共查询到20条相似文献,搜索用时 15 毫秒
1.
Production of plasmid DNA for human gene therapy using modified alkaline cell lysis and expanded bed anion exchange chromatography 总被引:3,自引:0,他引:3
Varley DL Hitchcock AG Weiss AM Horler WA Cowell R Peddie L Sharpe GS Thatcher DR Hanak JA 《Bioseparation》1999,8(1-5):209-217
We describe a process for the commercial manufacture of therapeutic grade plasmid DNA. The industrially scaleable unit operations employed in this process are: (i) optimized alkaline lysis; (ii) bag filtration; (iii) expanded bed anion exchange chromatography; (iv) ultrafiltration, and (v) size exclusion chromatography. These steps are scaleable alternatives to current approaches to plasmid DNA isolation such as high speed centrifugation for feedstock clarification and solvent precipitation for plasmid concentration, and an efficient alternative to conventional low through-put packed bed chromatography.The process produces plasmid DNA characterized by low level chromosomal DNA, RNA and endotoxin contamination without the use of flammable solvents or toxic reagents and is suitable for therapeutic administration. 相似文献
2.
A state-of-the-art in-line static mixer reactor (ISMR) was invented to lyse E. coli cells and neutralize the cell lysate continuously and efficiently for the extraction of plasmid DNA. It comprised two connected static dynamic mixers, each 0.01 m in diameter and 0.9 m in length, one for lysis and one for neutralization. Cells were lysed using concentrated alkaline with 1% SDS and the lysate was neutralized at feed rates of cell suspension:lysis solution:neutralization solution of 125:250:125, 250:500:250, and 500:1,000:500 mL/min. Distances for the mixtures to reach color homogeneity were dependent on feed rates. The higher the feed rates the shorter the mixing distances and times. However, complete cell lysis and neutralization were independent of color homogeneity. Lysate viscosity and neutralized floc size decreased and floc density increased, as distances and feed rates increased. High plasmid yields were obtained from both lysis and neutralization at feed rate ratios of 125:250:125 and 250:500:250 mL/min within mixing distances < or =0.6 m. Poor mixing performance and plasmid yield were obtained at a high feed rate of 500:1,000: 500 mL/min when residence and reaction times were less than 2 s and from mixing distances > or =0.6 m at all feed rates due to a longer exposure to strong alkali and shear flow. This invention showed excellent performance with scaleable potential for the commercial manufacture of plasmid DNA. 相似文献
3.
ChunSheng Hu XiaoChen Cheng YuXin Lu ZuZe Wu 《Preparative biochemistry & biotechnology》2016,46(8):844-849
The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24?±?0.41?g of pharmaceutical pUDK-HGF from 1.0?kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of >?95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications. 相似文献
4.
Kirsten Tschapalda Nadine Streitner Carsten Voß Erwin Flaschel 《Applied microbiology and biotechnology》2009,84(1):199-204
The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques
is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid
replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of
chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts
of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity
below the required level of 50 mg g−1 of plasmid DNA with almost complete plasmid recovery. 相似文献
5.
A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy
下载免费PDF全文
![点击此处可从《Biotechnology and bioengineering》网站下载免费的PDF全文](/ch/ext_images/free.gif)
With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. 相似文献
6.
Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last
years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms
during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids
(3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium
sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell
culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly
compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes.
The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets
(>3 weeks at 4°C, >12 weeks at −20°C) prior to processing. With alkaline lysates, however, storage at −20°C is mandatory to
avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at
room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work
are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current
knowledge on plasmid stability and process optimization.
Authors Freitas and Azzoni contributed equally to this work. 相似文献
7.
8.
