JPH03503212A - Affinity matrix of modified polysaccharide supports - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention Affinity matrix for modified polysaccharide supports) IJ U.S. Patent filed December 8, 1987 entitled “Affinity Matrix Comprising a No. 130.186, which is a continuation-in-part of application no. "Affinity Matrix Comprising a Support," filed January 27, 1987, U.S. Pat. This is a continuation-in-part of patent application no. No. 4,639,513, issued January 27, 1987, 198 titled ``Intrainjectable immunoglobulin G (IgG) and method for producing the same'' This is a continuation-in-part of U.S. Patent Application No. 656.922, filed on October 2, 2016. That U.S. patent application is currently U.S. Patent No. 4.663, issued May 5, 1987. ．． No. 163, filed February 2, 1984 entitled "Modified Polysaccharide Support" This is a continuation-in-part of U.S. patent application '5576,448, and the U.S. patent application is concurrently pending. filed February 14, 1983, entitled "Modified Polysaccharide Support," now abandoned. Continuation-in-part of U.S. Patent Application No. 466,114 The present invention relates to chromatography The present invention relates to carrier supports, such as supports for commercial use, and methods of preparation and uses thereof. More details The present invention relates to graft polymerization onto substrates for affinity chromatography media. A carrier support consisting of a polymer carrier prepared by the present invention may also allow the sample to flow radially or tangentially relative to the carrier support. Any supporting chemical structure related to chromatography equipment Highly selective by using recognition sites of biological macromolecules or biopolymers for Affinity chroma enables efficient separation of molecules with specific characteristics. Conventional technology uses materials with various chemical structures as supports. I've been using it. For example, agarose gels and cross-linked agarose gels are the most widely used It is a support material that has been utilized. These substances are hydrophilic and therefore do not cause non-specific binding. However, due to its compressibility, it is suitable for large-scale processing such as manufacturing. are not very attractive as carriers. Controlled pore glass (CPG) beads also It has been used in affinity chromatography. Filled with CPG It is possible to increase the throughput by using a column, but this carrier is made of agarose. More expensive than gel beads. Cellulose particles are also used for synthetic affinity solvents. It has been used by epidemiologists. However, compared to agarose gel , the formation of cellulose particles is more difficult and therefore the preparation of affinity solvents for enzymes has not received much attention. However, cellulose is the most common support material for all It is probably the cheapest of the Ricks. Two less frequently used supports The matrix consists of polyacrylamide gel beads, dextran and Ebichrome. The product name 5ephadex@ is a gel filtration material made from lohydrin. Beads from Le. Although convenient methods have been developed for using these , these beads have poor column packing properties due to their softness, and their low molecular weight Porosity reduces the availability of ligands for coordinating substances relative to solvents. I'm going to do it.

U.S. Pat. No. 4,281,233 to Coupek et al. Affinity polymer consisting of copolymer of rylate or methacrylate and crosslinkable monomer This shows a carrier for chromatography. This copolymer is covalently Contains linked monosaccharides or oligosaccharides. (Technically, oligosaccharides can contain up to 9 The carrier of bioactive substances having constituent monosaccharides is also the rice of Nakashima et al. It is disclosed in National Patent No. 4.352.884. This Nakash ima The carrier consists of a substrate coated with a copolymer. This substrate is one of various substances. Among them are inorganic materials such as glass, silica, alumina, synthetic polymers such as polystyrene, polyethylene, etc., and even cellulose. Contains eel natural polymer. Copolymers are hydroxy or alkoxyalkyl Hydrophilic acrylate or methacrylate such as acrylate or methacrylate monomer and an unsaturated carboxylic acid or amine that can be copolymerized. pace The substance or substrate is coated by conventional coating or application methods such as spraying, dipping, phase separation, etc. coated with this copolymer. This copolymer also contains glycidyl acrylate. A small amount of a crosslinking agent such as a methacrylate or a methacrylate may be present. This crosslinking agent is used in the coating process. It enables the subsequent cross-linking treatment and prevents elution from the coating layer (possibly due to the release of bioactive substances). ) will be prevented.

The amount of crosslinking agent is very small, between 0.5 and 1% by weight of the total weight of the copolymer. within the range of At such amounts of crosslinker, the copolymer's properties relative to the underlying substrate may be reduced. It is insufficient to cause qualitative covalent bonding or grafting. Thus Nakas The hima copolymer is essentially just a physical coating of the underlying substrate. Only. However, physical coatings come with a series of problems. The carrier contains A homogeneous distribution of the polymer cannot be expected, exhibiting a multilayered structure and lack of functional groups. Possible uniform distribution.

Another document of interest is Kraemer U.S. Pat. No. 4.070.348, It is a carrier of polysaccharides, enzymes, peptides, hormones and other biologically active substances. Copolymers of glycidyl- or amino-containing acrylates useful as Ru. The structure of Kraerner's final product is an acrylic copolymer chain comprising: Substances such as enzymes, proteins, etc. that are covalently denatured at multiple sites. This is what is happening.

The above discussion of the prior art, its advantages and disadvantages leads to the following conclusions: . Based on ion exchange chromatography and affinity chromatography A useful support for both low- and low-temperature purification, with a high degree of stability and a high degree of porosity. , low non-specific adsorption, high flow rate, incompressibility, and segmental gelation. There is a need for products that can be used for biological separation on an industrial scale. That is true. The shortcomings of the prior art mentioned above have the most important impact on meeting this need. It is especially in manufacturing at the industrial level that the effects are strongest.

On an industrial scale, fibrous Mar-IJ hooks with immobilized microparticles Molecular separation materials are disclosed in commonly assigned Crowder U.S. Pat. No. 4.384. ．． No. 957, the contents of which are incorporated herein by reference. We will incorporate it. This patent covers fine particles, refined small fiber valves, and long Formed by wet-laying sheets from an aqueous slurry of hard, soft fiber pulp. describes a composite fiber material made of fibers.

Using a matrix of fibrous/m particles, adding a cationic polymer to the slurry, By crosslinking this polymer to the matrix by drying in an oven, A filtration matrix is provided whose surface is coated with a positive charge. this A matrix with a charge such as It can be used to filter particles by adsorption. (For example, 0strei cher U.S. Pat. Nos. 4.007.113 and 4.007.114; See U.S. Pat. Nos. 4,305,782 and 4,309,247. Among these All contents are incorporated herein by this reference. ) cationic substances In the prior art wet slurry step using a slurry containing A distributed substance is obtained, and the multilayer coating is carried out in one place only, and in other places on the surface. It was inevitable that it would be exposed. Impurities that need to be removed The amount of the liquid is relatively small compared to the large volume of the liquid, and the uneven charge distribution makes the filter Due to the fact that the depth of the It is possible to use it. However, such products are sensitive to ion exchange It cannot be easily applied to processes. The throughput of such processes is The number of active sites as well as the accessibility of the active sites are important. For ion exchange materials The chemical functional groups used should not be buried close to the surface and should be , it must be displaced somewhat from the surface, perhaps by the side chains of the molecule. child One way to achieve this is by chemically modified silanes. This has been done by incorporating microparticles within a fibrous matrix. These scenes Runs can be performed using functional groups such as DEAE%CM or by affinity chromatography. It carries parts. They are mechanically stable, strong and non-swelling.

However, these are expensive, and the silica hydroxyl group makes them difficult to target proteins. It shows very strong non-specific adsorption.

Generally speaking, affinity techniques commonly used in laboratory-scale purification The support for chromatography is also fine particles (or modified fine particles for ion exchange). Fibrous matrices for chromatography or filtration, including They have been shown to be less useful for complex affinity purification processes.

Therefore, it is incompressible, adjustably expandable, has high exchange capacity, and exhibits high flow rates. affinity chroma on an industrial scale, which is flexible and relatively inexpensive to produce. Supports useful in tographic purification processes, as well as affinity matrices comprising such supports. There continues to be a need for ricks and equipment.

Summary of the invention Therefore, the object of the present invention is to develop a novel molecular support consisting of a conjugate of a polymeric carrier and a substrate. The goal is to provide the following.

Another object of the invention is to perform affinity chromatography or reverse phase chromatography. The purpose of this invention is to provide a useful molecular support for biochemistry.

Yet another object of the invention is to be useful in chromatographic operations on an industrial scale. An object of the present invention is to provide a support for chromatography.

Yet another object of the invention is affinity chromatography and reverse phase chromatography. The purpose of this invention is to provide an industrial process for tography.

Another object of the invention is to perform affinity chromatography and reverse phase chromatography. Method for preparing a support for affinity chromatography and a medium for affinity chromatography The goal is to provide the following.

Yet another object of the invention is to provide a radial or tangential alignment of the sample with respect to the carrier support. The object of the present invention is to provide a device that enables flow.

These and further objects of the present invention will become more apparent hereinafter. This was achieved by providing the following: That is, 1. A polysaccharide covalently bonded to a synthetic polymer; 2. The synthetic polymer has a) a polymer having a chemical group capable of being covalently bonded directly or indirectly to the polysaccharide; A compound that can be combined with b) the affinity of said polymerizable compounds) for said ligand or biologically active molecule; one or more polymerizable groups containing chemical groups capable of forming covalent bonds; made from compounds capable of A modified polysaccharide substance consisting of

A further object of the invention is that the modified polysaccharide substances described above are suitable for coupling of ligands. affinity, which is further modified to form a pre-activated intermediate compound, such as In providing molecular separation precursor compounds (hereinafter referred to as "preligand intermediates") more accomplished.

Another object of the present invention is to provide affinity molecular separation substances derived from the above-mentioned polysaccharide substances. This affinity molecular separation material was achieved by providing polysaccharide substrates and a synthetic polymer covalently bonded to a polysaccharide substrate and covalently linked to this synthetic polymer. Chromatography for binder affinity chromatography and reversed phase chromatography Capable of acting as a graphical support or reagent for biochemical reactants It consists of affinity ligands.

Yet another object of the invention is to provide a molecular separation process and/or production process using the above-mentioned substances. This was achieved by providing a chemical reaction process.

