The Potential of Circular Dichroism Spectroscopy for the Development of Therapeutic Mrna-lipid Nanoparticles

Circular dichroism (CD) is a powerful analytical technique that has become indispensable in developing biotherapeutic products and holds promise to similarly benefit the development of lipid nanoparticle (LNP) drug formulations.

What are the applications of Circular Dichroism Spectroscopy?

Spectroscopy is well-established for the structural characterisation of protein biotherapeutics, including mainly products based on monoclonal antibodies (mAbs) and derived constructs such as bispecifics, antibody-drug conjugates, or Fab fragments.

When it comes to the biopharmaceutical development of therapeutics based on nucleic acids, the application of CD spectroscopy is, so far, mostly limited to the characterisation of the drug substance itself. However, in this context, the technique is just as well established and routinely used.

In general, any insight that CD spectroscopy has been proven to provide about nucleic acids in an academic setting may as well be transferred to industry applications. For example, CD spectroscopy can be used to distinguish different G-quadruplexes and other nucleic acid secondary structures or differentiate between helical conformations of double-stranded oligonucleotides, which include the right-handed A-form, which is typical of RNA, the B-form, and the left-handed Z-form. As another example, the method has been used to quantify the fraction of RNA base pairs formed in self-complementary regions of single-stranded molecules or in annealed double-stranded RNA.

More importantly, thermal melting experiments that follow the CD signal can provide insight into RNA stability and allow the identification of folding transitions and their characterisation through thermodynamic parameters. As absorbance is usually acquired simultaneously with CD, melting curves can also be acquired based on hyperchromicity, and the absorbance can be used to confirm accurate quantification of the RNA concentration. Our CD systems can even provide rich datasets beyond CD and absorbance data, including orthogonal fluorescence data obtained with assays based on fluorescent dyes or tagged RNA if applicable.

What are LNPs?

A lipid nanoparticle (LNP) is a nanoparticle composed of a lipid-based shell that encapsulates a hydrophobic core commonly used as a carrier for drug delivery. LNPs have gained popularity in recent years due to their ability to encapsulate a variety of therapeutics, including RNA-based molecules, and their potential for targeted delivery to specific cells or tissues.

LNPs protect the drug cargo from degradation and allow for control of its release. They are also biocompatible and biodegradable, making them safe for pharmaceutical and biomedical applications. Additionally, LNPs can be tailored to specific applications and, therefore, be designed to achieve optimal drug delivery and targeting.

How can you use Circular Dichroism Spectroscopy in LNP Drug Development?

CD spectroscopy has already successfully been used in the development and quality control of therapeutic nucleic acids, including the drug substances of mRNA-based Covid-19 vaccines, in accordance with FDA, EMEA, and ICH guidance1.

On the one hand, the application of CD spectroscopy to characterise fully assembled mRNA-LNPs poses challenges not encountered with the drug substance alone. On the other hand, LNP drug products are undoubtedly more than the sum of their constituent components – and CD spectroscopy has long proven an ideal technique for monitoring structural changes arising from events in intra- and intermolecular interactions. Therefore, exploiting the potential of CD spectroscopy for a better understanding of the molecular interdependencies that govern the unique properties of LNPs and their stability as a complex appears pretty much predestined.

Make no mistake: dismissing CD spectroscopy as an asset for the characterisation toolset may be a fatal decision – no advances in the development of mRNA-LNPs would have been made over the last years without the will to explore new avenues and the motivation to face challenges head-on. It is true that the size of LNPs can cause issues with light scattering in CD measurements, and the fact that a CD spectrum will always be the sum of contributions from all CD-active components in a sample may make it difficult to isolate more discrete information. Even then though comparative studies, e.g., between different batches or formulations, can provide valuable insight into structural integrity and quality of an LNP drug product. The analytical approach of HOS comparisons, which has successfully been used for many years in the development of protein-based therapeutics, is completely entirely independent of the nature of the sample or of wavelength range, and therefore is perfectly applicable to any comparative study of mRNA-LNPs as well. Conclusions thus drawn can enable researchers to detect early signs of degradation and take steps to mitigate them, e.g., by making adjustments to the formulation and storage or processing conditions. This way, CD spectroscopy can be an invaluable tool in the development and quality control of LNP drug products and might even support advances towards a quality-by-design approach.

On a final note, we believe that any such advances can only be accelerated through collaboration and, just as holds true for protein therapeutics development, it will certainly always require a broad biophysical toolset to characterise mRNA-LNPs and understand them in a holistic approach to infer conclusions about trends and correlations between development process and product. In this spirit, we have teamed up with Malvern Panalytical and SINTEF on a new Application Note for you, which illustrates in more detail how CD spectroscopy and Differential Scanning Calorimetry can benefit the development of mRNA-LNPs.

Read the full application note titled “Multi-method Benchtop Analyses to Gain Novel Insight into Structure and Internal Stability of Lipid Nanoparticles for mRNA Delivery”, which was produced in collaboration with the SINTEF and Malvern Panalytical below:

LNP Application Note