Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography - A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography. - Bio

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Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography
A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography.


BioPharm International


ABSTRACT

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.


Avecia
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

Materials

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 Proteins

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


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