Mechanical disruption of bacterial cells for plasmid recovery

Details Mechanical disruption of bacterial cells for plasmid recovery Mechanical disruption of bacterial cells for plasmid recovery

1995-02-23

2002-09-24

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Cell membrane or cell surface is target

C435S173100, C435S173400, C435S259000

Reexamination Certificate

active

06455287

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods of cell disruption and plasmid extraction in the field of recombinant DNA technology. Specifically, it relates to mechanical methods of rupturing cells to release intact plasmids cloned within the cells.
BACKGROUND OF THE INVENTION
In the field of recombinant DNA technology, plasmid expression vectors are routinely employed to express foreign proteins. A number of recombinant proteins, including recombinant human insulin (HUMULIN®, Lilly), recombinant human erythropoietin (EPOGEN®, Amgen), recombinant tissue plasminogen activator (ACTIVASE®, Genentech), and recombinant &agr; interferon (ROFERON®, Roche), are now available for human pharmaceutical use, and commercial scale methods have been developed for recovery and purification of recombinant proteins from cell culture and/or microbial fermentation. For most of recombinant proteins produced in mammalian cell culture and for some recombinant proteins produced in microbes but secreted into the culture medium, cell disruption is not required for the recovery of these products. When cell disruption is required to release intracellular recombinant products from microbes, mechanical cell rupture methods are frequently used in such large recombinant protein recovery processes.
More recently, it has been shown that plasmid DNA may be useful as a non-viral nucleic acid delivery vehicle for clinical applications. (See, e.g., Wang et al.,
Proc. Nat'l Acad. Sci. USA
90:4156-4160 (1993); Ulmer et al.,
Science
259:1745-1749 (1993)). For such applications, which include gene therapy and genetic immunization, the plasmids themselves rather than the expressed proteins are the desired therapeutic product. Accordingly, there is a need for pharmaceutically acceptable large scale processes for recovery of intact plasmid DNA. For a number of reasons, mechanical cell disruption methods are preferred to chemical or enzymatical cell disruption methods if the yields are comparable.
Bacterial plasmids are double-stranded closed circular DNA molecules that range in size from about 1 kb to more than 200 kb. They are found in a variety of bacterial species, where they serve as accessary genetic units that replicate and are inherited independently of the bacterial chromosome. Plasmids can be produced via bacterial fermentation and recovered by cell disruption and plasmid recovery operations. Fermentation technology to produce plasmids is relatively well understood, and a number of laboratory scale methods useful for bacterial cultures ranging in size from 1 mL to 1 L have been developed to purify plasmid DNA from bacteria. (See Sambrook, Fritsch and Maniatis, Section 1.21, “Extraction and Purification of Plasmid DNA”,
Molecular Cloning: A Laboratory Manual
, Second Edition, Cold Spring Harbor Laboratory Press (1989).) These methods involve the growth of the bacterial culture and replication of plasmid; harvesting and lysis of the bacteria; isolation and purification of plasmid DNA.
Following growth of the bacterial culture, bacteria are normally recovered by centrifugation and lysed by one of a number of methods, including treatment with enzymes, nonionic or ionic detergents, organic solvents, alkali, or heat. The choice of lytic method is influenced by factors such as the size of the plasmid, the strain of bacteria used, and methods to be used subsequently to purify the plasmid DNA. Although well suited for small scale processes, enzymatic or chemical lysis are rather expensive. Chemical lysis also limits the choice of the downstream processing techniques used subsequently to purify the plasmid DNA. Enzymatic lysis frequently uses animal-derived enzymes such as lysozyme, which maybe accompanied with animal virus. Significant efforts to validate the removal of any possible viral contaminations are needed for this situation.
The currently published laboratory methods are in general unsatisfactory for large scale plasmid purification processes. Laboratory methods for isolation and purification of plasmids from bacterial culture frequently use dangerous organic solvents and chemicals such as cesium chloride and ethidium bromide, which are in general unacceptable for human pharmaceutical use. The few studies related to large scale plasmid recovery that have been reported, (See Chandra, G. et al.,
Analytical Biochemistry
203:169-172 (1992); Chakrabarti, A. et al.,
Biotechnology and Applied Biochemistry
16:211-215 (1992)), use chemical methods of cell lysis, i.e., alkaline-SDS lysis. However, SDS can cause significant problems in downstream purification.
SUMMARY OF THE INVENTION
In general laboratory scale plasmid purification methods were developed for gene cloning purposes, in which case, bacterial genomic DNA and tRNA or rRNA impurities as well as damaged plasmid are relatively unimportant. In contrast, plasmid DNA for pharmaceutical use must meet extremely high standards of identity and purity; this necessitates stringent limits on nucleic acid and protein impurities. Non-plasmid DNA, RNA, as well as plasmid DNA fragments may need to be removed in the downstream purification process. As intact plasmid DNA is ordinarily separable from both the larger intact host cell genomic DNA and from smaller cellular RNAs and DNAs on the basis of size and chemistry, it is important to avoid shearing either the plasmid DNA or the genomic DNA of the host organism. Linearized plasmid DNA and genomic DNA fragments similar in size to the intact plasmid product may be particular difficult to remove.
Bacterial plasmids for clinical applications typically contain large segments of product DNA (mammalian DNA for gene therapy applications or pathogen DNA for genetic immunization), as well as the expression vectors themselves, which contain the genes for selection in bacteria, the sequences for replication in bacteria, and the regulatory elements for expression in mammalian cells. Such plasmid molecules tend to be large, on the order of 10
6
-10
7
Daltons (5-20 kb), which is approximately two to three orders of magnitude larger than typical recombinant protein products (e.g., human growth hormone at 10
4
Dalton). Large plasmids (greater than 10 kb size) are particularly susceptible to damage, especially by physical forces that might be necessary to release the plasmid from the interior of the cell.
Although mechanical methods of cell disruption would be more economical and easier to carry out, and therefore preferred to enzymatic or chemical cell disruption methods for large scale processes, it is recognized that mechanical methods may damage DNA at the same time as the cells are broken. (Wheelwright, S. M.,
Protein Putification:Design and Scale up of Downstream Processing
, Oxford University Press (1991), in Chapter 6: Cell Disruption, p. 63. This presents a significant potential problem for pharmaceutical use, where intact, functional plasmid DNA is required and supercoiled plasmid DNA is preferred. Plasmid DNA which has been “nicked” but not cut through both strands, loses it supercoiled configuration and becomes “relaxed” circular DNA. Supercoiled plasmid DNA, which is smaller and more compact than relaxed closed circular plasmid DNA and less vulnerable to enzymatic degradation, expresses better than either relaxed circular or linear DNA. Although supercoiled plasmid DNA is preferred, both supercoiled and relaxed circular plasmid DNA are likely to express the gene of interest and are considered “intact” plasmid DNA. Although plasmid linearized using a selected restriction enzyme may constitute a functional expression unit, mechanical forces are likely to cut or break plasmid in a random manner. Randomly linearized plasmid DNA and broken or fragmented plasmid DNA are considered damaged and are likely to be ineffective or nonfunctional for pharmaceutical purposes. Not only is such damaged plasmid DNA ineffective, it will probably need to be removed in downstream processing to achieve a higher standard of purity. To permit recovery of intact plasmid DNA, processing conditions must be very mild, particularly

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