formation on the viscosity of concentrated monoclonal antibody formulations studied by neutron scattering. J Phys Chem B. 2016;120(2):278–91.PubMedCrossRefGoogle Scholar
  79. 79.
    Mason WP, et al. Measurement of shear elasticity and viscosity of liquids at ultrasonic frequencies. Phys Rev. 1949;75:936–46.CrossRefGoogle Scholar
  80. 80.
    Mason WP. Measurement of the viscosity and shear elasticity of liquids by means of a torsially vibrating crystal. Trans Am Soc Mech Eng. 1947;68:359–70.Google Scholar
  81. 81.
    Saluja A, Kalonia DS. Measurement of fluid viscosity at microliter volumes using quartz impedance analysis. AAPS PharmSciTech. 2004;5(3):68–81.CrossRefGoogle Scholar
  82. 82.
    Rodahl M, et al. Quartz-crystal microbalance setup for frequency and Q-factor measurements in gaseous and liquid environments. Rev Sci Instrum. 1995;66(7):3924–30.CrossRefGoogle Scholar
  83. 83.
    Rodahl M, Kasemo B. On the measurement of thin liquid overlayers with the quartz-crystal microbalance. Sens Actuators a-Phys. 1996;54(1–3):448–56.CrossRefGoogle Scholar
  84. 84.
    Saluja A, Kalonia DS. Application of ultrasonic shear rheometer to characterize rheological properties of high protein concentration solutions at microliter volume. J Pharm Sci. 2005;94(6):1161–8.PubMedCrossRefGoogle Scholar
  85. 85.
    Saluja A, et al. Application of high-frequency rheology measurements for analyzing protein-protein interactions in high protein concentration solutions using a model monoclonal antibody (IgG2). J Pharm Sci. 2006;95(9):1967–83.PubMedCrossRefGoogle Scholar
  86. 86.
    Saluja A, et al. Ultrasonic storage modulus 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(1):234–44.PubMedCrossRefGoogle Scholar
  87. 87.
    Patel AR, Kerwin BA, Kanapuram SR. Viscoelastic characterization of high concentration antibody formulations using quartz crystal microbalance with dissipation monitoring. J Pharm Sci. 2009;98(9):3108–16.PubMedCrossRefGoogle Scholar
  88. 88.
    Hunter RJ. Foundation of colloid science. Vol II. Oxford: Calderin Press; 1989, p. 675–873.Google Scholar
  89. 89.
    Belloni L. Interacting monodisperse and polydisperse spheres. In: Lindner P, Temb T, editors. Neutron, X-ray and light scattering. Amsterdam: Elsevier Science; 1991, p. 135–55.Google Scholar
  90. 90.
    Kaler E. Small-angle scattering from complex fluids. In: Brumberger H, editor. Modern aspects of small-angle scattering. Dordecht: Kluwer Academic Publishers; 1995, p. 239–353.CrossRefGoogle Scholar
  91. 91.
    Narayanan J, Liu XY. Protein interactions in undersaturated and supersaturated solutions: a study using light and X-ray scattering. Biophys J. 2003;84(1):523–32.PubMedPubMedCentralCrossRefGoogle Scholar
  92. 92.
    Zhang F, et al. Protein interactions studied by SAXS: effect of ionic strength and protein concentration for BSA in aqueous solutions. J Phys Chem B. 2007;111(1):251–9.PubMedCrossRefGoogle Scholar
  93. 93.
    Lilyestrom WG, Shire SJ, Scherer TM. Influence of the cosolute environment on IgG solution structure analyzed by small-angle X-ray scattering. J Phys Chem B. 2012;116(32):9611–8.PubMedPubMedCentralCrossRefGoogle Scholar
  94. 94.
    Mosbaek CR, et al. High concentration formulation studies of an IgG2 antibody using small angle X-ray scattering. Pharm Res. 2012;29(8):2225–35.PubMedCrossRefGoogle Scholar
  95. 95.
    Velev OD, Kaler EW, Lenhoff AM. Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen. Biophys J. 1998;75(6):2682–97.PubMedPubMedCentralCrossRefGoogle Scholar
  96. 96.
    Liu Y, et al. Effective long-range attraction between protein molecules in solutions studied by small angle neutron scattering. Phys Rev Lett. 2005;95(11):118102.PubMedCrossRefGoogle Scholar
  97. 97.
    Yearley EJ, et al. Small-angle neutron scattering characterization of monoclonal antibody conformations and interactions at high concentrations. Biophys J. 2013;105(3):720–31.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Andrea Allmendinger
    • 1
    Email author
  • Stefan Fischer
    • 1
  • Robert Mueller
    • 1
  1. 1.Pharmaceutical Development & Supplies, PTD Biologics EuropeF. Hoffmann-La Roche Ltd.BaselSwitzerland

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