|Title of Invention||
"A PROCESS FOR THE PREPARATION OF PHARMACEUTICAL GRADE PLASMID DNA FOR THERAPEUTIC APPLICATION"
|Abstract||The present invention relates to a method of producing pharmaceutical grade plasmid DNA from cultured bacterial cells, unicellular organism or from any cells using a series of filtration process and comprises of a method which is free from the use of any enzymes, organic solvents, chromatographic operation or multiple ultracentrifugation steps for the production of clinical grade plasmid DNA and a process which is scalable and provides a favourable protocol for the purification of plasmid DNA for vaccination or therapeutic application.|
|Full Text||Field of the invention
The present invention relates to a method of producing pharmaceutical grade plasmid DNA from cultured bacterial cells, unicellular organism or from any cells using a series of filtration process. The method described herein does not use any enzymes, organic solvents, chromatographic operation or multiple ultracentrifugation steps for the production of clinical grade plasmid DNA. The process is scalable and provide a favorable protocol for the purification of plasmid DNA for vaccination or therapeutic application.
With the development and progress of DNA vaccine, production of plasmid DNA has become important in recent days. To make the DNA vaccine more suitable for developing countries and to provide them in the cheapest rate, it is essential to produce and purify them in large quantities. In general, the DNA vaccine or plasmids are produced from unicellular microorganism by growing them in suitable environment and purify the genetic material of interest. The current methods available are too cumbersome and costly for preparation of plasmid DNA vaccine. Most of them are difficult to scale-up for large scale vaccination process as multiple centrifugation process are involved to recover pure pharmaceutical grade plasmid DNA. It is also essential that the methodology to produce plasmid DNA should be applicable to operate at very large scale and should comply with good manufacturing practices as required by the regulatory authorities. It should be convenient and should produce pharmaceutical grade DNA in cheapest way. Major problems associated with plasmid DNA purification from microbial culture are protein, RNA and genomic DNA contamination along with presence of high level of endotoxin. Multiple steps using expensive reagents, high speed centrifuges are used to eliminate these contaminants to obtain pure plasmid preparation but at a very high cost. The operational cost increases very high with the increase in volume of the pure plasmid. Thus, there
is a need to develop a process for preparation of pharmaceutical grade plasmid DNA without using RNase and multiple centrifugation steps.
Prior Art Relating to the Invention
Rapid development of the role of plasmid DNA from a molecule primarily used as a cloning vector to that of a potential biopharmaceutical (gene therapy, DNA vaccine etc.), necessitates the development of simple, robust and scalable purification process for large scale application of plasmid DNA (1). DNA vaccines are already in human clinical trials at various stages e.g. malaria DNA vaccine, HIV DNA vaccine and HBsAg DNA vaccine (2-6). DNA vectors to be used for in-vivo purposes should be purified under the strict cGMP guidelines. Hence, it is of paramount importance to develop a cost effective and a user-friendly method for plasmid DNA purification. Although, numerous methods are available in literature (7-10) most of them are for bench scale level or unsafe when considered in the context of direct gene transfer into an animal. Purification schemes that involve the use of large amounts of toxic organic chemicals (e.g. phenol, chloroform and ethidium bromide), ultra-centrifuges, "spin columns", while adequate for the generation of small amounts of research material, are not suitable for generating the quantities and qualities needed for biopharmaceutical applications (11). In addition, many current plasmid purification procedures involve the addition of RNase, typically of bovine origin. Materials derived from bovine sources are increasingly undesirable in the manufacture of pharmaceuticals due to concerns regarding bovine spongiform encephalopathies (BSEs) (12).
