Adeno-associated virus (AAV) vectors represent a significant advancement in the field of gene therapy with continued successes since the approval of Glybera (the first AAV1-LDL vector for treating the deficiency of lipoprotein lipase) in 2012.¹'² Their inherent safety profile, broad tropism (ability to infect a wide range of cells), and sustained transgene expression (long-lasting gene expression) make them promising tools for delivering therapeutic genes to target cells, potentially offering a cure for previously untreatable genetic disorders.¹ AAV vectors can also deliver genetic material encoding a desired antigen directly to host cells, leading to the production of specific antigen proteins. This stimulates a strong and long-lasting immune response, making them promising tools for developing vaccines.³

However, the therapeutic potential of AAV vectors hinges on their purity. For cellular uptake, contaminants within an AAV preparation, such as empty capsids devoid of therapeutic DNA, can compete with functional AAV vectors and pose a safety and efficacy risk.⁴ Conversely, highly purified AAV preparations ensure a greater concentration of functional vectors, enhancing the effectiveness and specificity of gene delivery.⁵ By minimizing off-target interactions and maximizing delivery to the intended cells, highly purified AAV vectors offer a more potent therapeutic effect with a reduced risk of adverse events, ultimately contributing to the success of AAV-based gene therapy.⁶

Ensuring the safety and efficacy of the final product requires meticulous cleansing of the viral harvest during the AAV purification process to eliminate unwanted cellular debris, empty capsids, and other impurities.⁷ This crucial stage enriches the product by selectively concentrating functional AAV vectors containing the therapeutic DNA. By implementing techniques such as chromatography or ultracentrifugation, researchers can achieve the desired level of AAV vector purity, meticulously tailored to the specific characteristics of the AAV serotype and the subsequent downstream applications.⁸

AAV purification process

A successful AAV purification process follows a series of well-defined steps to ensure that the final product is a highly purified and concentrated AAV vector suitable for safe and effective gene therapy applications.⁷

Harvesting of the AAV vectors

The method for collecting the viral harvest following AAV vector production within host cells depends on the specific AAV serotype. For serotypes like AAV8 and 9, the AAV vectors are present in the cell culture supernatant.⁹ In contrast, other serotypes necessitate cell lysis to release the encapsulated AAV vectors.

Clarification and Filtration

This step removes cellular debris and large contaminants from the harvest using techniques like centrifugation and filtration, resulting in a clarified solution containing the AAV vectors.

Concentration and Purification

This is the core purification step, where the clarified solution undergoes further processing through chromatography or ultracentrifugation-based techniques to concentrate and purify the AAV vectors. This step removes impurities like empty capsids and other unwanted components.

Formulation and Storage

The purified AAV vectors are then formulated with specific buffers and excipients to ensure stability and functionality during storage and delivery.¹⁰ Finally, the formulated AAV vector preparation is filled into vials and stored under controlled conditions until administration.

Maintaining aseptic conditions throughout the process is crucial to preventing contamination. Assays are also performed at various stages to monitor AAV vector yield and purity.

AAV Purification Methods

There are two main types of AAV purification techniques:

Ultracentrifugation-based methods

This method utilizes high-speed centrifugation to separate and isolate AAVs from the solution based on their buoyant density or sedimentation rates.

The two most common ultracentrifugation methods are:

Cesium Chloride (CsCl) Gradient Ultracentrifugation

In this traditional method, the CsCl solution forms a gradient of increasing density during high-speed centrifugation. AAV vectors, along with other particles in the sample like empty capsids and cellular debris, sediment at distinct positions within the gradient based on their individual densities, facilitating their separation.¹¹

Iodixanol Gradient Ultracentrifugation

Like CsCl, this method separates particles based on their buoyant density in a pre-formed gradient. However, it utilizes iodixanol, a synthetic, non-ionic gradient medium.¹²

This is a relatively safer technique due to the lower toxicity of iodixanol compared to CsCl. Iodixanol is less dense than CsCl, thus offering a gentler separation environment for AAV vectors.¹³ However, it may not be as efficient for some AAV serotypes as CsCl gradients.¹³

Chromatography-based methods

This method exploits the differential interaction of AAV vectors with a chromatography resin. These resins have a specific surface chemistry that selectively binds AAVs based on properties like size, charge, or affinity to specific ligands. AAVs are passed through a column packed with the resin. Contaminants with weak interactions flow through the column first, while AAVs bind to the resin. Subsequent elution steps with specific buffers or solutions detach the AAVs from the resin, achieving their purification.

