Characterising Lipid Nanoparticles for Vaccine Development

Lipid nanoparticles (LNPs) either loaded with nucleic acids or as liposomes containing an aqueous core, have received great interest from pharma as delivery vehicles for different therapeutic treatments for many different reasons.LNPs offer improved stability and delivery efficiency by protecting drug molecules from degradation by the body’s natural immune processes. Moreover, the LNP can be specifically targeted using customised ligands attached to its surface.

The breakthrough of mRNA-based vaccines

The fast pace of progress in mRNA vaccines (e.g. for COVID-19) would not have been possible without major recent advances in RNA encapsulation and delivery methods. Recent breakthroughs with mRNA-based highlight the potential of lipid-based particles as powerful and versatile delivery vectors for vaccines and gene therapies, to treat previously untreatable diseases. Extensive basic research into RNA and lipid and polymer biochemistry has made it possible to translate mRNA vaccines into clinical trials and has led to an astonishing pace of global vaccination. 

LNPs have been found to be the most effective mRNA formulation/delivery approach and function to protect the mRNA from degradation when injected into the patient and to promote entry of the mRNA into cells. LNPs typically consist of four components: an ionizable cationic lipid, which promotes self-assembly into virus-sized (~100 nm) particles and enables endosomal release of mRNA to the cytoplasm; lipid-linked polyethylene glycol (PEG), which increases the half-life of formulations; cholesterol, a stabilising agent; and naturally occurring phospholipids, which support the lipid bilayer structure.Inactive ingredients such as salts, sugars, and stabilizing acids are added to achieve formulation stability during transport and storage.

Analytical characterisation of these nanoparticles is critical to drug design, formulation development, understanding in vivo performance, as well as quality control during formulation and manufacture. The use of ever-more structurally complex molecules warrants a growing requirement for complementary and orthogonal analytics to ensure data quality and the reliability of research. 

How does Malvern Panalytical contribute to the characterisation of lipid nanoparticles

When developing LNP drug candidates, several critical quality attributes (CQAs) need to be addressed. Nanoparticle characterisation CQAs for particle size, particle size distribution and concentration can be measured using Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA). These techniques are both orthogonal and complimentary in addressing this. Surface charge, another CQA can be probed using Electrophoretic Light Scattering (ELS), a measure of particles colloidal stability CQA.

Particle size and stability using light scattering techniques

Light scattering techniques are used extensively in the characterisation of lipid nanoparticle and liposome research to measure particle size, stability, zeta potential and particle concentration. The Zetasizer range of light scattering instruments can be used to optimise lipid-based formulations and process conditions, such as monitoring stability, understanding surface modification, and developing formulations. Non-invasive backscatter (NIBS) optics enable reliable measurements of concentrated, turbid samples without the need for dilution. Delivering data in a short time frame, the Zetasizer allows users to implement this technique throughout the development pipeline. 

Aggregation/ encapsulation efficiency using Nanoparticle Tracking Analysis (NTA)

NTA allows you to visualise and size individual particles in the preparation, generating important information about nano-particle content. For instance, the presence of larger particles could represent either non-viral cell debris from the cell culture process or aggregates of virus particles containing many individual virions. In either case, such aggregates/contaminants represent a possible problem to the manufacturer. NanoSight helps vaccine developers devise a solution.

Biomolecular interactions using Isothermal Titration (ITC) and Differential Scanning Calorimetry (DSC)

Detailed characterisation of protein structure enables understanding of protein function. It is therefore among the activities central to academic and industrial research and development. ITC is used in quantitative studies of a range of biomolecular interactions, that work by directly measuring heat that is either released or absorbed during a biomolecular binding event. By providing a complete thermodynamic profile of the molecular interaction, ITC can explain mechanisms’ underlying interactions, and enable more confident decision-making in hit selection and lead optimisation.

DSC is used to characterise stability of a protein or other biomolecules directly in its native form and achieves this by measuring heat change associated with the molecule’s thermal denaturation. Precise and high-quality data obtained from DSC provides vital information on protein stability in process development, and in the formulation of potential therapeutic candidates. The ‘first principle’ nature and high resolution of DSC makes it a well-established technique for extended structural characterisation and stability profiling of biomolecules and viruses in solution. Due to its direct readout, broad temperature range and sensitivity to thermally-induced unfolding, DSC is also used as the gold standard technique for validation of data from higher throughput thermal stability assays.

How does the RedShiftBio (MMS technology) contribute to the characterisation of lipid nanoparticles

The nature and composition of a vaccine makes them inherently difficult to characterise. The active ingredient such as a toxoid or protein subunit is often in very low concentration. The adjuvant, such as alum, can often be in quite high concentration relative to the biologic, and its particle size can range from nanometers, to microns. Adding preservatives and antibiotics to the mix, the result is a material with such diverse properties that many typical analytical tools struggle to measure the formulated product.

Microfluidic Modulation Spectroscopy (MMS) combines mid-infrared laser spectroscopy with microfluidics to overcome many of the limitations of traditional spectroscopy-based technologies. Its ability to provide multiple attribute data reduces or eliminates the need for performing separate measurements across different tools. MMS offers ultrasensitive, highly reproducible, automated structural measurements of protein aggregation, quantitation, stability, structure and similarity – measurements that underpin drug safety and efficacy. This novel method can be used to examine protein secondary structure for a wide range of applications from mAb-based biotherapeutics to robust measurements of ADCs, AAVs, and mRNA. It provides direct, label-free measurements over the concentration range <0.1 mg/ml to >200 mg/ml. Real-time background subtraction eliminates need to dialyse samples. Even at low concentrations the AQS3Pro system allows detection of <2% change in secondary structure. 

How does Fluidity One (MDS Technology) contribute to the characterisation of lipid nanoparticles

Characterising membrane proteins and their interactions with lipids remains a major challenge. Traditional methods can involve use of detergents which often cause the loss of native lipids surrounding membrane proteins, which ultimately impacts structural and functional properties. Microfluidic Diffusional Sizing (MDS) is a new method that can used to determine protein size and concentration of protein samples for quality control purposes through laminar flow diffusion. MDS can also be applied to evaluate protein/ligand and protein/lipid interactions. This method enables users to detect virtually any primary amine-containing molecule, emphasising the versatility of the technique.

The Fluidity One-W uses MDS technology to study protein complexes and their formation in crude biological backgrounds such as cell lysates or blood plasma. Using small sample volumes (<10 μL) and in a relative short time (t < 15 min) MDS can be used to determine the diameter of lipid particles with high precision. These particles can then be confirmed by supportive DLS data using the Malvern Zetasizer system.

We can help manufacture your success 

ATA Scientific provides a range of physicochemical characterisation tools that are used from the initial characterisation of biological materials through to final manufacturing and quality control, and which deliver information essential to ensuring the stability and efficacy of the vaccine product.

Contact us for more information on the latest analytical technologies from Malvern Panalytical (DLS, NTA, ITC/DSC), Redshiftbio (MMS technology) and Fluidity One W (MDS technology).