Study of the reactione + e −→K + K − in the energy range1350 \leqq \sqrt s \leqq 2400 MeV

Thee ⁺ e ^?→K ⁺ K ^? cross section has been measured from about 750 events in the energy interval \(1350 \leqq \sqrt s \leqq 2400 MeV\) with the DM2 detector at DCI. TheK ^± form factor |F _F ^±| cannot be explained by the ρ, ω, ? and ρ′(1600). An additional resonant amplitude at 1650 MeV has to be added as suggested by a previous experiment.     

Chromatography of plasmid DNA

Liquid chromatography plays a central role in process-scale manufacturing of therapeutic plasmid DNA (pDNA) for gene therapy and DNA vaccination. Apart from its use as a preparative purification step, it is also very useful as an analytical tool to monitor and control pDNA quality during processing and in final formulations. This paper gives an overview of the use of pDNA chromatography. The specificity of pDNA purification and the consequent limitations to the performance of chromatography are described. Strategies currently used to overcome those limitations, as well as other possible solutions are presented. Applications of the different types of chromatography to the purification of therapeutic pDNA are reviewed, and the main advantages and disadvantages behind each technique highlighted.     

Titanosilicates as Supports for an Enzymatic Alcoholysis Reaction

Novel [Cr^III(amp)(bipy)(Cl)] (1) (H₂amp = N-(hydroxyphenyl)salicyldimine; bipy = 2,2-bipyridyl) and [Cr^III(app)(bipy)(Cl)]⁺ (2) (H₂app = N-(hydroxyphenyl)pyridine-2-carboxaldimine; bipy = 2,2-bipyridyl) complexes have been synthesized and characterized by physico-chemical methods. Complexes 1 and 2 have been employed as catalysts in the oxidation of both saturated and unsaturated hydrocarbons using tert-butylhydroperoxide (t-BuOOH). The significance of the results with respect to oxo-functionalization of C-H bonds both in unsaturated and saturated hydrocarbons is noted.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography

A hydrophobic interaction HPLC method was developed for the quantification of plasmid DNA and assessment of its purity in crude Escherichia coli lysates and other process streams. A Phenyl Sepharose Source (Amersham Biosciences) column was used to separate the double-stranded plasmid DNA molecules from the more hydrophobic impurities present in the process streams. The method is rapid (each analysis takes 7 min), reproducible, easy to perform and does not require previous digestion of RNA in samples with RNase or other pre-treatment. Furthermore, it is capable of handling heavily contaminated samples, with less than 5% of plasmid DNA thus constituting a good alternative to other less robust analytical techniques currently in use.     

Radiative decay ofJ/ψ into γ π+ π−

The radiative decayJ/ψ → γ π⁺ π^? has been studied using the 8.6 millionJ/ψ produced in the DM2 experiment at the DCIe ⁺e^? storage rings at Orsay. The π⁺ π^? mass spectrum shows a cleanf ₂ (1270) signal, and the possible presence of two other states at thef ₂ (1720) andf ₄ (2030) masses. For thef ₂ (1270), the branching ratio BR(J/ψ →γf)xBR(f→π⁺ π^?) is measured to be (7.50±0.30±1.12)×10^?4, and the spin analysis prefers theJ=2 assignment, with helicity parametersx=0.83±0.06 andy=0.01±0.06. The existence of higher mass states is discussed.     

Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.     

Non-planar smoulder propagation governed by diffusion

The steady propagation of a thin smouldering front in a half-spacehas been considered. A suitable coordinate transformation hasallowed the region near the leading edge of the front to beexamined for both a maintained planar surface and with surfacecollapse due to material shrinkage. The change in the oxidizerconcentration for a small increment in the propagation speedfor large time and surface collapse has been determined. Theinfluence of two types of nonlinear diffusion on the shape ofthe smouldering front has been found; other cases can be dealtwith in a similar manner.