TY - BOOK

T1 - Downstream processing of lentiviral vectors with focus on steric exclusion chromatography

AU - Labisch, Jennifer Julia

N1 - Doctoral thesis

PY - 2023

Y1 - 2023

N2 - Lentiviral vectors (LV) are widely used to deliver therapeutic genes for gene therapy and gene-modified cell therapy and have shown success in chimeric antigen T cell therapies. The ongoing market growth leads to an increasing demand for purified LV which requires efficient downstream processes. Due to the lower stability of LV, new demands are placed on the process. Existing unit operations must be greatly optimized and research into new, alternative methods is essential. In a holistic approach, the work focused on identified bottlenecks and presents new approaches for clarification, analytics, and chromatographic purification. In the first part of this work, a vacuum-based clarification method with diatomaceous earth was improved for LV which were produced by suspension cell culture. This clarification method allowed fast and high throughput clarification and improved handling by eliminating the centrifugation step and increasing filter capacity. Thus, clarification of LV with diatomaceous earth laid the foundation for subsequent chromatography studies. To improve the analytical sample throughput and accelerate process development the second part of this thesis deals with the development of a high throughput assay with automated readout and analysis for the determination of the infectious titer, which is the key process variable for enveloped viral vectors. For this purpose, transduced cells are quantified by immunological detection in a real-time live-cell analysis system using software-based image evaluation. Eventually, the third and fourth parts focused on steric exclusion chromatography. It could be demonstrated that process parameters like the buffer mixing strategy and flow rate are crucial for this thermodynamically driven process of depletion interaction between the LV and the membrane. Moreover, it was shown that an ideal PEG molecular weight and concentration must be identified. The visualization of the LV on the membrane showed that the LV were mainly found on the first membrane layer after loading. Therefore, the surface area-specific flow rate was crucial for scale-up. The mechanistic understanding of the process and the process optimizations enabled reproducibly high LV recoveries and removal of impurities.

AB - Lentiviral vectors (LV) are widely used to deliver therapeutic genes for gene therapy and gene-modified cell therapy and have shown success in chimeric antigen T cell therapies. The ongoing market growth leads to an increasing demand for purified LV which requires efficient downstream processes. Due to the lower stability of LV, new demands are placed on the process. Existing unit operations must be greatly optimized and research into new, alternative methods is essential. In a holistic approach, the work focused on identified bottlenecks and presents new approaches for clarification, analytics, and chromatographic purification. In the first part of this work, a vacuum-based clarification method with diatomaceous earth was improved for LV which were produced by suspension cell culture. This clarification method allowed fast and high throughput clarification and improved handling by eliminating the centrifugation step and increasing filter capacity. Thus, clarification of LV with diatomaceous earth laid the foundation for subsequent chromatography studies. To improve the analytical sample throughput and accelerate process development the second part of this thesis deals with the development of a high throughput assay with automated readout and analysis for the determination of the infectious titer, which is the key process variable for enveloped viral vectors. For this purpose, transduced cells are quantified by immunological detection in a real-time live-cell analysis system using software-based image evaluation. Eventually, the third and fourth parts focused on steric exclusion chromatography. It could be demonstrated that process parameters like the buffer mixing strategy and flow rate are crucial for this thermodynamically driven process of depletion interaction between the LV and the membrane. Moreover, it was shown that an ideal PEG molecular weight and concentration must be identified. The visualization of the LV on the membrane showed that the LV were mainly found on the first membrane layer after loading. Therefore, the surface area-specific flow rate was crucial for scale-up. The mechanistic understanding of the process and the process optimizations enabled reproducibly high LV recoveries and removal of impurities.

U2 - 10.15488/13334

DO - 10.15488/13334

M3 - Doctoral thesis

CY - Hannover

ER -