Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Analytical Biopharmacy Core University of Washington Seattle Washington 98195 United States of America

Analytical Biophysics and Materials Characterization Department of Chemistry and Biochemistry University of Arizona Tucson Arizona 85721 United States of America

Analytical Ultracentrifugacion and Light Scattering Facility Centro de Investigaciones Biológicas CSIC Madrid 28040 Spain

Beckman Coulter Inc Life Science Division Indianapolis Indiana 46268 United States of America

Biochemistry and Biophysics Center National Heart Lung and Blood Institute National Institutes of Health Bethesda Maryland 20892 United States of America

Biochemistry Cell and Molecular Biology Department University of Tennessee Knoxville Tennessee 37996 0840 United States of America

Biochemistry Core Facility Max Planck Institute of Biochemistry 82152 Martinsried Germany

Biochemistry Department University of Cambridge Cambridge CB2 1GA United Kingdom

Biophysics Core Facility Scientific Instrument Center Academia Sinica Taipei 115 Taiwan

Biophysics of Macromolecules German Cancer Research Center Heidelberg 69120 Germany

Central Instrument Center Mokpo National University Muan 534 729 Korea

Core Facility International Institute of Molecular and Cell Biology Warsaw 02 109 Poland

Department of Biochemistry and Biophysics University of California San Francisco San Francisco California 94158 United States of America

Department of Biochemistry and Biophysics University of California San Francisco San Francisco California 94158 United States of America; Tetrad Graduate Program University of California San Francisco San Francisco California 94158 United States of America

Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States of America

Department of Biochemistry and Molecular Biology Bio21 Instute of Molecular Science and Biotechnology University of Melbourne Parkville 3010 Victoria Australia

Department of Biochemistry and Molecular Biology Colorado State University Fort Collins Colorado 80523 United States of America

Department of Biochemistry and Molecular Biology Tel Aviv University Tel Aviv 69978 Israel

Department of Biochemistry La Trobe Institute for Molecular Science La Trobe University Melbourne Victoria 3086 Australia

Department of Biochemistry Uniformed Services University of the Health Sciences Bethesda Maryland 20814 United States of America

Department of Biochemistry University of New Hampshire Durham New Hampshire 03824 United States of America

Department of Biology Haverford College Haverford Pennsylvania 19041 United States of America

Department of Biophysics and Biophysical Chemistry Johns Hopkins University School of Medicine Baltimore Maryland 21205 United States of America

Department of Biophysics The University of Texas Southwestern Medical Center Dallas Texas 75390 United States of America

Department of Biotechnology Graduate School of Engineering Osaka University Suita Osaka 565 0871 Japan

Department of Chemical and Biomolecular Engineering University of Delaware Newark Delaware 19716 United States of America

Department of Chemistry and Biochemistry Center for Retrovirus Research and Center for RNA Biology The Ohio State University Columbus Ohio 43210 United States of America

Department of Chemistry and Biochemistry Concordia University Montreal Quebec H4B 1R6 Canada

Department of Chemistry and Center for Integrated Protein Science Technische Universität München 85748 Garching Germany

Department of Chemistry Mokpo National University Muan 534 729 Korea

Department of Microbiology University of Alabama at Birmingham Birmingham Alabama 35294 United States of America

Department of Molecular Genetics Biochemistry and Microbiology University of Cincinnati College of Medicine Cincinnati Ohio 45267 United States of America

Department of Pharmaceutical Sciences University of Colorado Denver Anschutz Medical Campus Aurora Colorado 80045 United States of America

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Prague Prague 12843 Czech Republic

Department of Physical Chemistry University of Murcia Murcia 30071 Spain

Department of Physiology and Biophysics School of Medicine Virginia Commonwealth University Richmond Virginia 23220 United States of America

Department of Structural and Molecular Biology Darwin Building University College London London WC1E 6BT United Kingdom

Diabetes Biophysics Novo Nordisk A S Måløv 2760 Denmark

Division of Biophysics Institute of Experimental Physics Faculty of Physics University of Warsaw Warsaw 02 089 Poland

Drug Design Laboratory Graduate School of Medical Life Science Yokohama City University Yokohama 230 0045 Japan

Dynamics of Macromolecular Assembly Section Laboratory of Cellular Imaging and Macromolecular Biophysics National Institute of Biomedical Imaging and Bioengineering National Institutes of Health Bethesda Maryland 20892 United States of America

