Wu Y, Xie G, Xu W, Yuan J, Xu W. W, Yu H. X, Jiang L. Y, Ruan J. J, Poon H. F. Optimizing AAV Filtration: Comparative Analysis of Membrane Performance and Recovery Rates. Biomed Pharmacol J 2024;17(4).

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Manuscript received on :26-09-2024
Manuscript accepted on :19-12-2024
Published online on: 31-12-2024

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Plagiarism Check: Yes
Reviewed by: Dr. Daya Shankar Gautam
Second Review by: Dr. Ahmed Salah
Final Approval by: Dr. Anton R Keslav

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YeQing Wu, GuoHao Xie, WeiXian Xu, Jun Yuan, Wayne Wenyan Xu, Henry Xiaoyu Yu, Lothar Yulin Jiang, Jeremy Junxi Ruan and H. Fai Poon^*

Department of Research and Development, QuaCell Biotechnology, Co., Ltd., Zhongshan, Guangdong, China.

Corresponding Author Email: fai@quacell.com

Abstract

Adeno-associated viruses (AAVs) have proven to be effective tools for gene therapy due to their ability to be engineered to deliver genetic material to target cells. This study investigates the performance of three different filtration membrane packs—Lepure, Cobetter, and Merck—in purifying the AAV8 serotype. We assessed the turbidity and AAV titer before and after filtration to evaluate the efficiency of each membrane. Before filtration, the AAV8 sample exhibited a turbidity of 173.6 nephelometric turbidity units (NTU) and a titer of 1.01 × 10¹¹ viral genomes per mL (vg/mL). Post-filtration, it is observed that the Lepure membrane achieved a turbidity of 6.65 NTU and an AAV titer of 4.48 × 10⁹ vg/mL, while Cobetter resulted in a turbidity of 3.44 NTU and a titer of 3.80 × 10⁹ vg/mL. Merck demonstrated the lowest performance with a turbidity of 0.49 NTU and an AAV titer of 9.70 × 10⁹ vg/mL. Notably, Lepure demonstrated the highest recovery rate at 13.3%, despite its higher turbidity, indicating minimal viral adsorption. These findings highlight the importance of selecting appropriate filtration systems to optimize AAV recovery while maintaining low turbidity levels, ultimately enhancing the efficiency of AAV as a vector for therapeutic applications. Further research is recommended to refine these filtration methods to improve the purification of AAV.

Keywords

AAV (adeno-associated virus); AAV vector purification; Gene therapy; member pack filtration; recovery optimization; serotype-specific purification; transduction efficiency

Introduction

Adeno-associated viruses (AAVs) have emerged as one of the most promising vectors for gene therapy due to their favorable safety profile, capacity for long-term gene expression, and minimal immunogenicity^16,18,19,21. AAV is a non-enveloped virus with a protein capsid that encases a small, single-stranded DNA genome—hundreds of unique strains has been identified across various species^10,24. Recombinant AAV (rAAV), lacking viral DNA, has proven to be an effective vehicle for certain gene therapy applications. As a protein-based nanoparticle^4,24, rAAV can penetrate the cellular membrane and deliver its genetic cargo directly to the control center of the cell, the nucleus^27,28.

However, the production of AAVs at a scale sufficient for clinical applications continues to pose significant challenges^8,18,24,28. Traditional methods often resulted in low yields, making the optimization of AAV production crucial for advancing gene therapy technologies^7,9,14. Recent advancements in bioprocessing techniques have focused on enhancing AAV yield through various strategies, including the optimization of cell culture conditions, vector design, and purification methods^3,12. One innovative approach that has gained traction in recent years is the use of specialized filtration systems during the purification process. Filtration can effectively remove impurities while concentrating the viral particles, leading to improved overall yields as a pivotal step in the manufacturing of biotherapeutic product². The implementation of specialized filters, such as tangential flow filtration (TFF) and depth filtration, has been shown to improve the clarity and purity of AAV preparations in the laboratory. These filtration techniques can selectively retain viral particles based on size and molecular characteristics, while allowing smaller contaminants, such as host cell proteins and DNA, to pass through¹³. By optimizing the pore size and surface properties of the filters, it is possible to enhance the recovery of infectious viral particles, thereby increasing the yield of AAV production^1,5. Moreover, recent studies have indicated that the use of filtration can significantly streamline the purification process, reducing the need for extensive chromatographic methodologies that are often time-consuming and resource-intensive^1,6. The combination of increased efficiency and yield makes specialized filtration systems a promising avenue for enhancing AAV production, optimizing both consistency and scalability. Such advancements will not only improve the manufacturing process, but contribute to the development of cost-effective and accessible gene therapies, ultimately expanding therapeutic opportunities for a wide range of genetic disorders^20,22. In this study, we aim to investigate the application of a novel filtration system in AAV production and its impact on yield enhancement. By examining the effects of filtration parameters on the recovery of AAV particles, we hope to provide valuable insights that could inform best practices for rAAV viral vector productions and contribute to the advancement of gene therapy.

Materials and Methods

The materials used in this study included filtration membrane packs, rubber tubing, a peristaltic pump, anhydrous ethanol, and 50 mL syringes—all purchased from Lepure (China). The primary filtration membrane packs included Lepure, Cobetter, and Merck (Table 1).

Table 1: Filtration membrane packs examined for comparative analysis of membrane performance and recovery rates.

