Lentiviral Vector Purification Using Genetically Encoded Biotin Mimic in Packaging Cell

Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging HEK293T cells were genetically engineered with a cyclical biotin-mimicking peptide displayed on a CD8α stalk, termed cTag8. LVs were modified with cTag8 by its passive incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. Expression of cTag8 on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As cTag8 binds streptavidin in the nanomolar range, the addition of micromolar concentrations of biotin resulted in the release of captured LVs by competitive elution, with overall yields of ≥60%. Analysis of eluted LVs revealed high purity with a >3-log and 2-log reduction in DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using monolith affinity chromatography, with an encouraging preliminary overall yield of 20%. This method will be of valuable use for both research and clinical applications of LVs. Lentiviral vectors (LVs) have recently witnessed an increasing demand in research and clinical applications. Their current purification processes represent the main bottleneck in their widespread use, as the methods used are cumbersome and yield low recoveries. We aimed to develop a one-step method to specifically purify LVs, with high yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging HEK293T cells were genetically engineered with a cyclical biotin-mimicking peptide displayed on a CD8α stalk, termed cTag8. LVs were modified with cTag8 by its passive incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. Expression of cTag8 on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As cTag8 binds streptavidin in the nanomolar range, the addition of micromolar concentrations of biotin resulted in the release of captured LVs by competitive elution, with overall yields of ≥60%. Analysis of eluted LVs revealed high purity with a >3-log and 2-log reduction in DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using monolith affinity chromatography, with an encouraging preliminary overall yield of 20%. This method will be of valuable use for both research and clinical applications of LVs.