AUC (Analytical Ultracentrifugation) capabilities for the analysis of gene therapy products

AAVs and LNPs are complementary gene transfer systems with respect to immunogenicity, tissue tropism and types of nucleic acids, which can be delivered.

 

AUC provides a wealth of orthogonal assays for their characterization allowing the determination of gene copy number and particle distribution as well as capsid titer and particle density.

 

Traditionally, AUC has been used in the biopharmaceutical industry to generate size distributions, which can be integrated like HPLC chromatograms and thus quantify protein aggregation. With the advent of high-concentration antibody formulations, also the determination of hydrodynamic and thermodynamic non-ideality parameters, like the second virial coefficient or kS and kD became of interest. The availability of powerful computers has added a second dimension of information which can be extracted from SV-AUC data. The two-dimensional fitting of these data yield size-and-shape distributions which contain diffusion (molecular weight) information for each individual species if sufficiently populated. This webinar will show how we can add a third and even a fourth dimension of information by collecting multi-wavelength data and vary the density of the experimental buffer system by adding either CsCl or D2O.

 

Data from band SV-AUC and analytical CsCl density gradient sedimentation equilibrium AUC (CsCl SE-AUC) will be compared with those from conventional boundary SV-AUC and MWL-SV-AUC experiments.

 

Finally, I will show how to determine the mRNA copy number from a series of SV-AUC experiments with varying D2O-concentrations. Their analysis yields a density distribution of the material under investigation which can be converted to a molecular weight distribution of the encapsulated nucleic acid.

 

Learning Objectives