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Inorganic monolithic sorbents for Biochromatography

#A-1214


Novel Inorganic Monolithic Sorbents for Chromatography of Biologically Active Substances

Tech Area / Field

  • BIO-CHM/Biochemistry/Biotechnology
  • CHE-POL/Polymer Chemistry/Chemistry
  • CHE-SYN/Basic and Synthetic Chemistry/Chemistry

Status
8 Project completed

Registration date
17.12.2004

Completion date
08.06.2009

Senior Project Manager
Safronova O N

Leading Institute
Scientific Research Chemical Technological Institute "CHEMTECH", Armenia, Yerevan

Collaborators

  • Xenogenetik GmbH, Austria, Vienna\nUniversité Louis Pasteur, France, Strasbourg\nNexttec GmbH Biotechnologie, Germany, Leverkusen\nBAM, Germany, Berlin

Project summary

At present monoliths are becoming an intensively studied subject due to their interesting hydrodynamic characteristics.

Monoliths are novel chromatography sorbents cast as a homogenous phase into chromatography columns as a single continuous piece containing highly interconnected channels in contrast to regular chromatographic sorbents which are packed as inpidual particles. Since the mobile phase is flowing through these channels, molecules to be separated are transported by convection rather than by diffusion. Due to their structure monolithic sorbents show flow rate independent separation efficiency and dynamic capacity as well as higher porosity when compared to conventional columns offering very fast analysis and rapid purification processes.

Compared to traditional sorbents monolithic materials combine a high selectivity and capacity, high flow rate of the mobile phase and mass transfer efficiency in a unique manner. Because of their physical properties, size and configuration they enable rapid separations of both small and large molecules such as proteins and nucleic acids with high resolution.

Despite all the advantages of monolithic sorbents, at present there are only a few commercially available monoliths for chromatography. In the main, there are monoliths on the basis of organic polymers (polymethacrylate, polyacrylamide, polystyrene, polyurethane, etc). They offer excellent biocompatibility, a wide effective working range of pH and stability in extreme media. However, the usage of polymeric monoliths in chromatography has several disadvantages. The general drawback of these materials, as is typical for all polymeric stationary phases, is their lower efficiency compared with silica gel phases. Most polymers, especially those with low degrees of cross-linking, swell or shrink in organic solvents (that could have undesirable effects upon the chromatographic performance), and they also have low mechanical stability.

Taking this into consideration, the perspective to create and to use new types of monolithic sorbents, e.g. on the basis of silica materials, devoid of the above mentioned disadvantages, becomes obvious. At present, monolithic silica columns are used in HPLC and capillary electrochromatography as capillaries. They are characterized by high efficiency of separation and mechanical stability, do not swell in solvents, have a rigid structure. On the other hand, they have a limited stability in acid and/or alkaline media, have low capacity.

Modern development of biotechnology requires the creation of new, relatively cheap, high performance and high capacity monolithic sorbents, possessing a complex of necessary reproducible properties. Monolithic sorbents, which combine useful properties of both polymeric and silica gel monoliths, could become such materials. Having a broad assortment of such monoliths (e.g. as discs, cylinders), a column with required properties for rapid isolation and purification of various biologically active substances from various biological objects can easily be constructed.

Objectives of the Project


- development of new relatively cheap, high-efficiency, high-capacity and selective organo-ceramic monolithic sorbents with bimodal porous structure, presented by throughpores and meso/macropores;

- functionalization of their surface by biospecific ligands (oligonucleotides, antibodies, lectines, etc.);

- study of their physico-chemical properties;

- their application in biochromatography for rapid separation, isolation and purification of various biologically active substances.


Technical Approach

In order to obtain organo-ceramic monolithic sorbents, synthetic multi-channel permeable ceramic discs and cylinders will be used as supports. Silica and alumo-silica coatings with predictable and reproducible porous structure will be applied on surface of such ceramic materials by sol-gel methods. The attractive side of sol-gel methods is the possibility to apply coatings with sorptive and ion-exchange properties on ceramic materials with different configuration. In addition, these methods provide:


- application and formation of coatings at relatively low temperatures;

- uniformity of coatings thickness;

- very fast formation of coatings;

- obtaining of coatings with specified porous structure, provided mainly by sizes of colloidal oxid particles.


Silica hydrosols with monodisperse spherical particles 10-80 nm in diameter will be used as raw materials for silica coatings application. In a number of cases alumo-silica sols, which ensure formation of coatings with increased ion-exchange properties will also be used. The main stages and conditions to form (alumo) silica coatings with different porous structure (including biporous structures with predictable composition of large and relatively small pores) will be studied. Application of silica and alumo-silica coatings will give an opportunity to:


- create the required porous structure in multichannel permeable ceramic monoliths;

- provide favorable conditions for modifying their surface by different substances with various functional groups.


In order to strengthen purposefully selective and sorption properties, to reduce to minimum the level of biomolecules nonspecific sorption, to impart necessary functionally-active ligands and to expand effective working range of pH, the obtained monoliths surface will be modified by high-molecular compounds, containing various functional groups and phases (RP-8, RP-18, carboxy-, epoxy-, amino-, amido-, other nitrogen-containing groups, etc.).

The choice of any functional groups will be based on the estimated value of interaction energy of concrete biologically active substances with the sorbent surface, on any property of separated biomolecules (their sizes, surface density of charge, chemical structure, hydrophobic properties, etc.). The conventional methods of (co)polymeric layers application, consisting in (co)polymerization of monomers and also in adsorption (chemosorption) of ready (co)polymers on silica-ceramic monoliths surface will be used.