In general, plasmid DNA is purified using standard alkaline hydrolysis method followed by alcohol precipitation (13-14). However for use of plasmid DNA as therapeutic agent there is a need for the development of efficient cost effective plasmid DNA purification process. More importantly the process should be scalable and should meet strictly the regulatory guidelines (10). It is also essential that the process of purification must result in absolutely pure plasmid
free of contaminating protein, RNA and endotoxins. In recent days new method of plasmid purification involving chromatography, salt precipitation and filtration membrane has been reported (1, 10). These methods involve use of ion exchange, (15, 16), and hydrophobic interaction chromatography (17). Purification protocols involving the use of chromatography columns are hampered by the low load capacities inherent to the operation. Plasmid purification based on triple helix interaction (18), ultra filtration (19, 20), precipitation methods (21) and by use of aqueous two phase partitioning (22) have also been developed. However most of these methods are not suitable for large volume processing and need either ultra-centrifugation steps or chromatographic steps for purification of plasmid DNA. QIAGEN / Novagen endofree giga and mega kits are commonly used in research laboratories for purifying in-vivo quality plasmid DNA but very large amount of plasmid to be purified and cost are the limitations with this kit. Thus there is a need to develop easy and scalable plasmid purification methods without involving extensive ultracentrifugation, chromatographic processes and finally RNAse free treatment process to reduce the overall cost of plasmid manufacturing. It also essential to show the method of plasmid purification results in low level of endotoxin which is accepted for human use. The present invention involves the method of plasmid DNA purification without using RNase, organic solvent and chromatographic based purification. It involves series of different filtration, ultra filtration and dia-filtration techniques to produce RNA and protein free plasmid DNA.
Objects of invention
The main object of invention is the development of a simple scaleable and cheap plasmid purification process to produce large amount of plasmid DNA suitable for therapeutic application including DNA vaccination. Plasmid DNA expressing any vaccine candidate or therapeutic protein of interest was grown in large quantities using a suitable bacterial host. Microbial cells were lysed to release the plasmid DNA of interest and were subsequently purified to homogeneity using series of filtration and dialysis process. The objective was to develop a process which
does not need expensive enzyme for cell lysis, centrifugation step for recovery as well as use of chromatographic separation steps. Depending on the size of the plasmid of interest, different filter membranes can be used to remove contaminating proteins, nucleic acids and other cellular contaminants. This purification method is scalable and can be used for large scale preparation of pure plasmid DNA for mass immunization. The method does not use enzymes, organic solvent based precipitation followed by ultracentrifugation and remove the endotoxin levels below the permissible limits for human uses. This method has been used to purify plasmid of different types. Purified plasmid form above mentioned method has all the characteristics preserved for transfection in animal cell.
Accordingly, the inventors have developed a process for purification of plasmid DNA for pharmaceutical application from cultured cells.
One object of invention is the purification of plasmid DNA expressing either a vaccine candidate or therapeutic protein from microbial cells mostly using series of filtration process. Initial separation of plasmid DNA from bacterial cell lysate is carried out is by coarse filtration followed by ultra-filtration. Purification of plasmid DNA is carried out by extensive dia-filtration using 1000 kDa molecular cutoff membrane filters. Final concentration of the pure plasmid is carried out by the use of 10 kDa molecular weight cutoff ultra-filtration membrane.
Another object of the invention is the development of a process for purification of plasmid DNA having high transfection ability in vitro.
Another object of invention is the development of a process for purification of plasmid DNA free of RNA contamination while not using RNase during the process.
Yet, another object of invention is the development of a process for purification of plasmid DNA having very low endotoxin contamination.
Another object of invention is the development of a process for preparation of plasmid DNA without using extensive centrifugation step or organic solvent based precipitation
Yet another object of invention is the purification of plasmid DNA using a series of steps which can be scaled-up easily for large scale production.
