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  Fed-batch fermentation process and culture medium for the production of plasmid dna in e. coli on a manufacturing scaleUSPTO Application #: 20050233421Title: Fed-batch fermentation process and culture medium for the production of plasmid dna in e. coli on a manufacturing scale Abstract: A process for producing plasmid DNA E. coli cells comprises a pre-culture and fed-batch process. The culture media of the batch phase and the culture medium added during the feeding phase are chemically defined. The culture medium of the feeding phase contains a growth-limiting substrate and is added, for at least a fraction of the feeding phase, at a feeding rate that follows a pre-defined exponential function, thereby controlling the specific growth rate at a pre-defined value. The process results in high yield and homogeneity of plasmid DNA. (end of abstract)
Agent: Michael P. Morris Boehringer Ingelheim Corporation - Ridgefield, CT, US Inventors: Hans Huber, Gerhard Weigl, Wolfgang Buchinger USPTO Applicaton #: 20050233421 - Class: 435091100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycoside, , Nucleotide, Polynucleotide (e.g., Nucleic Acid, Oligonucleotide, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20050233421. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This Application claims priority benefit from U.S. Provisional 60/568,857, filed May 7, 2004 and from EP 04 008 556.5, filed Apr. 8, 2004 the contents of which are incorporated herein. FIELD OF THE INVENTION [0002] The invention relates to the fermentation of Escherichia coli for the production of plasmid DNA (pDNA), in particular for pDNA intended for the use in gene therapy and DNA vaccination. [0003] Introduction [0004] The requirement for industrial fermentation of pDNA came up by the clinical success of gene therapy and DNA vaccination during the last decade. [0005] Gene therapy is the treatment or prevention of disease by the administration, delivery and expression of genes in mammalian cells. The ultimate goal of gene therapy is to cure both inherited and acquired disorders by adding, correcting, or replacing genes. Basically, there are two types of gene therapy vectors to achieve these goals, i.e. viral vectors based on inactivated viruses and non-viral vectors based on plasmid DNA. The present invention relates to the production of non-viral plasmid DNA. [0006] Since it was demonstrated that intramuscular injection of pDNA encoding an antigen elicits both a humoral and a cellular immune response, naked plasmid DNA has become of particular importance. [0007] The desired efficiency of a fermentation process for manufacturing plasmid DNA is characterized by a high yield of pDNA, either per volume fermentation broth (volumetric yield) or per biomass aliquot (specific yield). In the meaning of the present invention, yield is the concentration of plasmid DNA per volume or cell weight. Beyond a high yield, the plasmid has to be present in its intact covalently closed circular (ccc) or supercoiled form. In the meaning of the invention, the percentage of ccc form is termed "plasmid homogeneity". The concentration of other plasmid forms such as open circular (oc), linear and dimeric or multimeric forms, should be reduced to a minimum in the purified plasmid bulk, and are consequently not desired during fermentation. [0008] Therapeutic plasmids consist of three essential parts, i.e. the therapeutic gene (the "gene of interest") under the control of a eukaryotic promoter, mostly the cytomegalovirus (CMV) promoter, an origin of replication (or) for the autonomous propagation in the prokaryotic cell, and a selection marker, usually an antibiotic resistance gene. While the therapeutic gene is of clinical and medicinal relevance, both the ori and the selection marker play a crucial role during plasmid production, especially during fermentation. For construction of a therapeutic plasmid, a key factor is the choice of an origin of replication that replicates to a high number of plasmid copies per cell. Most therapeutic vectors bear the ColE1-type ori. Plasmids having a ColE1 origin derived from pBR322 may reach copy numbers of 50-100 plasmids per cell, plasmids derived from pUC can reach copy numbers of several hundred. [0009] The antibiotic selection marker and the use of antibiotics are necessary during transformation and selection of plasmid harboring cells. However, antibiotic selection pressure should be avoided during industrial manufacturing. It is therefore desirable to develop fermentation processes allowing a stable propagation of the vector without plasmid loss. [0010] The choice of the bacterial host strain is another important factor to be considered for fermentation of pDNA. Desirable host phenotypes include those with the ability to grow to a high cell density, to achieve high plasmid copy numbers, to generate a minimum of plasmid-free cells, to have a minimum potential for genetic alterations of the plasmid, the production of plasmids being predominantly supercoiled, and the compatibility with common purification procedures. Most strains of E. coli can be used to propagate pDNA, although the strain may have an effect on the quantity and quality of the obtained pDNA (Schoenfeld et al., 1995). Currently there is no consensus on the genotypic or phenotypic characteristics that would be ideal for bacterial strains used for pDNA manufacture. Frequently, the strain DH5-alpha was used before for fermentation of pDNA. BACKGROUND OF THE INVENTION [0011] A number of approaches have been described for fermentation of pDNA. The proposed methods differ with regard to the level of control imposed upon the cells and the numerous factors that influence fermentation. Low-level control simply allows plasmid-bearing cells to grow, whereas high-level tightly-controlled fermentations reach high yields of pDNA by specific measures which enhance replication. [0012] For pDNA production on a laboratory scale, cultivation of plasmid-bearing cells in shake flasks is the simplest method, which however normally achieves low yields. Plasmid yields obtained from shake flask cultivations are in the range of 1.5 to 7 mg per L culture broth (O'Kennedy et al., 2003; Reinikainen et al., 1988; O'Kennedy et al., 2000). In shake flask cultivations, several drawbacks such as poor oxygen transfer and the lack of possibility for pH value control, limit the pDNA yield. In U.S. Pat. No. 6,255,099 it was shown that, even in