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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Dynamic light scattering techniques can monitor viruses and virus-like particles in their native state.
Effective analysis of viruses and virus-like particles (VLPs) is crucial for the development of vaccines and drugs. VLPs are designed to mimic the activity of viruses via viral surface proteins, but they lack the infectious genetic material of viruses. Detection of VLPs can be a challenging task, and any detection method must be able to differentiate virus and VLP particles--which range from tens to hundreds of nm--from proteins. In addition, many sample preparation techniques have the potential to impact the structure and properties of virus particles and VLPs.
As a non-invasive method, dynamic light scattering (DLS) offers many advantages over more traditional approaches such as microscopy, which often require manipulations that can alter the particles, according to Sophia Kenrick, an application scientist with Wyatt Technology. “DLS provides the hydrodynamic size of molecules and particles in solution and consequently can measure the size of viruses and VLPs in their native state,” she adds.
The challenge of the native environment
Characterizing viruses and virus-like particles in their native environments is difficult, particularly when it comes to identification of crucial traits that are relevant to API development, production, and targeted applications. Particle size and morphology and the interactions that influence their self-assembly behavior, reactivity, and stability must be evaluated in the presence of many other types of molecules, according to Yuanming Zhang, chief application scientist for Brookhaven Instruments.
Determining the structures of virus particles and VLPs includes quantifying the total particle size, heterogeneity, and aggregate content in a final formulation solution, according to Kenrick, and all these aspects can be investigated with DLS. In batch mode (i.e., with an unfractionated sample) DLS provides an average hydrodynamic size for a given sample and some information about the size distribution. The DLS data can also be analyzed to determine if other species are present in solution in addition to the VLPs. DLS reveals the sizes of multiple species in the solution as long as there is approximately a five-fold difference in the hydrodynamic radii of the species. “Larger species may be contaminants or aggregates formed during the purification or formulation of the VLP. Researchers are very concerned about these aggregate species and want to find the formulation that minimizes their appearance and promotes the overall stability of the VLP,” Kenrick says.
“Non-invasive and label-free analytical techniques are highly preferable in order to minimize the disturbances/stresses induced on the particles by sample preparation and analysis,” observes Zhang. The best methods are also suitable for routine analysis of virus particles and VLPs for quality control (QC) purposes at various stages of the manufacturing process (upstream and downstream) and can be implemented in flexible platforms suitable for diverse field requirements.
Non-destructive and flexible
The non-invasive nature of DLS enables it to be flexibly implemented either in flow-through mode for in-line characterization or in batch-mode for offline analysis. “By taking advantage of the technological advances in lasers, photon-detectors, and fiber optics, DLS analyses can be performed even with a hand-held device, a versatile measurement format for monitoring manufacturing processes and performing product quality control analyses,” says Zhang.
In addition, DLS measurements are rapid-typically requiring less than 30 seconds-which allows researchers to measure the effects of a large number of formulation, processing, and environmental conditions on the size, conformation, and stability of VLPs quickly and efficiently, according to Kenrick. In particular, she notes that with a plate-based DLS instrument, all of these conditions may be arrayed in a 96-, 384-, or 1536-well plate and scanned in a single, automated experiment. The amount of sample needed for a typical DLS analysis is also quite small, 20–50 µL for typical 384-well plates or as low as 1 µL for cuvette-based instruments.
“DLS is appealing as an analytical technique because it covers a very wide range of particle sizes in a single measurement (~0.1 nm–1 μm). In the size range of virus and virus-like particles, DLS is very sensitive and can detect particles at weight fractions in the parts-per-million (ppm) range,” Zhang comments.
DLS measures the time-dependent fluctuation in scattered light intensity caused by the Brownian motion of particles in solution, such as virus and virus-like particles. The pattern of fluctuations is quantified using an autocorrelation analysis. This autocorrelation function is then fitted to provide the quantitative information on particle diffusivity (diffusion coefficient).
At a sufficiently low-particle concentration, particles diffuse independently from each other, and each particle’s hydrodynamic radius (Rh) can be calculated from its diffusivity using the Stokes−Einstein equation. At higher particle concentrations, particle-particle interactions (electrostatic, van de Waals, hydrogen bonding, hydrophobic forces, etc.) influence the diffusion behavior of the particles. The sum of these effects can be determined from the concentration dependence of the particle diffusivity and is quantified by the diffusion interaction parameter kD. A positive value for kD reflects net repulsive interactions between particles, which are generally favorable for a formulation, while a negative value indicates a net attractive force, which can lead to aggregation.
Batch DLS can also be used to quantify trends as a function of time, temperature, and concentration. Researchers often quantify the thermal stability of proteins and VLPs by measuring the temperature at which they unfold or aggregate, and then try to identify conditions to delay this behavior, according to Kenrick. Through use of DLS, these behaviors can be observed as an overall size change, a change in the polydispersity of the sample, or the appearance of a second large species.
