Handbook of Size Exclusion Chromatography and Related ...

The recovery of DNA fragments has been reported to be almost quantitative
(10,11).
4 PLASMIDS
Recently, there has been an increasing interest in the purification of plasmids for
use as vectors in gene therapy. Plasmid-mediated gene delivery systems, in which
plasmids are injected directly, should be a good alternative to viral-mediated gene
delivery systems, due to the potential safety and simple delivery of the gene. The
use of plasmids in clinical trials requires the reproducible and scalable production
process of highly purified plasmids to meet regulatory criteria for manufacturing
of biopharmaceuticals. The purification of plasmids has been traditionally
performed by extraction with toxic reagents and CsCl gradient centrifugation. The
purification process using SEC, however, would eliminate these undesirable
reagents for the clinical use of plasmids.
SEC has been applied to the purification of various forms of plasmids. It is
possible to obtain plasmid free of proteins, RNA, and chromosomal DNA from
cleared lysate of Escherichia coli cells. Figure 6 shows an example of the
purification of plasmid. Cleared lysate of E. coli cells containing amplified
Figure 6 Separation of cleared lysate of E. coli cells (A) and its phenol extract (B)
obtained on a TSKgel G6000PW two-column system (each column 60 cm 7.5 mm ID) in
0.1 M Tri–HCl buffer (pH 7.5) containing 0.3 M sodium chloride and 1 mM EDTA at a flow
rate of 1 mL/min. (From Ref. 21.)
© 2004 by Marcel Dekker, Inc.