Description of the invention:
The inventor has developed a simple inexpensive process for isolation and purification of pharmaceutical grade plasmid DNA from microbial cells. Use of plasmid DNA for vaccination or therapeutic application needs huge amount of plasmid DNA to be prepared using an easy process for its large scale use. Plasmid DNA purification method described herein results in the production of high purity plasmid DNA with minimal endotoxin and no RNA or protein contamination. The method in principle use series of filtration process using membrane or filters having different filtration characteristics. The described method does not use ultracentrifugation, RNase, protinase K, organic solvent or column chromatography. Model eukaryotic expression vector pEGFP expressing green fluorescent protein was isolated from E. coli cells using this method. This vector was transformed in to host bacterial E. coli DHoc using CaC^ method. Cells were concentrated from the medium by use of ultrafiltration using 0.45 |j,m molecular weight cutoff membrane and the plasmid was isolated using alkaline lysis method. Alkaline lysis was carried out 50 mM Tris-HCI, 5 mM EDTA, at pH 8 using 0.2 N NaOH /1 % SDS. Cell lysis was carried out for 5 minutes at room temperature after which 3 M potassium acetate solution was added and kept at room temperature for 10 minutes. The lysis mixture was clarified using four layer of sterile cheese clothe. The supernatant containing plasmid DNA was further clarified by passing the solution through 0. 45 (am ultrafiltration membrane. The
clear filtrate was diafiltered using 1000 kDa molecular weight cut off ultra filtration membrane. Dia-filtration was carried out in two steps. In first stage, dia-filtration was carried out in pH 10, Tris-EDTA buffer where as in second stage, dia-filtration was carried out in pH 8, Tris-EDTA buffer. Finally the dia-filtration was carried out with sterile MQ water (endotoxin free). During dia-filtration using 1000 kDa ultra-filtration membrane, most of the protein and RNA contaminants were filtered through the membrane leaving behind the pure plasmid. Dialyzed material was concentrated using the same 1000 KD membrane using ultrafiltration mode. Final concentration of the plasmid DNA was carried out in 10 kDa ultra filtration membrane to remove water. Concentrated samples were directly used for analysis. A maximum of 2.5-3 mg of pure plasmid DNA could be purified from I L of LB grown E. coli culture. Endotoxin level in the plasmid preparation was around 30 EU/mg and there was no residual protein or RNA contamination with the final preparation.
Preparation of high purity plasmid DNA using series of filtration process will be very useful for large scale therapeutic application of plasmid DNA. Such Process can be used for the preparation of plasmid DNA suitable for vaccination purpose for many infectious diseases.
In an embodiment the invention describes the process of purification of plasmid DNA expressing either a vaccine candidate or therapeutic protein from cells using series of filtration process.
In an embodiment the invention describe the process of purification of pharmaceutical grade plasmid DNA where the initial separation of plasmid from cell lysate is achieved by using coarse filter followed by filtration using 0.45 µm filter membranes.
In an embodiment the process describe the purification of plasmid DNA where the removal of contaminating protein and RNA molecule is achieved by filtration using 1000 kDa molecular weight cutoff ultra-filtration membrane.
In an embodiment of invention is the development of a process for purification of plasmid DNA having very low endotoxin contamination.
In an embodiment of invention is the development of a process for plasmid DNA purification without using organic solvent.
In an embodiment the invention describes the process for purification of plasmid DNA free of RNA contamination while not using RNase during the process.
In an embodiment, the DNA plasmid purified is made from CMV promoter expressing green fluorescent protein.
In an embodiment the plasmid DNA purified have size around 5-10 Kb .
In an embodiment the invention is the development of a process for purification of plasmid DNA having high transfection ability in vitro.
In another embodiment of the invention is the development of a process for preparation of plasmid DNA without using extensive ultra-centrifugation step.
Yet another embodiment of the invention is the purification of plasmid DNA using a series of steps which can be scaled-up easily for large scale production.
In an embodiment, the invention describes the process of plasmid DNA purification without using chromatographic separation steps.