Enzyme Research Group Concordia University Montreal Quebec H4B 1R6 Canada

ICS 6 Structural Biochemistry Research Center Juelich 52428 Juelich Germany

Institut Pasteur Centre of Biophysics of Macromolecules and Their Interactions Paris 75724 France

Institute for Biophysical Chemistry Hannover Medical School 30625 Hannover Germany

Institute for Bioscience and Biotechnology Research Fischell Department of Bioengineering University of Maryland Rockville Maryland 20850 United States of America

Institute of Biochemistry and Biotechnology Martin Luther University Halle Wittenberg 06120 Halle Germany

Institute of Biological Chemistry Academia Sinica Taipei 115 Taiwan; Biophysics Core Facility Scientific Instrument Center Academia Sinica Taipei 115 Taiwan

Institute of Molecular Biology and Department of Chemistry and Biochemistry University of Oregon Eugene Oregon 97403 United States of America

Institute of Structural and Molecular Biology Biophysics Centre Birkbeck University of London and University College London London WC1E 7HX United Kingdom

JG Brown Cancer Center University of Louisville Louisville Kentucky 40202 United States of America

Laboratoire de Médecine Régénérative et de Pharmacobiologie Ecole Polytechnique Fédérale de Lausanne Lausanne CH 1015 Switzerland

Laboratory of Biomolecular Research Department of Biology and Chemistry Paul Scherrer Institute 5232 Villigen PSI Switzerland

Laboratory of Chromatin Biochemistry Max Planck Institute for Biophysical Chemistry 37077 Göttingen Germany

Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda Maryland 20892 United States of America

Life Science Research Center Nihon University College of Bioresource Science Fujisawa 252 0880 Japan

Macromolecule and Vaccine Stabilization Center University of Kansas Lawrence Kansas 66047 United States of America

Materials Science and Engineering Laboratory National Institute of Standards and Technology Gaithersburg Maryland 20899 United States of America

Molecular Biophysics Suite Department of Biochemistry Oxford Oxon OX1 3QU United Kingdom

Molecular Interaction Analysis Shared Resource St Jude Children's Research Hospital Memphis Tennessee 38105 United States of America

National Centre for Macromolecular Hydrodynamics University of Nottingham School of Biosciences Sutton Bonington LE12 5RD United Kingdom

Pharma Research and Early Development Roche Innovation Center Basel F Hoffmann LaRoche Ltd Basel 4070 Switzerland

Physical Chemistry University of Konstanz 78457 Konstanz Germany

Program in Molecular Medicine University of Massachusetts Medical School Worcester Massachusetts 01605 United States of America

Protein Function and Interactions Novo Nordisk Foundation Center for Protein Research Copenhagen 2200 Denmark

Protein Interactions Core Department of Biochemistry University of Utah School of Medicine Salt Lake City Utah 84112 United States of America

Proteome and Protein Analysis University of Newcastle Newcastle upon Tyne NE1 7RU United Kingdom

Redox Biology Center University of Nebraska Lincoln Lincoln Nebraska 68588 United States of America

Research Complex at Harwell Rutherford Appleton Laboratory Oxfordshire OX11 0FA United Kingdom

Research School of Chemistry Section on Biological Chemistry The Australian National University Acton ACT 0200 Australia

Rudolf Virchow Center for Experimental Biomedicine University of Würzburg 97080 Würzburg Germany

School of Life Sciences University of Glasgow Glasgow G37TT United Kingdom

Structural Biology Platform IGBMC Illkirch 67400 France

Supramolecular Nanomaterials and Interfaces Laboratory Institute of Materials École Polytechnique Fédérale de Lausanne Lausanne CH 1015 Switzerland

Technology Facility Department of Biology University of York York YO10 5DD United Kingdom

Univ Grenoble Alpes IBS F 38044 Grenoble France; CNRS IBS F 38044 Grenoble France; CEA IBS F 38044 Grenoble France

Wadsworth Center New York State Department of Health Albany New York 12208 United States of America

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Svedberg T, Pedersen KO. The ultracentrifuge London: Oxford University Press; 1940.