The three main chromatography methods are:

Ion-exchange Chromatography (IEX)

This method separates particles based on their net surface charge. AAV capsids have a different type of surface charge distribution, which facilitates their effective separation from empty capsids and host cell proteins.¹⁴

IEX is a common choice for initial purification of AAV vectors as it is well-established, cheaper, and can handle large sample volumes, although it might not achieve high enough purity for all AAV-based gene therapy applications.²

Affinity Chromatography

This method offers highly specific separation based on the interaction between an immobilized ligand and a target molecule. In AAV purification, the ligand binds specifically to a protein on the AAV capsid.

Affinity Chromatography is highly efficient at separating AAV serotypes or removing empty capsids with similar charges to full vectors. However, the cost of developing and using specific ligands is expensive, and it may not be scalable for large-volume production.

Size-Exclusion Chromatography (SEC)

This method separates particles based on their size and shape. In AAV purification, it gently separates AAV capsids from residual aggregates or contaminants of different sizes, preserving AAV vector functionality. It is often used as a final polishing step after the main purification by IEX or affinity chromatography.¹⁵

Filtration techniques

Filtration techniques play a crucial role in the purification of AAV vectors by removing unwanted particles and contaminants at various stages of the process.

The two most common filtration techniques are:

Tangential Flow Filtration (TFF)

This is a pressure-driven process in which the feed stream flows tangentially across a semi-permeable membrane, allowing the filtrate (desired product) to permeate through while retaining larger particles and impurities in the retentate stream.¹⁶

This filtration technique is versatile enough to be used for clarification, concentration, and buffer exchange. It is adaptable for both small and large-scale AAV production and is relatively gentle on AAV vectors compared to other methods. However, the selection of membrane is crucial to ensure efficient AAV passage while retaining impurities, as an improper choice can otherwise lead to some AAV vector loss.¹⁶

Depth Filtration

This method employs a depth filter media with a tortuous path to capture impurities.¹⁷ During this process, the feed stream flows through the filter, and particles exceeding the size of the filter pores become trapped within the media.

Compared to TFF, depth filtration offers a simpler and less expensive approach, particularly suited for initial clarification steps due to its high capacity for capturing large particles.⁷ However, its versatility is limited as it primarily functions in this initial role. This technique is more susceptible to clogging, potentially requiring frequent filter changes.¹⁸ Additionally, compared to TFF, there is a greater risk of AAV vector loss due to adsorption onto the filter media.

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Factors Influencing AAV Purification

There's no single "best" method for AAV purification. The optimal approach depends on careful consideration of the specific vector characteristics, production scale, downstream processing needs, and other factors. By carefully evaluating these factors, researchers and manufacturers can select the most suitable purification techniques to achieve high-yield, high-purity AAV vectors for their specific applications.

Vector Characteristics

• Different AAV serotypes possess distinct physical properties, such as surface charges and affinity for cellular receptors. These variations can impact the selection of chromatography resins or membranes used for separation during purification.
• AAV vectors primarily exist as single particles, but aggregation can occur, forming particles of varying sizes. Size-exclusion chromatography can be effectively optimized to separate the predominant size of the AAV vector population from aggregates or other contaminants with different sizes.
• Stability considerations also play a role. Some AAV serotypes exhibit greater stability than others under specific conditions, such as exposure to certain buffers or pH ranges. This necessitates judicious selection of purification methods and protocols, as harsh conditions or prolonged processing times might not be suitable for less stable serotypes.¹⁹

Scale of Production

• Techniques like iodixanol gradient centrifugation or small-scale chromatography columns might be sufficient for research purposes or initial development.²⁰
• Scalability is crucial for commercial AAV production. Chromatography methods are generally preferred due to their better scalability compared to ultracentrifugation, which can be time-consuming for large volumes.²⁰

Downstream Processing Requirements

• If the AAV vector solution needs to be concentrated after purification, techniques like tangential flow filtration (TFF) can be integrated into the purification process.²¹
• The AAV vector might need to be suspended in a specific buffer for downstream applications like gene therapy. Diafiltration using TFF allows for buffer exchange while concentrating the AAV vector solution.²¹
• Sterile filtration might be required as a final step for clinical-grade AAV vectors.²² This ensures the absence of microorganisms in the final product.

