LAUSR

The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the lab to the clinic. The active deployment of these analytical technologies for process and product characterization has helped define critical quality attributes (CQA) and improve the quality of the clinical grade material. In this manuscript, we report an anion-exchange high-performance liquid chromatography (AEX-HPLC) method for relative and as well as absolute quantification of empty capsids (EC) and capsids encapsidating genetic material (CG) in purified preparations of AAV using serotype 5 as a model. The selection of optimal chromatographic buffer composition and step-gradient elution protocol offered baseline separation of EC and CG in the form of two peaks, as validated with the respective reference standards. The native amino acid-fluorescence based detection offered excellent linearity with a correlation coefficient of 0.9983 over two-log dilutions of the sample. The LOD and LOQ values associated with the total AAV5 capsid assay are 3.1E+09 and 9.5E+09, respectively. AEX-HPLC showed method comparability with the analytical ultracentrifugation (AUC) method for determination of the relative proportions of empty and capsids containing genetic material, supporting the reported HPLC method as an easy-to-access alternative to AUC with operational simplicity. Moreover, rapid and easy adaptation of this method to AAV8 material also demonstrated the robustness of the proposed approach.