Unlocking the Downstream Purification Process for Viral Vectors

Viral vectors are commonly used tools in molecular biology, designed to deliver genetic material into cells, which can occur in live organisms or cell cultures. The principle behind this involves utilizing the virus’s natural ability to deliver its genome into other cells to initiate infection. These vectors are widely applied in basic research, gene therapy, and vaccine-related products.

Currently, several viruses have been developed into clinically approved delivery systems for human drugs. The main viral vectors commonly used in clinical practice or clinical trials include adenovirus (AdV), adeno-associated virus (AAV), retrovirus (RV), lentivirus (LV), herpes simplex virus (HSV), among others. The downstream processing filtration process for these viral vectors is illustrated below:

Purifying viral particles is a challenging task. Downstream purification requires removing impurities such as host cell DNA (HCD), host cell proteins (HCP), plasmid DNA, and addressing challenges related to viral particle aggregation, envelope disruption, empty/full particles, and stability.

Adeno-Associated Virus (AAV) Vector

AAV is a non-pathogenic virus, known for its safety, broad host range, and physical stability. It is capable of stably expressing foreign genes for long periods, making it a widely used vector in gene therapy[1].

During virus harvest, physical or chemical methods are employed to lyse host cells, commonly using detergents like Tween or Triton. AAV is released from the lysed host cells and filtered through depth filters. Residual nucleic acid fragments are cleaved using nucleases, with careful control of temperature and duration to ensure nucleic acid fragments are cut to less than 200 bp. The process then moves on to Tangential Flow Filtration (TFF) for buffer exchange and concentration, with careful attention to avoid excessive concentration, which can cause viral particle aggregation. Typically, the concentration factor should not exceed 10 times, and low shear forces must be maintained to prevent damage to viral activity. BioLink’s TFFNOVA® Hollow Fiber Filter is ideal for this process due to its asymmetric structure, which reduces non-specific adsorption, increases filtration rate and flux, and reduces filtration time.

BioLink TFFNOVA® Hollow Fiber Filter

For AAV capture, MaXtar® AAV and Maxgo® AAV are recommended. Additives like arginine and glycerol can be included to prevent viral aggregation or adherence to the flow path walls. For polishing, MaXtar® Q can be used to remove impurities, including HCP and HCD, and to separate empty and full virus particles. The separation effectiveness of empty and full particles can be enhanced using additives like MgSO4 or Na2SO4. This step requires low sample conductivity to ensure complete binding of viral particles to the column, preventing breakthrough. MaXtar® Q is a strong anion exchange chromatography resin with superior performance compared to traditional resins, offering higher process flow rates and better dynamic binding capacity. The final stages include TFF for buffer exchange, followed by sterilizing filtration to complete the preparation of the final product.

Adenovirus (AdV) Vector

Adenovirus vectors are recombinant, replication-defective systems derived from adenoviruses. They offer high infection efficiency, infect both dividing and non-dividing cells, and have a large packaging capacity. They also pose no risk of random integration into the human genome, and they exhibit rapid transcriptional expression. Adenovirus vectors are easy to scale up for industrial production and are highly immunogenic, making them useful as vaccine vectors.

The purification process for adenovirus differs from AAV in the chromatography steps. Due to its larger particle size (typically 70-90 nm), the first chromatography step typically uses anion exchange chromatography with MaXtar® Q to capture the viral particles and remove some impurities. The second step involves polishing using multi-mode chromatography with MaXtar® COLL 400/700, which effectively removes HCD, HCP, and other impurities. MaXtar® COLL 400/700 is designed for larger molecules and can separate virus particles from smaller impurities with ease, offering improved performance in terms of flow rate and process scalability.

The final process includes TFF system for buffer exchange, sterilizing filtration, and preparation of the final viral stock.

Lentivirus (LV) / Herpes Simplex Virus (HSV) Vectors

Lentivirus vectors, derived from HIV-1, are highly efficient at delivering target genes into primary cells or cell lines, capable of infecting both dividing and non-dividing cells. HSV vectors, a type of oncolytic virus, can specifically infect and destroy tumor cells without affecting normal cells. However, both lentivirus and HSV vectors are enveloped viruses, and their envelopes are susceptible to shear forces during purification, which can significantly impact the viral activity. Additionally, these viruses have larger particle sizes, around 100-200 nm or even up to 300 nm.

Purification of these large viral particles requires specialized chromatography techniques. MaXtar® COLL 400/700, a multi-mode chromatography resin, is preferred to effectively separate large viral particles from smaller impurities.

Purification Case for Oncolytic Virus (OV)

In the TFF stage, due to the sensitivity of the viral envelope to shear forces, it is recommended to keep shear rates below 3000 1/s. Bio-Link’s TFFNOVA® Hollow Fiber Filter combined with the FiltraLinX® Benchtop Semi-automatic TFF System is ideal for this step.

FiltraLinX® Benchtop Semi-automatic TFF system

The above covers the key points and challenges of mainstream viral vector purification. When purifying different viral particles, it is important to flexibly select different resins and adjust the process sequence to ensure that the final product meets relevant quality control standards, including titer, purity, and impurity removal.

BioLink Suzhou R&D Center

BioLink R&D Center covers an area of 3,000 sqm and is equipped with R&D offices, cell culture laboratories, microbiology labs, purification labs, pilot-scale workshops, and analytical labs. The center is dedicated to providing customers with services such as upstream and downstream process development, small-scale and pilot-scale sample preparation, validation, and training.

Upstream Process:

• Bacterial/cell culture process development

• Culture process confirmation and transfer

• Lab scale and pilot scale fermentation sample preparation

Downstream Process:

• Purification process development

• Purification process confirmation and transfer

• Lab scale and pilot scale purification sample preparation

Validation and Training:

• Process characterization, resin lifespan validation

• Process training courses

• Operation training

References

【1】Wang J H , Gessler D J , Zhan W ,et al.Adeno-associated virus as a delivery vector for gene therapy of human diseases[J].Signal Transduction & Targeted Therapy, 2024, 9(1).DOI:10.1038/s41392-024-01780-w..

【2】张茗婧.腺病毒载体肺炎疫苗的构建及免疫评价研究[D].天津科技大学,2023.DOI:10.27359/d.cnki.gtqgu.2023.000296.

【3】房恩岳,张丽萍,马雪征,等.慢病毒载体系统及其安全性研究进展[J].病毒学报,2023,39(04):1181-1192.DOI:10.13242/j.cnki.bingduxuebao.004349.

【4】吴振,张紫怡,汪洋,等.重组Ⅱ型溶瘤单纯疱疹病毒纯化工艺的建立[J].中国生物制品学杂志,2018,31(05):552-554+562.DOI:10.13200/j.cnki.cjb.002184.