shake flask cultivations, a pDNA yield of up to 109 mg/L can be achieved with certain medium compositions and buffering conditions. [0013] To obtain higher quantities of plasmids, it has been suggested to cultivate the cells in controlled fermenters. A simple fermentation method, in which all nutrients are provided from the beginning and in which no nutrients are added during cultivation, is termed "batch-cultivation" or "batch fermentation". The application of batch processes in controlled fermenters has led to an increase of pDNA yield per volume. Depending on the plasmid/host combination and on the culture medium, the yield of pDNA obtained from such batch fermentations can vary strongly. Typical plasmid yields reported are in the range between 3.5 and 50 mg/L (O'Kennedy et al., 2003; WO 96/40905; U.S. Pat. No. 5,487,986; WO 02/064752; Lahijani et al., 1996). These cultivations were carried out with culture media containing so-called "complex components" as carbon and nitrogen sources. These components are obtained from biological sources; they include e.g. yeast extract, soy peptone or casein hydrolysate. [0014] Culture media consisting exclusively or predominantly of complex components are termed "complex media". Media that are composed of both a defined portion (defined carbon source, salts, trace elements, vitamins) and a complex portion (nitrogen source), are termed "semi-defined" media. According to U.S. Pat. No. 5,487,986, a very high amount of various complex components (50 g/L in total) was used. [0015] Culture media containing complex components have the disadvantage that these components originate from biological materials; therefore, the composition of the medium underlies normal natural deviations that make the cultivation process less reproducible. The same applies when a manufacturer changes the production process or when there is a change of supplier. Further disadvantages of using complex medium components are the uncertainty about the exact composition (presence of undesired substances), the impossibility to do stoichiometric yield calculations, the formation of undesired products upon sterilization, difficult handling due to poor dissolution, formation of dust as well as clumping during medium preparation. During fermentation, complex media more readily tend to foaming. Complex components of animal origin (meat extracts, casein hydrolysates) are in particular undesired for pDNA production due to the risk of transmissible spongiform encephalopathy and their use is therefore restricted by pharmaceutical authorities (CBER 1998). [0016] Because of the drawbacks of complex medium components, media have been developed that do not contain any complex components. Such culture media, which are termed "defined" or "synthetic" media, are composed exclusively of chemically defined substances, i.e. carbon sources such as glucose or glycerol, salts, vitamins, and, in view of a possible strain auxotrophy, specific amino acids or other substances such as thiamine. Chemically defined media have the advantage that their composition is exactly known. This allows better process analysis, fermentation monitoring and the specific addition of particular substances which enhance growth or product formation. The well-known composition allows to set up mass balance calculations, which facilitate the prediction of growth and the identification of possibly lacking nutrients. Compared to complex media, fermentations with defined media show enhanced process consistency and improved results during scale-up. Further practical aspects of defined media are better solubility, the absence of inhibiting by-products upon sterilization, and less foam formation during cultivation (Zhang and Greasham, 1999). Synthetic media, that were not specifically developed for pDNA production, such as M9 (Sambrook and Russel, 2001), may result in a low pDNA yield (WO 02/064752). In batch fermentations with defined culture media that were specifically designed for pDNA production, a higher yield of pDNA was obtained (Wang et al., 2001; WO 02/064752). The latter demonstrated that pDNA homogeneity was more than 90% ccc form. The enhanced yields of pDNA according to WO 02/064752 and Wang et al. (2001) were achieved by supplementation of amino acids that are biosynthetic building blocks of nucleosides, or by the direct addition of nucleosides. [0017] Although batch fermentations are usually simple and short, they have fundamental disadvantages that result in limited plasmid DNA yields. This is due to substrate inhibition and salt precipitation at high nutrient concentrations in the batch medium. Furthermore, the growth rate in batch fermentations cannot be controlled directly; it is therefore unlimited, while steadily changing during fermentation, and ceases only when one or more nutrients are depleted or if metabolic by-products (such as acetate) inhibit growth of the cells. [0018] Consequently, in order to increase biomass and plasmid yield in pDNA production, fed-batch fermentations have been developed. A fed-batch fermentation is a process in which, after a batch phase, a feeding phase takes place in which one or more nutrients are supplied to the culture by feeding. [0019] Different strategies have been pursued for fed-batch fermentation of E. coli to produce plasmid DNA: [0020] One method is the application of a feed-back control algorithm by feeding nutrients in order to control a process parameter at a defined set point. Feed-back control is hence directly related to cell activities throughout fermentation. Control parameters which have been used for feed-back control of fermentations include pH value, on-line measured cell density or dissolved oxygen tension (DOT). These methods have the benefit that high biomass concentrations can be obtained with a reduced risk of overfeeding the culture with the fed nutrient. [0021] For pDNA fermentation, a feed-back algorithm for controlling the dissolved oxygen tension at a defined set point by the feeding rate was used (WO 99/61633). [0022] When applying another, more complex algorithm, both the DOT and the pH were used as control parameters for a feed-back cultivation method (U.S. Pat. No. 5,955,323; Chen et al., 1997). In that method, the DOT was controlled by the agitation rate and feeding of a concentrated complex medium (glucose, yeast extract), whereby the pH was concomitantly maintained with ammonium hydroxide. Continue reading... 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