Recombinant adeno-associated viruses (AAV) are widely used as gene therapy vectors in human gene therapy. Reliable and accurate quantification of the physical particle titre is one of the parameters to be determined for precise dosing, which is of critical importance for the patients. In this report, we describe the validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the total AAV-2 physical particle titre through an international collaborative study organised by the Gene Therapy Working Group of the General European Official Medicines Control Laboratories Network co-ordinated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), which aims to develop and validate standard analytical methods for the quality control of gene therapy products. The method is based on a classical sandwich ELISA which uses monoclonal antibodies specific for a conformational epitope only present on the assembled capsid. The AAV-2 Reference Standard Stock Material was used as reference preparation. This study allowed validation of the sensitivity, accuracy and reproducibility of the method and definition of its working range. In addition, specificity and discriminatory capacity towards degraded preparations was also demonstrated.
Keywords: ELISA; European Pharmacopoeia; Gene Therapy Working Group; Physical particle titres; Reference Standard Stock; collaborative study.
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