By Sandrine Olivier, PhD, and John Stenson, PhD

Rapid advances in nanotechnology and nucleic acid chemistry are turbocharging pharmaceutical pipelines, speeding the development of treatments for previously incurable medical conditions. These advances include innovative vectors for the encapsulation and delivery of novel therapeutics.

Sandrine Olivier
Sandrine Olivier, PhD
Separations Specialist
Malvern Panalytical

Once a new vector is created, its potential as a delivery vehicle must be carefully assessed. For example, the introduction of a viral vector or a virus-like particle (VLP) typically prompts the determination of multiple 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 scientists in the pharmaceutical industry characterize their candidate molecules. With these technologies, particles in solution are first exposed to an incident laser beam. Some of the light is absorbed by the particle and reemitted in all directions. The intensity of the reemitted light relates to the size and molecular weight of the sample and can be measured by detectors set at different angles.

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

John Stenson
John Stenson, PhD
Product Manager, Nanomaterials
Malvern Panalytical

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 the different SLS technologies can provide and where their strengths lie.

This article demystifies SLS technologies and empowers investigators to choose the technology that is best suited to putting their vector under the spotlight.

What’s the best angle for your vector analytics?

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

SLS systems can measure any molecular mix by detecting scattered light at 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 viral vectors.

RALS and LALS systems are perfectly suited to viral and nonviral vector analytics, from early development through to manufacturing and quality control. The RALS detector, as its name suggests, collects scattered light at a right angle (90°) and is a highly sensitive and cost-effective way to measure 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, that is, the so-called anisotropic scattering molecules. Combining 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 as many as 21 angles. Once acquired, the measurements are modeled and extrapolated to elicit the final data. Accordingly, 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 reproducible data that meet global regulatory standards. But when clarity and visibility on other parameters are important for generating results and maximizing innovation, the simpler, lower-cost RALS and LALS systems can provide better solutions.

Each SLS detector requires calibration with standard materials, so calibrating a MALS system is more time consuming than setting up a RALS/LALS system. Added to this, the MALS detectors also require normalization using a sample of known molecular weight against the 90° reference detector. And as more signals are measured, the analysis becomes more complex. The analysis software in RALS and LALS systems is easier to use and provides opportunities for investigators to adapt analytical parameters without compromising data quality.

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

LALS is the best technique for accurate molecular weight measurements of large molecules. It provides top-quality data that outstrips data obtained from a MALS detector at a similarly low angle.

And in laboratories with a MALS setup, biochemists have realized that less is more. Regardless of the MALS capacity to measure multiple angles, they use only 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 provides the most robust and accurate data.

To be, or not to be … a MALS?

Despite the advantages of RALS and LALS, it is the MALS analysis that has become synonymous with absolute molecular weight measurements. Many innovators assume it is the only tool for the job. They often fail to realize that there is more to SLS technologies than MALS. Importantly, the other SLS tools can, in many cases, offer better solutions when it comes to measuring absolute molecular weight.

Confusion arises because the term “MALS” is mistakenly used by some investigators (and contributors to the scientific literature) to refer to absolute molecular weight analysis. The technologies differ in how many and which angles of scattered light they measure. (MALS, RALS, and LALS systems assess multi-, right-, and low-angle light scattering, respectively). Also, the technologies process data in fundamentally different ways when calculating absolute molecular weights.

The misunderstanding comes into sharp focus when customers who are considering purchasing an SLS system say, “I want a MALS detector,” when really, they want to measure absolute molecular weight. It’s rather like they are saying “I need an aspirin” when they want a painkiller. When it comes to molecular weight measurements, the confusion can create a costly 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 sufficient angles but minimizes complexity will give you the edge. But failing to grasp exactly which SLS technology is the best fit could result in suboptimal data and unnecessary spending on a system that fails to deliver value.

 

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