To complement batch analysis, DLS can also be combined with a fractionation technique, such as size-exclusion chromatography or field-flow fractionation, and multi-angle light scattering (SEC-MALS or FFF-MALS) to provide information about the conformation of a VLP. In this situation, the MALS analysis can provide the molar mass and root mean square radius (RMS radius or Rg) of the VLP, and DLS can simultaneously provide the hydrodynamic size. The combination of Rh and Rg can then be used to determine the shape of the VLP (i.e., rod-like, hollow sphere, filled sphere).
Because virus and VLPs are typically much larger than the rest of the assay ingredients, it is possible through use of DLS to pick up signals arising from very small populations of virus and VLPs among highly concentrated assay ingredients, including buffer chemicals and nutrient proteins, according to Zhang. He notes, however, that the diffusion characteristics of virus and VLPs can be significantly skewed and even completely overshadowed in the presence of a few impurities of larger sizes.
During sample preparation, proper care must be exercised to remove impurities like cells/cell fragments and dust contaminants to perform a valid DLS assay of virus and VLPs. Such sample preparation can typically be readily achieved with centrifugation and filtration tools available in an analytical lab, Zhang explains.
With proper sample preparation, DLS is appropriate for measuring the size, conformation, and thermal and colloidal stability of purified VLPs, and is particularly useful for quality assessment. Some examples include the comparison of multiple lots of a VLP production, understanding changes in size and aggregate content after stress testing, and ensuring a uniform sample distribution prior to analysis with another technique, according to Kenrick. “DLS is most appropriate for this type of analysis because the measurement is fast, non-destructive, and can be multiplexed using a plate-based instrument,” she adds.
DLS cannot, however, quantify the sizes of aggregated particles with radii greater than ~5 µm; these large sizes require a different characterization technique. DLS also cannot be used to count particles, although complementary light scattering techniques, such as SEC-MALS and FFF-MALS, can provide particle density values.
Enhancements in DLS
New developments in DLS technology are expanding its applicability. The availability of multiple DLS detection angles on a single measurement platform-forward (~15°) and backscatter (~173°), in addition to the standard 90°-allows practitioners to choose a desired detection angle with the click of a mouse. “Enabling exploration of the angular dependence of scattering from typical Mie scatterers, such as viruses and VLPs, makes it much more convenient to optimize DLS-detection conditions for targeted particles,” says Zhang.
Microrheology, in which probe particles of known sizes are used to determine the viscoelastic behavior of liquids containing virus and virus-like particles, is a technique now possible using DLS. “Compared to conventional viscometry and rheometry techniques, microrheology covers a much broader range of dynamic frequencies/shear rates (~10-3 to ~107 s-1), and only a very small amount of sample is needed. As a result, microrheology enables rheological studies on biological samples that previously were not possible due to the limited material availability,” Zhang observes.
For Kenrick, the adoption of DLS for high-throughput analysis using plate readers is an important development that expands the capabilities of this method for viruses and VLPs. The latest device from Wyatt, for example, can use standard 96-, 384-, or 1536-well plates, does not require any liquid handling during the measurement, and provides no opportunity for cross-contamination once the plate has been loaded into the detector. With the capability to heat samples to 85 °C, the system also expands the temperature range for which VLP unfolding and aggregation can be evaluated using DLS, according to Kenrick. The ability to take pictures of each well after completion of a DLS analysis also allows users to observe any bubble formation or precipitation that may have occurred during long time-course or high-temperature experiments and eliminate those test results.
Orthogonal methods complement DLS
The essential limitation of DLS, or light scattering in general, is that it does not distinguish the chemical nature of the scatterers. In addition, particle-size distribution calculated from the measured particles diffusivity determined using DLS reflects only a scattering intensity-weighted ensemble average over particles in the detection volume of a DLS measurement, according to Zhang. “From time to time, these limitations present challenges in terms of the interpretation of DLS results. Therefore, cross-validation of DLS assays is strongly recommended using analytical techniques that are orthogonal to DLS, such as spectroscopy, microscopy, or chromatography,” he comments.
Kenrick agrees that DLS alone is not sufficient to quantify the range of sizes and molecular weights present in a VLP solution. For example, the presence of doublets and triplets will increase the average radius measured by batch DLS, but these aggregates will not be resolved as separate populations. For this type of analysis, a fractionation technique, such as SEC or FFF, is required to separate the different sizes and quantify them individually. This separation process is then coupled with MALS to provide molar mass, root-mean-square radius, and particle-number density, as applicable, in addition to the hydrodynamic size from DLS.
“For these analyses, we recommend that DLS detection occur in the same measurement volume as the MALS detection so that the sample is not diluted by traveling from one detector to another, which can lead to broadening of eluting peaks,” Kenrick notes. “Dilution can break up reversible associations, so by avoiding dilution, researchers ensure that the size they obtain using DLS corresponds to the exact species for which they determined the molar mass and size using MALS,” she adds. Limiting the dilution effect is also crucial for ensuring an adequate DLS signal during an SEC or FFF separation because DLS is generally less sensitive than MALS.
Article DetailsBioPharm International
Vol. 28, No. 10
Citation: When referring to this article, please cite it as C. Challener, “Dynamic Light Scattering for Non-Destructive, Rapid Analysis of Virus-Like Particles,” BioPharm International 28 (10) 2015.