Monolithic Columns As Downstream Processing Solutions For Lentiviral Purification

By Mojca Tajnik Sbaizero, Dona Pavlovič, Kaja Bažec, Mirjam Krašna, Luka Bevc, Urh Černigoj, and Aleš Štrancar

Lentiviral vectors serve as powerful tools for delivering genes into both dividing and non-dividing cells. The third-generation lentiviral vectors, derived from HIV but modified to be replication-incompetent and self-inactivating, provide a relatively safe and efficient system for gene delivery, especially for accommodating larger genetic sequences compared to AAV vectors.

Although several lentiviral-based therapies have been approved or are undergoing clinical trials—mainly via ex vivo methods—challenges in downstream purification have limited their broader application, particularly for in vivo therapies.

To tackle these limitations, we investigated the use of CIM® monolithic columns for purifying lentiviral vectors. After extensive testing of various chemistries, three promising candidates were identified, with two selected for further refinement and optimization using CIMmic® and CIMmultus® monoliths.

Throughout process development, multi-angle light scattering (MALS) was utilized to detect viral particles, while ddPCR and infectivity assays were employed for analytical evaluation.

Additional optimization and validation are needed to determine the most effective chemistry for a complete downstream process. Implementing this approach successfully could greatly enhance the development and application of lentiviral therapies, particularly for in vivo use.