In addition, a further object of the invention is to chromatograph at least two components in a sample. This is a device for separating denatured polysaccharide substances using a roughy method. be configured to permit radial or tangential flow of material; A brief description of the drawing achieved by The invention will be appreciated by reference to the detailed description given below, considered in conjunction with the accompanying drawings. be better understood. In the accompanying drawings, Figure 1 shows one of the chromatography columns of the present invention. a partial sectional view of a side elevation of two embodiments; Figure 2 is an enlarged cross-sectional view taken along line 2-2 in Figure 1, and Figure 3 is an illustration of the solid stationary phase. The helically wound chromatography medium and the space between them a perspective view of the radial flow cartridge showing the spacer means; FIG. 4 shows another embodiment of the invention in which the chromatography column is disc-shaped. cross section, FIG. 5 is a top plan view of an inlet housing member of an embodiment of the invention that is disc-shaped; FIG. 6 is a top plan view of an outlet housing member of an embodiment of the invention that is disc-shaped; FIG. 7 is a top plan view of one embodiment of the stationary phase of a column of the invention in the form of a disc; FIG. 8 is a side elevational view of one embodiment of the stationary phase of the column of the invention in the form of a disc; FIG. 9 shows that prior to ultrasonic welding of the peripheral edge, multiple layers of separation media and this separation are applied. Disc-shaped invention showing spacer means interposed between adjacent layers of media a cross-sectional view of one embodiment of the stationary phase of the column, FIG. 10 is a cross-sectional view of a preferred shape of the disk-shaped ion exchange column of the present invention. In this configuration, the disc-shaped housing expands radially outward. forming a chamber with a portion of the spacer means removed for clarity. Figure 11 shows a spirally wound chromatograph with part of the solid stationary phase broken and removed. One of the tangential flow cartridges showing the feeding medium and spacer means and impermeable membrane. A perspective view of the embodiment, FIG. 12, shows one of the tangential flow cartridges within the cylindrical housing. A cross-sectional view of the embodiment, FIG. 13 shows one method used to construct a tangential flow cartridge, Figure 14 is a schematic diagram showing a comparison of radial and tangential flow of blood in the device. Mu, Figure 15 shows a book that provides tangential flow across two layers of chromatographic media. A cross-sectional view of another embodiment of the invention, FIG. 16, has a double layer of chromatographic media. shows a top plan view of a tangential flow cartridge, Figure 17 is a cross-sectional view of one turn of the double layer tangential flow car) illustrating the flow of the sample relative to the carrier and the spacer and channel means; Figure 19 shows the surface area vs. surface area for three media (A, protamine B1 and C). A graph of percent heparin removal is shown in Figure 20 for two media (A and Pro Shows a graph of percent removal of sulheparin versus contact time for (tamin) , Figure 21 shows the relationship between Gantt concentration (protamine) and Gantt concentration (protamine) for two surface area values. Figure 22 shows a graph of the amount of palin binding, and Figure 22 shows the flow rate for blood and physiological saline. shows a graph representing the pressure difference (Δp) between the inlet and outlet of a tangential flow cartridge. Figure 23 shows a graph of percent heparin removal versus filtered blood volume. show, Figure 24 shows the tangential flow cartridge input versus flow rate for blood and saline. FIG. 25 shows a graph representing the pressure difference (Δp) between the inlet and the outlet, and FIG. Graph of percent heparin removal versus blood volume filtered through the IJ. Fig. 26 shows the connection containing immobilized protein A for various flow rates. shows a graph representing the pressure difference (Δp) between the inlet and outlet of a linear flow cartridge; Figure 27 shows purification through a tangential flow cart containing immobilized protein A. 2 shows gel electrophoresis of IgG obtained from plasma and blood.

Brief description of the preferred embodiment The present invention covers a wide range of applications including affinity separations and chromatographic separations. as an insoluble support for applications in or as an insoluble support for bioreactors Concerns the discovery and development of useful materials.

This support material is based on a complex of organic synthetic polymers and polysaccharides. good In preferred embodiments, the complex itself is biologically inert. Organic combination The polysaccharides carry chemical groups that can bind to the polysaccharides, and of providing anchoring performance for affinity ligands or biologically active molecules in general. It carries chemical groups that can be used.

Strictly speaking, the polymer attached to the polysaccharide is either a copolymer or a homopolymer. Good too. A chemical group that can bind to a polysaccharide is an affinity ligand or biological When the same chemical group is useful as an anchoring unit for an active molecule, The mer is a homopolymer in this particular form. However, in another form In this case, the polymer is a copolymer, with a group capable of binding to the polysaccharide and an additional molecule. It also includes different groups that can function as anchoring groups. one In a preferred embodiment, the polymer is a homopolymer.

The present invention also provides affinity chromatography or bioreactive substances. by attaching affinity ligands or biomolecules, or by reverse phase chromatography. Modified polysaccharides by attaching hydrophobic substituents for matography It also relates to substances derived from

The invention also provides unmodified polysaccharides with modified or unmodified particulate matter. It also concerns mixtures of the substances mentioned above or their mixtures to provide various separation media. do.

The present invention may also be configured to provide radial or tangential flow of the sample relative to the support. The present invention also relates to a device made of the modified polysaccharide material of the present invention.

Material The term "polysaccharide" as used in the specification and claims refers to It is intended to include compounds consisting of hundreds or even thousands of constituent monosaccharides. this Their constituent monosaccharides are held together by glycosidic bonds. Its molecular weight is It is usually about 5000 or more and can range up to several million Daltons. So These are usually naturally occurring polymers, such as starch, glycogen, and sesame. Such as ululose, gum arabic, agar and chitin. This polysaccharide is one or more reactive hydroxyl groups. It is a straight chain or a branched chain. sell. The most useful polysaccharide for purposes of this invention is cellulose.

The polysaccharide is preferably completely unprotected, retaining all of its hydroxyl groups in a free state. are doing. For example, by acrylation or aminoacrylation, some of the hydroxyl groups can be It is possible to block something.

However, it is not preferable to excessively block the hydroxyl groups of the polysaccharide. because The polysaccharide thereby loses its hydrophilicity, a property that is chemically compatible with biomolecules. This is because it is necessary to bring about appropriate sexual interaction. Polysaccharides become hydrophobic Too much can lead to potential non-specificity due to negative interactions with molecules such as proteins. Binding and denaturation phenomena are introduced. Also, the hydroxyl groups of polysaccharides are excessively masked. , the reactivity of the resulting material with the polymer is greatly reduced.

For all these reasons, it is preferable to keep substantially all hydroxyl groups free. stomach. However, polysaccharides can be chemically activated as shown below. Wear.

Cellulose is a preferred polysaccharide. "Cellulose" makes wood valves, cotton, linen Convenient separators in commercially available forms of recycled formulations such as ramie or rayon. is intended to mean either lurose. A suitable form of cellulose There is no importance when it comes to choosing. Cellulose is a glucose unit It is a naturally occurring polysaccharide consisting of β (1>4) bonds. In its natural state, cellulose Adjacent chains of are extensively hydrogen bonded, forming microcrystalline regions. . These regions are interspersed with amorphous regions with fewer hydrogen bonds. limited By performing acid hydrolysis, preferential loss of amorphous regions occurs, resulting in so-called microcrystals. Cellulose is obtained. Cellulose useful in the present invention may be in its native state or in its finely divided state. It is cellulose in a crystalline state. In addition, the cellulose derived from cotton linters is better than cellulose obtained from bulbs, since the latter contains lignin. This is for the purpose of

The chemical reactions that attach polymers to polysaccharide materials typically proceed in microcrystalline regions. is difficult, but occurs more easily in amorphous regions.

Substitution of functional groups into cellulose, for example, has a destructive effect on its structure. Ru. When the reaction is run to complete completion, the cellulose matrix is destroyed. Finally, a water-soluble polymer is formed. A typical example of this phenomenon is Hydroxyethylcellulose and cellulose rubber are soluble in water. It then becomes a commonly used adhesive and bonding agent.

Each of the anhydrous constituent monosaccharides in the polysaccharide molecule has three or more reactive hydroxyl groups. do. Theoretically, it is possible to replace all three or more of these with polymers. can. However, the products of such reactions may contain three or more substitutions. In the case of ion exchange materials, they become soluble. Fully water soluble Even at sub-substitution levels, such polysaccharide derivatives are chromatographically It is no longer suitable as a support for feet. Therefore, substitution of polysaccharides is similar to that of amorphous regions. is confined to the reactive center and has a dry weight of approximately 1 mEQ/gm in fibrous form. It is rarely executed beyond the level.

At this level of substitution, the natural configuration of the polysaccharide structure is only slightly modified. large biomolecules have easy access to low-density, heterogeneous exchange sites. Ru.

The final structure of the molecular support of the present invention thus has multiple positions along the polysaccharide chain. It consists of polysaccharide chains that have been covalently modified with synthetic polymers.

Polymers that modify polysaccharides are either homopolymers or copolymers. homo The definition of polymer as a polymer or copolymer is defined as a polymerizable compound (a) and and (b) are different. In one form, the copolymer is a random copolymer. It can be a polymer, a block copolymer, or an alternating copolymer.

In one embodiment, the polymerizable compound (a) (if the polymer is a copolymer) Comonomer (a)) reacts with the hydroxyl groups of polysaccharides by forming covalent bonds. It has a group that can be Such polymerizable compounds are, for example, Kraem As defined in U.S. Pat. No. 4,070,348 to er et al. These compounds are Incorporated herein by this reference. The above chemical groups are polysaccharides At temperatures that initiate decomposition or depolymerization, such as temperatures from 0° to 120°, It is possible to react with the hydroxyl group in aqueous solution, thereby making the oxygen atom of the hydroxyl group form a covalent bond with. Water is always present in considerable excess relative to hydroxyl groups. Therefore, chemical groups that react spontaneously with water, such as incyanate groups, is less appropriate. The aqueous solution is pure water or water and water-miscible alcohol. , a mixture with one or more co-solvents such as ketones, etc.

The hydroxy reactive groups of comonomer (a) are preferably those known in peptide chemistry. activated carboxyl groups, or halogenated alkyl groups or epoxidized alkyl groups, such as O-alkylating agents such as alkyl groups. Typical examples of 0-alkylated comonomers are , acrylic or methacrylic anhydride, acryloyl methacryloyl N-hydroxy Succinimide, ω-iodoalkyl ester of acrylic acid or methacrylic acid and the alkyl group generally contains 2 to 6 carbon atoms, allyl chloride, chloro Romethylstyrene, chloroacetoxyethyl methacrylate, and glycidyl group It is a compound with The last one is glycidyl alcohol and unsaturated alcohol. or an unsaturated carboxylic acid, respectively. be. Glycidyl alcohol is aliphatic and cycloaliphatic alcohol and etherified alcohol. alcohol having 3 to 18 carbon atoms, and these carbon atoms are α, β- esterified with an unsaturated carboxylic acid, preferably acrylic acid or methacrylic acid, Alternatively, it is etherified with an olefinically or acetylenically unsaturated alcohol.

Typical compounds are glycidyl acrylate and methacrylate-1-14,5-epo Xypentyl acrylate, 4-(2゜3-epoxyprobyl)-N-butyl methacrylate), 9.10-epoxystearyl acrylate, 4-(2,3- epoxypropylene) - cyclohexyl methyl acrylate, ethylene glycol These are monoglycidyl ether acrylate and allyl glycidyl ether.

Preferred organic synthetic polymers include glycidyl acrylate and glycidyl methacrylate. Contains homopolymers of rylates.

One active monomer unit (a) is sensitive to hydroxyl groups and reacts with the provided polysaccharide. If not, they are transformed in the presence of water to form hydrophilic carboxyl groups, It becomes an aldehyde group or a hydroxyl group. The activated groups are therefore , generally present in greater numbers than necessary to bind the polysaccharide.

In another embodiment, the polymerizable compound (a) reacts directly with the hydroxyl groups of the polysaccharide. It is not a type of polysaccharide, but it is indirectly covalently bonded to a polysaccharide via a cross-linking compound. be able to. This is because the polysaccharide is first chemically activated by oxygen, etc., and can be polymerized. e.g. an epoxy group which can react with a suitable functional group of the functional comonomer (a). Or when reacting with a compound having a vinyl group.

