Lentiviral vectors (LVs) are indispensable part of gene therapy clinical trials due to their ability to transduce both dividing and non-dividing cells. Current cultivation methods produce titers of 105 to 107 TU/mL thus it is necessary to concentrate LV as well as to remove process and product related impurities. In this work we used a continuous producer cell line WinPac-RD for LV production. Recovery through the purification process was monitored by several orthogonal methods: an infectivity assay utilized GFP expression determined by flow cytometry, LV RNA genome was quantified via RT-qPCR using primers specific for the GFP gene, LV particles were detected with p24 ELISA and SYBR Green I-based product-enhanced reverse transcriptase (SG-PERT) assays. Using tangential flow filtration (TFF) we were able to remove significant amount of cell culture proteins, but LV recovery was only 20%. This led us to pursue a TFF-free nanofiber bind/elute step based on ion-exchange chromatography. The cellulose nanofiber based purification platform provides an accessible high surface area adsorbent well suited to viral vector sorption. Using this method, we were able to concentrate LV 100 fold while also achieving a 2 log removal of host cell protein and maintaining up to 90% yield of a functional vector.

Scalable nanofiber based lentiviral vector purification

255th National Meeting (2018)

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