Our team offers insights on depth filtration as a scalable, cost-effective clarification solution for AAV vectors. We discuss the gene therapy market, why clarification is a pain point, and the benefits of combining depth and membrane filters.
A rapidly expanding gene therapy market
The market for gene and gene-modified cell therapies is expanding rapidly. Hundreds of products are in the clinical pipeline, and several have already received approval from regulatory agencies around the globe.
According to the Alliance for Regenerative Medicine (ARM), more than 1200 clinical trials for cell, gene, and tissue-based therapies were underway at the end of 2020, and gene therapy investment increased by 73% that year. Two new approvals were also received in 2020, one for Orchard Therapeutics’ gene therapy Libmeldy by the European Medicines Agency (EMA) and the other for Kite Pharma’s Tecartus chimeric antigen receptor T-cell (CAR-T) therapy by the U.S. Food & Drug Administration (FDA). ARM also expects eight additional regulatory decisions in 2021 for new regenerative medicine products.
Most gene and gene-modified cell therapies in development and marketed today leverage viral vectors to deliver the gene of interest (GOI), in vivo delivery for gene therapies and ex vivo delivery for gene-modified cell therapies. While several different viral vectors have been employed, AAV is most widely used due to its ability to enter multiple cell types, low immunogenicity, and non-pathogenicity.
As candidate products move rapidly through the clinical phases to commercialization, and as gene therapies expand beyond rare diseases to indications with large patient populations, the need for robust, scalable production technologies is growing.
AAV clarification challenges
Like the manufacturing of recombinant proteins, the production of viral vectors occurs in cell culture (suspension or adherent) followed by clarification and downstream purification. Unlike proteins, viral vectors are commonly produced via transfection (and less often by coinfection) methods, though viral vectors can be toxic to some host cells.
AAV vectors are also generated within the cells rather than expressed into the cell culture media. Lysis of the cells enzymatically or with a surfactant is therefore required to release the viral particles. This process also generates significant amounts of cell debris and process-related contaminants including host-cell proteins and host-cell DNA. While digestion with Benzonase endonuclease reduces some of these contaminants.
Limitations of ultracentrifugation
Efficient clarification to reduce the high level of impurities is essential for improving the efficiency and effectiveness of downstream purification. Most notably, the feed stream must be sufficiently clean to ensure optimum performance of the much more expensive chromatography steps.
Most viral vector production processes have originated in academic laboratories where ultracentrifugation has traditionally been used for the clarification of the harvest. Unfortunately, this method may not be the best option for large-scale manufacturing. It carries high upfront capital costs, lengthy processing times, and is highly labor-intensive. Furthermore, it could be challenging to implement in a continuous manner and overall lacks scalability.
To transition from early- to late-stage development and commercialization, gene therapy developers must have access to a clarification method with performance that can scale up well.
Advantages of direct-flow filtration
For these reasons, direct-flow filtration using depth and sterilizing grade membrane filters has become the preferred method for larger-scale clarification of viral vectors. These single-use filters are easy to operate, readily scalable from lab to commercial production and have been shown to provide high yields and high filtrate quality when processing AAV feed streams.
So, what is a depth filter?
A depth filter has a greater depth of the porous media than membrane filters. Depth filter media mostly consists of a mixture of cellulose, a filter aid such as diatomaceous earth, and a binder resin. Depth filters exist in a broad range of pore sizes and chemistries and are designed to provide a tortuous path combined with some adsorptive capacity. These properties afford depth filters a large capacity when loaded with feed streams containing a wide range of particle sizes, as is seen in viral vector harvest applications.
The large number of filter options can be overwhelming and creates challenges in developing an optimal AAV clarification step. The key is finding the right combination of depth and membrane filters creating a filtration system that will meet the desired process performance goals within the constraints of the given system.
Libmeldy is a trademark of Orchard Therapeutics (Europe) Limited. Tecartus is a trademark of Kite Pharma, Inc. Any other trademarks are the property of their respective owners.