Opalescent Appearance of an IgG1 Antibody at High Concentrations and Its Relationship to Noncovalent Association
- 579 Downloads
Purpose. Therapeutic antibodies are often formulated at a high concentration where they may have an opalescent appearance. The aim of this study is to understand the origin of this opalescence, especially its relationship to noncovalent association and physical stability.
Methods. The turbidity and the association state of an IgG1 antibody were investigated as a function of concentration and temperature using static and dynamic light scattering, nephelometric turbidity, and analytical ultracentrifugation.
Results. The antibody had increasingly opalescent appearance in the concentration range 5-50 mg/ml. The opalescence was greater at refrigerated temperature but was readily reversible upon warming to room temperature. Turbidity measured at 25°C was linear with concentration, as expected for Rayleigh scatter in the absence of association. In the concentration range 1-50 mg/ml, the weight average molecular weights were close to that expected for a monomer. Zimm plot analysis of the data yielded a negative second virial coefficient, indicative of attractive solute-solute interactions. The hydrodynamic diameter was independent of concentration and remained unchanged as a function of aging at room temperature.
Conclusions. The results indicate that opalescent appearance is not due to self-association but is a simple consequence of Rayleigh scatter. Opalescent appearance did not result in physical instability.
Unable to display preview. Download preview PDF.
- 1.O. H. Brekke and I. Sandlie. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat. Rev. Drug Discov. 2:52-62 (2003).Google Scholar
- 2.M. Trikha, L. Yan, and M. T. Nakada. Monoclonal antibodies as therapeutics in oncology. Curr. Opin. Biotechnol. 13:609-614 (2002).Google Scholar
- 3.E. Andreakos, P. C. Taylor, and M. Feldmann. Monoclonal antibodies in immune and inflammatory diseases. Curr. Opin. Biotechnol. 13:615-620 (2002).Google Scholar
- 4.S. A. Marshall, G. A. Lazar, A. J. Chirino, and J. R. Desjarlais. Rational design and engineering of therapeutic proteins. Drug Discov. Today 8:212-221 (2003).Google Scholar
- 5.Package inserts or prescribing information for Herceptin (Genentech, San Francisco, CA, USA), Remicade (Centocor, Malvern, PA, USA), Simulect (Novartis, Basel, Switzerland), and Synagis (MedImmune, Inc., Gaithersburg, MD).Google Scholar
- 6.H. Schellekens. Bioequivalence and immunogenecity of biopharmaceuticals. Nat. Rev. Drug Discov. 1:457-462 (2002).Google Scholar
- 7.J. F. Carpenter and M. C. Manning. Rational Design of Stable Protein Formulations: Theory and Practice, Kluwer Academic/Plenum Publishers, New York, 2002.Google Scholar
- 8.K. E. van Holde. Physical Biochemistry, Prentice-Hall, Englewood Cliffs, NJ, 1985.Google Scholar
- 9.T. M. Schuster and T. M. Laue. Modern Analytical Ultracentrifugation, Birkhäuser, Boston, 1994.Google Scholar
- 10.G. Rivas, J. A. Fernandez, and A. P. Minton. Direct observation of the self-association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment and biological significance. Biochemistry 38:9379-9388 (1999).Google Scholar
- 11.J. Wen, T. Arakawa, and J. S. Philo. Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal. Biochem. 240:155-166 (1996).Google Scholar
- 12.H. G. Barth, B. E. Boyes, and C. Jackson. Size exclusion chromatography. Anal. Chem. 66:595R-620R (1994).Google Scholar
- 13.K. Monkos and B. Turczynski. A comparative study on viscosity of human, bovine and pig IgG immunoglobulins in aqueous solutions. Int. J. Biol. Macromol. 26:155-159 (1999).Google Scholar
- 14.E. J. Cohn and J. T. Edsall. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions, Reinhold, New York, 1943.Google Scholar
- 15.A. P. Minton. Quantitative characterization of reversible macromolecular associations via sedimentation equilibrium: an introduction. Exp. Mol. Med. 32:1-5 (2000).Google Scholar
- 16.European Pharmacopoeia. Fourth Edition., Directorate for the Quality of Medicines of the Council of Europe (EDQM), Fourth Ed., Strasbourg, France (2001).Google Scholar
- 17.R. Wirestam, V. A. Larsen, M. Stubgaard, C. Thomsen, B. Vikhoff, H. B. Larsson, F. Stahlberg, and O. Henriksen. Deuterium MR spectroscopy at 4.7 T. Quantification of tumour and subcutaneous tissue blood flow in animal models. Acta Radiol. 36:85-91 (1995).Google Scholar
- 18.C. Binder. Absorption of injected insulin. Acta Pharmacol. Toxicol. (Copenh.) 27(Suppl. 2):1-87 (1969).Google Scholar
- 19.J. Brange, D. R. Owens, S. Kang, and A. Volund. Monomeric insulins and their experimental and clinical applications. Diabetes Care 13:923-954 (1990).Google Scholar
- 20.D. L. Bakaysa, J. Radziuk, H. A. Havel, M. L. Brader, S. Li, S. W. Dodd, J. M. Beals, A. H. Pekar, and D. N. Brems. Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex. Protein Sci. 5:2521-2531 (1996).Google Scholar
- 21.J. A. Thomson, P. Schurtenberger, G. M. Thurston, and G. B. Benedek. Binary liquid phase separation and critical phenomena in a protein/water solution. Proc. Natl. Acad. Sci. U.S.A. 84:7079-7083 (1987).Google Scholar
- 22.C. Ishimoto and T. Tanaka. Critical behavior of a binary mixture of protein and salt water. Phys. Rev. Lett. 39:474-477 (1977).Google Scholar