LNP analysis and D2O Density Matching with UltraScan1)
1) Savelyev A, Gorbet GE, Henrickson A, Demeler B. Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment. PLoS Comput Biol. 2020 Jun 19;16(6):e1007942. doi: 10.1371/journal.pcbi.1007942. PMID: 32559250; PMCID: PMC7347214.
Presenters: Amy Henrickson, University of
Lethbridge, Canada
Description
In this workshop we will use
UltraScan to measure nucleic acid load in
lipid nanoparticles (LNPs). There are two orthogonal methods presented,
both of which are based on sedimentation velocity experiments (SVEs)
that can be used to characterize drug loading:
- Measuring density distributions based on D2O density matching
- Multi-wavelength AUC to characterize differential absorbances of
the liposome shell versus the nucleic acid absorbance spectrum.
Determining the amount of drug loaded into a LNP is of utmost importance
for many biopharma applications. Determining the precise loading with
nucleic acids is critical for achieving clinically relevant formulations,
and to avoid antigenic materials in vaccines or gene therapy formulations.
This can be challenging for many techniques since overall LNP size and
shape may not proportionally change with the cargo load. However, the
density, or partical specific volume of LNPs is a sensitive predictor of
loading state, regardless of particle size, and the absorbance profiles
of liposomes and nucleic acids is a unique characteristic that can be
followed by AUC.
SVEs can be used to determine the sedimentation and diffusion
coefficients, and partial concentrations of all solutes present in a
sample with high resolution. In this workshop we will demonstrate how
multiple SVEs performed in different D
2O:H
2O
ratios can be used to globally fit a partial specific volume (PSV)
distribution for samples that are heterogeneous in density, and to combine
this information with MW-AUC information to uniquely identify the LNP
loading state. Furthermore, we can combine the PSV distributions with
the corresponding sedimentation and diffusion coefficient distributions
to derive accurate molar mass, particle size and anisotropy distributions.
Software modules implemented in
UltraScan specifically addressing MW-AUC
and PSV distribution measurements by SVEs will be discussed.
This workshop will be presented on Monday from 14:30-16:30 (session 7).
