AAV Purification Services

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While adeno-associated viruses (AAVs) are quickly becoming the favored vector in gene therapy, Protheragen is a leading provider in the field with refined AAV purification services thanks to their many years of rigorous R&D work.

Overview of AAV Purification

The purification of adeno-associated virus (AAV) is a pivotal stage in the creation of AAV vectors, which are gaining attention for their application in gene therapy. In scientific terms, AAV purification encompasses the concentration and isolation of AAV particles while extracting empty capsids, host cell proteins, and other cellular debris. This purification process directly impacts the safety and effectiveness 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 compact, non-pathogenic viruses capable of reaching target cells to deliver genetic material. They have emerged as a leading platform for gene therapy because they can infect both dividing and non-dividing cells with minimal immunogenicity. The purification steps are complicated because functional AAV vectors containing the therapeutic DNA must be separated from empty or impure ones—these impurities are heterologous nucleotide sequences, proteins, or viral particles—that could undermine the safety and efficacy of the therapy.

Methodologies of AAV Purification

AAV purification incorporates multiple procedures, each having benefits and drawbacks. A thorough comprehension of these systems is critical in choosing the most suitable purification method 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, AAV purification services stand out for their scientific thoroughness and cutting-edge methods alongside customization to particular project requirements.

Ultracentrifugation Techniques

Cesium Chloride (CsCl) Gradient Ultracentrifugation: Based on multiple preclinical investigations using CsCl-purified AAV vectors for gene delivery, we confirmed that the process is refined to maximize recovery of pure AAV vectors.

Iodixanol Gradient Ultracentrifugation: This technique provides a safer, less viscous medium for AAV particles, which consequently results in milder separative processes that retain the integrity of the vectors.

Chromatography Techniques

Ion-Exchange Chromatography (IEX): We make use of IEX to free-separate particles according to their surface charge, which assists in the depletion of empty capsids and contaminants.

Affinity Chromatography: Our service in affinity chromatography utilizes specific ligands that interact with AAV capsid proteins, allowing for the selective capture of full vectors.

Size-Exclusion Chromatography (SEC): As a final polishing method in purification, SEC removes particles based on size, such that aggregates are separated from AAV vectors while ensuring smaller contaminants remain filtered out.

Filtration Techniques

Tangential Flow Filtration (TFF): TFF is utilized for the continuous recirculation of a sample stream across the membrane surface, retaining greater particulates and impurities while allowing the permeate to pass through.

Depth Filtration: Using filter media with a tortuous path to capture impurities, we offer depth filtration services. This approach is especially useful in the primary clarification stages since it guarantees that the feed stream is devoid of larger contaminants for downstream processes.

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.