Enhancing Lentiviral Purification: Combining CIM QA Monolithic Columns With Design of Experiments Approach

ABSTRACT Use of lentiviral vectors (LVs) has been steadily increasing over recent years, culminating with a recent focus on novel in vivo therapies. Despite an increased focus on LV therapies, the methods for their purification have not yet been standardised. Additionally, no major focus has been given to the problem of heterogeneity of LV harvest and removal of particle impurities. As such, novel methods for LV purification and a better understanding of factors influencing it have to be addressed. In order to address these issues, a purification method for LV has been developed using anion exchange chromatography, specifically, using quaternary amine (QA) Convective Interaction Media (CIM) monoliths. Using 96‐Well CIM QA monolithic plates and design of experiments (DOE) approach, we optimised downstream process from 20% recovery to 90%. The process was successfully transferred to a 1 mL CIMmultus QA (6 µm) column, where 70% average recovery was achieved. During purification, an average 3.3 log reduction of host cell protein impurities was achieved, as well as significant removal of particle impurities, with 31% reduction in ratio between total and transducing particles in elution, relative to clarified harvest. The CIM QA monolith column also demonstrated a very high binding capacity of 2.9 × 10 12 particles/mL of stationary phase. These conditions not only enhance purification efficiency but also hold significant promise for adaptation in large‐scale manufacturing processes.