Improving gene therapy analysis with multi-wavelength analytical ultracentrifugaton methods

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dc.contributor.authorHenrickson, Amy
dc.contributor.authorUniversity of Lethbridge. Faculty of Arts and Science
dc.contributor.supervisorDemeler, Borries
dc.date.accessioned2023-11-24T18:39:25Z
dc.date.available2023-11-24T18:39:25Z
dc.date.issued2023
dc.degree.levelPh.D
dc.description.abstractOver the past few years, research into gene therapies has dramatically increased, with thousands of drug candidates in clinical trials. However, only a few are available on the market today, highlighting the need for improved analysis methods that can help validate the drug development process. The challenges associated with gene therapy analysis vary with different vector types and compositions. The main challenges for lipid nanoparticles (LNPs), a non-viral vector, are their large size and inherent heterogeneity. The challenge for adeno-associated viruses (AAVs), a viral vector, is differentiating between the full AAV capsid and the product-related impurities due to their similar hydrodynamic radii and surface properties. For both vectors, these challenges prevent their accurate quantification and characterization by many chromatography and size-based techniques. We have employed analytical ultracentrifugation (AUC) to improve gene therapy analysis by characterizing solutes in a sample based on their molar mass, shape, and density. AUC provides high statistical certainty through bulk observations, resulting in the correct assessment of sample purity and loading states. To further improve the resolution of AUC results, we have incorporated multi-wavelength capabilities into our AUC methods, adding an orthogonal optical characterization dimension. This thesis presents considerations for the design, execution, and analysis of multi-wavelength (MW) AUC experiments, looking at cases where optical deconvolution is not possible and cases where it is. It also includes a section on using MW sedimentation velocity experiments to characterize protein-DNA interactions, providing an example of how stoichiometry can be determined from MW-AUC experiments. Further, we apply MW capabilities to several AUC methods to improve the quantification and characterization of AAVs and LNPs. This results in the precise quantification and characterization of vectors, product-related impurities, and other contaminants.
dc.identifier.urihttps://hdl.handle.net/10133/6634
dc.language.isoen
dc.proquest.subject0307
dc.proquest.subject0369
dc.proquestyesYes
dc.publisherLethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
dc.publisher.departmentDepartment of Chemistry and Biochemistry
dc.publisher.facultyArts and Science
dc.relation.ispartofseriesThesis (University of Lethbridge. Faculty of Arts and Science)
dc.subjectanalytical ultracentrifugation
dc.subjectadeno-associated viruses
dc.subjectlipid nanoparticles
dc.subjectmulti-wavelength
dc.subjectUltraScan
dc.subject.lcshUltracentrifugation
dc.subject.lcshDependoviruses--Biotechnology--Research
dc.subject.lcshLipids--Biotechnology--Research
dc.subject.lcshBiotechnology
dc.subject.lcshPharmaceutical technology
dc.subject.lcshGene therapy--Research
dc.subject.lcshMolecular spectra
dc.subject.lcshWavelengths
dc.subject.lcshDrug delivery systems--Research
dc.subject.lcshNanoparticles--Biotechnology--Research
dc.subject.lcshDissertations, Academic
dc.titleImproving gene therapy analysis with multi-wavelength analytical ultracentrifugaton methods
dc.typeThesis
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