Cancer Gene Therapy Using Plasmid DNA: Purification of DNA for Human Clinical Trials

Abstract

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/μg 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. A scaleable method for the production of highly purified plasmid DNA has been developed. The method takes into consideration all applicable regulations and guidelines that apply to the manufacture of an injectable drug substance. The unit operations are simple and reliable and can be used to generate high-purity plasmid DNA. The product is free of host Escherichia coli RNA. Contaminating E. coli DNA and endotoxins are reduced to trace levels without the use of toxic extraction techniques or animal-derived enzymes. The unit operations are an alkaline-mediated cell lysis, 2-propanol precipitation, ammonium acetate precipitation, PEG-8000 precipitation, column chromatography, and final aseptic processing to produce a sterile drug substance.