In particular the applicant has described a process for purification of plasmid DNA from culture bacterial cells with a purpose to produce large quantities suitable for biomedical application. Plasmid DNA is isolated from the cells by alkaline lysis and is further purified by using series of filtration process. The final plasmid preparation obtained using this method is free from protein and RNA and have very low level of endotoxin. The process is easy and can be used for large scale preparation of plasmids just by changing the capacity of filters used at different steps. Such plasmid preparation process can be used for obtaining he amount of plasmid DNA vaccine for many infectious diseases like malaria, HIV, tuberculosis, hepatitis, and many more DNA based vaccine for immunological and therapeutic actions.
The invention is described in detail with the aid of following examples and the accompanying drawings and tables. Various modifications that may be apparent to one in the art are intended to falls within the scope of the invention.
Description of the accompanying drawing
Figure 1. Schematic diagram of steps showing the process of plasmid purification from E. co//cells using filtration method. Initial separation is achieved by coarse filtration followed by filtration using 0.45 \xm membrane. Removal of protein and RNA is carried out using 1000 kDa molecular weight cutoff membrane. Final concentration is achieved using 10 kDa ultra filtration membrane which remove salt and water.
Figure 2. Agarose gel analysis plasmid preparations using ultra filtration and commercial kit. All variants of plasmid DNA were readily digested with Sal I, giving a single band corresponding to molecular size of 4.7kbp which is molecular size of pEGFP vector. Digestion profile clearly shows absence of any denatured plasmid purified by our new method. There is no evidence of shearing of plasmid DNA or RNA contamination also in agarose gel.
Figure 3 shows the pictures of fluorescent. Cos I cells under the fluorescent microscope transfected with EGFP vector purified by ultra-filtration method (Figure 3a) and QIAGEN method (Figure 3b). Transfection assay is very sensitive to contamination of RNA, proteins, or endotoxins so this assay clearly indicates that plasmid DNA is highly purified and can be used both for in-vivo purposes and for molecular biological manipulations.
Table 1. Comparison of the plasmid preparation using Qiagen kit and ultrafiltration method.
Example 1: Purification of green fluorescent protein expressing plasmid DNA from E. coli cells.
Numerous methods have been developed for the isolation and purification of plasmid DNA from bacterial cells (1, 8, 10). With increasing importance of genetic material as drug for human uses, there is need for preparation of pharmaceutical grade plasmid DNA meeting all the regulatory requirements. Classical methods such as alkaline lysis or CsCI dye buoyant density gradient ultracentrifugation are not suitable for large scale plasmid preparation. Chromatographic methods are time consuming and tedious for manufacturing of plasmid DNA free of impurities such as RNA, endotoxin and genomic DNA in large scale. The current method does not use any of the methods in general use for preparation of plasmid DNA from bacterial cell lysate. It does not involve ultracentrifugation step, organic solvent based precipitation, or RNase treatment to obtain plasmid DNA of pharmaceutical grade for human uses.
Materials and Methods
Plasmid DNA and its transformation into E. coli: pEGFP- N3 eukaryotic N-terminal proteins fusion vector was purchased from ClonTech Laboratories Inc. (CA, USA). It is a 4.7 kbp vector and expresses an enhanced green fluorescent protein (EFGP) under the control of CMV promoter. It also provides pUC origin of replication for propagation in E. coli and also expresses kanamycin resistance in
