Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

A Raman and infrared spectroscopic characterisation of the phosphate mineral phosphohedyphane Ca2Pb3(PO4)3Cl from the Roote mine, Nevada, USA

Phosphohedyphane Ca2Pb3(PO4)3Cl is rare Ca and Pb phosphate mineral that belongs to the apatite supergroup. We have analysed phosphohedyphane using SEM with EDX, and Raman and infrared spectroscopy. The chemical analysis shows the presence of Pb, Ca, P and Cl and the chemical formula is expressed as Ca2Pb3(PO4)3Cl. The very sharp Raman band at 975 cm−1 is assigned to the PO43-ν1 symmetric stretching mode. Raman bands noted at 1073, 1188 and 1226 cm−1 are to the attributed to the PO43-ν3 antisymmetric stretching modes. The two Raman bands at 835 and 812 cm−1 assigned to the AsO43-ν1 symmetric stretching vibration and AsO43-ν3 antisymmetric stretching modes prove the substitution of As for P in the structure of phosphohedyphane. A series of bands at 557, 577 and 595 cm−1 are attributed to the ν4 out of plane bending modes of the PO4 units. The multiplicity of bands in the ν2, ν3 and ν4 spectral regions provides evidence for the loss of symmetry of the phosphate anion in the phosphohedyphane structure. Observed bands were assigned to the stretching and bending vibrations of phosphate tetrahedra. Some Raman bands attributable to OH stretching bands were observed, indicating the presence of water and/or OH units in the structure.

Vibrational spectroscopic characterization of the sulphate-carbonate mineral burkeite: Implications for evaporites

Burkeite formation is important in saline evaporites and in pipe scales. Burkeite is an anhydrous sulphate-carbonate with an apparent variable anion ratio. Such a formula with two oxyanions lends itself to vibrational spectroscopy. Two symmetric sulphate stretching modes are observed, indicating at least at the molecular level the nonequivalence of the sulphate ions in the burkeite structure. The strong Raman band at 1065 cm−1 is assigned to the carbonate symmetric stretching vibration. The series of Raman bands at 622, 635, 645, and 704 cm−1 are assigned to the ν4 sulphate bending modes. The observation of multiple bands supports the concept of a reduction in symmetry of the sulphate anion from T d to C 3v or even C 2v.

Removal of herbicides from aqueous solutions by modified forms of montmorillonite

This investigation for the removal of agricultural pollutants, imazaquin and atrazine was conducted using montmorillonite (MMT) exchanged with organic cations through ion exchange. The study found that the adsorption of the herbicides was affected by the degree of organic cation saturations, the size of organic cations and the different natures of the herbicides. The modified clays intercalated with the larger surfactant molecules at the higher concentrations tended to enhance the adsorption of imazaquin and atrazine. In particular, the organoclays were highly efficient for the removal of imazaquin while the adsorption of atrazine was minimal due to the different hydrophobicities. Both imazaquin and atrazine were influenced by the changes of pH. The amphoteric imazaquin exists as an anion at the pH 5–7 and the anionic imazaquin was protonated to a neutral and further a cationic form when the pH is lower. The weak base, atrazine was also protonated at lower pH values. The anionic imazaquin had a strong affinity to the organoclays on the external surface as well as in the interlayer space of the MMT through electrostatic and hydrophobic interactions. In this study, the electrostatic interaction can be the primary mechanism involved during the adsorption process. This study also investigated a comparative adsorption for the imazaquin and atrazine and the lower adsorption of atrazine was enhanced and this phenomenon was due to the synergetic effect. This work highlights a potential mechanism for the removal of specific persistence herbicides from the environment.

