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

Gene therapy was born to treat genetic diseases, but with the development of technology, in recent years it is going beyond the scope of treating genetic diseases and gradually entering the field of major non-genetic diseases, and even the field of solid tumors. As the field of AAV gene therapy grows, its upstream and downstream production links also need to be improved. The entire industrial production process of AAV gene therapy products can be roughly divided into three parts: upstream culture, downstream purification and finished product packaging. Among them, upstream culture and downstream purification involve more production steps and the technology is more complex. Next, let us go into the upstream of AAV industrial production to find out!

The upstream production stage of AAV includes three main steps: plasmid production and preparation, cell culture, transfection and viral vector production. Each step has multiple process parameters that impact downstream purification pressure as well as final product quality. For example, the multiplicity of infection of the helper virus and production cells, cell density, infection time, and vector harvest time, etc.

The large-scale production of AAV at this stage includes adherent culture systems and suspension culture systems. Currently, about 65% of companies are building or planning to build suspension cell virus vector production platforms. It can be seen that the cell line culture for upstream AAV production is developing towards a suspension platform, but there are still many companies whose AAV production processes are mainly based on adherent culture. Both platforms require control and monitoring of parameters to ensure consistency during amplification.

Production And Preparation of Plasmids

Plasmids are the starting materials for AAV production, so a large number of GMP-compliant plasmids are needed every year to meet the needs of the downstream cell and gene therapy markets. The raw materials are usually obtained during the fermentation process of recombinant E. coli. The production and preparation process can be divided into steps such as fermentation, collection of bacterial cells, lysis of bacterial cells, solid-liquid separation, clarification, chromatography, and concentration.

Different from scientific research experiments, the biggest challenge facing industrial production is large-scale production amplification and purification, that is, maintaining the proportion of high superhelical plasmids and maintaining high purity. The above two points will directly affect the efficiency and quality of downstream virus production (such as reducing the empty shell rate, etc.). This requires greater attention to critical process steps.

➤ Oxygen required for bacterial fermentation

Insufficient oxygen supply will lead to abnormal bacterial metabolism, reduced plasmid stability, and reduced plasmid content with superhelical conformation, which will cause difficulties in the downstream purification process and indirectly increase production costs. In high-density bacterial fermentation culture, the oxygen supply efficiency of the system is particularly important. In large-scale plasmid fermentation systems, improving or optimizing oxygen supply efficiency is of great significance to improving plasmid yield.

➤ Alkaline lysis (most commonly used)

In the current large-scale plasmid purification process, the alkaline lysis method is widely used to lyse bacterial cells (other chemical methods include detergents, enzymes, and osmotic shock; physical methods include heating, shearing, stirring, ultrasonic waves, and freezing and thawing). As a key step in the plasmid purification process, it requires a long purification time and is very prone to problems. For example: poor pH control and excessive local alkalinity will cause irreversible damage to the plasmid.

This cleavage process requires irreversible denaturation of genomic DNA within a narrow pH range and the plasmid duplex needs to remain intact. In large-scale plasmid production, the process often has poor repeatability and is difficult to control. If the lysis time is too short, the bacterial cells will not be fully lysed or the bacterial genome will be denatured insufficiently, which adds difficulty and uncertainty to the subsequent steps of plasmid purification; if the lysis time is too long, the plasmid will be irreversibly damaged. The plasmid at this stage is very sensitive to shearing force, resulting in greater plasmid loss and easy loss of supercoiling, which affects the final downstream yield and quality.

This cracking method is relatively crude and there is still room for improvement. For example, some studies have improved the alkaline lysis mini-extraction method of plasmids. The improved method overcomes the problems of previous plasmid extraction methods such as RNA and other impurity contamination, cumbersome and time-consuming operations, and the inability to perform large-scale extraction under general conditions. The extracted plasmid DNA has a stable yield and good quality, and meets the requirements of most routine molecular biology experiments.

➤ Chromatography purification

As a commonly used method for large-scale plasmid purification, its purpose is to remove host DNA, RNA, proteins and endotoxins as well as non-supercoiled plasmid variants to meet the use requirements of the target product. Optimization of the purification process can increase plasmid yield and reduce costs. Most fillers currently on the market are mainly targeted at proteins rather than plasmid DNA. In terms of size, plasmid DNA is larger than protein and cannot enter the pores of most fillers, which will lead to a decrease in the adsorption performance of the filler to plasmids.

In addition, the purity of plasmids is one of the main challenges faced in large-scale production. Usually, the quality requirement of plasmid DNA purified by chromatography is GMP-grade plasmid DNA with a purity greater than 95% and no process-related impurities. At present, in order to improve the adsorption performance of fillers, some new chromatography fillers have been developed, which can exclude macromolecules above 700KD and flow through them directly.

Cell Culture Expansion

The currently widely used HEK293 cell adherent culture technology is often used to produce small doses of viruses in the laboratory. Simple operation can meet the needs of scientific research in most cases. However, it is slightly insufficient when applied to large-scale preparation of AAV. For example, the processing of adherent cells is more complicated, easily causes contamination during the culture process, and it is difficult to monitor and adjust culture conditions such as oxygen concentration, pH, etc.

Microcarrier culture technology solves some of the above-mentioned shortcomings of adherent cells to a certain extent, and the process scale-up is relatively easy. Suspension serum-free cell culture systems, such as HEK293, SF9, etc., make the process amplification of AAV production easier and reduce the burden on downstream purification of AAV.

This is mainly because the ingredients of animal-derived products (such as bovine serum) are too complex, and many ingredients are considered contaminants of viral vector products. It needs to be removed during the subsequent AAV preparation and purification process, which further increases the burden of the subsequent virus purification process. What's even more serious is that some of the potential virus-like microbial components are similar to viral vectors in size and some physical and chemical properties, making purification extremely difficult. Therefore, using non-animal-derived, more defined media in cell culture systems can significantly reduce the risk of contamination by exogenous contaminants.

Plasmid Transfection

The most commonly used transfection systems for packaging AAV include: three-plasmid transfection (adherent or suspension cells) system and baculovirus (insect cell) production system. Low transfection efficiency, high cost of preparation of plasmid DNA and high cost of transfection reagents are a major challenge for AAV vector production.

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There are currently a variety of plasmid transfection methods, but they all have their own limitations. For example, transfection using calcium phosphate, reagent purity and pH sensitivity can result in significant batch-to-batch variation in AAV production. Lipofectamine transfection efficiency is relatively high and cytotoxicity is low, but the reagents are expensive, especially in large-scale production of AAV.

In addition, in a three-plasmid transfection suspension cell culture system, the imbalance of plasmid distribution ratio can also lead to low transfection efficiency, which can further lead to obvious batch differences in the ratio of empty capsids. Constructing an AAV packaging cell system that stably integrates capsid gene sequences and other production-related gene sequences may be one of the ideal solutions.

The development of gene therapy requires the production of large quantities of viral vectors. Bioprocess engineers will put forward some requirements for the viral vector production process, such as high yield, scalability, robustness and commercial feasibility, etc. In the upstream process development of virus production, from cell amplification to transfection to virus production, the parameters of each step have the possibility of further optimization.