Malvern OMNISEC Emerges as the Ultimate tool for Accurate Quantification of Therapeutic Payload for LNP-based therapies

ATA Scientific Pty Ltd
Thursday, 14 December, 2023

Malvern OMNISEC Emerges as the Ultimate tool for Accurate Quantification of Therapeutic Payload for LNP-based therapies

Lipid-based nanoparticles (LNPs) hold great promise for the treatment, cure, and prevention of a range of challenging medical conditions, from genetic diseases to cancers. Not only do LNPs enable the efficient delivery of therapeutic payloads such as RNA, but unlike viral vectors, they can also be manufactured using cell-free production processes with the potential for rapid scaling, as demonstrated by the Micropore Pathfinder advanced cross flow technology. Understanding the therapeutic payload, particularly how much of the payload has been incorporated into the LNP, is critical to ensuring patients receive the correct therapeutic dose.

Although RNA quantitation has traditionally been performed using fluorescence measurement with RiboGreen dye, the analytical method has several downsides. The need for complex method development, where protocols are not easily transferable between different LNP formulations, in addition to the relative insensitivity of the assay and the presence of several commonly used salts, can compromise the accuracy of this popular assay.

Malvern Panalytical OMNISEC: the most advanced multi-detector GPC/SEC system for higher resolution and faster sample analysis.

Size exclusion chromatography (SEC) coupled to light scattering detectors

The Malvern Panalytical OMNISEC has emerged as a key approach to aid payload quantification when it comes to LNP-based therapies. SEC works by loading samples onto a column, which separates the sample components based on their hydrodynamic radius. Smaller components are retained in the column longer than larger molecules, meaning larger molecules elute from the column first. Unlike other SEC systems, the Malvern OMNISEC can be coupled to multiple advanced detectors, such as light scattering (right-angle and low-angle light scattering and multi-angle light scattering, RALS/LALS and MALS, respectively), UV/vis-PDA, and refractive index (RI) detectors and a differential viscometer enabling direct measurement of a range of parameters, several of which can be used to calculate payload information.

Figure 1: Schematic showing a typical SEC setup. For a larger image, click here.

Figure 2: An overview of the multi-detection pyramid, displaying what each SEC-coupled detector can directly measure and calculate.

Compositional analysis: calculating the weight fraction of your LNP payload

The compositional analysis method uses output from both the RI and UV/Vis-PDA detectors to determine the concentration of two components within a single sample (in this case, LNPs and the genetic payload). By using the data from two concentration detectors, advanced SEC system software can set up two equations to solve for the two unknowns: the concentration of component A (CA) and the concentration of component B (CB). The RI increment (dn/dc), which is a representation of the difference between the RI of the sample and the solvent together with the specific absorption coefficient (dA/dc), can be used to calculate the concentration of each component.

By comparing the concentrations of the two components, you can then obtain the weight fraction (%) of the LNP payload.

Figure 3: RI chromatograms of two mRNA-LNP samples, each overlayed with respective weight fractions of mRNA. For a larger image, click here.

SEC-LS: advantages and key considerations

SEC coupled to light scattering detectors (SEC-LS) has several benefits for LNP developers. As well as being a helpful tool to support LNP payload quantification, SEC-LS can be used to measure a range of other important sample parameters including size, molecular weight, and aggregation profile. Being able to measure multiple parameters without resorting to several different techniques delivers significant throughput benefits.

The measurements from SEC-LS are also highly accurate and reliable, and the technique does not require the difficult-to-acquire dedicated reagents needed in traditional methods. When it comes to getting the most out of SEC-LS for compositional analysis of LNPs there are some key considerations. The first is to ensure that a suitable chromatography column is used and that the LNP sample is adequately eluting from it. To minimise the risk of the sample sticking in the column, users should check the surface charge or measure the zeta potential of any samples to optimise the running buffer. This can done using Electrophoretic Light Scattering (ELS) as employed by the Malvern Zetasizer. LNP therapies prepared in physiological buffer can pose challenges when measuring zeta potential as they are highly conductive and can aggregate. Malvern’s diffusion barrier method protects the sample by keeping it away from the electrodes within the buffer while also reducing the amount of sample required for the measurement.

ATA Scientific offers a range of technologies to enable robust manufacture and characterisation of complex drug delivery systems and particularly LNP-based therapies. If you would like to learn more about how to better characterise your LNPs, reach out to our team of analytical specialists at +61 2 9541 3500 or


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