A production method has been developed for the purification of
pharmaceutical-grade plasmid
DNA for in vivo gene therapy. This method has been applied to the purification of
VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the
HLA-B7 protein. Purified
VCL-1005 is formulated with a cationic
lipid and injected directly into established
tumors of HLA-B7-negative patients with advanced
cancers to heighten the patient's immune response against the
cancer. The purification of
pharmaceutical-grade plasmid
DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of
recombinant protein pharmaceuticals. Defined
pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing
DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived
enzymes. The overall process yield of purified plasmid
DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli
DNA levels are reproducibly below 1% as measured by Southern analysis.
Endotoxin levels are less than 0.03
endotoxin units/micrograms plasmid
DNA and residual
protein is undetectable. This process was used to produce 100 mg of
VCL-1005 for use in an active clinical protocol.