Adeno-associated virus (AAV) is a naturally occurring, non-pathogenic virus that has become an indispensable vector for gene therapy. AAV carries beneficial genetic instructions into a patient’s cells to treat disease. To ensure the therapeutic dose is safe and effective, the raw AAV material produced in the lab must undergo an intensive purification process. This process, known as downstream processing, separates active viral particles from contaminants, transforming a crude biological mixture into a highly refined drug substance suitable for human use.
Producing the Raw Material and Initial Harvest
The manufacturing process begins in specialized bioreactors where host cells, typically human embryonic kidney (HEK293) cells, are genetically engineered to produce the AAV vectors in large quantities. These cells are provided with the necessary genetic components—including the therapeutic gene—to assemble the millions of viral particles. After a set period of growth and production, the AAV vectors must be collected from the cell culture, which marks the start of the harvest phase.
For many AAV serotypes, the virus remains trapped inside the host cells, requiring cell lysis to break the cells open and release the contents. Lysis is often achieved using chemical detergents or physical disruption methods, creating a complex mixture containing AAV, host cell proteins (HCPs), host cell DNA, and residual components. The mixture is then treated with an enzyme, such as a nuclease, to chop up long strands of host cell DNA and reduce viscosity, improving the efficiency of later filtration steps.
The next step is clarification, which removes large debris, such as broken cell fragments and aggregates, from the fluid. This is commonly achieved through centrifugation or depth filtration, which uses porous media to physically trap particulates. The resulting clarified solution is a cleaner starting material, but it still contains a high concentration of molecular impurities and a mix of active and inactive AAV particles. This material is now ready for high-precision separation technologies.
High-Resolution Separation Using Chromatography
The core of the purification process relies on chromatography, a technique that separates molecules based on differences in their physical or chemical properties as they travel through a specialized column. This step captures the AAV particles while shedding remaining process-related impurities. The initial capture step often uses Affinity Chromatography, which is highly selective because it employs a ligand that specifically binds to a protein on the AAV capsid surface.
This specific binding allows AAV particles to be separated from the bulk of host cell proteins and DNA. The AAV is then released from the column by changing the buffer conditions, yielding a concentrated, partially purified product. However, affinity chromatography does not distinguish between “full” capsids, which contain the therapeutic gene payload, and inactive “empty” capsids. Since empty capsids can trigger an immune response, their removal is necessary to maximize the therapeutic dose and ensure patient safety.
To remove these inactive empty capsids, a second, or “polishing,” chromatography step is performed, typically using Ion Exchange Chromatography. This technique separates particles based on slight differences in their electrical charge. Full AAV capsids have a different surface charge than empty capsids due to the negative charge contributed by the encapsulated DNA genome. By controlling the salt concentration and pH of the buffer, empty capsids are washed off the column before the desired full capsids are collected, often resulting in a product with 80% to over 95% full capsids.
Final Formulation and Concentration
Once the AAV vectors are purified, the volume is often too large, and the AAV is suspended in a buffer unsuitable for patient administration. This is addressed using Ultrafiltration/Diafiltration (UF/DF), typically carried out using Tangential Flow Filtration (TFF) equipment. TFF employs a semi-permeable membrane with pores sized to retain the large AAV particles while allowing smaller molecules and excess fluid to pass through.
The Ultrafiltration phase concentrates the AAV by reducing the fluid volume, forcing buffer and small contaminants through the membrane while the vector is recirculated. This process can concentrate the AAV solution by 10-fold or more. Diafiltration, a buffer exchange step, immediately follows or is integrated with UF. During diafiltration, a new formulation buffer—often a saline solution containing stabilizing agents—is continuously added to the AAV solution while the old buffer components are filtered out.
This buffer exchange is necessary to ensure the final drug product is stable, non-toxic, and compatible with the human body for injection. The final step in this sequence is often sterile filtration, where the concentrated, formulated AAV solution is passed through a fine filter (e.g., 0.2 \(\mu\)m pore size) to remove any remaining microbial contaminants, ensuring the product is sterile before it is filled into vials. The completed solution is now the drug substance, ready for final quality checks.
Verifying Safety and Purity
The success of the purification process is confirmed by a series of quality control (QC) tests performed on the final formulated product. These tests ensure the vector meets regulatory requirements for purity, potency, and safety before patient administration. One primary metric is Purity, which confirms the effective removal of process-related contaminants. This includes testing for residual host cell proteins (HCPs) and host cell DNA, which can trigger adverse immune reactions if present above defined limits.
A related aspect of purity is measuring the ratio of full to empty capsids to confirm the success of the polishing chromatography step. The concentration of active viral particles, known as Potency or Titer, is also measured to ensure the correct therapeutic dose. This involves quantitative methods, such as quantitative polymerase chain reaction (qPCR) to count genomic copies, and bioassays to confirm the vector is biologically active and successfully delivers the therapeutic gene into cells.
Finally, every batch must undergo Sterility testing to confirm the absence of microbial contamination, such as bacteria or fungi, which is required for an injectable product. The combination of these analytical tests validates the purification process, providing assurance that the final AAV drug product is consistently safe and therapeutically effective.