The AAPS Journal

, 10:481 | Cite as

A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation

  • Patrick H. BrownEmail author
  • Andrea Balbo
  • Peter Schuck
Research Article


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 words

analytical 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.


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Copyright information

© American Association of Pharmaceutical Scientists 2008

Authors and Affiliations

  1. 1.Dynamics of Macromolecular Assembly Section, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and BioengineeringNational Institutes of HealthBethesdaUSA

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