AAV Purification Services

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As the field of gene therapy continues to evolve, adeno-associated viruses (AAVs) have emerged as the vector of choice for many cutting-edge therapies. Protheragen is a leader in gene therapy manufacturing with many years of extensive R&D experience and is able to provide high-quality AAV purification services.

Overview of AAV Purification

Adeno-associated virus (AAV) purification is a critical step in the production of AAV vectors, which are increasingly recognized for their potential in gene therapy. From a scientific perspective, AAV purification involves the isolation and concentration of AAV particles while removing contaminants such as empty capsids, host cell proteins, and other impurities. This process is essential to ensure the safety, efficacy, and quality of the final AAV vector product.

The genome organization of wild-type AAV. Fig.1 AAV purification using AAVX affinity chromatography. (Florea M., et al., 2023)

AAV vectors are small, non-pathogenic viruses that can deliver genetic material to target cells. They have emerged as a leading platform for gene therapy due to their ability to infect a broad range of cell types, including both dividing and non-dividing cells, with minimal immunogenicity. The purification process is intricate, requiring the separation of functional AAV vectors containing the therapeutic DNA from those that are empty or contain impurities, which can compromise the therapeutic effect and safety.

Methodologies of AAV Purification

Several methods are employed for AAV purification, each with its advantages and limitations. Understanding these systems is crucial for selecting the most appropriate purification strategy for a given application.

Table 1. Methodologies for purification of synthetic AAV capsids. (El Andari J., et al., 2021)

Technology	Pros	Cons
Crude lysates	quick, cheap, and easy to generate compatible with any AAV capsid variant	various contaminants stocks usually not titrated unclear ratios of full: empty capsids
Iodixanol density gradients	Versatile decent purity enrichment of full capsids less toxic than CsCl	difficult to scale up open system prone to contamination time-consuming
CsCl (cesium chloride) density gradients	good separation of full: empty capsids high purity versatile	cumbersome hard to scale up open system prone to contamination time-consuming toxicity
IEC (ion-exchange chromatography)	ability to separate full and empty capsids on large scale easily scalable versatile	different physicochemical AAV properties require adaptation
IAC (immunoaffinity chromatography)	very specific easily scalable high yields and purity	serotype-selective (lesser concern with AAVX) antibody leaching no removal of empty capsids

Our Services

At Protheragen, our AAV purification services are distinguished by our commitment to scientific rigor, our use of state-of-the-art methodologies, and our ability to customize our services to meet specific project needs.

Ultracentrifugation Techniques

Cesium Chloride (CsCl) Gradient Ultracentrifugation: We have optimized this process to achieve high yields of pure AAV vectors, as evidenced by numerous preclinical studies that have utilized CsCl-purified vectors for successful gene delivery.

Iodixanol Gradient Ultracentrifugation: This method offers a non-toxic and less dense environment for AAV particles, leading to a gentler separation process that preserves vector integrity.

Chromatography Techniques

Ion-Exchange Chromatography (IEX): We utilize IEX to separate particles based on their surface charge, effectively removing empty capsids and other contaminants.

Affinity Chromatography: Our affinity chromatography service leverages specific ligands that bind to AAV capsid proteins, enabling the selective capture of full vectors.

Size-Exclusion Chromatography (SEC): As a final polishing step, SEC separates particles based on size, ensuring that our AAV vectors are free from aggregates and smaller contaminants.

Filtration Techniques

Tangential Flow Filtration (TFF): TFF is used for continuous circulation of the sample across a membrane, retaining larger particles and impurities while allowing the filtrate to pass through.

Depth Filtration: Our depth filtration service uses filter media with a tortuous path to capture impurities. This technique is particularly effective in the initial clarification steps, ensuring that the feed stream for downstream processes is free from large particles.

Our team of experts will work closely with you to design and optimize a purification strategy tailored to your specific AAV variant and application requirements. If you are interested in our services, please feel free to contact us for more details and quotation information of related services.

References

Florea, Michael, et al. "High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography." Molecular Therapy Methods & Clinical Development 28 (2023): 146-159.
El Andari Jihad, and Dirk Grimm. "Production, processing, and characterization of synthetic AAV gene therapy vectors." Biotechnology journal 16.1 (2021): 2000025.