Large-Scale cGMP Manufacture of a Plasmid Vector for Cystic Fibrosis Gene Therapy Clinical Trials.

Cai Y, Rodriguez, S, Nelson J, Batten T, Hebel H, Hyde SC, Gill DR

Molecular Therapy, Vol 20, S1, Abstract 761

The American Society of Gene and Cell Therapy Annual Conference, Philidelphia, 2012

Non-viral gene therapy employing a plasmid vector encoding the CFTR transgene has demonstrated its safety and efficacy. As opposed to DNA vaccines, low CpG content in the plasmid preparation is required to minimize inflammatory responses after administration. Small scale production of this CpG free plasmid to high purity has been demonstrated, however, a new set of obstacles arose during the cGMP manufacture at a large scale. Constructed with the R6K replication of origin, plasmid replication is regulated by both π protein expression from the host cell and Kanamycin resistance protein expression from the vector, resulting in low plasmid yields during fermentation. Pre-existing cell banks excluded the potential for improvements via strain or colony screening. The high impurity profile originating from low plasmid copy number impeded the performance of membrane and column chromatography. To address these challenges, process development was carried out to both upstream and downstream processing. Media optimization was able to double the specific plasmid yield from 0.25 g/kg cells to 0.5 g/kg cells in the simple batch mode. Improving the homogeneity of the cell lysate ensured consistency and notably higher recovery at a 1000 L scale. Greater than 70% RNA impurity was removed via salt precipitation and additional column guard pre-filtration, which enhanced column resolution and reduced processing time. These process improvements were effectively implemented to cGMP manufacture. In the end, 50 grams of plasmid DNA at clinical grade was successfully delivered on time for Cystic Fibrosis Gene Therapy Clinical Trials.