Scaleable purification process for gene therapy retroviral vectors.

Background: Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology.

Methods: A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 microm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold.

Results: Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77+/-11%). Up-scaling of the process resulted in high titer vector preparations, 3.2x10(8) infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination.

Conclusions: The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies.