Chromatographic behaviour and purification of N1.5-glycosyl enkephalins

Summary N ^1.5-Glycosyl enkephalins are potent analgesics. Two enkephalin-related glycosyl peptides: [DMet², Pro⁵] enkephalin (N ^1.5 -glucopyranosyl] amide and [DMet², Pro⁵] enkephalin [N ^1.5 -galactosyl] amide have been prepared following the liquid phase procedure. The chromatographic behavior of both the final glycosyl enkephalins and their synthetic intermediates on reversed-phase columns has been tested. Semipreparative, reversed-phase, HPLC conditions have been developed in order to obtain a simple and rapid procedure for the purification of the two synthetic pentapeptides. Thus, using isocratic elution, injections of 10mg of crude material after gel filtration gave homogeneous (99.9%) glycosyl enkephalins.     

Effect of salt on purification of plasmid DNA using size-exclusion chromatography

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5M, 1M, 2M, respectively) of two types of salt (NaCl and (NH(4))(2)SO(4)) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.     

Characterization of methacrylate monoliths for purification of DNA molecules

The suitability of methacrylate based anion exchange monolithic supports for the separation and purification of plasmid and genomic DNA has been explored. The effect of the size of the channels, ionic strength of the solution, and ligand density on the dynamic binding capacity has been investigated. The dynamic binding capacity was found to be flow independent, at least up to a linear velocity of 700 cm h(-1), and exceeded 9 mg mL(-1) for all types of DNA. The recovery depends on the pH value of the mobile phase and its ionic strength as well as on the density of the active groups. Under optimal conditions recoveries exceeding 80% were obtained even for genomic DNA. Finally, the suitability of this approach is demonstrated by purification of a real-life sample.     

DNA removal on different chromatography supports used for EPO purification by spike studies

Chromatographic techniques are used in the purification step of human recombinant erythropoietin production process to obtain a reliable product with high purity. Anion-exchange chromatography supports have proved high efficient in removing contaminants such as DNA. For that reason, the DNA removal was determined by spike studies, on three anion-exchange chromatographic supports: gel, membrane, and monolithic column, which is used in intermediate purification stage. This study showed that membrane and monolith columns have very good results in the removal of contaminants at this step. Log removal values (LRV) greater than 3.5 were obtained from DNA spike clearance studies. Monolithic column was determined as the best technological proposal, with more than 4 LRV, 7.72?mg DNA per milliliter of adsorbent and 85% protein recovery in nonspike run. The results of this study may be used as a guide in the selection of commercially available chromatography supports for intermediate purification steps in recombinant protein production.     

Analysis and purification of 2-hydroxyethyl methacrylate by means of thin-layer chromatography.

Poly(2-hydroxyethyl methacrylate) is nowadays accepted as a biocompatible, safe and stable hydrogel for medical use. In this paper, the use of thin-layer chromatography for the analysis and small-scale preparation of the initial monomer, 2-hydroxyethyl methacrylate, is described. Development on silica gel, with n-hexane-diethyl ether (1:1, v/v) and/or n-hexane-isobutyl methyl ketone-n-octanol (9:2:1, v/v; saturated with 25% nitric acid) is recommended for qualitative analysis. Preparative-scale work is preferably carried out on sulphuric acid-impregnated silica gel, with n-hexane-diethyl ether (1:1, v/v) as mobile phase. Inhibitors are detected by thin-layer chromatography and a drop-test procedure with diazotised sulphanilic acid. The nature of the contaminants present in several commercial samples of 2-hydroxyethyl methacrylate is discussed. n20D values are reported for the system 2-hydroxyethyl methacrylate-water.     

Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

Immobilization of glucoamylase onto novel porous polymer supports of vinylene carbonate and 2-hydroxyethyl methacrylate

Glucoamylase was immobilized onto novel porous polymer supports. The properties of immobilized glucoamylase and the relationship between the activity of immobilized enzyme and the properties of porous polymer supports were investigated. Compared with the native enzyme, the temperature profile of immobilized glucoamylase was widened, and the optimum pH was also changed. The optimum substrate concentration of immobilized glucoamylase was higher than that of native enzyme. After storage for 23 d, the immobilized glucoamylase still maintained about 84% of its initial activity, whereas the native enzyme only maintained about 58% of the initial activity. Moreover, after using repeatedly seven times, the immobilized enzyme maintained about 85% of its initial activity. Furthermore, the properties of porous polymer supports had an effect on the activity of the immobilized glucoamylase.     

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%.     

Performance of a non-grafted monolithic support for purification of supercoiled plasmid DNA

The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.     

Effect of urea on the behaviour of poly(2-hydroxyethyl methacrylate)-water mixtures

The effect of urea solutions on the equilibrium swelling of lightly crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) gels and on the viscometric behaviour of PHEMA, for various concentrations of urea and temperatures, has been studied. Urea raises the degree of swelling of gels and the intrinsic viscosity of PHEMA; the temperature coefficient of both quantities is negative. A thermodynamic analysis of the swelling data shows that a change in entropy is the driving force for the increase in swelling at low temperatures, while at higher temperatures (50–80°) a negative change in enthalpy prevails, both corresponding to the transport of PHEMA from the aqueous medium into urea solutions. The entropy and enthalpy of dilution parameters derived from viscometric measurements have negative values, and their absolute values decrease with increasing urea concentration. It has also been found that the PHEMA molecules in urea solutions under the θ-conditions are much more coiled than in an organic θ-solvent. The results are interpreted in terms of the existence of inter- and intramolecular hydrophobic bonds and by the destruction of hydrophobic clusters caused by urea.     

