Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions.

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.

Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations.

Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphology when examined by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clinically relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (w/w)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global analysis of multi-wavelength sedimentation velocity analytical ultracentrifugation, using density matching with heavy water, that not only measures the standard sedimentation and diffusion coefficient distributions of LNP mixtures, but also reports the corresponding partial specific volume distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial specific volume and have low to intermediate densities. Our method makes it possible to unambiguously measure the density of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured density is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable density and partial specific volume. An UltraScan software module was developed to implement this approach for routine analysis.

Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment.

Recent advances in instrumentation have moved analytical ultracentrifugation (AUC) closer to a possible validation in a Good Manufacturing Practices (GMP) environment. In order for AUC to be validated for a GMP environment, stringent requirements need to be satisfied; analysis procedures must be evaluated for consistency and reproducibility, and GMP capable data acquisition software needs to be developed and validated. These requirements extend to multiple regulatory aspects, covering documentation of instrument hardware functionality, data handling and software for data acquisition and data analysis, process control, audit trails and automation. Here we review the requirements for GMP validation of data acquisition software and illustrate software solutions based on UltraScan that address these requirements as far as they relate to the operation and data handling in conjunction with the latest analytical ultracentrifuge, the Optima AUC by Beckman Coulter. The software targets the needs of regulatory agencies, where AUC plays a critical role in the solution-based characterization of biopolymers and macromolecular assemblies. Biopharmaceutical and regulatory agencies rely heavily on this technique for characterizations of pharmaceutical formulations, biosimilars, injectables, nanoparticles, and other soluble therapeutics. Because of its resolving power, AUC is a favorite application, despite the current lack of GMP validation. We believe that recent advances in standards, hardware, and software presented in this work manage to bridge this gap and allow AUC to be routinely used in a GMP environment. AUC has great potential to provide more detailed information, at higher resolution, and with greater confidence than other analytical techniques, and our software satisfies an urgent need for AUC operation in the GMP environment. The software, including documentation, are publicly available for free download from Github. The multi-platform software is licensed by the LGPL v.3 open source license and supports Windows, Mac and Linux platforms. Installation instructions and a mailing list are available from ultrascan.aucsolutions.com.

Multi-speed sedimentation velocity simulations with UltraScan-III.

Recent developments in the UltraScan-III software make it possible to model multi-speed analytical ultracentrifugation sedimentation velocity experiments using finite-element solutions of the Lamm equation. Using simulated data, we demonstrate here how these innovations can be used to enhance the resolution of sedimentation velocity experiments when compared to single-speed experiments. Using heterogeneous systems covering as much as five orders of magnitude in molar mass and fivefold in anisotropy, we compare results from runs performed at multiple speeds to those obtained from single-speed experiments, fitted individually and analyzed globally over multiple speeds, and quantify resolution for sample heterogeneous in size and anisotropy. We also provide guidance on the design of multi-speed experiments and offer a program that can be used to deduce optimal spacing of rotor speeds and speed step durations when a few parameters from the experiment can be estimated. These include the meniscus position, the sedimentation coefficient of the largest species in a mixture, and a solute distribution. Our results show that errors observed in the determination of hydrodynamic parameters for system with great heterogeneity are markedly reduced when multi-speed analysis is employed.

Multi-wavelength analytical ultracentrifugation of human serum albumin complexed with porphyrin.

The new Beckman Coulter Optima AUC instrument, which features multi-wavelength detection that couples the hydrodynamic separation of colloidal mixtures to spectral deconvolution of interacting and non-interacting solutes present in a mixture, was used to analyze the composition of human serum albumin (HSA) bound to metallo-protoporphyrin. We present new methods implemented in UltraScan that permit Optima AUC-derived multi-wavelength data to be spectrally decomposed in the same fashion as has been made possible for the Cölfen detector earlier. We demonstrate this approach by spectrally separating sedimentation velocity experimental data from mixtures of apo-HSA and HSA complexed to different metallo-protoporphyrins. We further demonstrate how multi-wavelength AUC can accurately recover percentages of metallo-protoporphyrin-bound HSA and apo-HSA from mixtures and how multi-wavelength AUC permits the calculation of molar extinction coefficients for porphyrins bound to HSA. The presented method has broad applicability to other complex systems where mixtures of molecules with different spectral properties need to be characterized.

Multi-speed sedimentation velocity implementation in UltraScan-III.

