Challenges Faced by AAV Industrial Production And Coping Strategies (Downstream)

The downstream processes of AAV production mainly include: cell lysis and clarification, viral vector purification (capture purification, fine purification, ultrafiltration concentration, etc.), formulation/canning, and quality testing.

AAV particles obtained from the three upstream production methods all require further purification. The process involved is complex, and the removal efficiency of product-related impurities and process-related impurities needs to be examined. Downstream processing steps account for a large portion of the total cost of virus production and are very difficult, especially the purification process, so efficient methods for preparing high-purity viruses are important.

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One reason for the difficulty is that there are more than 100 different variant AAV capsids, and there are differences in the AAV proteins of different serotypes. Therefore, the different surface properties of various serotypes undoubtedly increase the difficulty of AAV purification. The second reason is the lack of an effective and reproducible platform detection method for process- and product-related impurities (including host cell material, DNA and empty capsids), especially intact capsids isolated from empty capsids.

The main purpose of the AAV downstream process is to purify the active ingredients from the raw solution produced by the upstream process so that the final product reaches a certain purity level to ensure its effectiveness and reduce patient safety risks.

Downstream process costs account for about 60% of the total cost. Therefore, an efficient and repeatable downstream production process is particularly important. The main challenges faced in the downstream process are: cell lysis, microfiltration and chromatography purification. Among them, the biggest challenge is the removal of AAV empty shell virus during the chromatography purification process. In addition to removing empty shell AAV through the purification process in the later stage, optimizing the upstream production process can also reduce the empty shell rate.

Because AAV is an icosahedron of 20-25nm, it is relatively small and very stable compared to other viruses. Therefore, for downstream processes, it has two significant advantages: first, the carrier can be sterilized and filtered through a 0.2μm filter, which facilitates the control of product sterility; second, AAV can withstand most harsh conditions, such as Shear, pH changes, high conductivity buffers, etc.

Cell Lysis

Early studies of AAV vectors typically used the AAV2 serotype, in which the amount of viral particles released into the culture medium was limited and cell lysis was required to obtain the product. Lysis methods include mechanical and chemical methods, and small laboratory-scale preparations often use repeated freezing and thawing, sonication, etc. In large-scale production and preparation, chemical methods (surfactants, organic solvents, etc.) are often used to break cells and release viral vectors from cells.

Among them, the use of surfactant TritonX-100 as a cell lysis method is often used in the AAV viral vector purification process (other methods include hypertonic solutions). Research has proven that this method is effective and low-cost, and can be easily applied to the large-scale production of AAV. However, according to recent studies, virus products treated with TritonX-100 can cause physiological toxicity such as eye damage, oral toxicity, and skin irritation. Therefore, it is particularly important to optimize existing methods or develop new methods for the cell lysis process in AAV technology.

Removal of Free Nucleic Acids

Since nucleic acids such as host DNA, RNA and other free DNA in the cell are released after lysing the cells, nucleases need to be used to digest and remove these nucleic acid impurities. While removing these impurities, it will also further reduce the viscosity of the feed liquid after cell lysis, which is beneficial to the subsequent purification process.

A large amount of cell debris is produced after cell lysis, which is difficult to remove through traditional dead-end filtration. Tangential flow filtration (TFF) technology uses a pump to push fluid through the surface of the filter membrane, scouring and removing trapped molecules on it, thereby minimizing fouling on the surface of the filter membrane. At the same time, the tangential flow will also generate pressure perpendicular to the filter membrane, pushing solutes and small molecules through the filter membrane. Using this method to separate biomolecules is more efficient and the concentration or diafiltration speed is faster. At the same time, it can effectively avoid the occurrence of clogging and is widely used in AAV downstream processes. In addition, during the microfiltration process, excessive shear force can cause aggregation or inactivation of AAV particles. Hollow fiber materials can effectively reduce the generation of shear force and have obvious advantages in AAV production.

After cell lysis and enzymatic treatment, the harvested material will be a highly turbid solution that includes AAV particles as well as process-related impurities such as DNA, proteins, media components, and cell debris. Removal of process-related impurities is typically performed by a combination of centrifugation and/or filtration with the goal of minimizing impurity and particle challenges prior to subsequent chromatography unit operations. Optimized harvesting steps will help increase yields and may also increase the life of the chromatography media and reduce the need for stringent cleaning protocols required for the chromatography media.

Clarification of Feed Liquid

Generally, the feed liquid after cell lysis needs to be clarified before further purification, and impurities such as cell debris are often removed through methods such as centrifugation or membrane filtration. Both centrifugation and membrane filtration are convenient at the laboratory scale. However, for the clarification of 50-2000 L feed liquid, disposable depth filtration is the preferred method in most cases to reduce the investment cost of hardware equipment as much as possible and facilitate scale expansion.

While stainless steel centrifuges are also suitable for large-scale production, their cleanability and operator exposure risk from aerosol formation are key factors to consider. In addition, although single-use centrifugation equipment such as Ksep and Unifuge exist on the market, both have limited scale options and may increase development time. Single-use technology reduces the initial investment cost of equipment, reduces water usage, and reduces the risk of cross-contamination. In addition, these technologies reduce product rotation times by reducing the need for cleaning and cleaning validation, which allows for increased production runs within a given time frame or shortened time between multiple procedures. This increases overall plant utilization and flexibility.

Preliminary Purification

Use affinity chromatography to remove impurities such as host cell proteins (HCPs) and culture medium serum proteins. Among them, HCP constitutes the main component of the AAV production process and process-related impurities. Its residual HCP content is usually considered a critical quality attribute of the product, because HCP may affect the safety and efficacy of the product, and the related risk is mainly immunogenicity. Therefore, it is a requirement of regulatory agencies to monitor the removal of residual HCPs from products during bioprocess development.

Risks associated with HCPs are typically assessed through a combination of downstream process capabilities, residual HCP levels, maximum dose, route of administration, frequency of administration, toxicology data, and clinical data. Risk control strategies can be developed by developing robust downstream purification bioprocesses to remove HCPs to the lowest possible levels. However, the detectability of residual HCP also depends on the sensitivity of the detection method.

Moderate Purification

Chromatography further removes major impurities.

Fine Purification

Trace impurities are removed through chromatography and the final purified product is obtained. Typical chromatography methods include affinity chromatography and ion exchange chromatography (IEC). Affinity chromatography has higher yield and purity, but it cannot distinguish between empty shell viruses and solid viruses. Empty AAV is usually produced by incomplete virus packaging during the production process. Empty capsids have a potential impact on the immunogenicity of the final product and will directly affect the effectiveness and safety of AAV vector products. Two common methods for removing empty capsid impurities include cesium chloride density gradient centrifugation and anion exchange chromatography (AEX).

Ion exchange chromatography (IEC) is currently used more often to separate and remove empty shell AAV. In IEC, there is obvious overlap between empty capsids and intact virion capsids in the peaks eluted from the chromatography, which means that to completely eliminate empty capsid AAV, some intact virions must be sacrificed, further reducing the yield of AAV. In addition, the elution conditions used, such as extreme pH and high conductivity, can cause damage to the AAV capsid and also require higher filler requirements, which is also an issue that needs to be solved.

Large-scale vector production capabilities with controllable quality are an important driving force for the development of the AAV gene therapy industry, and are also one of the keys to reducing R&D costs and gaining price advantages. After completing the above 6 steps, you need to can. At the same time, before AAV enters clinical use, the vector needs to be subject to very strict quality testing, such as AAV empty shell/solid ratio, impurity content, etc., so as to minimize the risk of accidents that may occur during clinical use of AAV vectors.