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Monolithic Chromatography And Its Modern Applications Pdf

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Hplc Ppt. High performance liquid chromatography is one of the most accurate methods widely used for the Stability indicating HPLC methods are used to separate various drug related impurities that are. By using an amide or amino bonded phase column, polar compounds can be retained by a normal phase or hydrophilic interaction chromatography retention mechanism. High Performance Liquid Chromatography HPLC HPL chromatographic separation is based on interaction and differential partition of the sample between the mobile liquid phase and the stationary.

Affinity monolith chromatography: A review of general principles and applications.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques.

Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process. Full view. The advantages of this procedure include high plasmid purity and the elimination of undesirable additives.

However, yields and consequently the productivity of the process are low. Previous work demonstrated that the most critical step of the process is the reverse phase chromatography, where conventional porous particle resins are used.

Therefore, to increase the process productivity alternative technologies such as membranes and chromatographic monoliths were tested as alternative options for this critical step. The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA.

Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree.

In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. Purchase full article. Separation of pDNA using traditional particle-based anion-exchange supports is usually slow and has a low capacity for pDNA due to steric exclusion effects.

Due to convective mass transfer properties, and large flow-through channels for binding large biomolecules, monoliths have been shown to provide a fast and efficient alternative for pDNA purification. Sample displacement chromatography SDC is a chromatographic technique that utilises different rela-tive binding affinities of components in a sample mixture and has been widely studied in the context ofpeptide and protein purification.

Here, we report a use of SDC to separate plasmid DNA pDNA isoformsunder overloading conditions, where supercoiled sc isoform acts as a displacer of open circular oc orlinear isoform. Since displacement is more efficient when mass transfer between stationary and mobilechromatographic phases is not limited by diffusion, we investigated convective interaction media CIM monoliths as stationary phases for pDNA isoform separation.

SD efficiency for pDNA isoformseparation was shown to be dependent on column selectivity for individual isoform, column efficiencyand on ammonium sulfate AS concentration in loading buffer binding strength. SD and negative modeelution often operate in parallel, therefore negative mode elution additionally influences the efficiencyof the overall purification process. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes,and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used.

This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, whichis compatible with continuous, multicolumn chromatography systems, and could therefore be used toincrease productivity of pDNA production in the future. The demand for high-purity supercoiled plasmid DNA to be applied as a vector for new therapeutic strategies, such as gene therapy or DNA vaccination has increased in the last years.

Thus, it is necessary to implement an analytical technique suitable to control the quality of the supercoiled plasmid as a pharmaceutical product during the manufacturing process.

The present study describes a new methodology to quantify and monitor the purity of supercoiled plasmid DNA by using a monolithic column based on anion-exchange chromatography. This analytical method with UV detection allows the separation of the plasmid isoforms by combining a NaCl stepwise gradient.

The specificity, linearity, accuracy, reproducibility and repeatability of the method have been evaluated, and the lower quantification and detection limits were also established. The main advantage achieved by using this monolithic column is the possibility to quantify the supercoiled plasmid in a sample containing other plasmid topologies, in a 4 min experiment.

This column also permits the assessment of the supercoiled plasmid DNA present in more complex samples, allowing to control its quality throughout the bioprocess. Therefore, these findings strengthen the possibility of using this monolithic column associated with a powerful analytical method to control the process development of supercoiled plasmid DNA production and purification for therapeutic applications.

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.

In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA. HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA pDNA purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column.

Three differently sized pDNA molecules of 3. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer.

The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles.

This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport.

In order to conduct further improvements, modelling of the downstream process was performed. A methodology based on process analysis tools, such as experimental design and modelling was established to identify factors with the highest influence on production cost and the amount of annual plasmid.

Taking into account that the pIDKE2 downstream process designed is in its initial stages of development, CIM technology was evaluated as a new manufacturing process on lab scale. Purity and recovery of CIM technology was better than porous particle matrix, thus SuperPro Designer was used in order to simulate the purification process.

Cost efficiency optimization of the pIDKE2 downstream process was done with the simulation model. Eicosapentaenoic and docosahexaenoic acids are important bio-active fatty acids in fish oils.

Monolithic HPLC columns both in the polymeric cation exchange silver-ion and RP formats were compared with corresponding packed columns for the isolation of these acids from tuna oil ethyl esters.

