Downstream Process Development for an Adeno-associated Virus Generated from HeLa Cell Line

Abstract

The goal of this work was to assemble a downstream purification process for adeno-associated virus (AAV) gene therapy product utilizing a HeLa cell line in Ultragenyx. HeLa cells were grown by upstream team and transfected with AAV. Harvest of cells resulted in gaining the intended gene therapy product, as well as several impurities that must be reduced or eliminated for drug safety. Additionally, an efficacious product must be enriched and concentrated. The downstream process consisted of several distinct unit operations to achieve these goals. Several platform processes and materials were adapted and evaluated, in addition to several new techniques, to develop a scalable downstream process. Clarification resulted in a new filter to be used, while AEX membrane filtration utilized a preexisting filter. Affinity chromatography also resulted in platform chromatography being used. Post heat inactivation, and after AEX chromatography resin selection, downstream unit operations resulted in a successful purification of this gene therapy product. Additionally, several unit operations yielded interesting findings that are then discussed, such as load conductivity impacts during clarification. The overall downstream process is discussed holistically, and suggestions are made for further possible improvements of this gene therapy product, with the hope of improving other AAV products within Ultragenyx.