A vibrational spectroscopic study of the anhydrous phosphate mineral sidorenkite Na3Mn(PO4)(CO3)

Sidorenkite is a very rare low-temperature hydrothermal mineral, formed very late in the crystallization of hyperagpaitic pegmatites in a differentiated alkalic massif (Mt. Alluaiv, Kola Peninsula, Russia). Sidorenkite Na3Mn(PO4)(CO3) is a phosphate–carbonate of sodium and manganese. Such a formula with two oxyanions lends itself to vibrational spectroscopy. The sharp Raman band at 959 cm−1 and 1012 cm−1 are assigned to the PO43− stretching modes, whilst the Raman bands at 1044 cm−1 and 1074 cm−1 are attributed to the CO32− stretching modes. It is noted that no Raman bands at around 800 cm−1 for sidorenkite were observed. The infrared spectrum of sidorenkite shows a quite intense band at 868 cm−1 with other resolved component bands at 850 and 862 cm−1. These bands are ascribed to the CO32− out-of-plane bend (ν2) bending mode. The series of Raman bands at 622, 635, 645 and 704 cm−1 are assigned to the ν4 phosphate bending modes. The observation of multiple bands supports the concept of a reduction in symmetry of the carbonate anion from D3h or even C2v.

Raman and infrared spectroscopic study of kamphaugite-(Y)

We have studied the carbonate mineral kamphaugite-(Y)(CaY(CO3)2(OH)·H2O), a mineral which contains yttrium and specific rare earth elements. Chemical analysis shows the presence of Ca, Y and C. Back scattering SEM appears to indicate a single pure phase. The vibrational spectroscopy of kamphaugite-(Y) was obtained using a combination of Raman and infrared spectroscopy. Two distinct Raman bands observed at 1078 and 1088cm(-1) provide evidence for the non-equivalence of the carbonate anion in the kamphaugite-(Y) structure. Such a concept is supported by the number of bands assigned to the carbonate antisymmetric stretching mode. Multiple bands in the ν4 region offers further support for the non-equivalence of carbonate anions in the structure. Vibrational spectroscopy enables aspects of the structure of the mineral kamphaugite-(Y) to be assessed.

Effective intercalation of sodium dodecylsulfate (SDS) into hydrocalumite: Mechanism discussion via near-infrared and mid-infrared investigations

The intercalation of an anionic surfactant, sodium dodecylsulfate (SDS), into hydrocalumite (CaAl-LDH-Cl) was investigated in this study. To understand the intercalation behavior, X-ray diffraction (XRD), mid-infrared spectroscopy (MIR), near-infrared spectroscopy (NIR) and scanning electron microscopy (SEM) were undertaken. The near-infrared spectra indicated a special spectral range from 6000 to 5600cm-1and prominent bands of CaAl-LDH-Cl intercalated with SDS around 8388cm-1. This band was assigned to the second overtone of the first fundamental of CH stretching vibrations of SDS, and it could be used to determinate the result of CaAl-LDH-Cl modified by SDS. Moreover, the results revealed that different adsorption behaviors were observed at different (high and low) concentrations of SDS. When the SDS concentration was around 0.2molL-1, anion exchange intercalation occurred and the interlayer distance expanded to about 3.25nm. When SDS concentration was 0.005molL-1, the surface adsorption of DS- was the major anion exchange event.

Raman and infrared spectroscopic characterization of the arsenate-bearing mineral tangdanite– and in comparison with the discredited mineral clinotyrolite

The minerals clinotyrolite and fuxiaotuite are discredited in terms of the mineral tangdanite. The mixed anion mineral tangdanite Ca2Cu9(AsO4)4(SO4)0.5(OH)9 9H2O has been studied using a combination of Raman and infrared spectroscopy. Characteristic bands associated with arsenate, sulphate and hydroxyl units are identified. Broad bands in the OH stretching region are observed and are resolved into component bands. These bands are assigned to water and hydroxyl stretching vibrations. Two intense Raman bands at 837 and approximately 734 cm−1 are assigned to the ν1 (AsO4)3− symmetric stretching and ν3 (AsO4)3− antisymmetric stretching modes. Infrared bands at 1023 cm−1 are assigned to the (SO4)2− ν1 symmetric stretching mode, and infrared bands at 1052, 1110 and 1132 cm−1 assigned to (SO4)2− ν3 antisymmetric stretching modes, confirming the presence of the sulphate anion in the tangdanite structure. Raman bands at 593 and 628 cm−1 are attributed to the (SO4)2− ν4 bending modes. Low-intensity Raman bands found at 457 and 472 cm−1 are assigned to the (AsO4)3− ν2 bending modes. A comparison is made with the previously obtained spectral data on the discredited mineral clinotyrolite.