Challenges and Solutions in AAV Purification

Despite the significant progress made in AAV purification techniques, several challenges remain that must be addressed to ensure the production of high-quality AAV vectors for clinical applications. A comprehensive understanding of these challenges and the implementation of effective solutions are crucial for the development of robust and cost-effective purification processes.

Balancing Product Yield and Purity

Balancing high yield and high purity during AAV purification remains a significant challenge. While a high yield facilitates dose optimization and broader clinical trials, achieving high purity is essential for efficient transduction, minimal immune response, and optimal biodistribution of the vector. Unfortunately, high-resolution purification techniques often come at the expense of yield by removing a larger portion of the AAV vector population, including some functional vectors.

Researchers address this challenge in two ways:

1. They employ multi-step approaches, such as an initial step like ion exchange chromatography to remove larger contaminants, followed by a high-resolution technique like size exclusion chromatography for final polishing.¹⁵'²³
2. They address inefficiencies during purification, such as incomplete capture or transfer losses, by refining parameters like buffer composition, resins, or centrifugation conditions.²⁴

Furthermore, ongoing research seeks to develop novel methods that minimize yield losses while maximizing purity.²⁴ Additionally, a high-quality viral harvest, achieved through optimized production cell lines and efficient clarification steps, contributes to a purer harvest with a higher concentration of functional AAV vectors, ultimately facilitating a more streamlined purification process.²⁵

Scalability

Scaling up AAV purification from research to commercial production presents several challenges:²⁶

• Multi-step purification protocols often struggle to maintain efficiency and minimize losses during large-scale processing. These protocols, optimized for smaller batches, may not translate well to larger volumes.
• Large-scale AAV production requires a sufficient viral harvest. Scaling up cell culture to generate enough AAV vectors can strain resources for media, bioreactors, and qualified personnel.
• Upgrading equipment is necessary to handle significant harvests. This necessitates investment in larger chromatography columns, high-throughput centrifuges, and other specialized processing equipment.
• Rigorous process validation for larger scales is crucial. This ensures consistent product quality and regulatory compliance.

Automation emerges as a critical solution by streamlining processes, minimizing errors, and potentially reducing labor costs, ultimately facilitating a more efficient and cost-effective path to large-scale production of high-purity AAV vectors.²⁷

Cost-effectiveness

AAV purification faces significant cost hurdles due to several factors:²⁸

• It requires the use of expensive buffers, chromatography resins, and other specialized consumables.
• Inefficient purification processes that result in high losses of AAV vectors further strain financial resources.
• Downstream processing steps such as formulation and filling also contribute to the overall cost burden.
• Upfront investment in large-scale facilities adds another layer of cost consideration.
• Labor costs associated with manual processing further exacerbate this issue.

These factors collectively contribute to a high cost per dose of the final gene therapy product.

Addressing these challenges necessitates a multi-pronged approach. Exploring cost-effective alternatives for consumables and developing continuous processing techniques offer promising solutions. Furthermore, standardizing AAV purification protocols and automating steps can minimize errors and improve efficiency, ultimately leading to cost savings.

Conclusion

In conclusion, AAV purification remains a critical yet intricate step in AAV-based gene therapy development. A thorough understanding of the available techniques, their strengths and limitations, and the factors influencing their selection is essential for achieving robust and efficient AAV vector production. As the field of AAV-based gene therapy continues to evolve, advancements in purification technologies and strategies will play a pivotal role in ensuring the successful delivery of safe and efficacious AAV-based therapies for a wider range of genetic diseases.

