A Robust And Scalable Suspension Platform For AAV Manufacturing


A major hurdle for manufacture of Adeno-Associated Virus (AAV) vectors is scalability, production yields, and a clean feed-stream to facilitate effective purification strategies. Using a DOE approach, Andelyn has developed a high yielding, scalable suspension platform for production of AAV; consistently producing high titers up to 4-5E11 vg/mL at 1L, 2L, AMBR250, 50L, 200L, 500L and 1000L scales in harvested media alone. Using FectoVIR®-AAV transfection reagent and a triple transient transfection approach, full factorial and mixture designs were developed to screen and optimize multiple factors including cell density, total amount of plasmid DNA, the ratio between the FectoVIR®-AAV and DNA, and the ratios between the plasmids. The optimized condition using FectoVIR®-AAV produced yields 2 – 10 times higher than those produced using PEI for all production scales for different serotypes.

We have also optimized downstream purification options and developed both an ultracentrifugation-based as well as an all-column chromatography-based procedure for purification of AAV. In addition, we have developed in-process analytics that track the purification, including an HPLC-based assay for determining the Full/Empty capsid ratios. Data from this work showcases recoveries at each stage of downstream purification.

As part of suspension platform development, Andelyn has also derived a suspension cell line capable of producing higher AAV yields. The Andelyn Suspension Cell line was derived as a clonal isolate by adaptation of the adherent SG293 cell line to suspension growth under serum free conditions and selected for higher AAV productivity. This cell line can be optimized for each process using a similar DOE approach.

Watch the webinar to learn more about the development of Andelyn’s Suspension Platform. Review the demands for AAV manufacturing using a suspension process and the DOE-based strategy used by Andelyn to achieve a scalable process both in terms production and quality of AAV purified.

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