Abstract
Analytical ultracentrifugation (AUC) and field flow fractionation (FFF) are 2 important biophysical methods for measuring protein aggregates. Both methods can separate protein monomer from its aggregate forms under a broad range of solution conditions. Recent advances in instrumentation and data analysis, particularly in the field of analytical ultracentrifugation technology, have significantly improved the capability and sensitivity of these biophysical methods for detecting protein aggregates. These advances have resulted in an increased use of these methods in the biopharmaceutical industry for characterization of therapeutic proteins. However, despite their many advantages over conventional methods, the difficulty in the use of the instrumentation and the complexity of data analysis process, have often hampered the widespread use and proper interpretation of data. This article reviews the recent progress in both technologies, and a few case studies are also presented to discuss their advantages and limitations.
Keywords: Analytical ultracentrifuge, sedimentation velocity, field flow fractionation, protein aggregates
Full Text
The Full Text of this article is available as a PDF (538.9 KB).
References
- 1.Schellekens H. Factors influencing the immunogenicity of therapeutic proteins. Nephrol Dial Transplant. 2005;20:vi3–vi9. doi: 10.1093/ndt/gfh1092. [DOI] [PubMed] [Google Scholar]
- 2.Hermeling S, Crommelin DJ, Schellekens H, Jiskoot W. Structure-immunogenicity relationships of therapeutic proteins. Pharm Res. 2004;21:897–903. doi: 10.1023/B:PHAM.0000029275.41323.a6. [DOI] [PubMed] [Google Scholar]
- 3.Robbins DC, Hirshman M, Wardzala LJ, Horton ES. High-molecular-weight aggregates of therapeutic insulin: in vitro measurements of receptor binding and bioactivity. Diabetes. 1988;37:56–59. doi: 10.2337/diabetes.37.1.56. [DOI] [PubMed] [Google Scholar]
- 4.Maislos M, Bialer M, Mead PM, Robbins DC. Pharmacokinetic model of circulating covalent aggregates of insulin. Diabetes. 1988;37:1059–1063. doi: 10.2337/diabetes.37.8.1059. [DOI] [PubMed] [Google Scholar]
- 5.Lebowitz J, Lewis MS, Schuck P. Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci. 2002;11:2067–2079. doi: 10.1110/ps.0207702. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Giddings JC. Field-flow fractionation: analysis of macromolecular, colloidal, and particulate materials. Science. 1993;260:1456–1465. doi: 10.1126/science.8502990. [DOI] [PubMed] [Google Scholar]
- 7.Calvin GJ. The Field-Flow Fractionation Family: Underlying Principles. New York, NY: John Wiley & Sons; 2000. [Google Scholar]
- 8.Reschiglian P, Zattoni A, Roda B, Michelini E, Roda A. Field-flow fractionation and biotechnology. Trends Biotechnol. 2005;23:475–483. doi: 10.1016/j.tibtech.2005.07.008. [DOI] [PubMed] [Google Scholar]
- 9.Laue TM, Stafford WF. Modern applications of analytical ultracentrifugation. Annu Rev Biophys Biomol Struct. 1999;28:75–100. doi: 10.1146/annurev.biophys.28.1.75. [DOI] [PubMed] [Google Scholar]
- 10.Schuster TM, Toedt JM. New revolution in the evolution of analytical ultracentrifugation. Curr Opin Struct Biol. 1996;6:650–658. doi: 10.1016/S0959-440X(96)80032-7. [DOI] [PubMed] [Google Scholar]
- 11.Stafford WF. Boundary analysis in sedimentation velocity experiments. Methods Enzymol. 1994;240:478–501. doi: 10.1016/s0076-6879(94)40061-x. [DOI] [PubMed] [Google Scholar]
- 12.Schuck P. Size-distribution analysis of macromolecules b sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J. 2000;78:1606–1619. doi: 10.1016/S0006-3495(00)76713-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hansen JC, Lebowitz J, Demeler B. Analytical ultracentrifugation of complex macromolecular systems. Biochemistry. 1994;33:13155–13163. doi: 10.1021/bi00249a001. [DOI] [PubMed] [Google Scholar]
- 14.Dam J, Velikovsky CA, Mariuzza RA, Urbanke C, Schuck P. Sedimentation velocity analysis of heterogeneous protein-protein interactions: Lamm equation modeling and sedimentation coefficient distributions c(s) Biophys J. 2005;89:619–634. doi: 10.1529/biophysj.105.059568. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Dam J, Schuck P. Sedimentation velocity analysis of heterogeneous protein-protein interactions: sedimentation coefficient distributions c(s) and asymptotic boundary profiles from Gilbert-Jenkins theory. Biophys J. 2005;89:651–666. doi: 10.1529/biophysj.105.059584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Correia JJ. Analysis of weight average sedimentation velocity data. Methods Enzymol. 2000;321:81–100. doi: 10.1016/s0076-6879(00)21188-9. [DOI] [PubMed] [Google Scholar]
- 17.Demeler B, Saber H, Hansen JC. Identification and interpretation of complexity in sedimentation velocity boundaries. Biophys J. 1997;72:397–407. doi: 10.1016/S0006-3495(97)78680-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Stafford WF. Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem. 1992;203:295–301. doi: 10.1016/0003-2697(92)90316-Y. [DOI] [PubMed] [Google Scholar]
- 19.Brookes E, Demeler B. Genetic Algorithm Optimization for Obtaining Accurate Molecular Weight Distributions from Sedimentation Velocity Experiments. In: Wandrey C, Cölfen H, editors. Progress in Colloid and Polymer Science-Analytical Ultracentrifugation. 8th ed. Heidelberg, Germany: Springer-Verlag; 2006. pp. 33–40. [Google Scholar]
- 20.Robert G. The Optima XL-A: A New Analytical Ultracentrifuge With a Novel Precision Absorption Optical System. London, UK: The Royal Society of Chemistry; 1992. [Google Scholar]
- 21.Allen F. The XL-I analytical ultracentrifuge with Rayleigh interference optics. Eur Biophys J. 1997;35:307–310. [Google Scholar]
- 22.Philo JS. An improved function for fitting sedimentation velocity data for low-molecular-weight solutes. Biophys J. 1997;72:435–444. doi: 10.1016/S0006-3495(97)78684-3. [DOI] [PMC free article] [PubMed] [Google Scholar]