Monolithic columns in both formats enabled rapid typically 5—10 min separations compared with packed columns 30 min. Baseline separation of eicosapentaenoic and docosahexaenoic esters was achieved on all RP columns. The results show that there is potential to use polymeric monolithic cation exchange columns for scaled-up preparation of eicosapentaenoic and docosahexaenoic ester concentrates from fish oils.

Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia HIT.

Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer.

This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution.

The elution profile of binding sequences was compared to that of a blank column no drug , and fractions with a chromatographic difference were analyzed via real-time PCR polymerase chain reaction and used for further selection. Sequences were identified by sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection.

This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care. Read full article. Plasmid DNA pDNA -based vaccines offer more rapid avenues for development and production if compared to those of conventional virus-based vaccines.

They do not rely on time- or labour-intensive cell culture processes and allow greater flexibility in shipping and storage. Stimulating antibodies and cellmediated components of the immune system are considered as some of the major advantages associated with the use of pDNA vaccines. This review summarizes the current trends in the purification of pDNA vaccines for practical and analytical applications.

Special attention is paid to chromatographic techniques aimed at reducing the steps of final purification, post primary isolation and intermediate recovery, in order to reduce the number of steps necessary to reach a purified end product from the crude plasmid. Recent advances in the field of RNA interference and new cost-effective approaches for large-scale double-stranded RNA dsRNA synthesis have fuelled the demand for robust high-performance purification techniques suitable for dsRNA molecules of various lengths.

To address this issue, we developed an improved dsRNA purification method based on anion exchange chromatography utilizing convective interaction media CIM monolithic columns. We demonstrated that linearly scalable CIM monolithic quaternary amine QA columns can be used as a fast and superior alternative to standard purification methods e.

LiCl precipitation to obtain highly pure dsRNA preparations. The impurities following Dicer treatment were quickly and efficiently removed with the QA CIM monolithic column, yielding siRNA molecules of high purity suitable for potential therapeutic applications. Moreover, baseline separation of dsRNA molecules up to 1 kb in non-denaturing conditions was achieved.

Gutierrez-Aguirre, L. Urbas, P. Kramberger, N. Mehle, D. Barut, M. Ravnikar, M. Potato spindle tuber viroid PSTVd is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only — nucleotides and no coding capacity.

Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contaminated hands, tools, machinery, etc. This was due to the fact that the binding of the viroid to the C4 matrix was less strong than to the highly charged anion-exchange matrix and could be easier and more completely eluted under the applied chromatographic conditions.

One-litre-water samples were mixed with different viroid quantities and loaded onto the column. The materials obtained were applied as the stationary phases in simple and robust technique — planar chromatography PLC. The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized.

The GMA—EDMA and BuMA—EDMA matrixes were successfully applied for the separation of low molecular weight compounds the mixture of several dies , as well as poly vinylpyrrolidone and polystyrene homopolymers of different molecular weights using reversed-phase mechanism.

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns.

Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis iTRAQ.

Affinity Chromatography: A Historical Perspective

Kolom monolith berbasis polimer organik poli- lauril metakrilat-co-etilen dimetakrilat disintesis secara in situ kopolimerisasi dalam kolom silicosteel dengan ukuran panjang 10 cm dan diameter dalam 1,02 mm. Pada penelitian ini, efisiensi pemisahan ditingkatkan dengan menggunakan kolom monolith poli- LMA-co-EDMA untuk memisahkan senyawa alkilbenzena melalui tiga parameter, yakni temperatur kolom, pemisahan secara isokratik dan pemisahan secara gradien. Pemisahan alkilbenzena yang lebih efisien ditunjukkan dari penggunaan mode gradien ditandai dengan nilai peak capacity , faktor retensi dan jumlah plat teoritis yang lebih baik. Perubahan waktu gradien berpengaruh terhadap faktor retensi dan peak capacity. Organic polymer-based monolithic column of poly lauryl methacrylate-co-ethylene dimethacrylate has been prepared by in- situ copolymerization inside of silicosteel column with the size of mm long x 1. This kind of monolith column used for separation of alkylbenzenes using reversed-phase high performance liquid chromatography HPLC.

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3 Comments

Newsmapoosong 27.05.2021 at 08:24

Protocol DOI:

Bladtefraser 28.05.2021 at 01:48

The internal structure of the monolithic column is created in such a way that many channels form inside the column.

Tony F. 28.05.2021 at 08:05

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques.

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