January 15, 2015 (Vol. 35, No. 2)

Daniel Some, Ph.D. principal scientist Wyatt Technology

Advantages of Utilizing a Light-Scattering Toolbox in Vaccine Development

Virus-like particles (VLPs) are biomolecular nanoparticles formed by controlled self-assembly of viral structural proteins. Lacking viral DNA for replication, VLPs are safe yet highly immunogenic, triggering an immune response without the risk of infection.

Following the success of blockbuster VLP-based vaccines against hepatitis B and human papillomavirus these bioparticles increasingly serve not only as immune stimulants but also as therapeutic gene delivery agents, modalities that take advantage of the DNA-encapsulation and -transport capabilities of VLPs.

In some ways, the  development of a VLP is similar to that of any biotherapeutic. Functional activity is assessed via the binding of target antibodies to the VLP, though unlike most protein-ligand interactions, the stoichiometry of the interaction may be unknown. Formulation development screens hundreds of combinations of buffer conditions and excipients to identify those most promising for maintaining long-term stability.

The top formulation candidates undergo accelerated and long-term stability tests. Comparability and lot-release tests of structure and efficacy must verify batch-to-batch consistency via high-resolution characterization and quantification of degradants; incomplete characterization could present a barrier to licensure in the face of tight regulatory control.

A unique challenge is the need to distinguish between unincorporated protomers (capsid proteins), capsomeres (VLP subunits), well-formed capsids (viral envelopes), and intermediate or malformed fragments. Capsids must exhibit specific size, shape, and mass values to present to the immune system an exterior similar to that of real viruses; partial or misformed VLPs may impact immune stimulus. Additionally, the use of VLPs for delivery of gene therapeutics requires analysis of the oligonucleotide payload inside a VLP scaffold.

Light-Scattering Toolbox

Light scattering for characterization of VLPs comes in three primary varieties. Multiangle static light scattering (MALS) measures absolute molar mass and size (radius of gyration, rg, or root mean square radius) from first principles. Dynamic light scattering (DLS) measures translational diffusion and hence hydrodynamic radius, rh. Electrophoretic light scattering (ELS) measures electrophoretic mobility.

The measurement ranges of MALS, DLS, and ELS are ideally suited to VLPs, which typically fall between 20 and 50 nm in radius. The combination of MALS, DLS, and ELS with different types of sample preparation and delivery creates a comprehensive toolbox of techniques for biophysical characterization.

MALS instrumentation couples to size-exclusion chromatography (SEC-MALS) or field-flow fractionation (FFF-MALS, described below) to characterize the distribution of populations in a VLP sample for structure, aggregation, and the presence of unassembled protomers or capsomeres. Adding online DLS detection to SEC-MALS or FFF-MALS further refines structural analysis: the relationships between the weight-average molecular weight (Mw), rh, and rg indicate conformation (shape and compactness).

Figure 1. FFF-MALS high-resolution separation and characterization over a small range, using less than 3 µg of VLP. Conversely, FFF may also be tuned to separate a very wide range of sizes.

Coupling a MALS detector to a composition-gradient delivery system (CG-MALS) creates a unique means for evaluating process assembly and degradation kinetics, and quantifying biomolecular interactions such as self-association or binding of antibodies to VLPs, without labeling or immobilization.

Since MALS determines absolute molar mass of the solute, it is particularly good at extracting binding stoichiometry (such as the number of antibodies per VLP, or complex assemblies) as well as affinity.
High-throughput dynamic light scattering (HT-DLS) can very rapidly assess hundreds of formulation conditions for gross aggregation as well as VLP dissociation during thermal or chemical stress tests. A microwell-plate-based, high-throughput in situ DLS plate reader such as the DynaPro® plate reader II is an ideal tool for time-sensitive development of stable formulations.

Rounding out the light-scattering toolbox, massively parallel phase analysis light scattering (MP-PALS, a high-sensitivity, high-throughput form of ELS) in conjunction with sample delivery by an autosampler, facilitates measurement and optimization of surface charge in formulation buffers for enhanced stability.

Little material is needed to characterize VLPs by light scattering—typically sub-microgram quantities for DLS, SEC-MALS, and FFF-MALS. CG-MALS requires several µg of VLP and about 100 µg of antibody, though more VLP material may be required to characterize weak self-association.

Figure 2. By combining UV, MALS, and refractive index (RI) detection with FFF, a 1 MDa DNA payload is distinguished from the 14.5 MDa protein envelope.

Separation by FFF-MALS

Unlike typical biotherapeutics, VLPs do not always fall within the range of separation by SEC. In many cases, they will not elute from a column, or elute at a time not representative of their true size, due to a variety of nonideal column interactions.

FFF overcomes many of the limitations of SEC, providing particle separation from 1 nm to >1,000 nm with no stationary phase and the ability to zoom in on specific ranges, or zoom out over the entire range, simply by modifying flow parameters. The commercial availability of FFF combined with sophisticated MALS and DLS detectors has proven great value in biotechnology, filling in the gaps left by industry standards such as unfractionated DLS and transmission electron microscopy (TEM).

Via FFF-MALS and FFF-DLS, unassembled protomeres, capsomeres, malformed VLPs, and aggregates may be separated and distinguished from well-formed, monomeric VLPs (Figure 1). FFF-MALS systems such as those developed by Wyatt Technology can quantify the variability across different preparations of VLPs before and after stresses (Figure 2).

A comprehensive array of tools based on light scattering supports the development of VLP-based vaccines through the characterization of essential biophysical properties as well as derived properties (Figure 3). This instrumentation draws on static, dynamic, and electrophoretic light scattering (MALS, DLS, ELS), combined with automated fractionation, composition gradients or high-throughput screening.

Researchers in vaccine discovery, process development, formulation development, and product characterization can leverage light-scattering technology to accelerate the VLP pipeline.

Figure 3. CG-MALS determines binding affinity of antibodies to VLPs, as well as absolute molecular stoichiometry. Hence it distinguishes between complexes where both arms of the antibody bind to a single VLP versus single-arm attachment that can lead to multi-VLP complexes.

Daniel Some, Ph.D., (dsome@wyatt.com) is principal scientist at Wyatt Technology.

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