Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

Ma, Jia; Metrick, Michael A.; Ghirlando, Rodolfo; Zhao, Huaying; Schuck, Peter

doi:10.1016/j.bpj.2015.07.015

Cited by 11 publications

(12 citation statements)

References 42 publications

(80 reference statements)

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“…The main characteristic of this data acquisition mode is that it can execute sedimentation steps serially at different rotor speeds and temperature, and initiate scans at each step at preset time intervals. Although the rules for the precise timing of speed changes and scans are nonobvious, in the previous communication on variable-field analytical ultracentrifugation (14) we have reported on a new utility function in SED-FIT that can convert between speedsteps.txt files and sedimentation equilibrium method .equ files that can be directly loaded into the analytical ultracentrifuge user interface (see tutorial video https://sedfitsedphat.nibib.nih. gov/tools/Protocols/TOSE_implementation.wmv).…”

Section: Resultsmentioning

confidence: 99%

“…The experimental rotor speed schedule was specified in speedsteps.txt files introduced previously in Ma et al (14), containing a table of elapsed times when rotor speed changes are planned to be initiated, the new target rotor speed, and the rotor acceleration (usually 280 rpm/s). For data analysis, this file will be automatically recognized by SEDFIT when it is located in the corresponding scan data folder, if scans acquired at different rotor speeds are loaded.…”

Section: Biophysical Journal 110(1) 103-112mentioning

confidence: 99%

“…Therefore, we incorporated a discretized approximation of the rotor acceleration into the explicit numerical finite element Lamm equation solutions (11)(12)(13) used in our SED-FIT software. Recently we have extended the consideration of time-varying rotor speeds from merely modeling the acceleration phase to the modeling of the approach to equilibrium in an arbitrarily changing centrifugal field, with the goal to calculate a rotor speed schedule minimizing the time required to attain sedimentation equilibrium at a target speed (14). While the previous work was in a largely diffusion-dominated regime, in this work we have focused on the sedimentation-dominated regime to simulate and model sedimentation velocity experiments of particles over a large size-range in time-varying fields.…”

Section: Introductionmentioning

confidence: 99%

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Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Zhao

Sandmaier

et al. 2016

Biophysical Journal

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Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by ∼100-fold with a quasi-continuous increase of rotor speed during the experiment. Such a gravitational-sweep sedimentation approach has previously been shown to be very effective in the study of nanoparticles with large size ranges. In the past, diffusion processes were not accounted for, thereby posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In this work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extend the macromolecular size range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitude of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar-sized macromolecular species, or in the study of broadly distributed nanoparticles.

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Section: Resultsmentioning

confidence: 99%

Section: Biophysical Journal 110(1) 103-112mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Zhao

Sandmaier

et al. 2016

Biophysical Journal

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“…The rotor was temperature equilibrated to a nominal temperature of 19.7°C while resting in the rotor chamber prior to the start of centrifugation. For SE, an overspeeding schedule was calculated to minimize the equilibration time (48); for SV the rotor was accelerated to full speed at once (40,000 rpm for VRC antibodies, 45,000 rpm for NISTmAb, 50,000 rpm for all other). Rayleigh interference optical data acquisition was used.…”

Section: Methodsmentioning

confidence: 99%

A Reappraisal of Sedimentation Nonideality Coefficients for the Analysis of Weak Interactions of Therapeutic Proteins

Chaturvedi

Schuck

2019

AAPS J

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The study of weak or colloidal interactions of therapeutic proteins in different formulations allows prediction and optimization of protein stability. Various biophysical techniques have been applied to determine the second osmotic virial coefficient B 2 as it reflects on the macromolecular distance distribution that governs solution behavior at high concentration. In the present work, we exploit a direct link predicted by hydrodynamic theory between B 2 and the nonideality of sedimentation, commonly measured in sedimentation velocity analytical ultracentrifugation through the nonideality coefficient of sedimentation, k S. Using sedimentation equilibrium analytical ultracentrifugation for independent measurement of B 2 , we have examined the dependence of k S on B 2 for model proteins in different buffers. The data exhibit the expected linear relationship and highlight the impact of protein shape on the magnitude of the nonideality coefficient k S. Recently, measurements of k S have been considerably simplified allowing higher throughput and simultaneous polydispersity assessment at higher protein concentrations. Thus, sedimentation velocity may offer a useful approach to compare the impact of formulation conditions on weak interactions and simultaneously on higher-order structure of therapeutic proteins.

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“…Acceleration to 26,000 rpm for apoferritin, 45,000 rpm for NISTmAb, or 50,000 rpm for BSA, respectively, was immediately followed by acquisition of radial scans using the Rayleigh interference optical system. SE experiments were carried out with 6 mm long solution columns, using time-optimized rotor speed profiles 68 to attain SE at 9,000 rpm. SE data were globally modeled in SEDPHAT using the INVEQ method by Ang and Rowe 69 to account for nonideality with the second virial coefficient.…”

Section: Methodsmentioning

confidence: 99%

Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity

Chaturvedi

Brown

et al. 2018

Nat Commun

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In concentrated macromolecular solutions, weak physical interactions control the solution behavior including particle size distribution, aggregation, liquid-liquid phase separation, or crystallization. This is central to many fields ranging from colloid chemistry to cell biology and pharmaceutical protein engineering. Unfortunately, it is very difficult to determine macromolecular assembly states and polydispersity at high concentrations in solution, since all motion is coupled through long-range hydrodynamic, electrostatic, steric, and other interactions, and scattering techniques report on the solution structure when average interparticle distances are comparable to macromolecular dimensions. Here we present a sedimentation velocity technique that, for the first time, can resolve macromolecular size distributions at high concentrations, by simultaneously accounting for average mutual hydrodynamic and thermodynamic interactions. It offers high resolution and sensitivity of protein solutions up to 50 mg/ml, extending studies of macromolecular solution state closer to the concentration range of therapeutic formulations, serum, or intracellular conditions.

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Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

Cited by 11 publications

References 42 publications

Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

A Reappraisal of Sedimentation Nonideality Coefficients for the Analysis of Weak Interactions of Therapeutic Proteins

Measuring macromolecular size distributions and interactions at high concentrations by sedimentation velocity

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