Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Cellular products or processes of preparing a cellular...
Reexamination Certificate
2002-10-28
2003-12-16
Foelak, Morton (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Cellular products or processes of preparing a cellular...
C210S656000, C210S681000, C521S134000, C521S139000, C525S328900, C525S329700, C525S330300, C525S333300
Reexamination Certificate
active
06664305
ABSTRACT:
The invention concerns a process of manufacturing of a chromatography material, a material obtainable according to the process an article comprising the material as well as a use of the material.
Chromatographic methods have been employed with great success whenever mixtures of substances have to be separated. Typically, the chromatographic processes are performed on a solid matrix. The quality of the separation and the respective reliability depends on the optimum chromatographic material. Mostly, the chromatographic matrices are porous matrices. By choosing the grade of porosity and/or the chemical nature of the surface of the matrices the chromatographic processes can be influenced and tailored for the respective separation problem. For example, if affinity chromatography is performed, an affinity ligand has mostly to be immobilized at the surface of the chromatographic material. Also in catalysis performed on solid matrix the porosity and general property of the surface of the matrix is important.
In these cases, for affinity chromatographic purposes as well as for solid phase catalysis, the ligand as to be immobilized on the respective surface of the matrix. Typically, as solid matrices grained materials having a certain particle size are used. The bond between the ligand, for example the affinity ligand, and the solid matrix has to be at least as stable as that the material survives the separation conditions. Furthermore, if the immobilization of a ligand has to be performed chemically, the reaction conditions have to be chosen in such a way, that the binding properties of the ligand are not adversely affected.
For example, immobilization of polypeptides on chromatographic media happens in an undirected manner (Turková, 1978). The immobilization can only imperfectly be influenced by adjusting the reaction conditions. In the case of immobilization of polypeptides, it can mostly only until a certain degree be determined which amino acids of the proteins can be linked to the matrix via activated moieties. It often happens that the amino acids being responsible for the affinity interaction between the ligand immobilized on the surface of the matrix and the molecule to be separated are not available anymore for specific reactions. Another difficulty may arise when the active part of the ligand cannot be reached by the binding partner in the mixture to be separated. Furthermore, due to the multipoint-attachment, the three-dimensional structure of the ligand bound to the matrix may be altered in such a way that the binding pocket will be deformed so that the substance to be separated cannot bind anymore (Walters, 1985).
In general the chromatographic media are designated in “gels” no matter whether or not they are solid or soft gels or are built from materials which are completely different to the properties of a gel (for example controlled pore glass). In solid gels such as Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden) trisacryl (BioSepra Inc. Marlborough, USA) or Macro-prep (BioRad, Richmond, USA) the gel forming macromolecules are arranged in bundles and the arrangement interacts with the fluidum of the mixture to be separated via capillary forces. In the soft gels (the genuine gels) like dextrane or polyacrylamid the polymer interacts with the fluidum directly such as a liquid. The gel can be interpreted as forming a single phase like a dynamic liquid (Jungbauer, 1994).
Typically, the following media are used in affinity chromatography: genuine gels from dextrane, agarose and polyacrylamid; silica material and chromatographic carriers which are stabilized via a ceramic skeleton as well as so called perfusion gels which consists from coated polystyrene (Afeyan, 1990). An important criterion for a chromatographic carrier or a carrier which can be used in the solid phase catalysis is its ability to be regenerated and, of course, an unspecific absorption as small as possible. Both criteria are determined by the chemical nature of the support but also by the method of the immobilization of the ligand on the surface of the carrier material as well as the ligand itself (review for regeneration of different chromatographic supports, Jungbauer, 1994). The method of regeneration of the matrix has to consider the chemical nature of the ligand and the method how the ligand was bound to the matrix.
Basically, there can be differentiated two methods for immobilization of ligands, one for inorganic carrier materials and a method for immobilization of ligands on organic carriers. In the prior art, the immobilization of a ligand has been performed during a multistep procedure. In particular:
1. introduction of an activatable moiety in or on the matrix, if necessary. The activatable moiety is normally a hydroxyl or a carboxyl group. In most cases, the introduction of an activatable group is necessary with inorganic substrates or supports. Also an activated moiety can be introduced. When bifunctional reagents are used for the activation, the activation of the matrix and the introduction of an activated “spacer” is performed in a one-step-reaction.
2. the activation of the carrier is the first step with the most organic chromatographic substrates if no spacer is introduced with a bifunctional reagent.
3. introduction of a spacer having a reactive or activatable group. When the spacer does not have a reactive group, a second step for activation has to be performed. If bifunctional reagents are used, step 2 and step 3 are reduced to a single step.
4. coupling of the ligand and
5. blocking of the remaining reactive groups.
If a spacer is used which has two reactive groups, an activation has not to be performed. In the case of organic substrate materials, the first step can be left out since free hydroxyl groups or other activatable groups are present.
Unfortunately, during coupling of a ligand on the matrix this happens typically in such a way that it leads to an inhomogeneous distribution of the ligand on the surface of the matrix (chromatographic substrate). For example the ligand concentration decreases from the outer to the inner portions of the particulate material. In this case the ligates (the substances interacting with the ligands) are in contact with a surface covered with the ligand with a density that is too high for efficient binding. On the other hand in the inner portions of the particular material the ligand density is too low. If block-polymers are utilized the problem may arise that during the immobilization process a gradient of concentration of the ligand is established. Due to this gradient, one will find an inhomogeneous distribution. Ligands for a protein or a biopolymer are very often not available since they are immobilized at sites which are not accessible for the proteins and other biopolymers.
It is very tedious to manufacture monoliths for affinity chromatography in high quantities with a reasonable batch to batch reproducibility. Each of the preactivated monoliths must be connected to a system equipped with at least one pump to deliver the reaction solution, such as ligand mixture, each piece of monoliths must be tested individually for its functionality such as dynamic binding capacity, ligand density, leakage and/or resolution.
Therefore, it is the object of the invention to provide a process of manufacturing of a chromatography material avoiding the above mentioned drawbacks and to improve the ligand density which is necessary for an optimal separation of mixtures or conversion of substrates, and to provide a material which can be used as a chromatographic carrier or substrate for conversion of substances.
According to the invention this object is achieved by a process of manufacturing of a chromatography material comprising the steps of
(i) reacting a polymerisable at least bifunctional monomer A with a ligand also having a functional group which binds covalently with one of the functional groups of said polymerisable bifunctional monomer A,
(ii) to a compound B comprising at least one polymerisable functional moiety
(iii) polymerizing the compound B
Barut Milos
Hahn Rainer
Jungbauer Alois
Podgornik Ales
Strancar Ales
BIA Separations d.o.o.
Foelak Morton
Jacobson & Holman PLLC
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