Mechanism of formation of short metal-ligand bonds in linear molecules CuCl2 and NiCl2 molecules

The electronic structures of the CuCl₂ and NiCl₂ linear molecules were analyzed in the framework of the extended version of the angular overlap model. The anomalously short metal-ligand bonds in these molecules were shown to be due to the strong sd-mixing of the orbitals of the metal ions.     

Flow behaviour of titanium dioxide dispersions in the presence of 2-hydroxyethyl cellulose

 The effects of 2-hydroxyethyl cellulose (HEC) of weight-average molecular weight 15,000, 90,000 and 7,20,000 on the rheological properties of TiO₂ dispersions were evaluated. For all three HECs, the maximum yield stress, τ_ymax, (occurred at zero zeta potential), decreased with increasing HEC concentration. Interestingly, the largest reduction in τ_ymax was observed with the HEC with the highest molecular weight. This reduction was attributed to steric interaction arising from adsorbed HEC. Adsorbed high-molecular-weight HEC formed an effectively thicker steric barrier because of its larger size and higher adsorption capacity. Bridging interactions that were expected to be important for the high-molecular-weight HEC were found to be unimportant here. In the flocculated regime, HEC enhanced the shear-thinning characteristics of the TiO₂ dispersions. Received: 8 November 1999 Accepted: 20 December 1999     

The symmetry behaviour of the first-order density matrix and its natural orbitals for linear molecules

The general results regarding the symmetry of density matrices constructed from wave functions of a given symmetry species and the consequences for the transformation properties of the natural p-states reviewed in a previous communication [1] are illustrated for the special case p=1 for linear molecules with C _v - or D _h -symmetry. It is shown that even if the wave function belongs to a degenerate species, the natural orbitals (NO's) can always choosen to be adapted to the effective symmetry group C . The role played by the symmetry-adapted natural orbitals (SANO's) and of the natural expansion for 2-electron wave functions in this case is discussed.

---

Zusammenfassung Die in einer früheren Arbeit [1] zusammengestellten Ergebnisse über das Symmetrieverhalten reduzierter Dichtematrizen, die aus Wellenfunktionen bestimmter Symmetrie konstruiert sind, sowie die Transformationseigenschaften der natürlichen p-Zustände werden am Beispiel von linearen Molekülen mit C _v - oder D _h -Symmetrie für den Fall p=1 illustriert. Es wird gezeigt, daß auch dann, wenn die Wellenfunktion zu einer entarteten Symmetriespecies gehört, die natürlichen Orbitale (NO's) der effectiven Symmetriegruppe C adaptiert sind.Die Rolle, die in diesem Fall die symmetrieadaptierten natürlichen Orbitale (SANO's) und die natürliche Entwicklung von 2-Elektronenwellenfunktionen spielen, werden diskutiert.

---

Résumé Les résultats généraux recensés dans une communication précédente [1] concernant la symétrie des matrices densités construites à partir de fonctions d'onde de symétrie donnée et ses conséquences sur les propriétés de transformation des états -p naturels, sont illustrés sur le cas spécial où p=1 dans les molécules linéaires à symétrie C _v ou D _h . On montre que même si la fonction d'onde appartient à une catégorie dégénérée, les orbitales naturelles (NO's) peuvent toujours être choisies pour être adaptées au groupe de symétrie effectif C . On discute le rôle joué par les orbitales naturelles adaptées à la symétrie (SANO's) et l'expansion naturelle des fonctions d'onde à deux électrons.

     

Effects of condensing agent and nuclease on the extent of ejection from phage lambda

We have recently demonstrated, that DNA ejection from bacteriophage lambda can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed -and hence for which there is no internal driving force for ejection- is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage lambda incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease (DNase I) or DNA condensing agent (spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximately 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside.     

The thermorheological complexity of photoelastic behaviour of 2-hydroxyethyl methacrylate — dodecyl methacrylate copolymers

The viscoelastic photoelastic behaviour of networks of 2-hydroxyethyl methacrylate — dodecyl methacrylate (DMA) copolymers in the main transition and rubberlike region was investigated. With increasing DMA content, photoelastic functions are quickly shifted to lower temperatures or shorter times; a detailed course of the functions suggests heterogeneity of the copolymers. Due to the existence of long side chains, optical function of all samples change the sign from positive to negative with increasing temperature. While the temperature dependences of the moduli of copolymers can be described by the two-phase Takayanagi model, the temperature dependences of optical functions cannot be described by using this model. It has been found, however, that the tempeature and time dependences of photoelastic functions can be described semiquantitatively by a three-phase model with a hypothetical statistical copolymer as the third component. The high values of the volume fraction of the hypothetical statistical copolymer found for the individual samples, suggest a considerable miscibility and a strong influence of the interphase boundary on the photoelastic behaviour of the copolymers.     

Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification

New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.     

Viewing of complex molecules of ethidium bromide and plasmid DNA in solution by atomic force microscopy

Tertiary structure changes in plasmid DNA, induced by ethidium bromide intercalation, have been observed in aqueous solutions by the use of an atomic force microscope. A relaxed closed circular pBR322 molecule became a positively supercoiled complex on the drug binding. The supercoiling always resulted in an interwound (or a plectonemic) form, but never a solenoidal (or a toroidal) form. A quantitative analysis of the compactness of such supercoiled complexes has been carried out.     

Chromatographic behaviour of closely related aromatic amines on phenol-impregnated thin-layers

Summary The TLC behaviour of closely related aromatic amines on silica gel plates impregnated with phenol, o-cresol, p-nitrophenol, quinol, catechol and pyrogallol has been studied. An attempt has been made to correlate the chromatographic behaviour of amines with the equilibrium constants of the adducts formed by the interaction of amines with absorbed phenol. Suitable adsorbent system and solvent systems for an efficient separation of closely related isomers of this class of compound of physiological importance have been developed.