A novel purification method was developed for recovering the pIDKE2 plasmid, which encodes a polyprotein encompassing amino acids 1–650 of the hepatitis C virus (HCV) polyprotein, from recombinant Escherichia coli. Bacterial cells were harvested and subjected to alkaline lysis. After centrifugation, the host contaminant RNA was removed from the clarified alkaline lysate using a highly loaded size-exclusion chromatography and the eluted fraction was applied to reverse-phase media: POROS R1 50. Finally, a second size-exclusion chromatography step was carried out to purify the plasmid DNA from other small molecular-weight contaminants. Analytical methods proved that the purified plasmid DNA had a purity of 95% after Sephacryl S1000. Plasmid identity was confirmed by restriction enzyme digestion. Biological activity of the purified plasmid was confirmed in vivo; immunized mice developed a positive antibody response against all HCV structural antigens. This procedure offers an alternative to traditional methods that use organic reagents, mutagenic and toxic compounds, and animal-derived enzymes. Although the yields are lower when using this method, it is scalable and free of animal-derived substances and organic solvents.
The final process has proved to be generally applicable and can be used from early clinical phases to market supply. Although the yields are lower, this method is scalable and free of animal-derived substances and organic solvents.
MATERIALS AND METHODS
Chemicals were purchased from Merck (Whitehouse Station, NJ) and Sigma (St. Louis, MO). Ultrafiltration equipment and membranes were provided by Sartorius (Goettingen, Germany). Design Expert Version 6.06 (DX6) software was obtained from Stat-Ease, Inc. (Minneapolis, MN).
Recombinant Co.120 and E1.340 were obtained from recombinant E. coli with >85% purity, by a combination of washed-pellet procedures and gel-filtration chromatography.4,5 Co.120 comprises the first 120 aa of the HCV nucleocapsid protein. E1.340 encompasses aa 192–340 in the viral polyprotein. E2.680 comprises aa 384–680 of the HCV polyprotein and is obtained from recombinant Pichia pastoris, also by a combination of washed-pellet procedures and gel-filtration chromatography.6