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.
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
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 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.