Reference

1. Wang JH, Gessler DJ, Zhan W, Gallagher TL, Gao G. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther. 2024;9(1):78.
2. Nass SA, Mattingly MA, Woodcock DA, Burnham BL, Ardinger JA, Osmond SE, et al. Universal method for the purification of recombinant AAV vectors of differing serotypes. Mol Ther Methods Clin Dev. 2018;9:33-46.
3. Nieto K, Salvetti A. AAV vectors vaccines against infectious diseases. Front Immunol. 2014;5:69616.
4. McColl-Carboni A, Dollive S, Laughlin S, Lushi R, MacArthur M, Zhou S, et al. Analytical characterization of full, intermediate, and empty AAV capsids. Gene Ther. 2024:1-0.
5. Ayuso E, Mingozzi F, Montane J, Leon X, Anguela XM, Haurigot V, et al. High AAV vector purity results in serotype-and tissue-independent enhancement of transduction efficiency. Gene Ther. 2010;17(4):503-10.
6. Münch RC, Muth A, Muik A, Friedel T, Schmatz J, Dreier B, et al. Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors. Nat Commun. 2015;6(1):6246.
7. Adams B, Bak H, Tustian AD. Moving from the bench towards a large scale, industrial platform process for adeno‐associated viral vector purification. Biotechnol Bioeng. 2020;117(10):3199-211.
8. Richter K, Wurm C, Strasser K, Bauer J, Bakou M, VerHeul R, et al. Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques. Eur J Pharm Biopharm. 2023;189:68-83.
9. Vandenberghe LH, Xiao R, Lock M, Lin J, Korn M, Wilson JM. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum Gene Ther, 2010;21(10):1251-7.
10. Srivastava A, Mallela KM, Deorkar N, Brophy G. Manufacturing challenges and rational formulation development for AAV viral vectors. J Pharm Sci. 2021;110(7):2609-24.
11. Burova E, Ioffe E. Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications. Gene Ther. 2005;12(1):S5-17.
12. Crosson SM, Dib P, Smith JK, Zolotukhin S. Helper-free production of laboratory grade AAV and purification by iodixanol density gradient centrifugation. Mol Ther Methods Clin Dev. 2018;10:1-7.
13. El Andari J, Grimm D. Production, processing, and characterization of synthetic AAV gene therapy vectors. Biotechnol J. 2021;16(1):2000025.
14. Heldt CL, Areo O, Joshi PU, Mi X, Ivanova Y, Berrill A. Empty and Full AAV Capsid Charge and Hydrophobicity Differences Measured with Single-Particle AFM. Langmuir. 2023;39(16):5641-8.
15. McIntosh NL, Berguig GY, Karim OA, Cortesio CL, De Angelis R, Khan AA, et al. Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering. Sci Rep. 2021;11(1):3012.
16. Grzenia DL, Carlson JO, Wickramasinghe SR. Tangential flow filtration for virus purification. J Memb Sci. 2008;321(2):373-80.
17. Griffiths IM, Mitevski I, Vujkovac I, Illingworth MR, Stewart PS. The role of tortuosity in filtration efficiency: A general network model for filtration. J Memb Sci. 2020;598:117664.
18. Mendes JP, Fernandes B, Pineda E, Kudugunti S, Bransby M, Gantier R, et al. AAV process intensification by perfusion bioreaction and integrated clarification. Front Bioeng Biotechnol. 2022;10:1020174.
19. Khanal O, Kumar V, Jin M. Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation. Mol Ther Methods Clin Dev. 2023;31.
20. Lock M, Alvira M, Vandenberghe LH, Samanta A, Toelen J, Debyser Z, Wilson JM. Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale. Hum Gene Ther. 2010;21(10):1259-71.
21. Miyaoka R, Tsunekawa Y, Kurosawa Y, Sasaki T, Onodera A, Sakamoto K, et al. Development of a novel purification method for AAV vectors using tangential flow filtration. Biotechnol Bioeng. 2023;120(11):3311-21.
22. Mandel RJ, Burger C, Snyder RO. Viral vectors for in vivo gene transfer in Parkinson's disease: properties and clinical grade production. Exp Neurol. 2008;209(1):58-71.
23. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999;6(6):973-85.
24. Kilgore R, Minzoni A, Shastry S, Smith W, Barbieri E, Wu Y, LeBarre JP, et al. The downstream bioprocess toolbox for therapeutic viral vectors. J Chromatogr A. 2023;1709:464337.
25. Benskey MJ, Sandoval IM, Manfredsson FP. Continuous collection of adeno-associated virus from producer cell medium significantly increases total viral yield. Hum Gene Ther. 2016;27(1):32-45.
26. Jiang Z, Dalby PA. Challenges in scaling up AAV-based gene therapy manufacturing. Trends Biotechnol. 2023;41(10):1268-1281.
27. Fu X, Williams A, Bakhshayeshi M, Pieracci J. Leveraging high-throughput purification to accelerate viral vector process development. J Chromatogr A. 2022;1663:462744.
28. Lyle A, Stamatis C, Linke T, Hulley M, Schmelzer A, Turner R, Farid SS. Process economics evaluation and optimization of adeno‐associated virus downstream processing. Biotechnol Bioeng. 2023.
29. Rodrigues GA, Shalaev E, Karami TK, Cunningham J, Slater NK, Rivers HM. Pharmaceutical development of AAV-based gene therapy products for the eye. Pharm Res. 2019;36(2):29.

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