Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

The influence of socio-cultural environments on the performance of nascent entrepreneurs:community culture, motivation, self-efficacy and start-up success

The importance of informal institutions and in particular culture for entrepreneurship is a subject of ongoing interest. Past research has mostly concentrated on cross-national comparisons, cultural values and the direct effects of culture on entrepreneurial behaviour, but in the main found inconsistent results. We add a fresh perspective to this research stream by turning attention to community-level culture and cultural norms. We hypothesize indirect effects of cultural norms on venture emergence: Community-level cultural norms (performance-based culture and socially supportive institutional norms) impact important supply-side variables (entrepreneurial self-efficacy and entrepreneurial motivation) which in turn influence nascent entrepreneurs' success in creating operational ventures (venture emergence). We test our predictions on a unique longitudinal dataset, tracking nascent entrepreneurs' venture creation efforts over a five-year time span, and find evidence supporting them. Our research contributes to a more fine-grained understanding of how culture, in particular perceptions of community cultural norms, influences venture emergence. Based on these findings, we discuss how venture creation efforts can be supported. Our research highlights the embeddedness of entrepreneurial behaviour and its immediate antecedent beliefs in the local, community context. © 2012 Copyright Taylor and Francis Group, LLC.

Methyl chloride purification for the silicone industry

This study experimentally investigated methyl chloride (MeCl) purification method using an inhouse designed and built volumetric adsorption/desorption rig. MeCl is an essential raw material in the manufacture of silicone however all technical grades of MeCl contain concentrations (0.2 - 1.0 % wt) of dimethyl ether (DME) which poison the process. The project industrial partner had previously exhausted numerous separation methods, which all have been deemed not suitable for various reasons. Therefore, adsorption/desorption separation was proposed in this study as a potential solution with less economic and environmental impact. Pure component adsorption/desorption was carried out for DME and MeCl on six different adsorbents namely: zeolite molecular sieves (types 4 Å and 5 Å); silica gels (35-70 mesh, amorphous precipitated, and 35-60 mesh) and granular activated carbon (type 8-12 mesh). Subsequent binary gas mixture adsorption in batch and continuous mode was carried out on both zeolites and all three silica gels following thermal pre-treatment in vacuum. The adsorbents were tested as received and after being subjected to different thermal and vacuum pre-treatment conditions. The various adsorption studies were carried out at low pressure and temperature ranges of 0.5 - 3.5 atm and 20 - 100 °C. All adsorbents were characterised using Brunauer Emmett Teller (BET), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) to investigate their physical and chemical properties. The well-known helium (He) expansion method was used to determine the empty manifold and adsorption cell (AC) regions and respective void volumes for the different adsorbents. The amounts adsorbed were determined using Ideal gas laws via the differential pressure method. The heat of adsorption for the various adsorbate-adsorbent (A-S) interactions was calculated using a new calorimetric method based on direct temperature measurements inside the AC. Further adsorption analysis included use of various empirical and kinetic models to determine and understand the behaviour of the respective interactions. The gas purification behaviour was investigated using gas chromatography and mass spectroscopy (GC-MC) analysis. Binary gas mixture samples were syringed from the manifold iii and AC outlet before and after adsorption/desorption analysis through manual sample injections into the GC-MS to detect and quantify the presence of DME and ultimately observe for methyl chloride purification. Convincing gas purification behaviour was confirmed using two different GC columns, thus giving more confidence on the measurement reliability. From the single pure component adsorption of DME and MeCl on the as received zeolite 4A subjected to 1 h vacuum pre-treatment, both gases exhibited pseudo second order adsorption kinetics with DME exhibiting a rate constant nearly double that of MeCl thus suggesting a faster rate of adsorption. From the adsorption isotherm classification both DME and MeCl exhibited Type II and I adsorption isotherm classifications, respectively. The strength of bonding was confirmed by the differential heat of adsorption measurement, which was found to be 23.30 and 10.21 kJ mol-1 for DME and MeCl, respectively. The former is believed to adsorb heterogeneously through hydrogen bonding whilst MeCl adsorbs homogenously via van der Waal’s (VDW) forces. Single pure component adsorption on as received zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) resulted in similar adsorption/desorption behaviour in similar quantities (mol kg-1). The adsorption isotherms for DME and MeCl on zeolite 5A, silica gels (35-70, amorphous precipitated and 35-60) and activated carbon 8-12 exhibited Type I classifications, respectively. Experiments on zeolite 5A indicated that DME adsorbed stronger, faster and with a slightly stronger strength of interaction than MeCl but in lesser quantities. On the silica gels adsorbents, DME exhibited a slightly greater adsorption capacity whilst adsorbing at a similar rate and strength of interaction compared to MeCl. On the activated carbon adsorbent, MeCl exhibited the greater adsorption capacity at a faster rate but with similar heats of adsorption. The effect of prolonged vacuum (15 h), thermal pre-treatment (150 °C) and extended equilibrium time (15 min) were investigated for the adsorption behaviour of DME and MeCl on both zeolites 4A and 5A, respectively. Compared to adsorption on as received adsorbents subjected to 1 h vacuum the adsorption capacities for DME and MeCl were found to increase by 1.95 % and 20.37 % on zeolite 4A and by 4.52 % and 6.69 % on zeolite 5A, respectively. In addition the empirical and kinetic models and differential heats of adsorption resulted in more definitive fitting curves and trends due to the true equilibrium position of the adsorbate with the adsorbent. Batch binary mixture adsorption on thermally and vacuum pre-treated zeolite 4A demonstrated purification behaviour of all adsorbents used for MeCl streams containing DME impurities, with a concentration as low as 0.66 vol. %. The GC-MS analysis showed no DME detection for the tested concentration mixtures at the AC outlet after 15 or 30 min, whereas MeCl was detectable in measurable amounts. Similar behaviour was also observed when carrying out adsorption in continuous mode. On the other hand, similar studies on the other adsorbents did not show such favourable MeCl purification behaviour. Overall this study investigated a wide range of adsorbents (zeolites, silica gels and activated carbon) and demonstrated for the first time potential to purify MeCl streams containing DME impurities using adsorption/desorption separation under different adsorbent pre-treatment and adsorption operating conditions. The study also revealed for the first time the adsorption isotherms, empirical and kinetic models and heats of adsorption for the respective adsorbentsurface (A-S) interactions. In conclusion, this study has shown strong evidence to propose zeolite 4A for adsorptive purification of MeCl. It is believed that with a technical grade MeCl stream competitive yet simultaneous co-adsorption of DME and MeCl occurs with evidence of molecular sieiving effects whereby the larger DME molecules are unable to penetrate through the adsorbent bed whereas the smaller MeCl molecules diffuse through resulting in a purified MeCl stream at the AC outlet. Ultimately, further studies are recommended for increased adsorption capacities by considering wider operating conditions, e.g. different adsorbent thermal and vacuum pre-treatment and adsorbing at temperatures closer to the boiling point of the gases and different conditions of pressure and temperature.