A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation
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Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Brown et al., Biomacromolecules, 8:2011–2024, 2007), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.
Key wordsanalytical ultracentrifugation Bayesian analysis hydrodynamic separation sedimentation velocity size-distribution trace aggregates
This work was supported by the Intramural Research Program of the National Institutes of Health, NIBIB. The authors wish to thank A. Pekar and his colleagues for providing access to preprints of their recent publication concerning trace detection during the preparation of this manuscript.
- 10.J. P. Gabrielson, M. L. Brader, A. H. Pekar, K. B. Mathis, G. Winter, J. F. Carpenter, and T. W. Randolph. 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. 96(2):268–279 (2007). Feb.PubMedCrossRefGoogle Scholar
- 14.T. Svedberg, and K. O. Pedersen. The ultracentrifuge, Oxford University Press, London, 1940.Google Scholar
- 16.P. Schuck. Sedimentation velocity in the study of reversible multiprotein complexes. In P. Schuck (ed.), Biophysical approaches for the study of complex reversible systems, Springer, New York, 2007, pp. 469–518.Google Scholar
- 19.P. Schuck. Measuring size-and-shape distributions of protein complexes in solution by sedimentation and dynamic light scattering. Autrans, France: Euroconference “Advances in Analytical Ultracentrifugation and Hydrodynamics”; 2002 8–11, June 2002.Google Scholar
- 20.P. Schuck. http://www.analyticalultracentrifugation.com/references.htm. 2007.
- 22.J. Philo. Characterizing the Aggregation and Conformation of Protein Therapeutics. Am Biotechnol Lab. 23:22–24 (2003). October.Google Scholar
- 23.J. P. Gabrielson, K. K. Arthur, B. S. Kendrick, T. W. Randolph, and M. R. Stoner. Common excipients impair detection of protein aggregates during sedimentation velocity analytical ultracentrifugation. J Pharm Sci. in press (2008) Apr 18.Google Scholar
- 25.P. Schuck. Diffusion-deconvoluted sedimentation coefficient distributions for the analysis of interacting and non-interacting protein mixtures. In D. J. Scott, S. E. Harding, and A. J. Rowe (eds.), Modern analytical ultracentrifugation: techniques and methods, The Royal Society of Chemistry, Cambridge, 2006, pp. 26–50.Google Scholar
- 28.A. Mohommad-Djafari. Bayesian inference and maximum entropy methods in science and engineering, American Institute of Physics, New York, 2001.Google Scholar
- 30.C. R. Smith, and W. T. Grandy Jr. Maximum-entropy and Bayesian methods in inverse problems, Reidel, Dordrecht, 1985.Google Scholar
- 32.P. Schuck www.analyticalultracentrifugation.com. 2007.
- 34.O. Lamm. Die Differentialgleichung der Ultrazentrifugierung. Ark Mat Astr Fys. 21B(2):1–4 (1929).Google Scholar
- 37.D. S. Sivia. Data analysis. A Bayesian tutorial, Oxford University Press, Oxford, 1996.Google Scholar