From vaccines to personalized cancer therapies, scientists are studying the use of mRNA across many clinical applications. However, there are several factors that are unique to mRNA drug product development that the biopharmaceutical industry must consider to manufacture these cutting-edge therapies. Lipid nanoparticles (LNPs) play a critical role for mRNA therapies because they protect the nucleic acid material from degradation as it is delivered to the targeted cell. Precise characterization of LNP components can be challenging; however, there are technologies available to help analyze the complex LNP systems used in delivering mRNA, such as the ultra-high-performance liquid chromatography-charged aerosol detector (UHPLC-CAD) method.
The UHPLC-CAD method developed through the Thermo Scientific
Chromeleon
Chromatography Data System (CDS) enables accurate molar ratio determination of lipid components with high sensitivity and reproducibility under compliance-ready conditions. By simplifying sample preparation and method development, this approach ensures efficient quality control and formulation development, streamlining the pathway for scalable production of LNP-based therapies.
The development of LNPs for mRNA delivery involves careful optimization to ensure that the particles are of the correct size and charge, enhancing their ability to effectively deliver mRNA to cells. The formulation of LNPs adds a further layer of complexity, as the process must balance multiple factors, including particle size, encapsulation efficiency, and stability. The optimal LNP formulation is one that can efficiently deliver mRNA while minimizing adverse immune responses and ensuring safety and efficacy in clinical applications.
Scientists recently studied how to optimize the formulation of LNPs, using precise calibration methods and chromatographic techniques to quantify lipid components and encapsulated payloads, to help ensure accurate formulation of LNPs for drug delivery applications.1 The lipids components used in this study were diluted in 99% ethanol to create stock concentrations (Table 1).
The payload used in this formulation was polyadenylic acid potassium salt (poly-A) and the LNPs were then formulated using the Thermo Scientific Ignite
NanoAssemblr
. The aqueous poly-A and organic lipids were added to separate syringes, which were then connected to the NanoAssemblr
system. Encapsulation efficiency was assessed using the RiboGreen
Assay and the size of the LNPs was measured using Dynamic Light Scattering (DLS).
After removing the formulations from the freezer, they were gently inverted several times and then placed in the UHPLC autosampler. For the calibration standards, stock solutions for both formulations were prepared by weighing lipid standards, dissolving them in ethanol, and sonicating them. A series of methanol dilutions were then performed to prepare the calibration standards, which were used to generate calibration curves for lipid quantification. Data acquisition and analysis were carried out using Chromeleon CDS 7.2.10 MUd software.
The initial attempt to separate the lipid components using a high concentration of organic solvent (75%) caused the LNPs to break through the column and resulted in poor data (Figure 1). A new method was developed to improve lipid separation and clarity, preventing interference from the LNPs and making the signals for each lipid more distinct. This method also allowed poly-A, encapsulated in the nanoparticles, to be detected as a separate peak, which requires further investigation.
Figure 1. Initial attempt to separate lipid components in formulations. (A) Formation #1. (B) Formation #2
Once a reliable method for separating the lipids was achieved, calibration curves were created for each lipid to measure their concentrations accurately (Figure 2). For the first formulation, scientists used a mix of four lipid standards and the concentration for each standard was adjusted accordingly. The calibration showed good accuracy with a high calibration coefficient (>0.998). For the second formulation, each lipid was measured individually using its highest concentration for the calibration. This process ensured that all the lipids could be quantified precisely.
Figure 2. Separation of formulations with optimized method. (A) Formulation #1. (B) Formulation #2.
In all, the study found that the UHPLC-CAD method was effective for measuring individual components of both LNP formulations. The measured molar ratios were in strong agreement with the original ratios specified for the samples, indicating the reliability and precision of the method. That consistency suggests that this approach can be reliably used and replicated for lipid quantification across various formulations.
These results are significant for mRNA vaccine research and manufacturing because all drug products require characterization to ensure safety and efficacy. The formulation is of equal importance. Some of the lipids used in LNP manufacturing are man-made for specific properties. These must be tested individually for purity and the final composition determined. The CAD detection is not only sensitive for these non-UV absorbing compounds, but it also gives the same molar response for all the species injected. This capability is important when looking at the molar composition as well as the degree of purity of the individual lipids used, especially when there may not be a standard available for each impurity detected.
The two current FDA-approved COVID-19 vaccines, mRNA-1273 and BNT162b, are notable examples of successful implementation of LNP technology, as they used LNPs to deliver antigen-coding mRNA. Today, many other lipid nanoparticle-oligonucleotide formulations have been developed and are under clinical evaluation for the prevention of other viral infections as well as the treatment of cancer and genetic diseases.
As scientists look to expand the use cases for mRNA therapies, the role of LNP delivery will continue to advance how biopharmaceutical companies approach the development of life-altering treatments, from vaccines to cancer therapies and treatments for infectious diseases. Overcoming challenges in the development of mRNA drug products through innovative and tech-forward approaches helps open the door to cutting-edge therapies.