In some cases, the surface of the obtained organo-ceramic monoliths will be exposed to additional functionalization by biospecific ligands (e.g. oligonucleotides, antibodies, metallocomplexes of porphyrins, etc.)

Thus, methods and conditions of modification (nature and composition of the solvent, temperature-temporal modes, chemical structure of monomers and polymers, porous structure of parent monolithic carriers, etc.), ensuring the required permeability of monolithic sorbents and their high separation capability will be studied and determined.

In order to study and determine the structure and properties of all parent, intermediate and finite substances, such modern methods of physico-chemical research, like scanning electron microscopy, IR-Fourier spectroscopy, mercury porosimetry, high-performance liquid chromatography (HPLC) and others will be used.

The approbation of the developed monolithic sorbents will be carried out on:


- separation and purification of nucleic acids from various biological objects (bacterial lysates, blood etc.);

- separation of oligonucleotides;

- retention of proteins, oligo- and polysaccharides, low-molecular organic substances and salts;

- multifunctional columns, packed with monoliths of different biological specificity.


Expected Results:

· Creation of new relatively cheap organo-ceramic monolithic sorbents for biochromatography, combining sorption-active meso/macropores with large throughpores and meeting the following requirements:


- high flow rates at pressures close to atmospheric (total porosity more than 70%);

- high sorption capacity (more than 1 mmol/g.) and pH stability (pH range 1-12);

- high efficiency of separation;

- high reproducibility and controllability of properties;

- ability to multifold regeneration and possibility of reuse.


· Creation of new inorganic monoliths, having simultaneously properties of microfilter, sorbent and ion-exchanger.
· Development of sol-gel process parameters, ensuring the formation of silica and alumo-silica coatings with predictable and reproducible porous structure on the surface of monolithic sorbents (specific surface: within the limits of 50-350 m2/g, pore size: 3-60 nm).
· Definition of optimal porous structure and chemical nature of the surface of modified monolithic sorbents in water-salt solvents in combination with properties of separated substances.
· Functionalization of monoliths activated surface by oligonucleotides, metallocomplexes of porphyrins and antibodies for affinity chromatography of proteins and nucleic acids.
· Creation of organo-ceramic monolithic sorbents with high capacity with respect to proteins, polysaccharides, low-molecular organic substances and salts and minimal nonspecific sorption with respect to nucleic acids.
· Study and installation of the mechanism of chromatographic separation of biopolymers.
· Determination of correlation among: polymerization variables – porous structure – permeability – selectivity of sorbents.
· Definition of the influence of alternative methods of modification of inorganic monoliths surface by polymers on structure of the formed polymeric layers.
· Development of technical notes on usage of the developed materials, specifically on construction of multifunctional columns.

The commercial value of the project results consists also in the possibility to use the developed monolithic sorbents, particularly, in one-stage process of DNA isolation and purification. It will enable to shorten significantly the isolation process, simplify its automation, reduce appreciably the expenses on isolation and decrease sharply the discharge into surrounding environment.

Besides, the results can be used in:

- creation of new types of bioreactors for biopolymers biotransformation;


- development of facilities and systems for purification (afterpurification ) of potable water;
- monitoring of the environment;
- creation of new types of catalysts for micro-heterogeneous catalysis of different reactions;
- creation of new systems for blood analysis and purification, etc.

The economic analysis of the developed materials will be carried out during the working process.

Meeting ISTC Goals and Objectives:


- Re-orientation of highly qualified weapons scientists and engineers towards peaceful activities;

- Supporting basic and applied research and technology development for peaceful purposes;

- Promoting integration of scientists of Armenia into the international scientific community.


The project will be realized by a group of highly qualified scientists and specialists from “Chemtech” Institute, who were previously involved in military programs of the Former Soviet Union. At different stages of the Project realization, experts from other interested Institutions could be involved as well.

Cooperation with foreign collaborators will be realized through joint experiments, seminars, testing of obtained materials, joint publications, mutual consultations, etc.

“Chemtech” Scientific Research Chemical Technological Institute has all necessary conditions for successful realization of the projected tasks: availability of modern research base, experience in this area of study and the results preliminary obtained.

The realization of the previous project: ISTC (# A-418, “New types of mineral-polymeric sorbents for chromatography of biologically active substances”) allowed, in particular, to develop powder sorbents for one-stage isolation and purification of nucleic acids from various biomaterials. Such sorbents were presented on World Fair "Analytika 2004" (Munich, Germany) and awoke the interest of many firms and companies. The performance of the presented project will make possible to develop such sorbents as relatively cheap monoliths. It will certainly contribute to the commercialization of the project results.

This project proposal is a integral part of the partnership project entitled “General principles for obtaining new types of organo-mineral monolithic sorbents for chromatography of biologically active compounds”, also presented in ISTC. The general purpose of the work will be reached at the realization of the tasks that are set in both projects (partner's and regular). The Partner Proposal is presented at the suggestion of one of the regular project collaborators (Nexttec GmbH Biotechnologie, Leverkusen, Germany). It includes some specific tasks, which are a special interest for the partner (in particular, application of polymeric layers with the help of olygomers with different hydrophilic-hydrophobic balance).

The initiators of the Project invite all interested scientific centers, research organizations, firms, companies, and inpidual experts from the USA, European community, Japan, Norway, Republic of Korea, and other countries, to cooperate within the framework of the Project. We also propose the realization of joint seminars and scientific investigations.


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