A lentivirus is a single-stranded RNA retrovirus widely used as a gene transfer tool in advanced therapy medicinal products based on its ability to infect both dividing and differentiated non-dividing cells.
Traditionally, lentiviral vectors (LVVs) are manufactured using an adherent cell production platform with serum, but serum-free suspension cell systems have also been developed as an alternative. At Porton Advanced, we have developed a proprietary suspension cell line, HEK293TG2S, through clonal screening, suspension adaptation, and viral vector production technologies.
Based on the concept of quality and design from advanced technologies, we have developed a HEK293T Lentiviral Vector production platform (LV-SMART®), which has been successfully used in more than a dozen client projects, has excelled in over 250 feasibility studies, and has contributed to 16 CMC projects. Porton Advanced also provides other commercial LVV suspension production platforms, as per our clients’ requests. Until now, we have successfully supported over 7 IND approvals globally using LV-SMART® and commercial LVV systems.
To learn more about this serum-free and animal-free suspension platform,visit: https://www.portonadvanced.com/platforms/lv-smart-lvvs-suspension-production-platform/
To obtain Lentiviral Vectors with high titer, low impurities, and low production cost, the experienced technical team at Porton Advanced conducts in-depth and comprehensive research on the upstream processing of LVVs. This includes screening and exploring factors such as basic culture medium, cell density, ratio of helper plasmids to GOI plasmids, quality dosage, transfection reagents, additives, and virus harvest time to determine the optimal process route, process parameter range, and most suitable raw materials. Currently, we have conducted feasibility tests for over 250 different CAR/TCR sequences and provided services for multiple CMC projects. The highest titer was above 3E+08 TU/mL.
To obtain LVVs with high purity and controlled production costs, the experienced technical team of Porton Advanced has conducted in-depth and comprehensive research on the development of our virus purification process. This includes the screening and exploring virus harvest solution clarification, enzyme digestion, column chromatography, UF/DF, sterilization filtration, and formulation to determine the optimal process route, process parameter range, and the most suitable raw materials. LVV produced based on the LV-SMART® platform has the advantage of good virus activity, low impurity residues, low production cost, simplified production process, and high virus recovery rate.
Our dedicated analytical development and testing team provides comprehensive services for viral vectors.
Porton Advanced has developed and optimized various methods for transducing cells with viral vectors, including T cells, NK cells, and stem cells. As a result, we have the capability to carry out infectious titer assays based on transduced target cells as requested by our clients.
Porton Advanced has a dedicated viral vector production team with decades of hands-on and management experience.
We have established robust production processes for adhesion and suspension production with a maximum of 200L per batch and 100 batches per year. Our GMP facility has an independent air conditioning system, a single-use and fully enclosed aseptic production process, and a unidirectional flow of personnel and logistics to minimize the risk of contamination and cross-contamination. The whole production operation is monitored by CCTV, which can be preserved for further traceability, and all the production equipment has completed CSV (Computerized Systems Validation). The facility design and quality system meet the requirements of GMP production management systems in China, the US, and the EU, allowing us to provide CDMO services for global clients.
Example process for Lentiviral vector suspension manufacturing
Porton Advanced provides a three-tiered cell banking service based on adherent and suspension cell lines that can be used for eCTD publishing and submissions to the FDA and NMPA. Cell lines include HEK293/T/F, HeLA, etc. Our technical team will establish a three-tiered cell bank in a GMP environment and perform programmed cooling and cryopreservation to maximize cell viability. We have successfully delivered several projects, one of which has moved to the commercial stage.
The LV-SMART® Platform has been used for different Lentiviral vectors' envelopes with different GOIs projects.
The crude titer for lentiviral vectors pseudotyped with VSV-G, BAEV, and Cocal and with different GOIs is over 5E07TU/mL.
Porton Advanced selects the appropriate chromatography resins, buffer composition, loading capacity, and column height based on the characteristics of the viral particles, such as their surface electric charge and the differences in molecular weight between them and impurities. Our goal is to maximize the interaction between viral particles and column resins. We employ multimodal chromatography and ion exchange chromatography as the purification steps to obtain higher purity of viral vectors.
Extended sample penetration can create a gel layer on the surface of the hollow fiber membrane, resulting in membrane polarization and protein precipitation. To mitigate the challenges associated with one-step high-fold concentration, a multi-step concentration approach is used. We begin with low-fold concentration, followed by diafiltration and exchange into the target buffer system before proceeding to the final concentration stage. Additionally, it is important to appropriately reduce the flow rate at the permeate end to decrease the transmembrane pressure (TMP), which helps slow the decline in flux. However, it is crucial to maintain a balanced flow rate between the permeate and retentate ends; otherwise, the desired resolution may not be achieved.
Porton Advanced prefers to use membrane materials with a low adsorption (such as 0.22 μm PES or PVDF) to lower non-specific absorption and trap bacteria and microbes. To avoid membrane blockage or flux reduction, we restrict maximum filtration volume (≤20L/m(2)), select a filter with an appropriate membrane surface area, gradually increase flow rate with constant pressure, and balance filtration efficiency and harvest rate to ultimately increase recovery rate at the aseptic step.
We typically use nuclease digestion to remove host cell DNA. The concentration, reaction time, and temperature are crucial factors for effective host cell DNA (HCD) removal. To further enhance the process, we employ single-step or multi-step filtration techniques to remove host cells and cell debris. Additionally, we optimize chromatography conditions to ensure host proteins are effectively absorbed by the resin, and we incorporate elution steps to achieve a high level of host cell protein (HCP) removal efficiency.
For large-scale manufacturing of lentiviral vectors, there are three main transient transfection methods available: Co-precipitating calcium phosphate, lipofection, and polyethyleneimine (PEI) transfection. Clients should select the most suitable transfection method based on their specific processes. The ideal transfection method should offer high efficiency and minimal cytotoxicity.
During the transfection process, it is essential to maintain the cells in optimal physiological condition for virus production. This includes ensuring high cell viability (greater than 90%) and keeping the cells in the log phase of the cell cycle. We can achieve this by using high-purity plasmid DNA, controlling the target gene sequence size (less than 2.5 kb), and extending the harvest duration (longer than 48 hours). Additionally, we can incorporate other reagents, such as sodium butyrate, to enhance the process.
Adherent production is easy to operate but challenging to scale up while achieving a high viral yield per unit. This method is generally suitable for small-scale production, typically less than 10 liters. In contrast, suspension production is easier to scale up and can be highly automated, requiring less labor. This makes it more appropriate for commercial production. The LV-SMART® Platform at Porton Advanced can produce lentiviral vectors with a high yield (greater than 5E8 TU/ml) on a large scale, exceeding 50 liters.
Using a stable cell line that integrates both the packaging genes and the target gene can help reduce batch-to-batch variation. However, developing stable cell lines typically takes a longer time and poses several challenges. In contrast, transient transfection is easier to perform, requires less time, and is low-cost, but it tends to produce higher batch-to-batch variation compared to stable cell lines. Establishing a stable producing cell bank, maintaining a high-purity plasmid library, and developing a mature manufacturing process can effectively control batch-to-batch variation.
The HEK293TG2S cell line has been adapted for use in suspension cultures, and the cell banks are registered with a US DMF number, making them ready for use and helps to shorten the CMC project timeline. The cell traceability information is clear and complete, which can meet the customer's declaration needs.
The filling and finishing process is conducted in an isolator (ISO Class 5). Equipment and consumables are sterilized using vaporized hydrogen peroxide (VHP), eliminating biological membrane contamination risk. Seal tests and aseptic process simulations are routinely executed to validate the reliability of sterile control.
