Demystifying static light scattering techniques

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By Sandrine Olivier, PhD, and John Stenson, PhD

Rapid advances in nanotechnology and nucleic acid chemistry are energizing pharmaceutical pipelines, accelerating the development of treatments for previously incurable medical conditions. These advances include innovative vectors for the encapsulation and delivery of new therapies.

Sandrine Olivier, PhD
separations specialist
Malvern analysis

Once a new vector is created, its potential as a delivery vehicle must be carefully evaluated. For example, the introduction of a viral vector or virus-like particle (VLP) typically prompts the determination of several critical attributes. These include virus titers, full/empty capsid ratios, and absolute molecular weight measurements.

For decades, static light scattering (SLS) technologies have been helping pharmaceutical scientists characterize their candidate molecules. With these technologies, particles in solution are first exposed to an incident laser beam. Part of the light is absorbed by the particle and re-emitted in all directions. The intensity of the re-emitted light is related to the size and molecular weight of the sample and can be measured by detectors set at different angles.

Three SLS technologies exist: multi-angle light scattering (MALS), right-angle light scattering (RALS), and low-angle light scattering (LALS).

John Stenson
John Stenson, Ph.D.
Product Manager, Nanomaterials
Malvern analysis

Choosing the best SLS system to characterize a candidate vector is critical to the success of any vaccine or gene therapy development program. But confusion exists among scientists around what different SLS technologies can deliver and where their strengths lie.

This article demystifies SLS technologies and allows investigators to choose the best technology to put their vector in the spotlight.

What is the best angle for your vector analysis?

Increasingly, biochemists and vaccinologists are using SLS technologies to study the stability of viral vectors and measure polysaccharide degradation and structural changes that follow protein conjugation.

SLS systems can measure any molecular mixture by detecting scattered light from a different angle. When coupled with other techniques, such as gel permeation chromatography (GPC) or size exclusion chromatography (SEC), an SLS system is an essential tool for characterizing complex samples such as vectors viral.

The RALS and LALS systems are ideally suited for the analysis of viral and non-viral vectors, from early development through manufacturing and quality control. The RALS detector, as its name suggests, collects scattered light at right angles (90°) and is a very sensitive and economical means of measuring the molecular weight of proteins and protein conjugates. It also offers the best signal-to-noise ratio of the three systems.

A true LALS detector collects scattered light at 7°, making it the most accurate way to measure the absolute molecular weight of larger molecules, i.e. the so-called anisotropic scattering molecules. The combination of RALS and LALS detectors in one system provides the perfect solution for measuring both smaller molecules and larger, more complex molecules.

Unlike a single-detector RALS or LALS system, or a two-detector RALS/LALS system, a MALS system typically has at least three detectors. Indeed, MALS systems can be configured to collect scattered light from up to 21 angles. Once acquired, the measurements are modeled and extrapolated to obtain the final data. As a result, data acquisition, processing, and analysis are more complex and time-consuming with MALS than with the alternatives, RALS and LALS.

When an SLS system is needed, less is often more

All SLS technologies are robust and generate highly accurate and repeatable data that meets global regulatory standards. But when clarity and visibility into other metrics are important for driving results and maximizing innovation, simpler and less expensive RALS and LALS systems can provide better solutions.

Every SLS detector requires calibration with standard materials, so calibrating a MALS system takes longer than setting up a RALS/LALS system. Additionally, MALS detectors also require normalization using a sample of known molecular weight relative to the 90° reference detector. And the more signals are measured, the more complex the analysis becomes. The analysis software for RALS and LALS systems is easier to use and offers researchers the ability to tailor analytical parameters without compromising data quality.

RALS offers the most sensitive and accurate measurements for small molecules, such as proteins that scatter light without angle dependence, providing a single measurement at 90° of scattering. It is only when measuring larger macromolecules (such as polysaccharides or viruses) that the intensity of the scattered light begins to vary with the angle of measurement. This variation must be taken into account to ensure accurate data. In this case, a LALS or MALS system can really deliver results.

LALS is the best technique for accurate measurements of the molecular weight of large molecules. It provides higher quality data that exceeds data obtained from a MALS detector at an equally low angle.

And in labs with a MALS setup, biochemists have realized that less is more. Regardless of the MALS’ ability to measure multiple angles, they only use one detector, the 90° signal, to accurately measure their protein samples. For protein analyses, a RALS system offers advantages. And with the best signal-to-noise ratio, it delivers the most robust and accurate data.

To be, or not to be… a MALS?

Despite the advantages of RALS and LALS, it is MALS analysis that has become synonymous with absolute molecular weight measurements. Many innovators assume it’s the only tool for the job. They often don’t realize that SLS technologies are more than MALS. It is important to note that other SLS tools can, in many cases, offer better solutions when it comes to measuring absolute molecular weight.

The confusion arises because the term “MALS” is incorrectly used by some researchers (and contributors to the scientific literature) to refer to absolute molecular weight analysis. The technologies differ in the number and angles of scattered light they measure. (MALS, RALS, and LALS evaluate multi-angle, right-hand, and low-angle light scatter, respectively). Additionally, the technologies treat data fundamentally differently when calculating absolute molecular weights.

The misunderstanding arises when customers considering purchasing an SLS system say, “I want a MALS detector,” when in reality they want to measure absolute molecular weight. It’s a bit like saying “I need an aspirin” when they want a painkiller. When it comes to molecular weight measurements, confusion can create an expensive headache.

Choose the best tools for the job. SLS technologies are invaluable for the accurate characterization of your viral vector to regulatory standards. And choosing a system that measures enough angles but minimizes complexity will give you the edge. But not understanding exactly which SLS technology is best suited could result in suboptimal data and unnecessary expense on a system that doesn’t deliver value.

Sandrine Olivier, PhD, is a separations specialist and John Stenson, PhD, is a product manager, nanomaterials, at scientific instrument supplier Malvern Panalytical.

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