Reference
1. Characterization of lipid components in lipid nanoparticle (LNP) formulations – Application Note AN001928 – an-001928-bt-lipid-components-LNP-an001928-na-en.pdf (SECURED)
Ken Cook, PhD, Manager, Application Scientists, Analytical Instruments, and Sissi White, Senior Product Applications Specialist, Analytical Instruments, Thermo Fisher Scientific.
The post The Characterization of Lipid Components in Lipid Nanoparticle Formulations appeared first on GEN - Genetic Engineering and Biotechnology News.
The UHPLC-CAD method developed through the Thermo Scientific
Challenges in lipid nanoparticle formulation
The development of LNPs for mRNA delivery involves careful optimization to ensure that the particles are of the correct size and charge, enhancing their ability to effectively deliver mRNA to cells. The formulation of LNPs adds a further layer of complexity, as the process must balance multiple factors, including particle size, encapsulation efficiency, and stability. The optimal LNP formulation is one that can efficiently deliver mRNA while minimizing adverse immune responses and ensuring safety and efficacy in clinical applications.
Scientists recently studied how to optimize the formulation of LNPs, using precise calibration methods and chromatographic techniques to quantify lipid components and encapsulated payloads, to help ensure accurate formulation of LNPs for drug delivery applications.1 The lipids components used in this study were diluted in 99% ethanol to create stock concentrations (Table 1).
The payload used in this formulation was polyadenylic acid potassium salt (poly-A) and the LNPs were then formulated using the Thermo Scientific Ignite
After removing the formulations from the freezer, they were gently inverted several times and then placed in the UHPLC autosampler. For the calibration standards, stock solutions for both formulations were prepared by weighing lipid standards, dissolving them in ethanol, and sonicating them. A series of methanol dilutions were then performed to prepare the calibration standards, which were used to generate calibration curves for lipid quantification. Data acquisition and analysis were carried out using Chromeleon CDS 7.2.10 MUd software.
Significance of results
The initial attempt to separate the lipid components using a high concentration of organic solvent (75%) caused the LNPs to break through the column and resulted in poor data (Figure 1). A new method was developed to improve lipid separation and clarity, preventing interference from the LNPs and making the signals for each lipid more distinct. This method also allowed poly-A, encapsulated in the nanoparticles, to be detected as a separate peak, which requires further investigation.
Figure 1. Initial attempt to separate lipid components in formulations. (A) Formation #1. (B) Formation #2
Once a reliable method for separating the lipids was achieved, calibration curves were created for each lipid to measure their concentrations accurately (Figure 2). For the first formulation, scientists used a mix of four lipid standards and the concentration for each standard was adjusted accordingly. The calibration showed good accuracy with a high calibration coefficient (>0.998). For the second formulation, each lipid was measured individually using its highest concentration for the calibration. This process ensured that all the lipids could be quantified precisely.
Figure 2. Separation of formulations with optimized method. (A) Formulation #1. (B) Formulation #2.
In all, the study found that the UHPLC-CAD method was effective for measuring individual components of both LNP formulations. The measured molar ratios were in strong agreement with the original ratios specified for the samples, indicating the reliability and precision of the method. That consistency suggests that this approach can be reliably used and replicated for lipid quantification across various formulations.
These results are significant for mRNA vaccine research and manufacturing because all drug products require characterization to ensure safety and efficacy. The formulation is of equal importance. Some of the lipids used in LNP manufacturing are man-made for specific properties. These must be tested individually for purity and the final composition determined. The CAD detection is not only sensitive for these non-UV absorbing compounds, but it also gives the same molar response for all the species injected. This capability is important when looking at the molar composition as well as the degree of purity of the individual lipids used, especially when there may not be a standard available for each impurity detected.
mRNA technology in action—today and going forward
The two current FDA-approved COVID-19 vaccines, mRNA-1273 and BNT162b, are notable examples of successful implementation of LNP technology, as they used LNPs to deliver antigen-coding mRNA. Today, many other lipid nanoparticle-oligonucleotide formulations have been developed and are under clinical evaluation for the prevention of other viral infections as well as the treatment of cancer and genetic diseases.
As scientists look to expand the use cases for mRNA therapies, the role of LNP delivery will continue to advance how biopharmaceutical companies approach the development of life-altering treatments, from vaccines to cancer therapies and treatments for infectious diseases. Overcoming challenges in the development of mRNA drug products through innovative and tech-forward approaches helps open the door to cutting-edge therapies.
Reference
1. Characterization of lipid components in lipid nanoparticle (LNP) formulations – Application Note AN001928 – an-001928-bt-lipid-components-LNP-an001928-na-en.pdf (SECURED)
Ken Cook, PhD, Manager, Application Scientists, Analytical Instruments, and Sissi White, Senior Product Applications Specialist, Analytical Instruments, Thermo Fisher Scientific.
The post The Characterization of Lipid Components in Lipid Nanoparticle Formulations appeared first on GEN - Genetic Engineering and Biotechnology News.