Search

Lentiviral vectors (LVs) are widely used in clinical trials of gene and cell therapy. Low LV stability incentivizes constant development and the improvement of gentle process steps. Steric exclusion chromatography (SXC) has gained interest in the field of virus purification but scaling up has not yet been addressed. In this study, the scaling up of lentiviral vector purification by SXC with membrane modules was approached. Visualization of the LVs captured on the membrane during SXC showed predominant usage of the upper membrane layer. Furthermore, testing of different housing geometries showed a strong influence on the uniform usage of the membrane. The main use of the first membrane layer places a completely new requirement on the scaling of the process and the membrane modules. When transferring the SXC process to smaller or larger membrane modules, it became apparent that scaling of the flow rate is a critical factor that must be related to the membrane area of the first layer. Performing SXC at different scales demonstrated that a certain critical minimum surface area-dependent flow rate is necessary to achieve reproducible LV recoveries. With the presented scaling approach, we were able to purify 980 mL LVs with a recovery of 68%.

Steric exclusion chromatography (SXC) is a purification method that is based on steric exclusion effects from the surface of the target and a hydrophilic stationary phase after the addition of polyethylene glycol (PEG), which leads to an association of the target with the stationary phase without direct binding, such as covalent, electrostatic, and hydrophilic/hydrophobic interactions. The gentle nature of the method has led to an increased focus on sensitive targets such as enveloped viruses with potential for other sensitive entities, e.g., extracellular vesicles and virus-like particles. SXC is related to PEG-mediated protein precipitation, but investigation of further process parameters was crucial to gain a better understanding of the SXC method. After explaining mechanistic fundamentals and their discovery, this review summarizes the findings on SXC from its first reference 11 years ago until today. Different applications of SXC are presented, demonstrating that the method can be used for a wide variety of targets and achieves high recovery rates and impurity removal. Further, critical process parameters for successful process implementation are discussed, including technical requirements, buffer composition, and scalability.

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process.