E. coli. This vector was transformed into the host bacteria E.coli DH5a, according to CaCl2 method (13).
Purification of plasmid DNA
Plasmid purification using ultra-purification method: Plasmid purification from E. coli cell lysate was carried out using series of filtration processes. Different types of ultra filtration membranes as described were purchased from Millipore, India. The 2.5 L E. coli cell pellet was used to isolate and purify EGFP plasmid using ultra-filtration. Plasmid DNA was isolated from E. coli using alkaline-lysis method of Birnboin and Dolly (14) with slight modification as indicated. Cell pellet from 2.5L culture was suspended thoroughly in 125 ml of 50 mM Tris/5mM EDTA ; pH-8.0 and incubated at room temperature for 5 minutes. And then 125 ml of freshly made 0.2N NaOH / 1%SDS was added to the mixture, mixed gently and left at room temperature for 5 minutes. Finally 125 ml of chilled 3M potassium acetate solution (pH-4.8) was added to the above mixture and mixed very gently and left for 10 minutes at room temperature. The cell lysate suspension was collected and clarified using four layer of sterile cheesecloth. The plasmid DNA in supernatant was further clarified using 0.45 ^im ultra filtration membrane (Pellicon mini polyvinylidene fluoride UF membrane, Millipore, India). The filtrate from the ultra filtration membrane was dia-filtered extensively using 1000 kDa molecular cutoff membrane (Ultracell cellulose membrane, Millipore India). Dia-filtration was carried out in two steps. In the first step, the plasmid was dia-filtered using Tris-EDTA buffer (10 mM Tris-HCI, 10 mM EDTA) at pH 10, followed by dia-filtration at Tris-EDTA buffer (10 mM Tris-HCI, 10 mM EDTA) at pH 8. The plasmid solution was again dia-filtered using sterile MQ water (endotoxin free) and was concentrated. Final concentration of the plasmid solution was carried out by 10 kDa molecular weight cutoff ultra filtration membrane (Pellicon XL, Millipore India). Dialyzed material was collected and directly used for analysis and transfection studies. The detail flow sheet of plasmid purification process is given in figure 1.
Purification of plasmid DNA using Qiagen kit
Purification of EGFP vector using QIAGEN kit: Endofree Giga plasmid purification kit was purchased from QIAGEN (Germany). This kit is suitable for purifying high copy number endotoxin free plasmid from 2.5 L of E. coli culture. This kit is based on a modified alkaline lysis procedure and further purification using a DEAE based column. This kit also provides endotoxin removal buffer to remove the maximum amount of endotoxin so that DNA can be used for in-vitro and in-vivo experiments. EGFP transformed E. coli was grown in 5L of Luria Bertani broth in shake flask (500 ml media in 1L flask) in presence of kanamycin (20 ng/ml) at 200 rpm at 37 ° C for 12-16 hrs. Cells were centrifuged and half of pelleted cells were kept separately for plasmid purification by ultra-filtration method. Remaining centrifuged cells were processed for plasmid DNA purification according to instructional manual provided by QIAGEN. The plasmid purified from QIAGEN method is finally dissolved in 5 ml of endotoxin free MQ-water.
In- Vitro Transfection Assay:
Transfast ™ , a cationic lipid based transfection reagent, was purchased from Promega Corp. (USA). Cos I cell line were grown in DMEM medium supplemented with 10 % FCS initially in tissue culture flask and then plated in 24 well plates with 4x 10 6 cell density in each well and after 24 hrs. 0.2|4.g purified EGFP plasmid DNA, purified by both QIAGEN method and ultra-filtration method, were transfected separately in Cos I cells according to instructions provided by Promega manual provided with the Transfast ™. reagent. Negative control was also set up in the experiment in which transfection was done in same way except DNA was not added in the well. After 48 hrs, cells were analyzed for presence of fluorescence under the inverted fluorescence microscope (Olympus).
Limulus Amoebocyte Lysate (LAL) Test:
LAL test kit was purchased from Sigma Chemical Corp. (USA). For each plasmid preparations, their concentration were adjusted in such a way that 10mg DNA is present in 10ml of endotoxin free water and log2 dilution was made for each sample till 1/128 times using endotoxin free water. Endotoxins standards were also made to quantify the endotoxin level in the samples. In test and as well as in standard 10ml LAL reagent was added, mixed properly and incubated at 37 ° C for exactly one hour and then each tube was analyzed for presence of hard gel which indicates positive for endotoxin. Endotoxin level in EU/mg of plasmid DNA was calculated using end point titration method according to the instructions provided with kit. Other analytical tests to measure purity of plasmid: For each plasmid DNA preparations, purity and yield were calculated by measuring O.D. at 260 nm and at 280 nm (13). Proteins contamination in each plasmid preparation (Img plasmid in a well) was checked on 12 % SDS-PAGE using the silver staining method (23). To analyze structural integrity of the purified plasmid purified by both methods were linearized using Sal I restriction enzyme (from Promega Corp. USA).
