Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

Abstract

Sedimentation velocity analytical ultracentrifugation (SV-AUC) coupled with direct computational fitting of the observed concentration profiles (sedimentating boundary) have been developed and widely used for the characterization of macromolecules and nanoparticles in solution. In particular, size distribution analysis by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample preparation, data acquisition, and data analysis. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.

Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-Coding RNAs

“…We used an RNA that was smaller than our constructs (74nt tRNA), and another, which was larger, (23S rRNA) to represent a relative difference in RNA size ( Figure 2B), (Table 1). We further employed SV experiments using analytical ultracentrifugation to determine the composition and anisotropy of biomolecules in solution [56,61,62]. Figure 2 presents the sedimentation coefficient distribution of SEC-purified RNA preparation.…”

A systematic mutational analysis identifies a 5‐residue proline tag that enhances the in vivo immunogenicity of a non‐immunogenic model protein

“…In this study, we used AUC for characterizing the aggregation states of the mutants, because it is considered as a powerful and accurate method that reliably detects aggregates without the need of interacting matrices. Indeed, size exclusion chromatography (SEC) often fails to detect the actual aggregate contents due to their interaction with the SEC-column matrix [34,35].…”

Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-coding RNAs

“…The resolution of the denaturing gel was not enough to discern the difference in molecular weights between MVEV (~32 kDa) and PowV (~37kDa) RNA constructs (Table 1). Therefore, we employed sedimentation velocity experiments using analytical ultracentrifuge which determines the composition and anisotropy of biomolecules in solution [50,54,55]. Figure 3 presents the sedimentation coefficient distribution of SEC-purified RNA preparation.…”