Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-11-21
2003-12-16
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S089000, C435S252800, C435S259000, C435S243000
Reexamination Certificate
active
06664078
ABSTRACT:
The present invention relates to the production of biomass for the isolation of ccc plasmid DNA comprising culturing a bacterial transformant in a bioreactor containing an antibiotic-free batch medium under batch-conditions and, at the end of the batch phase, feeding under feed-back conditions a portion of a feed medium after the rise of DO above a threshold-set point. Said feed medium comprises besides a carbon and a nitrogen source, a magnesium salt, preferably in concentrations above 20 mM. Preferably, the bacterial transformant is harvested after the end of the culture and frozen or freeze-dried. Also preferred is that ccc plasmid DNA is, optionally directly, isolated after harvesting the bacteria.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
With the advent and progress of recombinant DNA technology into a variety of fields such as food stuff production and medical therapy, the desire for large quantities of highly pure DNA has constantly risen. Traditional methods of purifying genomic or plasmid DNA (see, e.g., Sambrook et al., “Molecular Cloning, A Laboratory Manual”, CSH Press, 2
nd
edition, 1989, Cold Spring Harbor N.Y.) usually require sophisticated methodology if the DNA is to be free from RNA and other contaminating organic compounds. In particular, methods for obtaining ccc plasmid DNA in pure form regularly suffer from the disadvantage that other plasmid topologies also produced have to be separated from the desired product. For example, Lahijani et al., Human Gene Therapy 7 (1996), 1971-1980 have reported that high yields of pBR322-derived plasmids intended for human gene therapy may be obtained when plasmids comprising a temperature-sensitive single-point mutation that affects the negative regulation of replication from the ColE1 origin of replication are employed. Using this process, a yield of 2.2 g of plasmid DNA from a 10 liter-fed batch fermentation were reported. However, the bacterial transformants were grown in the presence of the antibiotic kanamycin which would render them unsuitable for registration and subsequent use in humans. Other approaches have tried to avoid the use of antibiotics; see, e.g., Chen et al., J. Industrial Microbiology and Biotechnology 18 (1997), 43-48. In this report, an automated fed-batch fermentation with feed-back controls based on dissolved oxygen and pH for the production of supercoiled plasmid DNA is disclosed. This DNA is suggested to be useful for DNA vaccines. However, the results reported, for example in
FIG. 4
, do not support the suggested suitability of the plasmid DNA for vaccination purposes. This is due to the fact that besides ccc plasmid DNA a variety of other plasmid forms are produced under these conditions. Furthermore, this method leads to high contaminations with genomic DNA and, comparatively, only small plasmid amounts can be obtained.
Accordingly, ccc plasmid DNA produced by the prior art methods is unsuitable for a variety of purposes such as medical purposes due to the heterogeneity of the product obtained and/or due to the employment of antibiotics in the production process. The technical problem underlying the present invention was therefore to provide a method that overcomes these prior art difficulties and allows for the production of ccc DNA without the concomitant production of other plasmid forms and which is, moreover, suitable for medical purposes. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Thus, the present invention relates to a method for the production of biomass for the isolation of ccc plasmid DNA comprising
(a) culturing a bacterial transformant in a bioreactor containing an antibiotic-free batch-medium comprising
(aa) a carbon source;
(ab) an inorganic salt mixture;
(ac) a nitrogen source;
under batch-culturing conditions;
(b) feeding under feed-back conditions to said culture of (a) at the end of the batch phase, after rising of DO above a threshold-set point, a portion of a feed-medium comprising
(ba) a carbon source; and
(bb) a magnesium salt; and
(c) allowing the bacterial transformant to metabolize said feed-medium.
The term “biomass”, as used in the context of the present invention, relates to any biological material that is or arises from cells or organisms that are capable of reproduction.
The term “ccc plasmid DNA” refers to a plasmid isoform that is a circular plasmid which is typically but not necessarily underwound relative to a relaxed molecule. This results in a more compact conformity of the molecule which is described as a supercoiled covalently closed circle of the plasmid DNA. In
E.coli
cells, two enzymes regulate the supercoiling of DNA. The gyrase introduces negative superhelical turns into the molecule while the topoisomerase I relaxes the DNA by introducing single-strand breaks. It is most preferred in accordance with the method of the invention that ccc plasmid DNA in monomeric form is produced. Indeed, the method of the invention provides this particularly desired type of plasmid usually in an amount of more than 90% of overall plasmid production. Also useful, although less preferred, is the production of dimeric ccc plasmid DNA.
The term “batch-medium” refers to the medium used in batch cultivation, i.e., in discontinuous cultivation of bacterial transformants or other microorganisms. This discontinuous cultivation is characterized by a single inoculation into fresh medium (batch medium) at the start of the cultivation until nutrients and substrates have been exhausted.
The term “feed-back conditions” relates to the supplementation of medium concentrate during fed-batch cultivation depending on cultivation parameter(s) like, e.g., DO, pH, etc., which are correlated with the growth of the microorganism.
The term “threshold-set point” in the context of the present invention is intended to mean a defined value for a parameter that is monitored during fed-batch cultivation. In case of over-reaching or under-reaching of that value a monitor signal initiates the response in the regulation of the cultivation. The person skilled in the art is in the position to determine such defined values on the basis of his common knowledge and the teachings of this invention, see, for example, example 1.
The term “DO” (concentration of dissolved oxygen) refers hereby to the amount of oxygen in a liquid in per cent of the saturation concentration.
Under the above-defined feed-batch conditions, the concentration of dissolved oxygen (DO) rises during cultivation of bacterial transformants after the consumption of nutrients such as contained in the feed-medium. Therefore, the invention relates in a preferred embodiment to a method wherein the feeding of a bacterial transformant culture comprises repeated feeding-cycles after each rising of the DO above a threshold-set point. It is well known to the person skilled in the art that feeding can be controlled and/or measured via other control parameters, like medium pH, specific growth rate of bacterial transformants, respiration coefficients or others. Nevertheless, all these parameters are interrelated and indicative of the DO. Therefore, irrespective of which parameter is actually employed as a read-out system, it allows a direct (or indirect) conclusion on the DO value. Accordingly, measurement of any of said parameters is covered by the invention as long as it allows a conclusion with respect to the rising of DO above a threshold-set point.
The term “allowing the bacterial transformant to metabolize said feed-medium” relates to the partial or complete metabolization of said feed-medium and preferably to the essentially complete metabolization of said feed medium.
In accordance with the present invention it has been found that the here disclosed method leads to the production of high plasmid concent
Flaschel Erwin
Friehs Karl
Schleef Martin
Schmidt Torsten
Guzo David
Lambertson David A.
Qiagen GmbH
Wesolowski Michael R.
Yankwich Leon R.
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