A framework for the global analysis of multi-speed analytical ultracentrifugation sedimentation velocity experiments is presented. We discuss extensions to the adaptive space-time finite element fitting methods implemented in UltraScan-III to model sedimentation velocity experiments where a single run is performed at multiple rotor speeds, and describe extensions in the optimization routines used for fitting experimental data collected at arbitrary multi-speed profiles. Our implementation considers factors such as speed dependent rotor stretching, the resulting radial shifting of the finite element solution's boundary conditions, and changes in the associated time-invariant noise. We also address the calculation of acceleration rates and acceleration zones from existing radial acceleration and time records, as well as utilization of the time state object available at high temporal resolution from the new Beckman Optima AUC instrument. Analysis methods in UltraScan-III support unconstrained models that extract reliable information for both the sedimentation and the diffusion coefficients. These methods do not rely on any assumptions and allow for arbitrary variations in both sedimentation and diffusion transport. We have adapted these routines for the multi-speed case, and developed optimized and general grid based fitting methods to handle changes in the information content of the simulation matrix for different speed steps. New graphical simulation tools are presented that assist the investigator to estimate suitable grid metrics and evaluate information content based on edit profiles for individual experiments.

Spectral and Hydrodynamic Analysis of West Nile Virus RNA-Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge.

Interactions between nucleic acids and proteins are critical for many cellular processes, and their study is of utmost importance to many areas of biochemistry, cellular biology, and virology. Here, we introduce a new analytical method based on sedimentation velocity (SV) analytical ultracentrifugation, in combination with a novel multiwavelength detector to characterize such interactions. We identified the stoichiometry and molar mass of a complex formed during the interaction of a West Nile virus RNA stem loop structure with the human T cell-restricted intracellular antigen-1 related protein. SV has long been proven as a powerful technique for studying dynamic assembly processes under physiological conditions in solution. Here, we demonstrate, for the first time, how the new multiwavelength technology can be exploited to study protein-RNA interactions, and show how the spectral information derived from the new detector complements the traditional hydrodynamic information from analytical ultracentrifugation. Our method allows the protein and nucleic acid signals to be separated by spectral decomposition such that sedimentation information from each individual species, including any complexes, can be clearly identified based on their spectral signatures. The method presented here extends to any interacting system where the interaction partners are spectrally separable.

Next-Generation AUC: Analysis of Multiwavelength Analytical Ultracentrifugation Data.

We describe important advances in methodologies for the analysis of multiwavelength data. In contrast to the Beckman-Coulter XL-A/I ultraviolet-visible light detector, multiwavelength detection is able to simultaneously collect sedimentation data for a large wavelength range in a single experiment. The additional dimension increases the data density by orders of magnitude, posing new challenges for data analysis and management. The additional data not only improve the statistics of the measurement but also provide new information for spectral characterization, which complements the hydrodynamic information. New data analysis and management approaches were integrated into the UltraScan software to address these challenges. In this chapter, we describe the enhancements and benefits realized by multiwavelength analysis and compare the results to those obtained from the traditional single-wavelength detector. We illustrate the advances offered by the new instruments by comparing results from mixtures that contain different ratios of protein and DNA samples, representing analytes with distinct spectral and hydrodynamic properties. For the first time, we demonstrate that the spectral dimension not only adds valuable detail, but when spectral properties are known, individual components with distinct spectral properties measured in a mixture by the multiwavelength system can be clearly separated and decomposed into traditional datasets for each of the spectrally distinct components, even when their sedimentation coefficients are virtually identical.

Characterization of size, anisotropy, and density heterogeneity of nanoparticles by sedimentation velocity.

A critical problem in materials science is the accurate characterization of the size dependent properties of colloidal inorganic nanocrystals. Due to the intrinsic polydispersity present during synthesis, dispersions of such materials exhibit simultaneous heterogeneity in density ρ, molar mass M, and particle diameter d. The density increments ∂ρ/∂d and ∂ρ/∂M of these nanoparticles, if known, can then provide important information about crystal growth and particle size distributions. For most classes of nanocrystals, a mixture of surfactants is added during synthesis to control their shape, size, and optical properties. However, it remains a challenge to accurately determine the amount of passivating ligand bound to the particle surface post synthesis. The presence of the ligand shell hampers an accurate determination of the nanocrystal diameter. Using CdSe and PbS semiconductor nanocrystals, and the ultrastable silver nanoparticle (M4Ag44(p-MBA)30), as model systems, we describe a Custom Grid method implemented in UltraScan-III for the characterization of nanoparticles and macromolecules using sedimentation velocity analytical ultracentrifugation. We show that multiple parametrizations are possible, and that the Custom Grid method can be generalized to provide high resolution composition information for mixtures of solutes that are heterogeneous in two out of three parameters. For such cases, our method can simultaneously resolve arbitrary two-dimensional distributions of hydrodynamic parameters when a third property can be held constant. For example, this method extracts partial specific volume and molar mass from sedimentation velocity data for cases where the anisotropy can be held constant, or provides anisotropy and partial specific volume if the molar mass is known.