Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment

Analytical Chemistry, vol. 95, pp. 8478–8486

Abstract

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Authors 8

1. Christoph Gstöttner Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands

2. Andrei Hutanu

   Roche (Switzerland)

   Affiliation as printed

   Pharma Technical Development Analytics, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland

3. Sacha Boon Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands

4. Aurelia Raducanu
   Affiliation as printed

   Pharma Technical Operation Cell- and Gene Therapy, Roche Diagnostics GmbH, 82377 Penzberg, Germany

5. Klaus Richter
   Affiliation as printed

   Coriolis Pharma Research GmbH, 82152 Planegg, Germany

6. Markus Haindl
   Affiliation as printed

   Pharma Technical Operation Cell- and Gene Therapy, Roche Diagnostics GmbH, 82377 Penzberg, Germany

7. Raphael Ruppert
   Affiliation as printed

   Pharma Technical Operation Cell- and Gene Therapy, Roche Diagnostics GmbH, 82377 Penzberg, Germany

8. Elena Domínguez‐Vega corresponding Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands