Molecular detection platform offers new insights into gene drug manufacturing

Molecular detection platform offers new insights into gene drug manufacturing

Nature Communication (2022). DOI: 10.1038/s41467-022-33157-4″ width=”800″ height=”530″/>
Mechanisms of determination of payload capacity and distribution of mRNA LNPs by PEG content. a, b Hypothetical assembly processes and formulation characteristics of LNP with high concentration of PEG mol% (a); or low PEG mol% concentration (b) and compositional drift during dialysis from pH 4.0 (left) to pH 7.4 (right). Labeled population fractions are actual formulation data with PEG mol% = 1.5% (a) or 0.5% (b). a Each number label represents population behavior during dialysis: 1, fractionation of empty LNPs; 2, stabilization of empty NLPs; 3, cleavage of lipophilic complexes with initially high mRNA payload; 4, remaining a same mRNA payload for lipophilic complexes with an initially low or intermediate payload; 5, fusion of empty LNPs with mRNA complexes; 6, fusion of non-lipophilic complexes. The cross represents the finding that the mRNA payload of lipophilic complexes does not increase during dialysis due to the lack of fusion in this condition. b Labels are: 1, fusion between lipophilic complexes; 2, fusion of empty LNPs with mRNA complexes; 3, fusion of non-lipophilic complexes; 4, Fractionation of empty LNPs. Credit: Nature Communication (2022). DOI: 10.1038/s41467-022-33157-4

An important component of vaccines protecting people against the SARS-CoV-2 virus and its variants are lipid nanoparticles, or LNPs. These circular particles carry therapeutic mRNA payloads, the snippets of genetic material that trigger our immune system’s defense against COVID-19.

Even with their success, some characteristics of the particles, such as payload distribution, are unknown. Researchers and the Food and Drug Administration want to learn more about these characteristics to improve reporting on metrics in pharmaceutical manufacturing.

A new molecular detection platform developed by two professors from the Whiting School of Engineering answers the FDA’s call. Hai-Quan Mao and Tza-Huei (Jeff) Wang want to determine how many mRNA molecules an LNP can carry and whether the mRNA is evenly packed inside the particle to help researchers design treatments and vaccines more efficient and effective.

“Our platform processes molecules at the single nanoparticle level, but unlike current imaging methods for mRNA LNPs, our approach is based on fluorescent spectroscopy and gives us the ability to see through particles,” said Wang, a professor in the Departments of Mechanics. Engineering and Biomedical Engineering at the Whiting School, and Principal Investigator at the Institute for NanoBioTechnology.

The ability to peer inside nanoparticles allows researchers to differentiate and measure empty LNPs that contain no mRNA, LNPs with mRNA, and floating mRNAs in a sample.

Their platform, called Cylindrical Illumination Confocal Spectroscopy, or CISC, works by labeling mRNA and LNP components with fluorescent signals of up to three colors and passing the sample through a detection plane. The detection plane reads the fluorescent signals and measures their intensity before comparing the strength of the intensities with that of a single mRNA molecule.

Analyzing the data with an algorithm called deconvolution tells the team both how many mRNA copies are inside the LNP – if any – and their distribution in the sample. The team’s platform overcomes contrast limitations and increases the throughput of analyzing samples, which are observed in cryotransmission electron microscopy, the current gold standard for imaging mRNA LNPs.

Tests performed using this detection platform revealed that from a reference solution of mRNA LNPs used in academic research studies, more than 50% of LNPs are not loaded with mRNA molecules, and LNPs filled with mRNA, most contained two to three mRNA molecules per particle.

“Being able to quantitatively resolve the payload characteristics of mRNA LNPs at the single-particle level has never been done before. We are intrigued by the substantial presence of empty LNPs, and by changing the formulation conditions, a single nanoparticle can load as few as one to up to ten mRNA molecules,” said Mao, a professor in the departments of Materials Science and Engineering and Biomedical Engineering at the Whiting School and director of the Institute. for NanoBioTechnology.

The team’s results are published in Nature Communication.

“There are a lot of groups researching the LNP,” Wang said. “However, when they find a formula that might work well, it has been difficult to relate those findings to the composition and payload distribution of the nanoparticles. Using this platform, we can provide a deeper understanding full picture of what happens at the single-particle level.”

Further research is needed to find out how many mRNA molecules per capsule of LNP are optimal for the most effective treatment. However, the empty LNPs revealed by the new platform show that there is a need to improve mRNA packaging methods inside LNPs.

Mao and Wang say their platform shows it has the potential not only to be used in all stages of LNP-related research and development, but also in the development of other drug delivery systems. drugs and quality control measures at the manufacturing stage. The team has filed a patent application covering the technique and is working with collaborators to use the platform to analyze other types of therapeutic cargoes in various nanoparticle systems to treat different diseases.

“The FDA has recently responded to the need for higher quality measurements in nanoparticle design in the pharmaceutical industry,” said Michael J. Mitchell, a leading LNP research scientist and assistant professor of innovation. Skirkanich at the university’s bioengineering department. of Pennsylvania.

“This will become increasingly important as LNP mRNA technology expands beyond vaccines to new therapies delivered into the bloodstream, which have very stringent requirements. The new detection platform developed by the team of Drs. Mao and Wang is a potentially significant advance in addressing the needs during the research and regulatory phase, and can potentially aid in the development of LNP mRNA technology in- beyond vaccines.


New platform could make delivery of gene drugs easier and more affordable


More information:
Sixuan Li et al, Payload distribution and capacity of mRNA lipid nanoparticles, Nature Communication (2022). DOI: 10.1038/s41467-022-33157-4

Provided by Johns Hopkins University

Quote: Molecular detection platform provides new insights into gene drug manufacturing (2022, September 23) Retrieved September 23, 2022 from https://phys.org/news/2022-09-molecular-platform-insights-gene- medicine.html

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