title: A bayesian approach for quantifying trace amounts of antibody aggregates by sedimentation velocity analytical ultracentrifugation.
abstract: 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 approximately 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.
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892-5766, USA. brownpatr@mail.nih.gov
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Brown
authors.forename: Andrea
authors.initials: A
authors.lastname: Balbo
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
mesh_terms: D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001499:Bayes Theorem; D001702:Biopharmaceutics / Q000379:methods / Q000706:statistics & numerical data; D003198:Computer Simulation; D007074:Immunoglobulin G / Q000302:isolation & purification; D008962:Models, Theoretical; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000592:standards; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000379:methods / Q000706:statistics & numerical data
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins