An optical alignment system improves precision of soluble aggregate quantitation by sedimentation velocity analytical ultracentrifugation

Doyle, Brandon L.; Budyak, Ivan L.; Rauk, Adam P.; Weiss, William F.

doi:10.1016/j.ab.2017.05.018

Cited by 10 publications

(8 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4 b). Using again the relative amounts of monomer and dimer, it can be seen that the minimum is not at zero (a similar observation was made by Doyle et al ( 2017 ), where they were using total aggregate amount as the metric) (Fig. 5 ).…”

Section: Resultssupporting

confidence: 74%

“…Beyond this level of alignment precision, other limitations in the instrumentation such as temperature control and accuracy of data capture can become more significant limitations. As the accuracy in these areas also improves (Zhao et al 2014 ), optical alignment procedures and the great precision they offer (Doyle et al 2017 ) will then become increasingly significant.…”

Section: Discussionmentioning

confidence: 99%

“…1 ), and Spin Analytical (Berwick, ME USA). In addition, an optical alignment method of comparable precision—and the potential for even greater precision—has been developed by researchers at Eli-Lily (Doyle et al 2017 ), but this is not yet commercially available. These researchers investigated the total amount of aggregate predicted from SEDFIT in a monoclonal antibody system, at misalignment angles ranging from − 2.5° to + 2.5° as a possible metric for alignment.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

et al. 2018

View full text Add to dashboard Cite

In establishing the sources of data variability within sedimentation velocity analysis in the analytical ultracentrifuge and their relative importance, recent studies have demonstrated that alignment of the sample cells to the centre of rotation is the most significant contributing factor to overall variability, particularly for the characterisation of low levels of protein aggregation. Accurate mechanical and optical alignment tools have been recently designed. In this study, we (1) confirm the effect of misalignment observed by others on the estimated amounts of bovine serum albumin (BSA) monomer and dimer, and the sedimentation coefficient value for the BSA dimer; and (2) demonstrate the high performance of a mechanical alignment tool and the usefulness of a simple and complementary enhanced manual alignment protocol which should be useful for situations where these tools are not available.

show abstract

Section: Resultssupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Cell misalignment has been linked to convective flow which ultimately leads to artifacts materialized in a higher apparent aggregate content. 21,[33][34][35] An illustrative example in Figure 1 (taken from 35 ) shows how purposefully and systematically misaligning cells in the rotor impacts aggregate quantitation. The graphs in Figure 1B and Figure 2 clearly show the impact of misalignment on SV-AUC aggregate quantitation.…”

Section: Cell Alignmentmentioning

confidence: 99%

Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation

Bou-Assaf¹,

Budyak²,

Brenowitz³

et al. 2022

Journal of Pharmaceutical Sciences

Self Cite

View full text Add to dashboard Cite

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.

show abstract

“…These issues have been addressed by implementing new manufacturing practices for higher-quality centerpieces (Gabrielson et al 2010) and development of a mechanical alignment tool ), a commercially available analog of which is available from Spin Analytical and is now routinely employed for high-precision aggregates measurement. Recently, a new custom-made optical tool allowing additional improvement of cell alignment has been described (Doyle et al 2017). Generally, in relation to aggregates quantification, AUC data are analyzed using the sedimentation coefficient distribution c(s) model implemented in the SEDFIT program.…”

Section: Approaches For Aggregate Quantification By Sv-aucmentioning

confidence: 99%

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

2017

View full text Add to dashboard Cite

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.

show abstract

An optical alignment system improves precision of soluble aggregate quantitation by sedimentation velocity analytical ultracentrifugation

Cited by 10 publications

References 14 publications

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

Contact Info

Product

Resources

About