Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode

Article Article scientifique Article Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode eng 2022-10-15 2022-10-15 author Aebischer Megane Klothylde 871151 author Gizardin-Fredon Hugo 0000-0002-3566-7206 941062 author Lardeux Honorine 936134 author Kochardt Dominik author Elger Carsten author Haindl Markus author Ruppert Raphael author Guillarme Davy 0000-0001-7883-5823 623637 author D'Atri Valentina 0000-0002-2601-7092 846921 12332 10.3390/ijms232012332 36293189 1422-0067 PMC9604245 https://www.mdpi.com/1422-0067/23/20/12332 <p>Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.</p> Anion-exchange chromatography Full/empty ratio Gene therapy Recombinant adeno-associated virus Step gradient Dewey 615 31 Section des sciences pharmaceutiques 17 false 881 Sciences analytiques International journal of molecular sciences 23 20 af3212f1-5ac6-40b2-967c-c083f071f42b Article (Published version) Public Article-scientifique-from-unpaywall.pdf 3006046 application/pdf CC BY-4.0 openAccess Faculté des sciences / Section des sciences pharmaceutiques

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Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode	International journal of molecular sciences			2022	228	168

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Gizardin-Fredon, Hugo