Novel Chromatography Matrices for Fast Isolation of Plasmid DNA

Detta är en avhandling från Division of Pure and Applied Biochemistry, Lund University, Sweden

Sammanfattning: As new therapeutic areas in which genetic material is employed, i.e. gene therapy and DNA vaccination, are expanding, a substantial increase in the demand for gene vectors, e.g. plasmid DNA, is foreseen. Chromatography, being a high-resolution method, is considered essential for the purification of gene vectors when high purity is required. Plasmids are large molecules, which cannot penetrate the narrow pores in most contemporary chromatography matrices. Thus, new chromatography matrices are required. The development of three novel support materials is described in this thesis. First, an anion-exchange material based on fibres is described. The small diameter of the fibres provides the material with a plasmid-binding capacity of about 1 mg/ml bed volume. It was shown that with this material, the columns can be operated with maintained adsorption properties at flow rates of up to 1800 cm/h, equivalent to 0.5 column volumes per second. Second, the development of a superporous anion-exchange material is described. The walls of the superpores provide additional surface area for plasmid binding, thus increasing the plasmid-binding capacity of the adsorbent. Significantly, the superporous agarose beads are shown to have 4-5 times higher binding capacity (3-4 mg/ml bed volume) than corresponding homogeneous agarose beads. Third, the development/design of a RVC-Cytopore composite material is described. Cytopore is an easily compressible, macroporous, cellulose-based anion-exchange material. Reticulated vitreous carbon (RVC) is a strong, non-compressible matrix material. The composite combines advantageous properties of both these materials, making it suitable for chromatography. The composite was shown to withstand very high flow rates. The dynamic binding capacities (10% breakthrough) were determined for several compounds, including DNA and RNA, and were found to be 13-17 mg/ml bed volume. When developing these novel plasmid-binding materials, important insights were gained regarding the recovery of the adsorbed plasmids. Based on these findings, the following factors were identified as being important for the recovery of DNA from the matrices: i) the type of amine employed as anion-exchange ligands (primary amines bind most strongly), ii) the nucleic acid structure (plasmid DNA may bind more strongly than genomic DNA), iii) the influence of charge on the pKa value of the ligands (the pKa was seen to increase substantially in the presence of plasmids), and iv) the solid support itself (steric factors can lead to kinetically stable complexes).