The experimental procedure was as follows. The rubber tubing was first rinsed with sterile ultrapure water on both the inner and outer surfaces, dried, and then immersed in anhydrous ethanol¹⁵. Using a syringe, the tubing was filled with anhydrous ethanol and allowed to soak for 15 minutes to ensure complete disinfection. Next, the filters were connected in series, and the rubber tubing was attached to the peristaltic pump, which was set to a rotation speed of 10 revolutions per minute (rpm). The air inside the membrane pack was then expelled, and the dead volume of the system was calculated based on the first drop of liquid that flowed out from the end, along with the flow rate at the same pump speed. The AAV sample to be filtered was then introduced into the system, and the collection of the filtered AAV sample began once half of the dead volume was expelled from the end¹⁷. After filtering 150 mL of the sample, the system was flushed with phosphate-buffered saline (PBS) at a fixed volume of 300 mL, approximately equivalent to four times the system’s dead volume. Finally, the turbidity and titer of the samples were measured before and after filtration to assess the filtration efficiency of the membrane²³. The recovery efficiency was calculated by the ratio of the number of AAV particles recovered to the number of AAV particles before recovery, thereby quantifying the effectiveness of the filtration process.

Results And Discussion

The experimental results demonstrated significant differences in the performance of the various filtration membrane packs. Before filtration, the AAV8 serotype exhibited a turbidity of 173.6 NTU and an AAV titer of 1.01 × 10¹¹ vg/mL. After filtration, the results varied among the three brands assessed (Table 2). Filter 1 had a dead volume of 78.24 mL, a flow rate of 15.5 mL/min, the highest turbidity of 6.65 NTU, and an AAV titer of 4.48 × 10⁹ vg/mL. Filter 2 showed a dead volume of 77.34 mL, a flow rate of 23.5 mL/min, a turbidity of 3.44 NTU, and an AAV titer of 3.80 × 10⁹ vg/mL. In contrast, Filter 3 had the highest dead volume of 81.37 mL, a flow rate of 23.8 ml/min, a significantly lower turbidity of 0.49 NTU, and an AAV titer of 9.70 × 10⁵ vg/mL.

Table 2: Post-filtration quantitative measurements of the filter membrane packs examined for comparative analysis of membrane performance and recovery rates

Interestingly, while filter 1 exhibited the highest turbidity post-filtration, it also demonstrated the lowest viral adsorption, indicating that such filtration parameter allowed more particulate matter, such as cellular debris or residual host cell DNA, to pass through while retaining a higher number of AAV particles. The recovery rates calculated from the experiments revealed that filter 1 achieved a statistical significant recovery rate of 13.3 ± 0.3% when compare to recovery rate of 11.3 ± 0.2% of filter 2 (p-value < 0.05, Studet’s t-test); and the recovery rate of filter 3 is statistically significant when compare to that of filter 1 and fiilter 2 (Fig 1). The minimal viral adsorption observed with filter 1, Lepure is significant as it implies a more efficient recovery of viral vectors, despite an increased presence of impurities. These findings suggest that the choice of filtration membrane plays a crucial role for optimizing AAV recovery and minimizing loss during the purification process^1,13,26. Additionally, the images taken during the filtration process revealed the visual differences between the filtered products to be subtle. however, the products obtained via filter 1 appeared darker compared to the nearly colorless product obtained via filter 3. This observation agrees with the turbidity measurements, where filter 1’s higher turbidity suggests a greater presence of particulate matter post-filtration.

Figure 1: Comparison of filter membranes for different commercial filters. The recovery rates calculated from the experiments revealed that filter 1 achieved a statistical significantly higher recovery rate of 13.3 ± 0.3%.   Click here to view Figure

Conclusion

Overall, these results underscore the importance of selecting appropriate filtration systems to maximize AAV recovery while maintaining an acceptable level of turbidity²⁵, playing an essential role for effective gene therapy applications. The examination on the effects of filtration parameters on the retention of AAV particles in this study has proven to yield more promising results, as opposed to traditional methods such as using chromatographic filtration^11,12,16. Further optimization and analysis of these filtration methods could enhance the purification processes for AAV production, building the foundation for clinical success in the treatment of various human diseases. It is also pivotal to conduct further research on the compatibility of filtration membrane packs across various AAV serotypes, in order to support the development of universal vectors that meet the high standards of purity, efficacy, and safety^12,18,22,28.

Acknowledgement

The authors would like to acknowledge the Zhongshan City Innovation and Entrepreneurship Cooperation. The writing and reference was assisted by you com and jenni.ai.

Funding Sources

The study was supported by Zhongshan City Innovation and Entrepreneurship Research Team Award: Entrepreneurship Team for the R&D and Commercialization of Critical Raw Materials for Antibody Drugs (CXTD2020004).

Conflicts of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Authors’ Contributions

YeQing Wu: Conceptualization, Methodology, Data Collection, Writing – Final Approval of the Manuscript

GuoHao Xie:  Conceptualization, Methodology, Data Collection, Writing – Final Approval of the Manuscript

WeiXian Xu: Data Collection, Analysis, Writing – Final Approval of the Manuscript

Jun Yuan: Data Collection, Analysis, Writing – Final Approval of the Manuscript

Wayne Wenyan Xu: Data Collection, Analysis, Writing – Final Approval of the Manuscript

Henry Xiaoyu Yu: Analysis, Writing – Final Approval of the Manuscript

Lothar Yulin Jiang: Analysis, Writing – Final Approval of the Manuscript

Jeremy Junxi Ruan: Analysis, Writing – Final Approval of the Manuscript

Fai Poon: Conceptualization, Methodology, Data Collection, Writing – Final Approval of the Manuscript

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