Towards a scalable purification protocol of GaLV-TR pseudotyped lentiviral vectors.

Lentiviral vectors (LV) that are used in research and development as well as in clinical trials are in majority VSVg-pseudotyped. The predominance of this pseudotype choice for clinical gene therapy studies is largely due to a lack of purification schemes for pseudotypes other than VSVg. Here we report for the first time the development of a new downstream process (DSP) protocol allowing high yield production of stable and infectious GaLV-TR-LV particles. We identified critical conditions in tangential flow filtration (TFF) and chromatographic steps for preserving the infectivity/functionality of LV during purification. This was done by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and secondly by defining the optimal order of these steps. A three-step process was developed for GaLV-TR-LV purification consisting of one TFF, and two chromatographic steps (ion exchange and size exclusion chromatography) permitting recoveries of >27% of infectious particles. With this process, purified GaLV- pseudotyped LV vectors enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin-1 peptide, whereas in the same conditions non-purified vector transduced only 9% of the cells (MOI 20). Our protocol will allow for the first time the purification of GaLV-TR-LV that are biologically active, stable and with sufficient recovery in the perspective of preclinical studies and clinical applications. Obviously, further optimizations are required to improve final vector yields.