Purification of plasmid DNA

Details Purification of plasmid DNA Purification of plasmid DNA

2000-07-21

2001-11-06

Ketter, James (Department: 1636)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100

Reexamination Certificate

active

06313285

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for purification of plasmid DNA. More specifically, a method is provided that is simple and scalable and utilizes tangential flow filtration, resulting in higher yields of highly pure plasmid than the classical alkaline-lysis-based method.
2. Description of Related Disclosures
Purification of plasmid DNA from cell cultures is a prerequisite for many studies and pharmaceutical uses. In general, plasmid purification methods may be considered as two-stage processes involving an initial cell disruption/plasmid isolation step followed by one or more subsequent purification steps. The most common methods for initial isolation are modified versions of two approaches: one based on release of plasmid by boiling (Holmes and Quigley,
Anal. Biochem
., 114: 193-197 (1981)) and the second based on alkaline pH and detergent-mediated solubilization of the bacterial cell membranes (Birnboim and Doly,
Nucleic Acids Res
., 7: 1513-1523 (1979)). Both of these methods result in the release of plasmid DNA from its cytosolic location.
In addition to the common use of ultracentrifugation through cesium chloride gradients (Clewell and Helinski,
Proc. Natl. Acad. Sci. USA
, 62: 1159-1166 (1969)), downstream purification has typically involved either selective precipitation of plasmid from contaminants (Lis and Schleif,
Nucleic Acids Res
., 2: 383-389 (1975); Ishaq et al.,
Biotechniques
, 9: 19-24 (1990); Kondo et al.,
Anal. Biochem
., 198: 30-35 (1991); Chakrabarti et al.,
Biotech. Appl. Biochem
., 16: 211-215 (1992)) and/or the use of column chromatography (Horn et al.,
Human Gene Ther
., 6: 565-573 (1995); U.S. Pat. No. 5,707,812; Chandra et al.,
Anal. Biochem
., 203: 169-172 (1992); Marquet et al.,
BioPharm
: 26-37 (1995); Johnson and Ilan,
Anal. Biochem
., 132: 20-25 (1983); Vincent and Goldstein,
Anal. Biochem
., 110: 123-127 (1981)). Column chromatography protocols rely on reverse-phase (Edwardson et al.,
Anal. Biochem
., 152: 215-220 (1986); Johnson et al.,
Biotechniques
, 4: 64-70 (1986); van Helden and Hoal in
New Nucleic Acid TechniQues
, Walker, Ed. (Humana Press: Clifton, N.J. 1988), pp. 69-74)), normal-phase (Marko et al.,
Anal. Biochem
., 121: 382-387 (1982)), ion-exchange (Perbal in A
Practical Guide to Molecular Cloning
(Wiley: New York, 1984), pp. 165-175; Colman et al.,
Eur. J. Biochem
., 91: 303-310 (1978); Garon and Petersen,
Gene Anal. Tech
., 4: 5-8 (1987); Kim and Rha,
Biotech. Bioeng
., 33: 1205-1209 (1989); Ohmiya et al.,
Anal. Biochem
., 189: 126-130 (1990)), size-exclusion (van Helden and Hoal, supra; Perbal, supra; Cornelis et al.,
Plasmid
, 5: 221-223 (1981), Micard et al.,
Anal. Biochem
., 148: 121-126 (1985); Moreau et al.,
Anal. Biochem
., 166: 188-193 (1987); Raymond et al.,
Anal. Biochem
., 173: 125-133 (1988); Hansen and Rickett,
Anal. Biochem
., 179: 167-170 (1989)), and mixed-mode (Flanagan et al.,
Anal. Biochem
., 153: 299-304 (1986)) methodologies.
Alternatives to these approaches include the use of 0.2-micron membranes as a substitute for a centrifugation step during alkaline lysis in a 96-well plate format (Ruppert et al.,
Anal. Biochem
., 230: 130-134 (1995)), the use of aqueous two-phase separation (Cole,
Biotechniques
, 11: 18-24 (1991)), and the use of ion-exchange membranes (van Huynh et al.,
Anal. Biochem
., 211: 61-65 (1993)) for plasmid purification. Typically, these methods have required additional purification steps involving either organic solvent-based extraction (e.g., phenol/chloroform) or precipitation (e.g., isopropanol, ethanol) steps, as well as the addition of exogenous enzymes (e.g., RNase, Proteinase K) to produce plasmid of adequate purity.
Additional techniques for plasmid DNA purification involve polyethylene-glycol-based DNA purification methods (Lis and Schleif, supra; U.S. Pat. No. 5,707,812 wherein a short-chain polymeric alcohol is added to the lysate so that the lysate will bind to the column or membrane used for purification); acid-phenol purification of plasmid DNA (Zasloff et al.,
