For the treatment of
melanoma DNA vaccines are a promising therapeutic approach. In our institute a plasmid encoding a
melanoma-associated
epitope (MART-1) and an immunostimulatory sequence (
tetanus toxin fragment-c) termed pDERMATT was developed. In a phase I study the plasmid will be administered intradermally using a newly developed
tattoo strategy to assess the toxicity and efficacy of inducing
tumor-specific T-cell immunity. To facilitate this study a Good Manufacturing Practice (GMP)-compliant plasmid manufacturing process was set up and a
pharmaceutical dosage form was developed. Each batch resulted in approximately 200mg plasmid
DNA of a high purity >90%
supercoiled DNA, an A260/280 ratio 1.80-1.95, undetectable or extremely low residual
endotoxins, Escherichia coli host cell
protein,
RNA, and
DNA. In the manufacturing process no animal derived
enzymes like
RNase or potentially harmful organic
solvents are used. After sterile filtration the concentration of the plasmid
solution is approximately 1.1mg/mL. For the scheduled phase I study a concentration of 5mg/mL is desired, and further concentration of the
solution is achieved by lyophilisation. The formulation
solution is composed of 1mg/mL pDERMATT and 20mg/mL
sucrose in Water for
Injections. Upon reconstitution with a five times smaller volume an isotonic
sucrose solution containing 5mg/mL pDERMATT is obtained. Lyophilised pDERMATT is sterile with >90%
supercoiled DNA, an A260-280 ratio 1.80-1.95, content 90-110% of labeled, and residual water content <2% (w/w). The product yields the predicted profile upon restriction-
enzyme digestion, is highly immunogenic as confirmed in an in vivo mouse model, and stable for at least six months at 5 degrees C. We have not only developed a reproducible process to manufacture
pharmaceutical grade plasmid
DNA but also a stable
dosage form for the use in clinical trials.