SEM, EDX and Raman and infrared spectroscopic study of brianyoungite Zn3(CO3,SO4)(OH)4 from Esperanza Mine, Laurion District, Greece

The mineral brianyoungite, a carbonate–sulphate of zinc, has been studied by scanning electron microscopy (SEM) with chemical analysis using energy dispersive spectroscopy (EDX) and Raman and infrared spectroscopy. Multiple carbonate stretching modes are observed and support the concept of non-equivalent carbonate units in the brianyoungite structure. Intense Raman band at 1056 cm−1 with shoulder band at 1038 cm−1 is assigned to the CO32− ν1 symmetric stretching mode. Two intense Raman bands at 973 and 984 cm−1 are assigned to the symmetric stretching modes of the SO42− anion. The observation of two bands supports the concept of the non-equivalence of sulphate units in the brianyoungite structure. Raman bands at 704 and 736 cm−1 are assigned to the CO32− ν4 bending modes and Raman bands at 507, 528, 609 and 638 cm−1 are assigned to the CO32− ν2 bending modes. Multiple Raman and infrared bands in the OH stretching region are observed, proving the existence of water and hydroxyl units in different molecular environments in the structure of brianyoungite. Vibrational spectroscopy enhances our knowledge of the molecular structure of brianyoungite.

A Raman and infrared spectroscopic study of the sulphate mineral aluminite Al2(SO4)(OH)4·7H2O

The mineral aluminite has been studied using a number of techniques, including scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDX) and Raman and infrared spectroscopy. Raman spectroscopy identifies multiple sulphate symmetric stretching modes in line with the three sulphate crystallographically different sites. Raman spectroscopy also identifies a low intensity band at 1069 cm−1 which may be attributed to a carbonate symmetric stretching mode, indicating the presence of thaumasite. The observation of multiple bands in this ν4 spectral region offers evidence for the reduction in symmetry of the sulphate anion from Td to C2v or even lower symmetry. The Raman band at 3588 cm−1 is assigned to the OH unit stretching vibration and the broad feature at around 3439 cm−1 to water stretching bands. Water stretching vibrations are observed at 3157, 3294, 3378 and 3439 cm−1. Vibrational spectroscopy enables an assessment of the molecular structure of aluminite to be made.

Near infrared spectroscopy of selected natural magnesium carbonates: implications for geosequestration

The approach to remove greenhouse gases by pumping liquid CO2 several kilometres below the ground implies that many carbonate containing minerals will be formed. Among these minerals, the formation of hydromagnesite, dypingite and nesquehonite are possible, thus necessitating a study of such minerals. These minerals with a hydrotalcite-related formulae have been characterised by a combination of infrared and near infrared spectroscopy. Layered double hydroxides (also known as anionic clays or hydrotalcites) are a group of layered clay minerals described by the general formula: [M1–x2+Mx3+(OH)2]x+[An–]x/n∙mH2O. The infrared spectra of the minerals are characterised by OH and water stretching vibrations. Both the first and second fundamental overtones of these bands are observed in the NIR spectra in the 7030–7235 cm–1 and 10,490–10,570 cm–1 spectral ranges. Intense (CO3)2– symmetrical and anti-symmetrical stretching vibrations confirm the distortion of the carbonate anion. The position of the water bending vibration indicates water is strongly hydrogen-bonded to the carbonate anion in the mineral structure. NIR spectroscopy offers a method for quickly analysing such materials.