Impact of the Membrane Structure of the Stationary Phase on Steric Exclusion Chromatography (SXC) of Lentiviral Vectors.

For steric exclusion chromatography (SXC), hydrophilic stationary phases are used to capture the target molecule in the presence of polyethylene glycol. The influence of the structure and pore size of the stationary phase on the process requirements are not yet well understood. To better understand the SXC process, membranes with different pore sizes that served as a stationary phase were compared for the purification of lentiviral vectors (LVs). A design of experiments (DoE) was performed to assess the combined impact of PEG concentration and membrane pore size on the purification performance. A visualization experiment showed that the LVs were captured on the first membrane layer for a pore size up to 2.2 µm, and for a pore size larger than 2.2 µm, LVs were also partly found on the second and third membrane layers. Moreover, we could observe that increasing membrane pore size requires a higher PEG concentration to achieve comparable LV recoveries. Using five membrane layers as a stationary phase was sufficient to achieve good performance, supporting the visualized capture results. In conclusion, we could show that each stationary phase has its optimal PEG buffer compositions for SXC, depending on the membrane structure and pore size.

Scaling Up of Steric Exclusion Membrane Chromatography for Lentiviral Vector Purification.

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%.

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

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.