Schuck P. Analytical ultracentrifugation as a tool for studying protein interactions. Biophys Rev. 2013; 5: 159–171. 10.1007/s12551-013-0106-2 PubMed DOI PMC

Aragon SR. Recent advances in macromolecular hydrodynamic modeling. Methods. 2011; 54: 101–114. 10.1016/j.ymeth.2010.10.005 PubMed DOI PMC

Ortega A, Amorós D, García de la Torre J. Prediction of hydrodynamic and other solution properties of rigid proteins from atomic- and residue-level models. Biophys J. 2011; 101: 892–898. 10.1016/j.bpj.2011.06.046 PubMed DOI PMC

Byron O. Construction of hydrodynamic bead models from high-resolution X-ray crystallographic or nuclear magnetic resonance data. Biophys J. 1997; 72: 408–415. 10.1016/S0006-3495(97)78681-8 PubMed DOI PMC

Perkins SJ, Nan R, Li K, Khan S, Abe Y. Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling. Methods. 2011; 54: 181–199. 10.1016/j.ymeth.2011.01.004 PubMed DOI

Rai N, Nöllmann M, Spotorno B, Tassara G, Byron O, Rocco M. SOMO (SOlution MOdeler) differences between X-Ray- and NMR-derived bead models suggest a role for side chain flexibility in protein hydrodynamics. Structure. 2005; 13: 723–734. 10.1016/j.str.2005.02.012 PubMed DOI

Ebel C. Sedimentation velocity to characterize surfactants and solubilized membrane proteins. Methods. 2011; 54: 56–66. 10.1016/j.ymeth.2010.11.003 PubMed DOI

Silvera Batista CA, Zheng M, Khripin CY, Tu X, Fagan JA. Rod hydrodynamics and length distributions of single-wall carbon nanotubes using analytical ultracentrifugation. Langmuir. 2014; 30: 4895–4904. 10.1021/la404892k PubMed DOI

Carney RP, Kim JY, Qian H, Jin R, Mehenni H, Stellacci F, et al. Determination of nanoparticle size distribution together with density or molecular weight by 2D analytical ultracentrifugation. Nat Commun. 2011; 2: 335 10.1038/ncomms1338 PubMed DOI PMC

Liu J, Andya JD, Shire SJ. A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation. AAPS J. 2006; 8: E580–E589. 10.1208/aapsj080367 PubMed DOI PMC

Berkowitz SA. Role of analytical ultracentrifugation in assessing the aggregation of protein biopharmaceuticals. AAPS J. 2006; 8: E590–E605. 10.1208/aapsj080368 PubMed DOI PMC

Gabrielson JP, Brader ML, Pekar AH, Mathis KB, Winter G, Carpenter JF, et al. 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. 2007; 96: 268–279. 10.1002/jps.20760 PubMed DOI

Gabrielson JP, Arthur KK. Measuring low levels of protein aggregation by sedimentation velocity. Methods. 2011; 54: 83–91. 10.1016/j.ymeth.2010.12.030 PubMed DOI

Philo JS. A critical review of methods for size characterization of non-particulate protein aggregates. Curr Pharm Biotechnol. 2009; 10: 359–372. PubMed

Pekar AH, Sukumar M. Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: practical considerations that affect precision and accuracy. Anal Biochem. 2007; 367: 225–237. 10.1016/j.ab.2007.04.035 PubMed DOI

Berkowitz SA, Engen JR, Mazzeo JR, Jones GB. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov. 2012; 11: 527–540. 10.1038/nrd3746 PubMed DOI PMC

Zhao H, Mayer ML, Schuck P. Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity. Anal Chem. 2014; 18: 3181–3187. 10.1021/ac500093m PubMed DOI PMC

Zhao H, Casillas E, Shroff H, Patterson GH, Schuck P. Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation. PLoS One. 2013; 8: e77245 10.1371/journal.pone.0077245 PubMed DOI PMC

Kingsbury JS, Laue TM. Fluorescence-detected sedimentation in dilute and highly concentrated solutions. Methods Enzym. 2011; 492: 283–304. 10.1016/B978-0-12-381268-1.00021-5 PubMed DOI

Gabrielson JP, Arthur KK, Stoner MR, Winn BC, Kendrick BS, Razinkov V, et al. Precision of protein aggregation measurements by sedimentation velocity analytical ultracentrifugation in biopharmaceutical applications. Anal Biochem. 2009; 396: 231–241. 10.1016/j.ab.2009.09.036 PubMed DOI

