posted on 2024-05-16, 16:05authored byAdriana
Coll De Peña, Daniel Zimmer, Everett Gutterman-Johns, Nicole M. Chen, Anubhav Tripathi, Christina M. Bailey-Hytholt
Lipid nanoparticles (LNPs) are clinically advanced nonviral
gene
delivery vehicles with a demonstrated ability to address viral, oncological,
and genetic diseases. However, the further development of LNP therapies
requires rapid analytical techniques to support their development
and manufacturing. The method developed and described in this paper
presents an approach to rapidly and accurately analyze LNPs for optimized
therapeutic loading by utilizing an electrophoresis microfluidic platform
to analyze the composition of LNPs with different clinical lipid compositions
(Onpattro, Comirnaty, and Spikevax) and nucleic acid (plasmid DNA
(pDNA) and messenger RNA (mRNA)) formulations. This method enables
the high-throughput screening of LNPs using a 96- or 384-well plate
with approximate times of 2–4 min per sample using a total
volume of 11 μL. The lipid analysis requires concentrations
approximately between 109 and 1010 particles/mL
and has an average precision error of 10.4% and a prediction error
of 19.1% when compared to using a NanoSight, while the nucleic acid
analysis requires low concentrations of 1.17 ng/μL for pDNA
and 0.17 ng/μL for mRNA and has an average precision error of
4.8% and a prediction error of 9.4% when compared to using a PicoGreen
and RiboGreen assay. In addition, our method quantifies the relative
concentration of nucleic acid per LNP. Utilizing this approach, we
observed an average of 263 ± 62.2 mRNA per LNP and 126.3 ±
21.2 pDNA per LNP for the LNP formulations used in this study, where
the accuracy of these estimations is dependent on reference standards.
We foresee the utility of this technique in the high-throughput characterization
of LNPs during manufacturing and formulation research and development.