Result and discussion
The result for DNA yield, purity, endotoxin level and presence of protein contamination in plasmids purified by both methods are summarized in Table 1. We analyzed plasmid purity according to target specifications and methods usually follow in industry. The yield obtained by our newly developed method is slightly lower than the one purified by QIAGEN method. This can be attributed to chances of loss of small amount of supercoiled form of plasmid during diafiltraion. However, this can not be the hindrance factor for using this novel technique in future as difference is small and moreover ultrafiltration method will be more suitable for producing large amount of plasmid DNA. Few 100 mgs of pure plasmid can be purified using this method just by changing the number of membrane without compromising the purification efficiency. Again in this method
the same ultra filtration membranes can be used repeatedly for many cycle of plasmid purification unlike the commercial one time use columns. There are strong indications that the size of the plasmid DNA to be used will grow. Thus, for DNA vaccines, their effectiveness might be increased by, for example, incorporating genes for signaling molecules (e.g. cytokines) into antigen-carrying plasmids. This together with the expectations of the progression of gene therapy to metabolic and other multigeneic diseases will lead to further increase in vector size. Thus efficiency of our novel method in terms of quantity of plasmid DNA finally purified will be augmented for larger size vector.
The purity of the plasmid was analyzed by agarose gel electrophoresis and was comparable to that obtained using commercial kit, there was no shearing of the plasmid during the process (Figure 2). This emphasizes the suitability of the ultra-filtration based purification process for getting pure plasmid for biomedical application. The plasmid also showed high transfection ability using Cos I cells (Figure 3, a and b), this show that plasmid have good quality for in vivo application.
A 2607 A 280 ratio, a criterion to measure the plasmid purity (13), of purified plasmids by both methods is in the range of target specification that indicates its good quality. SDS-PAGE after silver staining (data not shown) does not show any band that indicates absence of any protein contamination in plasmid purified by our new method. As we know there is no single polypeptide chain in biological system having molecular weight even near about 1000KDa, all will be of much lower molecular weight than 1000 KDa. However, there are chances of large protein complex being in this molecular weight if they are in native form but during alkaline lysis procedure most of large proteins get precipitated and some small proteins remain in crude lysate of plasmid but they will be denatured so will be far below the 1000 KDa range in molecular weight. Thus dia-filtration removes any remaining protein contamination present in lysate and makes final product protein-free. Endotoxins, also known as lipopolysaccharides or LPS, are cell
membrane components of Gram negative bacteria (e.g. E.coli). A single E. coli cell contain about 2x10 6 LPS molecules (24), each consisting of a hydrophobic lipid A moiety, a complex array of sugar residues and negatively charged phosphate groups. Endotoxin contamination is of great concern in plasmid purification as they strongly influence transfection of DNA into primary cells and sensitive cultured cells, and increased endotoxin levels lead to sharply reduced transfection efficiency (25). Furthermore, it is extremely important to use endotoxin-free plasmid DNA for gene therapy or DNA vaccine applications, since endotoxins cause fever, endotoxic shock syndromes, and activation of the complement cascade in animals and humans (26). Endotoxins also interfere with immune cells (macrophages and B cells) by causing nonspecific activation of immune response. Therefore, it is important to use plasmid DNA having endotoxin within the permissible range ( Example 2
Preparation of HBsAg plasmid DNA from E. coli cells