The polymerizable comonomer (c) is a carrier material in the case where ra) is different from (a). They vary depending on the final use. The ultimate use of carrier materials is affinity ligands. comonomer (b) is an affinity ligand, i.e. ” chemical group capable of producing a covalent bond of this comonomer (b) to the group is held. Most affinity ligands have hydroxy, amino, or thiol groups. , retains nucleophilic moieties such as carboxyl groups, so it does not react with such nucleophilic moieties. There may be some electrophilic group present in the comonomer etc. that can Wear. Such electrophilic groups have the ability to react with the hydroxyl groups of cellulose. Activating groups include, but are not limited to, the groups previously described. do not have. They also include carbonyl chloride, carboxylic anhydride and carboxylic acid azide. activated group used in peptide chemistry for the formation of peptide bonds, such as Phenyl esters used to form ruboxyl groups as well as Schiff (imine) bases and aldehydes.

Also useful are the carboxylic acids of the hydroxylamino derivatives of formula (1) There is salt; In the formula, R is an α,β-unsaturated polymerizable radical, and Ro is a C0 to C6 atom. alkyl or alkanoyl group, phenyl group or hydroxyphenyl group. R” is a direct bond (-) or a C2 to C3 alkyl or alkanoyl group.

Typical compounds of this type include:

Other compounds with activated carboxyl groups include acryloyl chloride and methyl chloride. Tacryloyl, acrylic and methacrylic anhydride, maleic anhydride, phenyl Acrylates and methacrylates, glycidyl acrylates and methacrylates , 4-iodobutyl acrylate and methacrylate, and 2-isopropenyl -4,4-dimethyloxacylone-5. The last compound shown is a protein can react directly or via transformation with the terminal amino group of the amino group.

Very useful potential in comonomers useful for binding affinity ligands, etc. Reactive groups that are electrophilic can be activated with reagents such as cyanogen halides to make them electrophilic. It is a group that can become . Technically, cyanogen halides are 1,2-diols It is known that an activated structure having the following formula (3) is produced by reacting with the following formula (3).

This structure can then react with the nucleophilic portion of the affinity ligand. como Among the preferred 1,2-diols present in polymer (b) are glucose, polymer, Monosaccharides such as glucose and galactose, trisaccharides such as lactose and maltose It contains a variety of carbohydrates, including trisaccharides such as raffinose, or glycosides in general. be caught. This 1,2-diol-containing functional group can be used for esterification, amide formation, Attaching to the polymerizable comonomer (b) by reaction such as esterification or other Can be done. Among the most preferred of these are glycidyl acrylate or methacrylate reacts with carbohydrates to produce ether-containing comonomers, etc. There is.

If the ultimate use of the carrier material is as a carrier for biological molecules, comonomers It is also possible to use any of the anchoring groups mentioned for mer (a) or ra). It is. Other types of activating groups are also used, including aldehydes and amines. be able to.

The polymerizable comonomer (b) may be of substantially one type or one or more types. It can be a mixture of types.

Preferably, the polymerizable unsaturated monocompound etc.) is a polymerizable compound of formula (4) and R’ In the formula, R1 is hydrogen or methyl, A is CO or S02, X is 011, OM (M is a metal ion), or OR” (R” is a linear or branched chain C1 to cps alkyl group), 0R3011 (R' is straight chain or branched C2 c67JL, g) Lt or ha aromatic group'), ONR'R' or haON"R'R' R' (R' is the same as or different from R5, and this are hydrogen, R2 or R3011).

AX can also be of the form of formula (5), where Y? ;! -C02-1-C112C 02-1-so, -1-CH, 503-1-0PO, H-1-CI (2PO, 1 l-1-C1,N　(CH2COO-)　2,-CI+2-NR'R'Also bar C I+, -N"R'R'R', it, Iha the free acid corresponding to the above, It is a stellate or partial ester group. In these formulas, the groups R4, R'; ', R6, or R51R8 forms a 5- to 7-membered heterocyclic ring with a nitrogen atom. can be done. R4, R5 and R6 are as defined above.

When this material is used as a scaffold for affinity ligands or biomolecules, A is CD or SO, and X is most preferably 0-CI+2-CH(Oll)- CHz - Carbohydrate, in which case "-carbohydrate" is a monosaccharide, trisaccharide or polysaccharide, and the affinity cyanide halides to react with nucleophilic reactive groups on reactive ligands or biomolecules. Therefore, it has a group that can be activated. Como more preferred as a sticking substance Nomer et al.) described the reaction of glycidyl acrylate or methacrylate with glucose. It is a comonomer obtained from

The average molecular weight of a polysaccharide-modified polymer is a function of the amount of monomer mixture present in it. be. Covalent bonds can only form throughout the amorphous region of the polysaccharide surface. It is necessary to have at least a sufficient number of comonomer (a) units of .

The number of comonomer (b) units must not be too small. because otherwise , exchange ability, or fixation/interaction ability are insignificant. Ko The number of monomer (5) units should not be too large. Because it is a comonomer ( This is because it causes great difficulty in the reaction between the reactive group in a) and the polysaccharide. good Preferably, the polysaccharide-modified copolymer contains between 1 and 500 units of comonomer (a) and most preferably between 20 and 100 comonomer units). be. This is between about 100 and 100,000, preferably between 1,000 and 10 ．． Corresponding to a molecular weight between 000 and 000.

If desired, comonomers different from the comonomers indicated by (a) or (a) above may be used. It is also possible to add other neutral comonomers (C) to the polymer. This comonoma - is a neutral chemical group such as a hydroxy group, an amide group, an alkyl group, an allyl group, etc. It can be a polymerizable unsaturated compound having Preferred among comonomers (C) are C1 to C6 alkyl acrylates or methacrylates, or the corresponding hydroxyalkyl acrylate or methacrylate. Comonomer (C ) function is to increase the abundance of hydrophobic or hydrophilic residues in the polymer, The goal is to provide the desired balance of hydrophilic and hydrophobic groups where necessary.

The minimum proportion of comonomer (a) to the total content of comonomers is important. synthesis The polymer is conjugated to the polysaccharide to allow for substantial covalent bonding of the polymer to the polysaccharide. It should have sufficient amount of comonomer (a). Comonomers present in polymers If there are too few ■ in mar (a), grafting will be difficult if not impossible. be. Generally, for the sum of (a) plus et al.) and (C) (if any) About 4-12, preferably 5-10% by weight of comonomer (a) is required. about In amounts of 0.5 to 1 or 2% by weight, substantially grafting to the polysaccharide It merely cross-links the polymer without any additional cross-linking.

The upper limit of the amount of comonomer (a) in the polymer is determined by the desired stiffness and functionality. and can vary up to 99.9% by weight depending on the degree of hydrophilicity. Ru. Preferably, comonomer (b) is predominant over comonomer (a). Ko Monomer (C) may be up to 20% by weight of the total of (a) plus (b) plus (C). Vine exists in quantity.

The weight ratio of polysaccharide to modifying polymer can be adjusted freely, and The amount of comonomer varies from 0.1 to 5 parts by weight.

Ionizable chemistry in which comonomer (b) can provide cation exchange capability Unless some degree of cross-linking is carried out, the substance is Flexibility in solution tends to contribute to the formation of micelle-type aggregates, It was found that the force was slowly lost. Therefore, these types of modified polysaccharides Therefore, it is a preferred embodiment of the present invention to perform polymer crosslinking. The bridge is , such as mono- and polyethylene glycol dimethacrylates and diacrylates (polyethylene glycol residues contain up to 6 ethylene groups), ethylene glycol residues methacrylate, ethylene diacrylate, tetramethylene dimethacrylate, Tetraethylene diacrylate, divinylbenzene, triallyl cyanurate, At least two Polymerizes a small amount of polyunsaturated comonomer with two polymerizable α,β-carbon double bonds. It can be brought about by incorporating it into a combination formula.

Another type of crosslinking agent is copolymers made from aminoalkyl comonomers (aminoalkyl comonomers, etc.) Applicable to Due to the presence of a lone pair of electrons on the aminoalkyl nitrogen atom, nitrogen Crosslinking can be carried out with bifunctional reagents that react with elementary lone pairs of electrons.

Among them are halogens such as Hal-CO-(CHi)nCo-tlal. diacyl or an alkyl halide such as Hal-(CHa)nl1al , where 1al is a halogen such as chlorine, bromine or iodine, and n is 2 or and 12. Other bifunctional reagents that can react with nitrogen atoms are also available. can be used.

The advantage of these bifunctional reagents is that they simultaneously crosslink the copolymer while It also imparts a cationic charge to the nitrogen center, thereby ionizing the substance. It is about doing.

The amount of crosslinking agent is best determined by experimentation. polymer over a long period of time It is considered sufficient to maintain a constant exchange capacity, but If expansion is prevented and too much stiffness is obtained, it is considered too much. It will be done. Ideally, in the range of 5 to 20 mole percent per unit of synthetic polymer. The amount of crosslinking agent is sufficient.

As used throughout this application and claims, the term "affinity ligand" The term can be immobilized on a stationary phase, affinity chromatography some small fraction used to purify complementary binding molecules from the solute phase by It is intended to include molecules of molecular weight or high molecular weight. Ligands are e.g. inhibitors , cofactors, prosthetic groups, or macromolecular substrates, all of which are enzymes. It is useful for purifying enzymes and holoenzymes. Other ligand/coordinate pairs include enzyme/macromolecule inhibitors; - heavyweight nucleic acids/complementary strand nucleic acids: habten or antigens/antibodies; ;Antibody/Protein or Polysaccharide: Monosaccharide or Polysaccharide/Lectin or Receptor: Lectin/ Glycoprotein or receptor; small target compound/binding protein; binding protein /There is a small target compound. Antigen/antibody pair as a ligand/coordinate pair When used, this technique is called "immunoafinity" chromatography. It has a special name.

"Biologically active molecules" that can be bound to the carrier of the present invention include enzymes, enzyme groups, etc. substances, inhibitors, hormones, antibiotics, antibodies, antigens, peptides, carbohydrates, nucleic acids, etc. I can see it. The only requirement for these molecules is that they are suitable for anchoring synthetic polymers. It has a reactive group that can be covalently bonded to a chemical group.

Of particular interest are hydrolases, isomerases, proteases, and amylases. This is the immobilization of enzymes such as enzymes. These immobilized enzymes are otherwise is well known in the art and can be used in biochemical reactors. can.

The term reversed phase chromatography or “hydrophobic interaction chromatography” The use of chromatography is used to adsorb hydrophobic components present in a mixture. is intended to include. Such components include lipids, cell fragments, etc. is included. In this example, comonomer (b)(2) typically comprises C, Acrylate or methacrylate esters of straight or branched chain alcohols of cps , or acrylates or methane of aromatic alcohols such as phenol or naphthol. It is a tacrylate ester.

As such, the carrier material of the present invention is in the same state as other polysaccharide-based carrier materials of the prior art. It can be used in various ways. Alternatively, and in a preferred embodiment The polysaccharide material, preferably in fibrous form after denaturation, is subjected to affinity chromatography. such as fibrous sheets with graphic properties, bioreactivity or reverse phase properties. A self-supporting fiber matrix) can be formed by IJ Fuchs. This modified fiber The fiber-purchased polysaccharide fibrous media can also incorporate unmodified fibers of a variety of different dimensions. Additionally, modified or unmodified particulate matter can be incorporated.

