Method of plasmid DNA production and purification

Details Method of plasmid DNA production and purification Method of plasmid DNA production and purification

1999-06-10

2003-01-07

Benzion, Gary (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S384000, C435S404000, C424S124000, C536S026500, C536S026500, C530S417000

Reexamination Certificate

active

06503738

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the production of highly purified plasmid DNA, and in particular to production and isolation of pharmaceutical grade plasmid DNA for use in gene therapy.
BACKGROUND OF THE INVENTION
A variety of methods are available for isolating and purifying plasmid DNA. In general, these methods take advantage of the physical differences between chromosomal DNA and plasmid DNA. In terms of size, chromosomal DNA is larger than plasmid DNA. When cells are lysed, the larger chromosomal DNA becomes linearized and entangled in the cellular debris and may be separated from the cell lysate.
Prior art methods of isolating and purifying plasmid DNA include lysis by boiling (Holmes and Quigley, Anal. Biochem. 114, 193 (1981)), lysis with alkali (Birnboim and Doly, Nuc. Acids Res. 7, 1513 (1979)), and lysis with detergent (Godson and Vapnek, Biochem. Biophys. Acta 299, 516 (1973)). PCT publication no. 95/21250 discloses a method of isolating plasmid DNA using detergent in combination with alkali treatment. Prior art methods also use highly toxic chemicals to extract and isolate the plasmid DNA, such as ethidium bromide, cesium chloride, phenol, and chloroform. Moreover, these methods are most effective with smaller plasmids; e.g. plasmids less than approximately 8-10 kb. As plasmid size increases, plasmid DNA isolation becomes more difficult using the existing prior art methods.
In general, methods that employ alkali quickly add a standard amount of sodium hydroxide to the cellular suspension (Birnboim and Doly, supra). The pH of the resulting solution rises rapidly, in some cases, to over 13, which results in much degradation of the plasmid DNA. In addition, the concentration of the cells in suspension is dilute (i.e., of the order of an optical density (OD) of 1-3 units at a wavelength 600 nm for a 1 cm light path) to maximize recovery of plasmid DNk
An object of the invention is to provide a method of preparing pharmaceutical grade DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host RNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bactenal host protein.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host chromosomal DNA.
Another object of the invention is to provide a plasmid DNA preparation which is substantially free of bacterial host endotoxins.
Another object of the invention is to provide a method of isolating and purifying relatively large plasmid DNAs.
Another object of the invention is to provide a scalable method of isolating large amounts of plasmid DNA in sufficiently pure form for use in gene therapy.
Another object of the invention is to maximize the yield of plasmid DNA from a host cell/plasmid DNA combination.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a method for producing and isolating highly purified plasmid DNA. The plasmid DNA produced and isolated by the method of the invention contains very low levels of contaminating chromosomal DNA, RNA, protein, and endotoxins. The plasmid DNA produced according to the invention is of sufficient purity for use in vivo or ex vivo gene therapy.
Thus, the invention encompasses a process for producing and isolating highly purified plasmid DNA that includes the step of alkaline lysis in which the pH of the solution is monitored and raised in a controlled manner such that it does not rise above a predetermined pH value which is equal to 0.1 pH units below the irreversible alkaline denaturation value of the plasmid DNA.
Preferably, the predetermined pH value is equal to 0.2 pH units below the irreversible alkaline denaturation value of the plasmid DNA which is being isolated.
The predetermined pH value is within the range of 8.0 to 14.0 and is preferably within the range of about 81.0 to 13.1 and most preferably about 12.1 to 12.9.
The controlled rise in pH according to the method of the invention results in exceedingly low degradation of plasmid DNA, and thus permits higher yield of plasmid DNA.
As used herein, the terms “denature”, “denatured DNA” and “denaturation” are defined as conformations of DNA in which the hydrogen bonds between strands of double stranded DNA are ruptured. The term “irreversible alkaline denaturation value” is defined as the pH value at which no more than about 50% of the alkaline denatured plasmid DNA fails to renature as determined by standard agarose gel electrophoresis. Determination of the irreversible alkaline denaturation value is described hereinbelow.
According to the invention, the alkaline lysis step is performed on cells harvested from a fermentation which has been grown to a biomass of cells that have not yet reached stationary phase, and are thus in exponential growth, about 2-10 g dry weight/liter.
In a preferred embodiment, the alkaline lysis step is performed on cells harvested from a fermentation which has been grown to a high biomass of cells that have not yet reached stationary phase and are thus in exponential growth.
As used herein, a “high biomass” or “high density” is defined as a cellular concentration of approximately 10-200 g dry weight per liter, and preferably 12-60 g dry weight per liter. As used herein, the term “exponential growth” refers to that portion of the cellular growth cycle between the lag phase and the stationary phase when cells are doubling at a logarithmic rate.
The term “exponential growth” is also meant to encompass the late lag phase (i.e., the early stationary phase) which occurs between the logarithmic growth phase and stationary phase, when the cell growth rate is slowing, and therefor encompasses an extended exponential growth phase. Therefore, “stationary phase” refers to horizontal growth, i.e., when the cells have essentially stopped dividing and have reached a quiescent stage with respect to cell doubling. According to the invention, the combination of controlled pH increase and lysis at high cell density from cells harvested during exponential growth produces a high yield of intact and highly pure plasmid DNA from a single batch of cells.
The invention also encompasses a method for determining the optimum lysis conditions for lysing host cells containing plasmid DNA, comprising the steps of a) growing a culture of bacterial host cells to a cell density within the range of about 12 g to about 60 g per liter dry weight units; b) lysing the bacterial cells during exponential growth at a pH of said culture sufficient to cause cell lysis and to cause denaturation of no greater than 50% of plasmid DNA contained in said cells; and c) selecting a pH value for optimum lysis conditions which is about 0.1 pH units below the pH of step b).
Preferably, the lysing step b) is performed at a pH sufficient to cause denaturation of no greater than 90-95% of plasmid DNA, and the pH selected in step c) is about 0. 17-2.0 pH units below step b) pH.
Determining an optimum lysis pH or an optimum sodium hydroxide concentration for cell lysis according to the invention permits a longer time period during which cell lysis may occur, which in turn allows for a) destruction of larger amounts of undesirable endotoxin which may be present in the plasmid DNA preparation, b) denaturation of larger amounts of chromosomal DNA, and c) precipitation of larger amounts of chromosomal DNA, without concommitant loss of plasmid DNA in terms of quantity or loss of high quality plasmid DNA produced according to the invention.
The invention also thus encompasses a fermentation process which maximnizes yield of plasmid DNAs from large scale cultures of transformed host cells. The fermentation process includes controlling the growth rate such that the supply of metabolites essential for growth is adequate to permit growth to a high biomass, but is not in excess so as to inhibit such growth. It is critical to this aspect of the invention that growth is not reduced by supplying inhibitory concentrations of metabolites and catabolites. However,

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