Plasmid expressing hepatitis B surface antigen (HBsAg) in E.coli was purified using the above method. The plasmid was a kind gift from DNA vaccine corporation curtsey Dr. Robert Whalen. The plasmid pRC/CMV-S (around 5618 base pair) was purified to homogeneity using the above described filtration
method. E. coli cells were grown in presence of ampicillin (100 |ag/ml) and lysed using alkaline lysis method. The lysed cells were filtered using sterile cheese clothe to remove high molecular weight contaminated and cell debris. The supernatant was filtered through 0.45 µm membrane filter to get clear supernatant which contain the plasmid of interest. The plasmid containing solution was dialfiltered using 1000 kDa molecular cut off membrane (Ultracell cellulose membrane, 1000 kDa, Millipore India) in two steps to remove proteins and RNA. In first step, the dia filtration was carried out using 10 mM Tris-EDTA buffer at pH 10, where as in the second step the dia-filtration was carried out using 10 mm Tris-EDTA buffer at pH 8. The pure plasmid was concentrated using 10 kDa membrane. Around 2 mg of pure plasmid could be purified using the above methods. The immunogenicity of the plasmids was tested using Wistar rats. Animals were bled at different time interval through retro-orbital plexus and the serum was analyzed for anti-HBs Ag antibody titers by AUSAB assay using ABBOT Lab, ELISA Kit. Plasmid purified using above methods elicited antibody titers thus proving the suitability of the separation method for large scale production of plasmid DNA for vaccination process.
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Table 1. Comparison of pDNA yield from E. co//cells using ultrafiltration method and commercial kit of Qiagen column
1. A process for the purification of plasmid DNA from cultured bacterial cells using
series of filtration steps comprising the steps of:
(a) cells lysis using alkaline method incorporating SDS as detergent;
(b) treating the lysed cells with precipitating solution made of potassium acetate; characterized by
(c) filtration of the complete cell lysate using sterile cheese cloth followed by ultra filtration using 0.45 µm membrane;
(d) dia-filtration of the clear solution using 1000 kDa ultra filtration membrane at pH 10 followed by at pH 8 using 10 Mm Tris-EDTA buffer;
(e) concentration and dia-filtration using the above membrane in presence of sterile Milli Q water;
(f) final concentration of pure plasmid preparation using 10 kDa molecular weight cutoff ultra filtration membrane.
2. A process as claimed in claim 1, wherein the initial clarification of plasmid DNA from the cell lysate is achieved by using 0.45 µm ultra filtration membrane without using any organic solvent based precipitation step.
3. A process as claimed in claim 1, wherein the removal of RNA and protein from the plasmid mixture is achieved by use of extensive dia-filtration using 1000 kDa molecular weight cutoff cellulose based ultra filtration membrane.
4. A process as claimed in claim 1, wherein the plasmid DNA preparation is free from protein or RNA contamination.
5. A process as claimed in claim 1, wherein the plasmid DNA preparation is substantially free from endotoxin contamination.
6. A process as claimed in claim 1 wherein the Plasmid DNA is circular plasmid DNA suitable for transfection.
7. A process as claimed in claim 1, wherein the effective filtration area is changed during different filtration steps to vary the amount of pure plasmid obtained.
8. A process as claimed in claim 1, wherein the cultured bacterial cells are cultured E. coli cells.
9. A process for the preparation of pharmaceutical grade plasmid DNA made from CMV promoter through a series of filtration processes.
10. A process as claimed in claim 9, wherein the plasmid DNA is an eukaryotic expression vector.
11. A process as claimed in claim 9, wherein the plasmid DNA expresses green fluorescent protein.
|Indian Patent Application Number||335/DEL/2003|
|PG Journal Number||09/2011|
|Date of Filing||21-Mar-2003|
|Name of Patentee||NATIONAL INSTITUTE OF IMMUNOLOGY|
|Applicant Address||ARUNA ASAF ALI MARG, NEW DELHI-110 067, INDIA.|
|PCT International Classification Number||C07H 21/04|
|PCT International Application Number||N/A|
|PCT International Filing date|