A fibrous medium consists of a porous matrix of fibers in which books Because of the features of the invention, the fibers are effective in molecular reactions.

This matrix is substantially homogeneous with respect to each component. selectively, granules It doesn't matter if there are children. If such particulates exist, it is also possible to separate molecules Alternatively, it is preferable to modify it so as to make it effective for the reaction. such fine particles the fibrous phase in an amount effective to achieve the desired separation or reaction. Should.

Overall, the medium is substantially inert and dimensionally stable.

Preferred microparticles that can be used are those provided in finely divided form. chromatographic functionality, i.e. effective molecular separation and Contains all substances that can react and/or react.

Mixtures of such compositions may also be utilized. Examples of such particulates are silicon clay such as mosquito, alumina, zirconium oxide, diatomaceous earth, perlite, vermiculite, Carbon such as activated carbon, modified polymer fine particles such as other ion exchange resins, microcrystalline cells There are loins, molecular sieves, etc., whose surfaces are treated by conventional methods. can be managed. These substances include Biosila, Hi-Plosi l, Li Chroprep Si.

Cropak Si, Nucleosil, Partis, Poras i], 5pherosil, Zorbaxcil, Corasil, Pa1l osil, Zipax, Bondapak, LiChrosorbHyp ersil.

Zorbax, Perisorb, Fractosil, Corning Such as Porous Glass, Dowex1Amberlite resin, etc. It is commercially available under various trade names.

An example of literature describing microparticles useful for separating molecules is the U.S. patent of Miller No. 3.669.841, U.S. Pat. No. 3.722.181 to Kirkland et al. No. 3,795,313 to Kirkland et al. U.S. Patent No. 3,983,299 to Chang, U.S. Patent No. 4.029.58 to Chang No. 3, Ste. U.S. Pat. No. 3,664.967, Krekeler U.S. Pat. No. 4.053.565 and 1her U.S. Pat. No. 4.105.426 be. The entire disclosures of all these documents are incorporated herein by this reference. I will do so.

The particle size of the particulates is not critical, but the size of the particles is not critical, but This will have some effect on the flow rate. For radial flow applications, approximately 5 microns Large, uniform particle sizes are preferred, with approximately 10 to 100 microns being practical. It is within the scope of ability. Tangential flow where the sample crosses the matrix material rather than through it For this application, a somewhat smaller particle size can be used. The amount of fine particles is , can vary widely up to about 80% by weight of the stationary phase. The optimal particle concentration is , depending on the desired molecular separation.

A fibrous medium is one that is capable of immobilizing the fine particles contained therein, i.e. It should be possible to prevent substantial loss of particulates from the phase.

In the case of a medium configured for radial flow, the medium is a medium through which the fluid passes. It should have sufficient porosity. However, the modified cellulose material of the present invention Even if it is self-bonding and does not require extra fibers or binders, such It is possible to use minute fibers or binders. Other fibers that can be used in this medium Includes polyacrylonitrile fibers, nylon fibers, wool fibers, rayon fibers and polyester fibers. Polyvinyl chloride fibers, other cellulose fibers such as wood bulbs and cotton, and cellulose acetate This includes the following:

One embodiment of the present invention includes a fibrous medium comprising two different types of cellulose. One of them is the modified cellulose according to the present invention, and the other is the modified cellulose according to the present invention. is unmodified cellulose.

Another embodiment of the present invention that can be attached to the cellulose described above is Good structural integrity both in the as-built condition and in the final dry condition. It is useful for providing sheets with good properties and also allows for handling during processing. It is an unrefined structural fiber that is suitable for its final intended use. like this These fibers are typically relatively large, with commercially available fibers ranging from 6 to 60 microns in diameter. Ru. Wood bulbs can also be used, with fiber diameters ranging from 15 to 25 μm. , having a fiber length of about 0.85 to about 6.5M. This unrefined self-bonding structural fiber The fiber typically has a Canadian standard freeness of +400 to +800 m1'. child These long self-bonding fibers may constitute more than 50% by weight of the fiber medium. preferably 60-100%, most preferably 100% of the fibrous medium. Ru. Selectively, +100 to 1600mj! Refined to Canadian standard freeness between small quantities of cellulose bulbs with normal dimensions (+400 to +800 m f) Can be incorporated into most cellulose bulbs. In particular, about 1 to about 20 % purified valve, and about 50% to about 90% unrefined cellulose in matrix can be included in the Microparticles can also be added.

When the particulate matter is millimicrons in size, the applicant's U.S. Pat. No. 4.48 Addition of a mixture of cationic and anionic resins as described in No. 8.969 It is desirable to use the Alternatively, in addition to millimicron-sized particles, No. 4,578, which has oxygen atoms along the polymer backbone. ．． Neutral organic polymer resins such as those described in No. 150 and No. 4.596.660 It is also possible to use a medium containing

Also of particular interest in the present invention is U.S. Pat. No. 3.9 to Regnier. No. 83.299, Kirkland et al., U.S. Pat. No. 3.795.313, Ki U.S. Pat. No. 3,722,181 to Rkland et al. U.S. Pat. No. 4.034.139 to Ta1ley et al., U.S. Pat. No. 4.118 to Ta1ley et al. ．． No. 316, tlo ChaB et al., U.S. Pat. No. 4,029.583 or Reg. Modified inorganic supports such as those described in U.S. Pat. The method is to use a modified cellulose fibrous medium carrying the material. These The entire disclosures of all documents are incorporated herein by this reference.

In particular, it is possible to induce siliceous particles with silane, and various It is possible to attach groups for ion exchange or anchoring. In this example Next, it modifies both the cellulosic fibers and the siliceous particles and stimulates their interaction. The use results in increased fixation and/or ion exchange capacity. particulate matter The addition helps increase the stiffness and strength of the fibrous media, making it suitable for industrial Applications, especially those involving high pressure, should be easily available.

Preparation method The polymer-modified polysaccharide materials of the invention can be prepared in a variety of ways. . Generally, in some embodiments, the preparer first prepares the polymer and then converts it into its Condensation to polysaccharide via reactive hydroxyl groups (if available). Or another state , the preparer first reacts the polysaccharide with a hydroxy-reactive comonomer (a), Comonomer (b) and optionally other comonomers (e.g. crosslinking comonomers) are then added. polymer, hydrophobic comonomer, etc.). Therefore, these reactions involve two There are different types. 1) binding of the polysaccharide to the reactive hydroxyl group of comonomer (a), and 2 ) is the polymerization of polymerizable unsaturated compounds. In a preferred embodiment, a highly capable This reaction is preceded by polymerization before covalent bonding, with the intention of imparting strong adsorption functionality. carried out under conditions.

Furthermore, as a third method of (indirectly) attaching synthetic polymers to polysaccharides, There is a prior chemical activation of the polysaccharide. For example, periodate, hydrogen peroxide, cerium The polysaccharide can be treated with an oxidizing agent such as aluminum or other metallic oxidizing ions.

Reacting the activated polysaccharide with a polymerizable vinyl chloride compound containing amino groups or by reacting with epoxy group-containing vinyl resin. Polysaccharide derivatives carrying unsaturated functionality along the chain are usually be forced to These unsaturated functionalities are then used for further conjugation of polymers. It can function as an attachment point.

Another type of chemical activation of polysaccharides includes diepoxide or shrimp chlorohydride. This includes reactions with compounds such as phosphorus, thereby converting epoxy groups or other groups into A polysaccharide derivative is provided which is carried along the chain. These epoxies or other The groups then serve as attachment points in the polysaccharide chain.

Polymerization of comonomers includes radical chain (free radical) polymerization, step reaction polymerization, It can be carried out by ionic polymerization and coordination polymerization.

Particularly useful is free radical polymerization.

Free radical addition polymerization of radically polymerizable comonomers involves initiation, addition and completion. The reaction is carried out with a free radical initiator using well-known steps for oxidation. one A common procedure utilizes substances that generate radicals that can react with monomers. It is to be. Perhaps the simplest of all polymerization initiators is organic peroxide compounds and azo compounds. These substances at temperatures between 50 and 140°C It decomposes spontaneously to free radicals at a finite rate in common organic solvents. example Benzoyl peroxide decomposes into two benzoyloxy radicals at 60°C. do. Another example is provided by the azo compound azobisisobutyronitrile. It similarly decomposes into radicals at readily available temperatures. Become.

The required energy can also be provided by irradiating the initiator system with ultraviolet light. Offered vine. For example, initiation can be performed using the initiator system with benzophenone and its derivatives, benzophenone, of photopolymerization initiators such as alkyl esters or derivatives, or acetophenone. This can be done by irradiating with ultraviolet light in the presence of In that case, the amount of initiator It is necessary for the absorption to take place in the spectral region in which the radiation is irradiated. like this and at relatively lower temperatures than when purely thermal excitation is used. Radicals can be generated at a limited rate. Biomolecular reactions also initiate polymerization. Generates radicals that can be used to Particularly important are redox reactions. , which occurs in an aqueous medium and involves the process of accepting and passing electrons. For example, iron (1 1) Plus hydrogen peroxide or silver (1), plus 820°'- system is based on monomer lamination. It is particularly important for initiating radical polymerization. Due to the low starting temperature, the main Redox initiators or photochemically induced initiators are particularly preferred.

The initiator is in an amount sufficient to initiate the polymerization reaction. Polymerization is essentially This is done until all the monomers or comonomers are incorporated into the polymer chain. child This is easily verified by simple analytical tests on the reaction mixture.

Preferably, this polymerization occurs substantially simultaneously with the covalent attachment of the polymer to the polysaccharide, or is achieved just before. Binding and polymerization are preferably carried out in the same aqueous phase. Yes.

In one embodiment, the hydroxyl group of the polysaccharide of the monomer (or comonomer (a)) Whether the condensation is carried out before or after polymerization, the temperature of the reaction mixture is usually or by adding suitable acid/base catalysts.

The most preferred way to carry out this process is to use hydroxy-reactive polymers (or in the "-container" system using nomer (a)). All desired monomers and polysaccharides is added to an inert solvent system such as water, alcohol, or organic. polysaccharide and molybdenum The monomer is treated under conditions that initiate polymerization of the monomer. This is an example For example, ammonium persulfate and sodium thiosulfate may be added to a well-stirred mixture. by adding an aqueous solution of an initiator and initiating polymerization at about 15°C to 40°C. Accomplished. Alternatively, the polymerization can be initiated by photochemical means by adding a photoinitiator. It is also possible to have the for a sufficient period of time to allow the polymerization to proceed to completion. After stirring, the temperature of the reaction mixture is raised to a temperature sufficient to cause condensation. By increasing the binding of the formed polymer to the hydroxyl groups of the polysaccharide takes place . When the linking group on the polymer is a glycidyl group, such temperatures are typically 80- It is around 100℃. The reaction then proceeds to completion at the second temperature. or allowed to proceed for a sufficient period of time to denature the polysaccharide to the desired capacity. I can't stand it. The product is filtered, washed and dried and prepared for further processing if necessary. available. Unreacted monomers are preferably washed away with alcohol and unreacted catalysts are washed away with alcohol. The unreacted polymer is removed with an aqueous medium and with methanol or ethanol.

