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.
Gene therapy and DNA immunization are becoming important alternatives for developing successful preventive and therapeutic
treatments for many diseases. With thousands of people now receiving plasmid DNA (pDNA), it therefore must be produced by
scalable manufacturing processes that meet stringent quality criteria in terms of purity, potency, efficacy, and safety. Plasmids
are circular, double-stranded molecules that comprise approximately 1% of the total content of the host bacterial cell. They
are normally isolated by an alkaline procedure, designed to disrupt the host cells and denature proteins and chromosomal DNA
(chDNA), while preserving plasmid's structural integrity.1 Although commonly used in laboratories, pDNA isolation methods that use elements such as organic reagents, mutagenic and
toxic compounds, and animal-derived enzymes, are an additional concern for regulatory agencies, and therefore, must be avoided.2,3 Another challenge in purifying plasmids is eliminating contaminant cellular components from the host, generally E. coli, which induce immunological and biological responses. RNA removal presents a challenge in producing genetic therapeutics
because of the similarity in chemical composition and structure with pDNA and its high abundance in crude plasmid preparation.
This article describes a new pDNA purification method that uses a nonenzymatic approach to RNA removal based on size-exclusion
chromatography on Sepharose CL 4B, using a buffer containing 1.5 M (NH4)2SO4. The pDNA was pooled and directly applied on the POROS R1 50 media, which is a rapid alternative to conventional chromatography.
For the last purification step, another size-exclusion chromatography method was chosen, because it can achieve two objectives
in one operation—further purification and buffer exchange. Following these steps, the immunogenicity of the plasmid obtained
was evaluated. Functionality of purified plasmids was confirmed in vivo; all immunized animals developed anti-HCV antibodies.
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.
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