Alkaline-cell lysis through in-line static mixer reactor for the production of plasmid DNA for gene therapy

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.     

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.     

Proposal for a better integration of bacterial lysis into the production of plasmid DNA at large scale

The paper addresses the question of how to achieve bacterial lysis in large-scale plasmid DNA production processes, where conventional alkaline lysis may become awkward to handle. Bacteria were grown in shaker flasks and a bioreactor. Suboptimal growth conditions were found advantageous for stable plasmid production at high copy numbers (up to 25mg/L could be achieved). Cells were harvested by filtration in the presence of a filter aid. A linear relationship between the biomass and the optimal filter aid concentration in terms of back pressure could be established. Bacteria-containing filter cakes were washed with isotonic buffer and lysis was achieved in situ by a two-step protocol calling for fragilisation of the cells followed by heat lysis in a suitable buffer. RNA and other soluble cell components where washed out of the cake during this step, while the plasmid DNA was retained. Afterwards a clear lysate containing relatively pure plasmid DNA could be eluted from the cake mostly as the desired supercoiled topoisomer, while cell debris and genomic DNA were retained. Lysis is, thus, integrated not only with cell capture but also with a significant degree of isolation/purification, as most impurities were considerably reduced during the procedure.     

Effects of growth medium selection on plasmid DNA production and initial processing steps

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.     

Biochemical characterization of phi X174-protein-E-mediated lysis of Escherichia coli

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage phi X174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of beta-galactosidase in the supernatant fraction of protein-E-lysed cells. Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein-E-mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.     

Inducible cell lysis system for the study of natural transformation and environmental fate of DNA released by cell death.

Two novel conditional broad-host-range cell lysis systems have been developed for the study of natural transformation in bacteria and the environmental fate of DNA released by cell death. Plasmid pDKL02 consists of lysis genes S, R, and Rz from bacteriophage lambda under the control of the Ptac promoter. The addition of inducer to Escherichia coli, Acinetobacter calcoaceticus, or Pseudomonas stutzeri containing plasmid pDKL02 resulted in cell lysis coincident with the release of high amounts of nucleic acids into the surrounding medium. The utility of this lysis system for the study of natural transformation with DNA released from lysed cells was assessed with differentially marked but otherwise isogenic donor-recipient pairs of P. stutzeri JM300 and A. calcoaceticus BD4. Transformation frequencies obtained with lysis-released DNA and DNA purified by conventional methods and assessed by the use of antibiotic resistance (P. stutzeri) or amino acid prototrophy (A. calcoaceticus) for markers were comparable. A second cell lysis plasmid, pDKL01, contains the lysis gene E from bacteriophage phi X174 and causes lysis of E. coli and P. stutzeri bacteria by activating cellular autolysins. Whereas DNA released from pDKL02-containing bacteria persists in the culture broth for days, that from induced pDKL01-containing bacteria is degraded immediately after release. The lysis system involving pDKL02 is thus useful for the study of both the fate of DNA released naturally into the environment by dead cells and gene transfer by natural transformation in the environment in that biochemically unmanipulated DNA containing defined sequences and coding for selective phenotypes can be released into a selected environment at a specific time point. This will allow kinetic measurements that will answer some of the current ecological questions about the fate and biological potential of environmental DNA to be made.     

Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Plasmid DNA for biopharmaceutical applications is mainly produced in E. coli cells. The first and most crucial step for recovering the plasmid is the cell lysis. Governed by the physico-chemical properties of the polynucleotide, alkaline lysis has been the lysis-method of choice. This chemical disintegration technique was initially developed for the lab scale and non-pharmaceutical applications. A continuous, fully automated and closed system combining alkaline lysis, neutralization and clarification in one gentle and generic operation was developed. This system consists of a three units. One unit controls mixing and contact time during the alkaline treatment, another one controls the neutralization and the concurrent formation of flocs and a third one the separation of flocs and pDNA containing lysate. Based on optimization experiments the selected process parameters resulted in yields up to 100% and homogeneities comparable to that obtained by gentle manual lysis. The process does not need enzymes and it is scalable and routinely used for cGMP-production of pharmaceutical grade plasmid DNA from 200 L fermentations.     

Transforming activity of plasmid and chromosomal DNA inEscherichia coli

An auxotrophic strain ofEscherichia coli with therecB recC sbcB genotype was transformed by chromosomal DNA of the prototrophic strain and by plasmid DNA carrying genes for antibiotic resistance (R1drd 19). The donor plasmid DNA obtained by cell lysis in the presence of Triton X-100 and subsequent centrifugation in a caesium chloride-ethidium bromide gradient was shown to have a circulaf molecule and to retain its completeness after penetration into the recipient. Experiments with mixtures or plasmid and chromosomal DNA indicate a competition between these two DNA types during the transformation reaction in the given system.     

重组大肠杆菌碱裂解方法的改进

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。     

Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis.

The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222. Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG). A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222. Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG. The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.     

Isolation and characterization of lysozyme-sensitive mutants of Staphylococcus aureus.

Four lysozyme-sensitive mutants were isolated after nitrosoguanidine mutagenesis of a lysozyme-insensitive strain of Staphylococcus aureus. One mutant was sufficiently effective for the isolation of macromolecules, such as plasmid deoxyribonucleic acids, from a cell after lysozyme-induced cell lysis.     

尼泊尔马桑根瘤内生菌纯培养物的质粒检测

采用胶内裂解法快速检测了21株马桑根瘤内生菌纯培养物和4株弗兰克氏菌参考菌株的质粒,其中有5株马桑分离菌株和1株参考菌株含有质粒。除马桑菌株和参考菌株各有1株携带2个质粒外,其它菌株均只含有1个质粒。这些质粒的分子量约为13～20kb。根据所含质粒的大小和数目,将21株马桑分离菌株划分成4个质粒类群。实验还对菌丝体生长,细胞酶解和裂解等条件对质粒检测效果的影响进行了探讨。     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.     

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.     

The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products.

Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained. These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification.     

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.     

Evaluation of the interaction of phi X174 gene products E and K in E-mediated lysis of Escherichia coli.

Gene K of bacteriophage phi X174 was cloned, and its gene product was localized in the cell envelope of Escherichia coli. Compared with the sole expression of the phi X174 lysis gene E, the simultaneous expression of the K and E genes had no effect on scheduling of cell lysis. Therefore, a direct interaction of proteins E and K could be excluded. In contrast, phi X174 infection of a host carrying a plasmid expressing gene K resulted in a delayed lysis and an apparent increase in phage titer.