Further reaction of the modified polysaccharide is carried out by cross-linking.

Another further reaction of modified polysaccharide materials is to model affinity ligands or biologically active molecules. It is to adhere to the scaffolding group of monomer (or comonomer (b)). child The reaction involves attaching the affinity ligand or biological substance to be immobilized in a suitable solvent, usually aqueous. The body molecules and modified polysaccharides are mixed and conditions are sufficient to form a covalent bond between them. This can be easily achieved by wiping down and performing the fixation for a period of time. Monomer (also activates the polysaccharide groups on comonomer (b)) with substances such as cyanogen halides, Furthermore, it is necessary to treat this activated polysaccharide with affinity ligands or biomolecules. The key point. In this example, thousands of affinity ligands or biologically active molecules are used. (or comonomer (b)) and then the resulting polymer. Preferably, the reamer or copolymer is attached to the polysaccharide.

The reaction between the affinity ligand or biologically active molecule and the scaffolding group of the polymer Usually carried out at temperatures between 0°C and 50°C, catalysts such as acids or bases, or gold or other substances such as carbodiimides, or trifunctional substances such as glutaraldehyde. The method may include the addition of functional crosslinking agents. the resulting ligand or biological The modified polysaccharide containing active molecules is washed under suitable conditions and prepared for further processing if necessary. available.

Affinity matrices are created by grafting a polymeric carrier onto a substrate and then attaching an affinity matrix. By attaching the Gand to this grafted and covalently bonded synthetic polymer, It is made by This pre-bound structure is referred to below as the pre-ligand structure.

Suitable substrates include those mentioned above, i.e. polysaccharides such as cellulose. It will be done. Cellulose is again the preferred substrate material used.

The poly7- grafted onto the substrate is a precursor of various functional groups such as amine and thio. may contain a functional group that acts as a functional group. This type of modification is suitable for specific applications. provides a means for subsequent guidance. Typical monomers include glycidyl urea Ethylenically unsaturated oxirane-containing monomers such as acrylates and methacrylates containing an ethylenically unsaturated hydroxyl group such as mer, hydroxyethyl methacrylate Monomers, ethylenically unsaturated amide group-containing monomers such as acrylamide included. Glycidyl methacrylate-1-(GMA) and glycidyl acrylate (GA) is a preferred grafting monomer.

Satisfactory affinity especially when used in radial diversion devices) IJ hook preparation To achieve this, the pore size of the grafted polymer can be adjusted to Enough space for molecules to freely pass through so that they can even bind to the ligand. It is important to ensure that there is However, such a large pore size is not required. The requirements often give rise to questions about mechanical stability. Affinity Ma I -Currently available products marketed as Ux to provide structural rigidity. It relies on a carefully controlled degree of crosslinking. However, these lightly cross-linked The mixed materials are extremely brittle and often deteriorate even under normal mixing techniques. . Physical stability can be increased by increasing the degree of crosslinking. However, increased crosslinking reduces the porosity of the material.

One embodiment of the invention balances structural stiffness and porosity requirements. This problem of making cellulose or other natural polymers form a three-dimensional framework a second network of acrylic polymer is formed within and around it. The problem is solved by a two-stage interpenetrating network. Cellulose or Other natural polymer substrates provide the necessary stiffness, and thus acrylic polymers Only light crosslinking is required, and this lightly crosslinked polymer is not susceptible to the required chemical reactions. The material has a wide and porous property. Polymerization conditions similar to those of Examples 3 and 4 described below were used. Cross-linking occurs under certain conditions.

The synthetic poly 7- is formed using any technique common in the art. suspension polymerization is the preferred polymerization technique, selectively using surfactants, the monomers are mixed with the reaction mixture. The mixture is kept suspended by continuous stirring. Free to dissolve in the monomer phase A radical initiator is used and polymerization is carried out by thermal fragmentation of the catalyst. be exposed. Prior to completion of polymerization, reaction conditions are adjusted to facilitate the coupling reaction. is modified, whereby the hydrocarbon chain of the polymerization product has an accompanying functional group attached to it. It is grafted onto a natural polymer substrate while still in situ. The latter of this polymerization In the step, polymerization and coupling proceed simultaneously.

GMA grafted onto cellulose represents one of the preferred affinity matrices. vinegar. GMA provides three functions; the oxirane group of the GMA monomer to form a covalent bond with the hydroxyl group on the surface of cellulose; Ran groups provide cross-linking ability for synthetic polymer networks, and the remaining The oxirane group functions for subsequent ligand binding.

Described below are the physical and chemical properties of the GMA-cellulose affinity matrix. It is a characteristic of

1. Mechanical rigidity Cellulose fibers provide good structural strength as a solid support member. these The fibers are further strengthened by the strong hydrogen bonding forces between the polysaccharide units of the cellulose fibers. The high degree of crystal structure provides additional strength. additional mechanical stiffness The properties are provided by the crosslinks that occur between the cellulose and the synthetic polymer.

2,7 chromoporous By carefully selecting the fiber diameter, length, degree of fibrillation and degree of cross-linking. This results in highly controlled macroporosity.

3. Hydrophilicity Hydroxyl groups in the cellulosic structure provide a high degree of hydrophilicity. Also GMA polymer oxidation of the glycidyl group to a diol or its combination with a monomer containing a hydroxyl group. Polymerization further contributes to the hydrophilicity of the matrix.

4. Chemical resistance Cellulose has low solubility in common solvents. Also GMA polymer By crosslinking the glycidyl groups of cellulose with each other and with the hydroxyl groups of cellulose, the solvent Provides resistance to.

5. Structural integrity Due to the grafted crosslinkable monomers, there is no expansion or contraction of the matrix. Small enough to see. Additional cross-linking with difunctional molecules provides additional stability sex is brought about.

6. Low degree of non-specific adsorption A grafting step is available to further purify the cellulosic raw material and perform non-specific binding. Reduce the number of parts.

7. Chemical reactivity Grafting to create multiple oxirane groups for ligand coupling Weight increases of up to 200% were obtained after. Furthermore, these coordinating groups can be For example, they can be separated by "spacer arms". Excellent flow properties, i.e. The high degree of control afforded over structure and porosity results in superior flow performance. Ta. Additionally, this matrix can be completely dried between uses. , thereby enhancing its flow properties.

This cellulose-GMA affinity matrix was prepared as shown in Table I below. A chemically modified vine. The purpose of this chemical denaturation is to create a matrix that binds to the ligand. The first step is to prepare a complex or preligand structure.

Table I Represents the substrate and the moiety number covalently bonded to it.

1 of Table I above. Suitable oxidizing agents include perchlorate, sulfur trioxide and There are iodate salts, and periodate salts are preferred.

2 of Table I above. Preferred aminating agents for NH3-R-NO3 include the structure NH3-R-NO3. Included are compounds having the formula where R is a direct bond or (Cz)n.

Thiolation is carried out using compounds such as Na5H or SH.

Table 1 below briefly summarizes the oxidation, amination and thiolation reactions.

Table II Aldehyde reactions produced as a result of oxidation with periodate as shown in Table ■ above. The reaction product is further processed to form a boronated affinity adsorbent according to the following reaction equation: can be formed.

Amination with diamines such as hydrazine proceeds according to the following formula, where hydrazine is This results in the formation of molluscum.

This hydrazine substituted product is converted to the azide form according to the following formula. This azide is by reaction with a suitable ligand, e.g. lysine, according to the following formula: NH converted into an affinity matrix.

! where R1 and R2 are C1 to C4 alkyl.

As shown in Table 1 and ■ above, the GMA-cellulose matrix has N a It can be thiolated with S It. This thiol group is then subjected to the following reaction According to the formula, it can be activated by reaction with 2,2'-dipyridine disulfide. can.

This substance reacts with the sterically accessible SH groups of proteins according to the following formula: .

Similarly, the substrate can be modified to provide an amide functionality, and its The amide functionality is further reacted with a diamine according to the following formula.

The resulting hydrazine is subsequently treated as in the above amination of the oxirane ring. and process it.

Substrates modified with synthetic polymers and containing reactive hydroxyl groups can be prepared using suitable materials such as cellulose. Hydroxylethyl methacrylate 01EMA) or hydroxyl methacrylate on the substrate It is produced using compounds such as lupropyl methacrylate (HPMA). adjacent The hydroxyl group can be activated using CNBr according to the following formula.

The starting point for designing an affinity matrix to remove a particular enzyme is the to investigate the structure and, in particular, of the inhibitors of that enzyme. enzymes and their The complex that forms with the inhibitor is determined by the way the enzyme interacts with a particular protein structure. give the best picture of how they interact. Enzyme inhibition is always It is competitive and reversible as shown in Eq.

E (enzyme) + ■ (inhibitor) = EI complex Usually, the residue at the reaction site of the inhibitor is the enzyme Combines with the hollow structure part. For kallikrein and trypsin, kallikrein Those that interact with the nucleophilic hydroxyl group of the active site of serine or arginine It is the basis. Animal enzyme inhibitors are often extracted from plants.

Therefore, an effective affinity matrix binds botanical inhibitors for enzyme adsorption. Vine created by. Plasma kallikrein inhibitors are found in potatoes and beans. It is known to be isolated from nuts.

The substrate product thus obtained can further improve the amine functionalization and glycidyl functionalization of proteins. By using the glycidyl group as a precursor, it is possible to attach another moiety via the glycidyl group. It is also possible to This newly combined part is as shown in the table ■ below. It is activated by various activation mechanisms for proteins.

Table III Killing Protein A via metal ions Pc region IgG binding polyphenol to Coupling proteins through polyphenols are non-coupled to polyphenols. Always reactive. The basic reaction involves the formation of polyphenyls in the quinone form via the following pathway: It is the addition of some nucleophilic residue to the protein.

Method 1 Cellulose-GMA matrix For binding antibodies or enzyme inhibitors to solid matrices, antigen or enzyme In order for the ligand to have maximum affinity for the matrix, it must be bound to the matrix. It is known that it is important to maintain the active conformation even after It is being Antibody molecules exist in their active form in only a few conformations. Exists. Functional affinity varies greatly depending on binding to solid surfaces It is. Therefore, matrix and ligand interactions due to forces such as hydrogen bonding and hydrophobic interactions The non-covalent interactions between antibodies have an obvious effect on antibody conformation. It has a slight influence. Antibodies are structurally bulky, so they have limited surface area and pores. The physical properties of IJ Fuchs, such as the dispersion of This is something to consider. For example, rgG bound to 5epharose■ is about 3 At levels greater than -4/g, further binding of IgG becomes effective as a ligand. Not. Presumably, higher levels of IgG bind to 5epharose■. As the antibody progresses, the activity of the antibody actually decreases due to the crowding of IgG that interferes with the antibody's action. It will probably decrease to By using cellulose as a substrate, cellulose Maximum activity was achieved at a high degree of substitution of 7 mg/g of Ru. However, the active binding site between the substrate and the active binding site, e.g. by the use of "spacer arms" Additional binding capacity can be provided by increasing the spacing between becomes.

