The Purification of Plasmid DNA for Clinical Trials Using Membrane Chromatography - Membrane chromatography ensures purity at high flow rates. - BioPharm International

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The Purification of Plasmid DNA for Clinical Trials Using Membrane Chromatography
Membrane chromatography ensures purity at high flow rates.


BioPharm International
Volume 23, Issue 2

ABSTRACT

Membrane chromatography offers a good solution to the challenge of developing an efficient chromatographic procedure for plasmid DNA purification. The large convective pores of anion exchange membranes allow plasmid DNA to access all the anionic binding sites of the membrane at high flow rates. Here we demonstrate that the pIDKE2 plasmid can be purified from a recombinant Escherichia coli lysate using a Sartobind D membrane combined with size exclusion chromatography to render material with 95% purity and an average yield of 50%. This process yields therapeutically suitable plasmid DNA that meets all regulatory requirements.


(SARTORIUS AG)
Gene therapy and DNA immunization are important and promising possibilities to successfully develop preventive and therapeutic strategies for various diseases. Thousands of people have already received plasmid DNA (pDNA) without serious adverse effects. In the last decade, an increasing number of clinical trials for gene therapies and vaccines based on pDNA have reached the final phases. However, the amount of highly purified pDNA that will be required for these products, should they ultimately reach the market, has been largely underestimated. Commercial-scale processes for plasmid production must be able to manufacture grams or even several kilograms of purified pDNA per batch while meeting the quality standards required by the health authorities.

The chromatographic supports used in purifying such plasmids play a major role these processes, the development of which can be challenging.1 In particular, RNA removal is a challenge because its chemical composition and structure are so similar to those of pDNA, and because it is so abundant in crude plasmid preparations.2–4 Also, the large pDNA molecules adsorb only on the outer surface of the particulate supports, and consequently their capacity is very low—usually on the order of only hundreds of micrograms of plasmid per milliliter of chromatographic support.5

Here we describe a procedure for purifying the pIDKE2 plasmid, which encodes the hepatitis C virus (HCV) core, E1, and E2 structural proteins, using a Sartobind D membrane (Sartorius-Stedim Biotech, Goettingen, Germany) at high flow rates. The quality of the pDNA produced using this membrane-based purification process was demonstrated using the analytical methods recommended in relevant US FDA guidance.6 The procedure delivers the pIDKE2 plasmid with an average yield of 50% and with 95% purity. The biological activity of the purified plasmid was confirmed in vivo: vaccinated mice developed a positive antibody response against all HCV structural antigens—86.6% and 60% for the core and E2 proteins, respectively.

MATERIALS AND METHODS

Materials

All the reagents used to make the buffers were purchased from Merck (Darmstadt, Germany) and Sigma (St. Louis, MO). G25 coarse chromatography medium, Sepharose CL-4B, and Sephacryl S-1000 were purchased from Amersham BioSciences (GE Healthcare, Piscataway, NJ).

Recombinant proteins

Recombinant Co.1207 and E1.3408 were obtained from E. coli. Co.120 comprises the first 120 amino acids (aa) of the HCV nucleocapsid protein, whereas E1.340 encompasses aa 192–340 of the viral protein.9 E2.680 comprises aa 384–680 of the HCV polyprotein and is obtained from recombinant Pichia pastoris.

Production of pIDKE2

E. coli DH10B cells harboring pIDKE2, a plasmid for DNA immunization expressing the first 650 aa of the HCV polyprotein from the 1b-Cuban isolate genotype,10 were grown overnight, at 37 °C, in 100 mL shake flasks containing 50 μg kanamycin/mL, at 250 rpm. The engineered E. coli was cultured in a 5-L fermenter (Marubishi, Tokyo, Japan) using a complex media containing 50 g/L of yeast extract in fed-batch mode.


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