Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches

Article Article scientifique Article Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches eng 2023-05-09 2023-05-09 author Aebischer Megane Klothylde 871151 Section des sciences pharmaceutiques (FARMA) author Bouvarel Thomas 0000-0001-9128-3488 974216 author Barrozo Emmalyn 825360 author Kochardt Dominik author Elger Carsten author Haindl Markus author Ruppert Raphael author Guillarme Davy 0000-0001-7883-5823 623637 Section des sciences pharmaceutiques (FARMA) author D'Atri Valentina 0000-0002-2601-7092 846921 Section des sciences pharmaceutiques (FARMA) 8503 10.3390/ijms24108503 1422-0067 https://www.mdpi.com/1422-0067/24/10/8503 <p>The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis–sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.</p> Adeno-associated virus Liquid chromatography Step gradient Multi-isocratic conditions Gene therapy Viral particles Dewey 615 31 Section des sciences pharmaceutiques 17 false 881 Sciences analytiques International journal of molecular sciences 24 10 d224503b-f0cc-4d95-8bd9-e50830dc8dd5 Article (Published version) Public Article-scientifique-from-crossref.pdf 2054645 application/pdf 79c0cbdcb839979e9be934c4e9f9f712ac8240c0b7444883fea1c55a1470c93c openAccess Faculté des sciences / Section des sciences pharmaceutiques

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Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches	International journal of molecular sciences			2023	125	167

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Barrozo, Emmalyn