Therefore, by introducing a hydrophilic spacer arm according to the reaction below, antibodies can be It becomes possible to increase the activity of

As indicated above, the substrates of the present invention modified with synthetic polymers Tailor to specific needs by cross-linking and/or substrates be able to. Crosslinking can be achieved, for example, with mono- and polyethylene glycol dimethacrylate. diacrylates (polyethylene glycol residues contain up to 6 ethylene groups) ), ethylene dimethacrylate, tetraethylene diacrylate, diviny Rubenzene, triallyl cyanurate, methylenebisacrylamide or methylene at least two polymerizable α,β-carbons such as bismethacrylamide, etc. Inclusion of small amounts of polyunsaturated comonomers with double bonds in the polymer formulation Done by Vine.

Another type of cross-linking agent is the This method uses the existence of child pairs. In this case, the crosslink reacts with this nitrogen lone pair of electrons. It is carried out using bifunctional reagents that include Ha l-C0-(CH2) n Diacyl halide such as -C0-11a I, or tla l-(C1l 2) Includes alkyl civalides such as n-Ha1. Here it is called Hal. is a halogen such as chlorine, bromine or iodine, and n is a number from 2 to 12. be. Compounds similar to this in which the -(CL)n- moiety is substituted with phenyl are also can be considered.

Crosslinker bonds are best determined experimentally. The polymer has the desired structural integrity and If porosity is achieved, it should be considered sufficient. Ideally, synthetic Amounts of crosslinking agent of 5-20 mole percent per polymer unit are sufficient.

Typical ligands include DNA blood group antigens, anti-alpha fetoprotein, C1O, Protein A1 protein G, polylysine, mucopolysaccharides such as heparin, methylation Albumin, tryptophan, phenylalanine, concanavalin A and others However, it is not limited to these. Removes proteolytic enzymes from LgG In order to Sancarboxylic acids, trasylol and latent inhibitors are among the most porous.

For removal of mucopolysaccharides such as heparin, low molecular weight salts such as protamine sulfate are recommended. Basic protein (protamine) is useful. Removal of kallikrein and PKA Benzamidine is effective for this purpose.

Polymyxin B sulfate is effective for removing endotoxin.

As one skilled in the art will appreciate, ligands suitable for the practice of the present invention include the all that remain biologically active while being immobilized by the affinity matrix. such ligands include, but are not limited to, the following common ligands: Indicated by rilas.

Amino acids, avidin-biotin, carbohydrates, glutathione, hydrophobic matrix , immunoglobulin, insoluble protein, lectin, nucleotide, polyamino acid and polynucleic acids, and specific ligands.

Typical amino acids suitable for affinity ligands include: 1. -alanine, -arginine L-asparagine, L-aspartic acid, cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L- Lysine, L-methionine, L-phenylalanine, L-proline, L-serine, -threonine, L-thyroxine, D-) liptophan, L-) ribtophan, Includes L-tyrosine and valine.

Typical avidin-biotin ligands include avidin, biotin, and desthiobiotin. These include tin, diaminobiotin and 2-iminobiotin.

Typical carbohydrates include glucosamines, glycopyranoses, and galactosamines. , fucosamines, fucopyranosylamines, galactosylamines, glycopi Contains lanosides and others.

Typical glutathione ligands include glutathione, hexylglutathione, and and sulfobromophthalein-3-glutathione.

Typical hydrophobic matrices include aminoallyl, butyl, decyl, dodecyl, Contains ethyl, hexyl, methyl, octyl, pentyl, phenyl and propyl It will be done.

Typical immunoglobulins include anti-goat 18G, anti-human IgG, and anti-mouse IgG. , anti-rabbit IgG, goat 1gG, human IgG, rabbit IgG, etc. Contains immunoglobulin and antiglucose-6=phosphate dihydrogenase .

Typical insoluble proteins include aprotinin, fetuin, gelatin, and globulin. bottle, glycophorin, hemoglobin, insulin, lactalbumin, valve Albumin, protamine, protein A1 protein G1 ribose binding protein and trypsin inhibitors.

Typical nucleotides include adenosine mono- and syric acid, cytidine and triphosphate, flavin mononucleotide, guanosine mono-, di- and triline acids, and uridine mono-, di- and triphosphates.

Typical polyamino acids and polynucleic acids include DNA, polyadenylic acid, and polynucleotides. dilic acid, polylysine, polyriboadenylic acid, polyribocytidylic acid, polyribogua Includes nilic acid, polyriboinosinic acid and polyuridylic acid.

The affinity matrix of the present invention has a resiliency with respect to the substrate and the synthetic polymer matrix. Includes combining guns. Typically immobilized on a stationary phase and affinized. used to bind complementary molecules from the solute phase by tea chromatography Any ligand that can be used is considered.

The affinity matrix of the present invention is a device that allows radial or tangential flow of the sample. configured within. “Radial” flow is where the sample flows through the affinity matrix. It is intended that “Tangential” flow is when the sample flows through the surface of the affinity matrix. It is intended for cases where it will not peel or penetrate.

General preparation techniques By copolymerizing vinyl monomers and subsequently covalently bonding them to cellulosic substrates. The composite material thus formed can be made in the form of paper of various thicknesses. Created like this The affinity matrices are then punched into disks, columns, and cartridges. Or mounted on a suitable holder such as a plate.

A typical affinity polymer (IJ sock) of the present invention is a GMA amination reaction as described above. those containing primary amino groups formed by reaction (“A type”), peroxides CrG type containing aldehyde groups formed by oxidation of GMA by ) is included. If additional spacer arm length is desired, e.g. Any desired length of space can be created using alternating combinations of aldehydes and diamines. It is also possible to create a saarm.

Ligand immobilization procedure for A-type and G-type affinity matrices and will be explained in detail below.

Ligand immobilization method for A-type affinity matrix A-type matrices are free at the ends of hydrophilic spacer arms (>10A). - has a primary amino group. This can be used to immobilize glue gantts on a small scale. It is very useful for affinity purification of proteins, enzymes, antibodies, etc.

These A-type matrices are polysaccharides, i.e. synthetic on cellulosic substrates. It consists of a composite material formed by covalent bonding of polymers. Amine functionality is an example For example, by covalent attachment of 1,6-diamithexane to this activated complex. M) It has been introduced into the IJx. The matrix thus formed is originally It is hydrophilic and the degree of crosslinking is appropriately controlled, making it dimensionally stable. Ru. Dimensional changes under pH and salt conditions are negligible. This ma Trix structures are stiff enough to withstand large fluxes for high-velocity flows. through a matrix that has and is highly porous and configured for radial flow. Makes it possible to transport large quantities. The free primary amino group is an acrylazide group, a carbon Ligands with carbonyl and aldehyde groups can be attached. present invention One example of consists of a preligand intermediate with a free primary amino group. .

Aldehyde groups are generated in the matrix by reaction with glutaraldehyde .

The aldehyde group thus generated undergoes a binding reaction with a ligand containing an amino group. be involved in Another example contemplates aldehyde-terminated intermediates.

Glutaraldehyde-activated amino groups of A-type matrices, preferably fatty A-type polymers are suitable for immobilizing ligands containing group-primary amino groups. Activate Trix with glutaraldehyde.

A. Material 1. $l buffer solution A-sodium phosphate (0.1 M5 pH 7.3).

2. Buffer B - Sodium phosphate containing 0.25M sodium chloride ( 0.05M) (final pH 7.6).

3. Buffer C-0. Sodium phosphate containing 25 M sodium chloride (0. 05M) (Final pH 6.5>.

4. Glutaraldehyde - 10% glutaraldehyde in buffer A.

5. Glycine ethyl hydrochloride ester - 1% solution in buffer C. Adjust pH to 6.5 . Solution 100r! Add 100 mg of NaCNBHs per dl.

B. Activation 1. Type A Ma) IJ Fuchs Equilibrate with buffer A at room temperature.

2. Circulate the prepared glutaraldehyde solution (see A, 4. above) at room temperature for 6 hours. make a circle

3. Remove excess glutaraldehyde by washing with buffer A.

To determine whether glutaraldehyde has been removed, use Shipp's reagent solution. Observe the fluid. Once all excess glutaraldehyde has been removed, the matrix The group is now ready to bind to a ligand containing an amino group. Or The matrix can also be stored at 4°C.

Binding of C2 ligand 1. Contains NaCNBHs at pH 6 to 8.5 (i.e. 1/2 in the ligand solution) d) Prepare amino functional ligand solution using appropriate buffer 0 and store at room temperature or 4°C. Circulate through the matrix for 6 to 16 hours at a time.

*Contains amino functional moieties (e.g. Tris, glycine, ethanolamine, etc.) Buffers that contain

2. Remove unbound ligand by washing with the same buffer until baseline is obtained. do.

3. Glycine hydrochloric acid at a pH of about 6.5 in the presence of NaCNBH for about 4 hours at room temperature. Excess active groups are inactivated by circulating lysine ethyl ester I0. do.

Dissolve 1 g of glycine ethyl hydrochloride in Kimoto 100-buffer C. Adjust the pH section. Subsequently, NaCNBIls was added to a final concentration of 1/W11.

4. Any desired buffer containing 0.25 to 0.5M sodium chloride Reagents are removed by washing with solution (pH 5 to 7.6).

mouth, measurement of bound ligand The amount of ligand bound to the matrix is determined by the amount of unbound ligand left in the buffer solution. It is calculated indirectly by measuring the amount of the index (step C,2,). measure Dialyze the remaining ligand solution to remove potentially interfering reaction byproducts. It is necessary to

Ligand immobilization method for G-type affinity matrix G-type matrices are suitable for immobilization of ligands and proteins on a small scale. It is very useful for affinity purification of proteins, enzymes, antibodies, etc.

These G-type matrices are synthesized on polysaccharide, i.e. cellulosic substrates. It consists of a composite material formed by covalent bonding of polymers. Aminofunctional ligand Functional groups capable of binding bonds are introduced into this composite structure in the form of polymers. It will be done. Typically, GMA-grafted cellulose is reacted with perchlorate, Produces hydroxylated intermediates. Upon further reaction with periodate, a yields a dehyde terminus. The matrix thus formed is hydrophilic in nature; Since the degree of crosslinking is appropriately controlled, it is also dimensionally stable. pH and salt The dimensional changes under these conditions are negligible. This matrix structure The body is stiff enough to withstand large fluxes for high velocity flows, and The matrix is porous and allows for the transport of large amounts through the matrix. This ma Trix has many vicinal hydroxyl groups that are susceptible to oxidation by periodate. Easy to use. The aldehyde group thus generated is a free primary atom. It participates in the bonding reaction with a mino group, preferably an aliphatic amino group. Low molecular weight or high content Which ligand [L] (!J amine p-aminobenzamidine, protein A, albumin, IgG, etc.) can also be bonded to the affinity matrix with high binding efficiency. fixed to the base. Typical affinity matrix) Flowchart representation of IJ Fuchs preparation Final affinity product below A. Material 1. 0.05M sodium phosphate buffer containing 0.25M sodium chloride . Optimal PI (6,0-8,0° The preferred pH value for binding is albumin -7,0~ 8.0 IgG -6,5-8,0 p-Aminobenzamidine-6,2 Note: Relaxation agents containing amino-functional moieties (e.g. Tris, ethanolamine, etc.) Buffer solutions should not be used.

