AAV Gene Therapy Manufacturing Process

Introduction to AAV Production Process

The production process of AVV is similar to the production of antibodies and vaccine drugs, mainly including upstream culture, downstream purification and formulation.

Downstream Purification

The second half of the virus production process requires purification of viral particles from process- and product-related impurities. These downstream processing steps can account for a large portion of the total cost of virus production, so it is important to efficiently produce high-purity virus. General downstream operations mainly include:

• Use detergent, mechanical action, hypertonicity or freeze-thaw operation to lyse cells to release the virus;
• Nucleic acid removal, the lysate is digested with endonuclease to reduce nucleic acid contaminants;
• Remove cell debris and fragments by centrifugation or microfiltration before chromatographic purification;
• Affinity chromatography to remove host cell proteins (HCPs) and any serum protein impurities;
• Use cesium chloride gradient ultracentrifugation to separate the whole-gene infectious virus from empty virus and non-infectious virus.

Cell Lysis

The basic mechanical technique for releasing AAV vectors from cells is repeated freezing/thawing followed by low-speed centrifugation steps. However, this technique is difficult to scale up for production. Mechanical homogenization, such as French press, is another lysis method in which cell membranes are ruptured under high-pressure shear forces. Although this method is scalable, it often results in product loss due to aggregation and precipitation caused by shear stress. Chemical cleavage methods, such as Triton X-100, have higher overall yields in the viral vector purification process and are easy to scale up. But it also has its limitations. Studies have shown that Triton X-100 causes some acute oral toxicity, eye damage, skin irritation and chronic aquatic toxicity. Therefore, this detergent was listed as a substance of very high concern by the European Chemicals Agency in 2016.

Filter

Filtration is the most expensive unit operation in AAV downstream processing. During the filtration process, AAV particles can aggregate or lose function due to shear stress. Optimizing filtration to achieve high-throughput recovery of adenovirus remains a challenge as it also depends on the AAV serotype being processed. Currently, continuous filtration as a separation technology may reduce filter clogging, but the industry has not yet implemented this technology. In addition, large hold-up volumes and product losses during the filtration process are also a problem.

Purification

Typical purification methods include affinity chromatography and ion exchange chromatography (IEC). Although affinity chromatography is capable of producing highly pure AAV, it cannot differentiate between empty and intact viral capsids. One of the biggest challenges in downstream purification is meeting the purification methods specific to each AAV serotype to achieve optimal yields while maintaining product potency and integrity.

Solutions to AAV Production Challenges

Comprehensive introduction to the three process stages of AVV preparation, it can be seen that downstream processing can account for a large part of the total cost of virus production, and it is also very difficult, especially the purification process.

One reason is that there are differences in the AAV proteins of different serotypes, as there are more than 100 different variant AAV capsids. Therefore, the surface characteristics of various serotypes make AAV purification difficult.

The second reason is the lack of an efficient and reproducible platform method for process- and product-related impurities (including host cell material, DNA, and empty capsids), especially to isolate intact capsids from empty capsids.

Therefore, in order to solve the above problems, pharmaceutical companies are committed to the development of new purification products in order to achieve:

• Achieve separation of virus particles
• Reduce product-related impurities
• Maintain efficiency and output goals

Anti-AAV Antibody ELISA Kit
AAV Antibodies and Titration ELISA