Arthur KK, Gabrielson JP, Kendrick BS, Stoner MR. Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): Sources of variability and their relative importance. J Pharm Sci. 2009; 98: 3522–3539. 10.1002/jps.21654 PubMed DOI

Krayukhina E, Uchiyama S, Nojima K, Okada Y, Hamaguchi I, Fukui K. Aggregation analysis of pharmaceutical human immunoglobulin preparations using size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity. J Biosci Bioeng. 2013; 4: 104–110 10.1016/j.jbiosc.2012.07.021 PubMed DOI

Taylor JF. The determination of sedimentation constant with the Spinco ultracentrifuge. Arch Biochem Biophys. 1952; 36: 357–364. 10.1016/0003-9861(52)90421-9 PubMed DOI

Errington N, Rowe AJ. Probing conformation and conformational change in proteins is optimally undertaken in relative mode. Eur Biophys J. 2003; 32: 511–517. 10.1007/s00249-003-0315-x PubMed DOI

Cecil R, Ogston AG. The accuracy of the Svedberg oil-turbine ultracentrifuge. Biochem J. 1948; 43: 592–598. PubMed PMC

Ghirlando R, Balbo A, Piszczek G, Brown PH, Lewis MS, Brautigam CA, et al. Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references. Anal Biochem. 2013; 440: 81–95. 10.1016/j.ab.2013.05.011 PubMed DOI PMC

Henrion M, Fischhoff B. Assessing uncertainty in physical constants. Am J Phys. 1986; 54: 791 10.1119/1.14447 DOI

Shulman S. The determination of sedimentation constants with the oil-turbine and spinco ultracentrifuges. Arch Biochem Biophys. 1953; 44: 230–240. 10.1016/0003-9861(53)90028-9 PubMed DOI

Miller GL, Golder RH. Sedimentation studies with the Spinco ultracentrifuge. Arch Biochem Biophys. 1952; 36: 249–258. 10.1016/0003-9861(52)90409-8 PubMed DOI

Zhao H, Ghirlando R, Piszczek G, Curth U, Brautigam CA, Schuck P. Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation. Anal Biochem. 2013; 437: 104–108. 10.1016/j.ab.2013.02.011 PubMed DOI PMC

Jakoby W. Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation. Anal Biochem. 2013; 437: 103 10.1016/j.ab.2013.02.017 PubMed DOI

Zhao H, Balbo A, Metger H, Clary R, Ghirlando R, Schuck P. Improved measurement of the rotor temperature in analytical ultracentrifugation. Anal Biochem. 2014; 451: 69–75. 10.1016/j.ab.2014.02.006 PubMed DOI PMC

Ghirlando R, Zhao H, Balbo A, Piszczek G, Curth U, Brautigam CA, et al. Measurement of the temperature of the resting rotor in analytical ultracentrifugation. Anal Biochem. 2014; 458: 37–39. 10.1016/j.ab.2014.04.029 PubMed DOI PMC

Kar SR, Kingsbury JS, Lewis MS, Laue TM, Schuck P. Analysis of transport experiment using pseudo-absorbance data. Anal Biochem. 2000; 285: 135–142. 10.1006/abio.2000.4748 PubMed DOI

Brown PH, Balbo A, Schuck P. On the analysis of sedimentation velocity in the study of protein complexes. Eur Biophys J. 2009; 38: 1079–1099. 10.1007/s00249-009-0514-1 PubMed DOI PMC

Schuck P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J. 2000; 78: 1606–1619. 10.1016/S0006-3495(00)76713-0 PubMed DOI PMC

Laue TM, Shah BD, Ridgeway TM, Pelletier SL. Computer-aided interpretation of analytical sedimentation data for proteins In: Harding SE, Rowe AJ, Horton JC, editors. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Cambridge: The Royal Society of Chemistry; 1992. pp. 90–125. 10.1007/s00249-009-0425-1 DOI

Waugh DF, Yphantis DA. Rotor temperature measurement and control in the ultracentrifuge. Rev Sci Instrum. 1952; 23: 609–614. 10.1063/1.1746108 DOI

Perlman GE, Longsworth LG. The specific refractive increment of some purified proteins. Am Chem Soc. 1948; 111: 2719–2724. 10.1021/ja01188a027 PubMed DOI

Zhao H, Brown PH, Schuck P. On the distribution of protein refractive index increments. Biophys J.; 100: 2309–2317. 10.1016/j.bpj.2011.03.004 PubMed DOI PMC