2. Sodium metaperiodate (NalO4): freshly prepared aqueous solution (1 , 5%).

3. Glycine ethyl hydrochloride 1% aqueous solution.

1. Assemble affinity multi-IJ hook using tube, small laboratory pot connection to the amplifier.

2. Wash the Mul-'Jx with 100 Inl of deionized water (5 times). new A specially prepared Na104 (1.5%) aqueous solution was added at room temperature for 0.5 to 2 hours. 4-6 Allow to pass for d1 minute. This oxidation reaches its maximum activity if it continues for 2 hours. Sexualization occurs.

3. Wash 7 Trix with 200 rnl of deionized water (10 times) and clean the Nal port. .

remove. To determine the removal of Na104, the conductivity of the eluate and 0. D , check 28°.

4. IJ Fuchs flattened with a suitable phosphate buffer (as described in the materials section) balance.

5. Prepare a ligand solution (10 mg/m) in equilibration buffer. This Riggan solution in the presence of NaCNBII3 (final concentration in ligand solution 1 /mj! For ligand solutions less than 0.010ml, a minimum of 10μ of NaCNBH - is required. ) Circulate at 4-5 m12/min (4-10 hours).

*50 μl of NaCNBH− to a final concentration of 1 μl/r in the ligand solution Dissolve NaCNBfls in buffer to be sufficient to make rLl. N Since the aCNBIIa reagent is prone to decomposition during storage, NaCNBIIs The addition of 5 (lμl) solution is repeated two to three times during the binding process.

6. Remove unbound ligand by washing with phosphate buffer until absorbance stabilizes at baseline. remove the code.

7゜ Glycine ethyl hydrochloride solution at pH 6.5 in the presence of NaCNBH- Excess active groups were inactivated by rJJ cycling 00 for about 4 hours at room temperature. Ru.

Dissolve 1 g of woody plant in 100 ml of equilibration buffer.

Adjust pH to 6.5. Then add NaCN to a final concentration of 1/- Add BHs.

8. High and low pi containing 0.25 to 0.50M sodium chloride, respectively. Wash the matrix alternately (four times) with buffer solution to remove excess reagent and remaining unbound. The combined ligand is finally removed.

9. The sample is then applied to produce the desired product using standard established techniques. Elute.

10. The matrix bound with the ligand was treated with 0.9% sodium chloride at 4°C. 0.02% sodium azide containing thorium or a suitable preservative (pH 6, 0-7,0).

Measurement of C0-bound ligand The amount of ligand bound to the matrix is determined by the amount of unused ligand left in the buffer solution (step 6.). Calculated indirectly by measuring the bond gantt. low molecular weight ligands (e.g. p-aminobenzamidine, lysine, etc.), this measurement is appropriate. This is done using a color reaction or ultraviolet light. Macromolecules (e.g. protein A1 antibodies) ), unbound ligand is added to remove reaction by-products that interfere with measurements. It is necessary to dialyze the solution.

Enzyme removal using affinity matrix 1, Plasminogen from IgG The metabolism of blood coagulation that exists in the human body occurs through two opposite processes. Ru. That is, the fibrin formation process associated with the blood coagulation system and the fibrinolysin system This is the fibrin removal process that takes place. Under normal physiological conditions, these two One system is essentially inert. However, in emergency cases, blood endogenous A significant amount of plasmin is suddenly activated from an inactive precursor by the offspring, and the system is non-equilibrium. Clinical research has shown that the blood of people who suddenly die due to stress is liquid and clottable. It has been shown that it is not solid. This condition is now known as plasminogen This study is based on the proteolytic elution of fibrin by activated plasmin from is known. Therefore, it is possible to remove fibrinolysin effects and cleave IgG. To avoid this, plasminogen must be removed from IgG.

Both plasminogen and plasmin act as if they were gamma globulins. behave. The isoelectric point of plasminogen is estimated to be at pH 5.6. Ru. It is probably a glycoprotein and contains a small amount of phosphorus. different researchers The molecular weight is reported to be 143.000 or 84.000 by .

This molecule behaves as if it were an asymmetric shape with an axial ratio of 9 to 16. human The chemical properties of plasmin are similar to those of plasminogen, but activation Due to the separation of some molecules during the process, the molecular weight is slightly lower. Plasminnow The conversion of gen to plasmin changes from an asymmetric shape to a more compact spherical type. It involves changing the shape of something.

Removing proteolytic enzymes from IgG requires that the solution be bound by enzyme inhibitors. This is done by passing the sample through a stained affinity matrix. Plasmino Of all possible inhibitors of the -gen family, the following three are the most effective: It was discovered that

(1) ε ~ Ruaminocaprone or lysine (2) Methyl-p-aminocyclohe Trasylol xanecarboxylic acid (3) In a preferred embodiment, the inhibitor, ε-aminocaproic acid, inhibits CNBr activity. bonded to the cellulose-GMA matrix using oxidation.

Additionally, certain quaternary amines have been found to exhibit plasmin activity and Since plasminogen has an isoelectric point of 5.6, QAE and DEAEMA Removal of plasminogen by Trix also occurs to some extent.

A technique developed to measure the concentration of plasminogen in human plasma. is based on the proteolytic activity of plasmin. Plasminogen is directly It cannot be measured directly and is converted to plasmin through activation by urokinase. and then transfer this plasmin to its protein content on some substrate. Measured from deactivation. The physiological substrate of plasmin is fibrinogen; For better sensitivity and reproducibility, synthetic substrates such as casein are preferred.

The principle of the casein method is that within a given period of time, expressed as plasminogen activity, The purpose of this study is to analyze the ability of plasminogen in plasma to digest casein. . This activity is due to casein hydrolysis based on the proteolytic action of plasmin. can be measured by the amount of tyrosine equivalents released. The standard method is About Trompol Reagent National Heart In5titu te) was established.

2. Cold ethanol of affinity matrix Cohn for removal of kallikrein IgG created by fractional distillation has prekallikrein activator (PKA) activity. undesirable and harmful external factors such as sex, activated clot factors, and esterase activity. It is known to contain sexual activity. These exogenous activities [produce a hypotensive response] Intravascular administration to patients for treatment J Alving et al., New England dJ, Med, 299:66 (1978). Enzyme inhibition The harmful agent as a ligand, e.g. a cellulose substrate, preferably modified as described above. protea such as kallikrein by binding to treated cellulose. -ase is removed from the plasma. Kallikrein, like other serine proteases, , by synthetic compounds such as p-carboethoxyphenyl epsilon guanidine caproate. It is also inhibited. For the removal of kallikrein, it binds to cellulose-GMA. benzamidine is a preferred affinity ligand.

Hydrophobic comonomers ( b) (ii) is added to the copolymerization mixture. This is followed by cleaning with alcohol. be exposed.

Preferably forming a self-supporting fibrous medium from the modified polysaccharide materials of the present invention includes: It can be carried out immediately after polymerization and modification of the polysaccharide. In this embodiment, the affinity link Gand or biomolecule anchoring may be performed on the formed sheet itself. Ru. Alternatively, a fibrous medium is formed after fixation of affinity ligands or biomolecules. It will be done. A preferred method is to form a fibrous medium after modification of the polysaccharide and apply it on a sheet. to carry out further reactions such as fixation to.

Preferably, the self-supporting fibrous media using the modified polysaccharide of the present invention comprises fibers and If desired, vacuum filter the aqueous slurry of additional resin and modified or unmodified particulates. It can be created by This results in a high degree of uniform porosity and Fine pore structure with excellent flow properties and practical properties for fibers, resins and particulates. A sheet with qualitative homogeneity is formed.

Vacuum filtration takes place on a porous surface, usually a woven wire mesh, which is actually 50 mesh to 200 mesh, each mesh opening is 28 0 micrometer to 70 micrometer. A finer mesh is Not suitable due to locking problems and/or structural unsuitability.

All components (modified fibers, other fibers, fine particles, modified fine particles, other resins, etc.) are The order of addition to the slurry is such that during the mixing process the slurry is added to the U! 4-section hydrodynamic If the slurry is subjected to significant shear forces, it is usually relatively unimportant, e.g. prepared with a concentration of 4% and then subjected to the appropriate conditions required for vacuum filtration and sheet formation. It is diluted by adding water to the desired concentration. This appropriate density means that the sheet Depends on the type of equipment used to form it. Typically, the slurry It is cast onto a porous surface, vacuum filtered and dried in the usual manner.

The flat, dimensionally stable sheet can be of any thickness as desired; It is then cut into appropriate dimensions depending on the type of use. In one embodiment The wet sheet is then dried and then sized to form a disc. disconnected. This disk is loaded into a cylindrical column of appropriate dimensions to achieve the desired It is possible to form a medium that The disc and cylinder should preferably have an interference fit; This forces the disk into the cylinder without distortion, but it falls due to gravity. There is no gap between the disc and the cylinder. Fill the column dry. Once loaded, a pump is used to pump the solvent through the stacked elements within the column. It is possible to use a In some embodiments, the element swells to form a wall of the cylinder. forming a substantially tight end seal against the Individual elements are dimensionally stable Columns have common characteristics to other chromatography media, such as orientation and handling. is not sensitive to the problem of oxidation, especially when using gel-like media. but However, this medium is equally suitable for use in sheet form. It is.

In another embodiment, the sheet-like stationary phase is Used as stationary phase in chromatography columns. of such a column One is described in US Pat. No. 4,496.461 to Leeke et al. The entirety is incorporated herein by this reference.

In one embodiment shown in FIG. 1, a cartridge-shaped radial flow column is used. The overall number is 10. This consists of a cylindrical stationary phase 12 and a cylindrical stationary phase 12. A cylindrical tube 1 forming a cylindrical chamber 14 acting as a housing 3. The solid stationary phase 12 is formed from glass, metal or polymer. The tube or cylinder 13 is insertable into the stationary phase chamber 14 . It has a diameter somewhat larger than the outer diameter of. Regular analytical and preparative columns A suitable fluid reception, collection and monitoring system can be used with this column as in Can be used together. Stationary phase 12 is located within chamber 14 and is preferably Alternatively, it has a longitudinal axis 16 concentric with the axis of the cylindrical chamber 14. selectively, multiple arranging several cartridges with different contours in a single housing; can also be used, thereby allowing parallel and/or serial flow between cartridges. Yes (not shown). The solid stationary phase has chromatographic functionality and Effective for separation by identity chromatography and reversed phase chromatography. be.

Referring to the embodiment shown in FIGS. 2 and 3, the stationary phase 12 is a swellable matrix. It is composed of Rix 18, which is swellable and sheet-shaped. , is an active medium for chromatographic separations. In one embodiment, this matrix Kusu is fibrous. The sheet-like chromatography medium 18 is a single non-woven It is sandwiched between the mesh 22 or a plurality of meshes. chromatography The medium 18, such as a composite sheet with a non-woven mensch 22, has a longitudinal axis 16. are wound around a cylindrical core 24 and form a plurality of layers around the shaft 16. There is. Core 24 is provided with a plurality of longitudinally and axially oriented channels 21. and directs fluid into surrounding channels 23 in fluid communication with core 24. I'm wearing it. Because of its openness and thickness, the mesh 22 allows each layer of the media 18 to be acting as a spacer means between and allowing controlled swelling of the medium. This also improves the distribution of the sample flowing through the stationary phase 12. peripheral channel A cylindrical core 24 is provided with a cylindrical core 24 to allow the sample to flow from the tube 23 into the hollow interior of the core. An opening 26 is provided near the top.