Nitrile metabolizing enzymes are of great industrial interest for the selective bio-transformation of nitriles and surface modification of synthetic polymers under mild reaction conditions. In the present work, isolated strain Amycolatopsis sp. IITR215 was cultivated in the bench top bioreactor for the recovery of maximum biomass of whole cell catalyst. Effect of different lyoprotectants was studied on nitrile metabolizing enzyme from Amycolatopsis sp. IITR215 in which sorbitol proved to be an efficient lyoprotectant. In physical and mechanical methods, only 30% activity was recovered while 85% activity was achieved in the enzymatic method using 2 g/l lysozyme. Very less activity was recovered during stationary phase when cells were grown in mineral base media containing 1 g/l yeast. Therefore, recovery of intracellular enzymes was enhanced by using different concentrations of sodium cholate and deoxycholate. 相似文献
9.
Nurçin Çelik Öztürk Dilek Kazan Aziz Akın Denizci Altan Erarslan 《Engineering in Life Science》2012,12(6):662-671
Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first‐order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ΔGi, and ΔHi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔSi values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ΔG≠, ΔH≠, , and values of enzyme were lower, but there was no change in ΔS≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing. 相似文献
10.
Eijiro Watanabe Susumu Inamoto Mi-Hion Lee Song Uk Kim Teru Ogua Hirotada Mori Sota Hiraga Makari Yamasaki Kazuo Nagai 《Molecular & general genetics : MGG》1989,218(3):431-436
Summary The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning. In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene. The purity of the final preparation was more than 95%, as determined by densitometry. The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene. Therefore, it was concluded that the protein was the sopB gene product. Using anti-SopB serum, the SopB protein was detected in the cell lysates of F+, F, and Hfr strains. The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322. 相似文献
11.
12.
The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite. 相似文献
13.
DNA polymerase (Pol) lambda is a DNA repair enzyme involved in base excision repair, non-homologous end joining and translesion synthesis. Recently, we identified Pol lambda as an interaction partner of cyclin-dependent kinase 2 (CDK2) that is central to the cell cycle G1/S transition and S-phase progression. This interaction leads to in vitro phosphorylation of Pol lambda, and its in vivo phosphorylation pattern during cell cycle progression mimics the modulation of CDK2/cyclin A. Here, we identify several phosphorylation sites of Pol lambda. Experiments with phosphorylation-defective mutants suggest that phosphorylation of Thr 553 is important for maintaining Pol lambda stability, as it is targeted to the proteasomal degradation pathway through ubiquitination unless this residue is phosphorylated. In particular, Pol lambda is stabilized during cell cycle progression in the late S and G2 phases. This most likely allows Pol lambda to correctly conduct repair of damaged DNA during and after S phase. 相似文献
14.
The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression 总被引:1,自引:0,他引:1
BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors. 相似文献
15.
Mamoru Tsukuda Izumi Mochimatsu Miki Sakumoto Yasukazu Mikami Seiichiro Yuyama Shunsuke Yanoma 《Biotherapy》1993,6(3):167-174
Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD
cluster differentiation
- IFN
interferon
- IL
interleukin
- JRU
Japanese Reference Unit
- LAK
lymphokine activated killer
- mAb
monoclonal antibody
- MLTC
mixed lymphocyte tumor cell culture
- PBMC
peripheral blood mononuclear cells
- TILs
tumor infiltrating lymphocytes 相似文献
16.
17.
Genetics of lactobacilli: Plasmids and gene expression 总被引:20,自引:0,他引:20
18.
19.
Application of plasmid DNA as pharmaceutical to be used in gene therapy and vaccination has been investigated intensively in recent years. To be able to provide sufficient material that is in accordance with quality of pharmaceutical grade it is mandatory to gain comprehensive process knowledge which is even requested by regulatory agencies. Regarding plasmid DNA production the specific growth rate has been identified as one of the key parameters. The reduction of specific growth rate results in an increase of plasmid DNA formation. However, quantitative explanations that allow for efficient process development and design are still missing. The presented study proposes a model that clearly demonstrates the relationship between specific growth rate and plasmid formation due to identification of the specific plasmid production rate as relevant key parameter. In addition the model is proved to serve as a useful tool in process development and design. 相似文献
20.
The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means. 相似文献