Nucleic Acids Res
., 5: 1139-1153 (1978)); and different methods for relatively small-scale purification of plasmid DNA for research use (Sambrook et al.,
Molecular Cloning: A Laboratory Manual
, 2
nd
ed. (Cold Spring Harbor Laboratory Press: New York, 1989); Ausubel et al., eds.,
Current Protocols in Molecular Biolocy
, (John Wiley & Sons: New York, 1989)). Techniques for DNA-RNA separations are reviewed in Roman and Brown,
J. Chromatogr
., 592: 3-12 (1992).
Tangential flow filtration (TFF), or cross-flow filtration, is a separation technique whereby flow is directed across the membrane surface in a sweeping motion (Gabler,
ASM News
, 50: 299 (1984)). This sweeping action helps to keep material retained by the membrane from creating a layer on the filter surface, a condition known as concentration polarization. TFF is used to concentrate and/or desalt solutions retained by the membrane (retentate) or to collect material passing through the membrane (filtrate). Materials smaller than the pore size (or nominal-molecular-weight cutoff (NMWC)) are able to pass through the membrane and may be depyrogenated, clarified, or separated from higher-molecular-weight or larger species. Materials larger than the pore size or NMWC are retained by the membrane and are concentrated, washed, or separated from the low-molecular-weight species. The principles, theory, and devices used for TFF are described in Michaels et al., “Tangential Flow Filtration” in
Separations Technology, Pharmaceutical and Biotechnology Applications
(W. P. Olson, ed., Interpharm Press, Inc., Buffalo Grove, Ill. 1995). See also U.S. Pat. Nos. 5,256,294 and 5,490,937 for a description of high-performance tangential flow filtration (HP-TFF), which represents an improvement to TFF; and WO 87/04169 for a description of tangential flow affinity ultrafiltration, which involves mixing the solution to be purified with an affinity gel that selectively binds to the substance to be purified and then subjecting the liquid to TFF so that all components except the bonded material pass through the filter.
Additional methods for purification of viruses, nucleic acid, bacteriophage, and other biological materials using physical separation such as TFF or other cross-flow filtration techniques are set forth in various publications (Richards and Goldmintz,
J. Virol. Methods
, 4: 147-153 (1982); Fernandez et al.,
Acta Biotechnol
., 12: 49-56 (1992); Matsushita et al.,
Kagaku Kogaku Ronbunshu
, 20: 725-728 (1994); Rembhotkar and Khatri,
Anal. Biochem
., 176:373-374 (1989); WO 98/05673 published Feb. 12, 1998; EP 307,373; Sekhar et al.,
Hum. Gene Ther
., 7: 33-38 (1996)).
With the increasing utilization of plasmid DNA as biopharmaceuticals in gene therapy applications rather than as a cloning vector, a growing need exists for simple, robust, and scalable purification processes that can be used in the isolation of both intermediate and large amounts of this molecule from transformed prokaryotes. The use of plasmid purification methods that are currently available for the purpose of generating large amounts of research material, or for supplying a clinical trial, is limited for many reasons. Purification schemes that involve the use of large amounts of flammable organic solvents (e.g., ethanol and isopropanol), toxic chemicals (e.g., ethidium bromide, phenol, and chloroform). Ultracentrifuges and “spin-columns,” while adequate for the generation of small amounts of research material, are not suitable for use in generating the quantities of material needed for biopharmaceutical applications.
In addition, many current plasmid purification procedures involve the addition of RNase, typically from bovine origin. Materials derived from bovine sources are increasingly undesirable in the manufacture of pharmaceuticals due to concerns regarding bovine spongiform encephalopathies (BSE) (Hill et al.,
Nature
, 389: 448-450 (1997)). In general, it is desirable to avoid the addition of enzymes

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