Referring to FIG. 1, the wound composite sheets 18 and 22 and core 24 are connected to a stationary phase. End camps 32 and 34 are capped. The stationary phase end cap of this subassembly 32 and 34 are thermoplastic to the core 24 and the ends of composites 18 and 22. Sealed by welding. Consisting of 18.22.24.32 and 34 The subassembly is then placed into chamber 14. Then the cylindrical end cap is A cap 36 is thermoplastically welded to the top end 31 of the cylinder 13. Fluid immediate The sample 42 is thus passed through the solid stationary phase into the hollow channel of the core 24 from the outside. can flow radially through the tube 21, which has a solid interior and exterior. Completely separated by stationary phase and separated by stationary phase end caps 32 and 34. That's because it's sealed.

Preformed stationary phase end caps 32 and 34 attach to the cylindrical solid stationary phase 12. In contrast, this stationary phase end camp covers the inner surface of the thermoplastic stationary phase end cap. Sufficient to form a thermoplastic seal with core 24 and composite sheets 18 and 22. By heating the stationary phase end cap to a temperature sufficient to soften it by It is preferable to apply it as follows. All edges are now in this softened material. embedded. The softened material is then cured, typically under ambient conditions. , the sealing surfaces of the stationary phase end caps 32 and 34, and the core 24 and solid stationary phase 12. forming a thermoplastic sealing relationship with the ends of the material to form a leak-tight seal. child The method of applying stationary phase end caps is well known in the filtration art. For example, Meyering et al., filed on May 28, 1982, Miller U.S. Patent Application No. 383,383 and U.S. Patent Application No. 383,3 See No. 77.0 Optionally, the stationary phase end cap is placed on the solid stationary phase. It can also be integrally molded on-site.

Thermoplastic stationary phase end caps are preferred due to ease of bonding; Thermosetting resin can be welded with thermoplastic, i.e. softened by heat. It is also possible to use it in any possible polymerization stage. The resin is then cured. , an inseparable structure is created. Such structures must have a liquid-tight seal. , without risk of destroying solid stationary phase 12 and stationary phase end caps 32 and 34. Can be autoclaved. For biomedical applications, disinfectant Heat with a softening point high enough that it will not soften under the conditions of autoclaving Plastic resins are preferred. Examples of plastic materials that can be used are polyolefins. be.

Referring to FIG. 1, one preferred column 10 includes a subassembly 18°22.24 ．． At one end of 32 and 34 which is not opened to the outside and is sealed, a fixed It has a constant phase end cap 34. This stationary phase end cap 34 is attached to the cylinder 13. can fit into the bottom end wall 44 while passing around the outside of the stationary phase 12 to Allowing sample 42 to flow into chamber 14 . That is, the subassembly 18.22.24 ．． Stationary phase end cap 34 on this underside of 32 and 34 covers the bottom end of cylinder 13. in spaced relation from wall 44 and thus prevents the flow of sample 42 into chamber 14. This is allowed.

The upper end of cartridge 40 is in fluid communication with channel 21 of cylindrical core 24 has a cylindrical end cap 36 that extends from the outer periphery of the cylindrical core 24. 9 cylindrical end cap allowing fluid flow to the outside of the cylindrical end cap 36; 36 includes a fitting for providing fluid coupling via coupling means (not shown). 48 is molded.

Referring to FIG. 2, chromatographic media 18 is wound around core 24. The outer surface of core 24 is completely covered with textile material 20 prior to being woven. additionally , after winding the chromatographic medium 18 around the core 24, its outer surface The surface can be completely covered with mesh material 22.

Figures 4 to 10 show other embodiments of the chromatography column of the present invention. In this example, the column is disc-shaped, and in this case the same sign is used. The numbers indicate similar parts.

Referring to FIGS. 4 to 10, the column is disc-shaped and has an overall diameter of 11 0, an inlet housing member 112 and an outlet housing member 114. , a stationary phase 116.

The inlet housing member 112 includes a sample inlet means 118, a baffle means 120, sample dispensing means 122. The sample inlet means 118 is connected to the sample dispensing means 122. It's communicating.

Sample distribution means 122 includes a plurality of radial distribution channels or grooves 130 and a plurality of concentric a distribution channel 140, a radial distribution groove 130 and a concentric distribution channel 14. 0 are in communication with each other and with the inlet means 118.

The radial distribution groove 130 is defined by line P+ in FIG. 1O and P1′ in FIG. The distribution groove bottom and distribution groove wall portions 134a and 134b are placed in the plane shown. The concentric distribution channel 140 includes a concentric distribution channel bottom 142 and a concentric distribution channel bottom 142. It includes channel walls 144a and 144b and a concentric distribution channel top 146.

Optionally, the inlet housing member 112 may include venting means 150; Its function and operation will be described below. The venting means communicates with the chamber 152. ing. Chamber 152 includes inlet housing member 112 and outlet housing member 1 14 (see FIGS. 4 and 10), the chamber 152 is formed by A stationary phase 116 is included.

Outlet housing member 114 includes sample collection means 154 and sample exit means 156. , sample collection means 154 is in communication with sample outlet means 156.

Sample collection means 154 includes a plurality of radial collection grooves 160 and a plurality of concentric collection channels. The radial collection groove 160 and the concentric collection channel 170 each include a It also communicates with sample outlet means 156.

The radial collection groove 160 is defined by line P8 in FIG. 4 and p, l in FIG. A radial collection groove bottom placed in the plane shown and radial groove walls 164a and 1 The concentric collection channel 170, including 64b, has a concentric collection channel bottom 172 and a concentric collection channel bottom 172. cardiac collection channel sidewalls 174a and 174b; and a concentric collection channel top 176. including.

The stationary phase 116 has chromatographic functionality and Effective for distance. 7 and 8, and in particular FIG. 8, the stationary phase 116 is Sheet with chromatographic functionality and effective for chromatographic separation a plurality of layers of matrix 180 and optionally this swellable matrix 1 and spacer means 182 between each of the 80 adjacent layers. This is best shown in FIG. 9, which is a cross-sectional view of one embodiment of O. This matrix The wax can be a swellable matrix. In one preferred embodiment, the expansion The wettable matrix is fibrous.

If swellable, matrix 180 is hydrophilically swellable and chlorinated. 0 selective spacer means 182 containing an active medium for chromatographic separation. , typically a woven or non-woven mesh, as shown in FIGS. 2 and 3 above. Similar to mesh 22 in the figure, this will be discussed further below. Immediately Due to the openness and thickness of the mesh, there are gaps between each layer of the matrix 180. Acts as a spacer means to allow controlled expansion without clogging the porous structure of the media. of the sample flowing radially through the stationary phase 116. Improve distribution.

As can be observed in FIG. 8, the method of forming the stationary phase 116 is ``sandi'' with alternating layers of swellable matrix and layers of spacer means. The periphery of this sandwich body is compressed to form a liquid-tight structure 184. There is a particular thing. Typically, a swellable matrix 180 and alternating discs The periphery of the spacer means 182 and the spacer means 182 are joined together. Preferably, matrix 180 contains or has a thermoplastic polymeric material bonded therein. Similarly preferred In a new embodiment, the spacer means 182 is also made from a thermoplastic polymeric material. or contains it. In this configuration, the ends are selectively heated Uniform bonding is achieved by suitable means such as processing, ultrasonic welding, or the like. In Figure 4 As can be seen, in the preferred embodiment, the liquid-tight perimeter structure 184 is itself , the respective mating positions of the inlet housing member 112 and the outlet housing member 114. It is contained in a liquid-tight, gas-tight seal formed by ends 186 and 188.

In this way, the sample introduced through the inlet means 118 passes through the outlet means 156. It must pass through a fixed layer 116 before being discharged through it.

The disk-shaped chromatography columns shown in FIGS. 4 to 10 are conventional and well-known can be formed using the following manufacturing techniques. a stationary phase 116, a swellable matrix and Pre-formed "sand inches" by alternating layers of spacer means selectively Inlet housing member 11 with peripheral edge sonic welded and configured as shown in FIG. 2 and on which the outlet housing member 114 is placed. After that, the entrance The mating end 186 of housing member 188 and outlet housing member 190 are coupled together. An air-tight and liquid-tight seal is formed. If ultrasonic welding is desired, connect Branson 5onic Power Compan in Danbury, Cutt. using the technology described in the 1971 information sheet PH-3 of the contents of which are incorporated herein by this reference. and.

The venting means 150 described above represents an optional configuration of the disc-shaped embodiment of the column. , the purpose of which is to allow the air within the column to be evacuated from the column during use.

Typically, the venting means 150 becomes occluded when all air is removed from the system. configured so that In another embodiment, the venting means 150 removes the hydrophobic medium. No. 4,113., the contents of which are herein incorporated by reference. As disclosed in No. 627, it allows the passage of gas but not liquid.

In another embodiment shown in FIG. 10, chamber 152 expands radially outwardly. ing. The term "radially expanding outward" means that the volume inside the chamber is This means that the volume is smaller than the volume of the periphery of the chamber. In this configuration, Referring to FIG. 10, inside d.

The distance between the dispensing means 112 and the collecting means 114 of the dispensing means 1 at the periphery d, 12 and the collection means 114.

If the stationary phase 116 is hydrophilically swellable, the sample solution in contact with the separated phase 116 causes swelling of the separated phase. As the separated phase swells, the inlet side of the separation medium and The pressure difference between the outlet sides increases, thus restricting sample flow. housing in front As described above, i.e. by configuring it to expand radially outward, the stationary phase input The pressure difference between the inlet and outlet sides decreases towards the periphery, thus increasing the stationary phase clamp. Maximize the use of chromatographic separation capabilities to increase the adsorption capacity of a given unit. substantially increase. The housing configuration described above is suitable for non-swellable stationary phases. However, it is still possible to employ the applicant.

In another embodiment, also shown in FIG. 10, a series of concentric distribution channels 1 40 and the volume of each of the concentric collection channels 170 is within the chromatography column. It increases from the periphery to the periphery. Thus, the swelling of the hydrophilic and swellable stationary phase Blockage of the channel is prevented, thus ensuring uniform distribution of sample and maximum utilization of column capacity. use is promoted.

In the example shown in Figure 10, lines A, A'', C and C' are chromatographic A line representing a cross-section in a parallel plane perpendicular to the longitudinal axis of the column. be. Lines B and B'' are a concentric distribution channel 140 and a concentric collection channel, respectively. A line representing a cross-sectional view of a plane substantially tangent to vertices 146 and 176 of B and β' make angles α and α' with lines A and A''. Lines B and B′ that make angles α and α′ with respect to lines A and Ao, respectively, are defines a radially outwardly expanding chamber 152, which also This defines a limit to the expansion of the stationary phase 116. As mentioned earlier, the radius The optimal shape for embodiments that extend outward in the direction is such that the stationary phase 116 is at the outermost apex. It is as if it is just in contact with 146 and 176. At this time, the stationary phase 116 If the stationary phase is swellable, in its most swollen state, the stationary phase It just contacts the outer vertices 146 and 176. Are the angles α and α′ the same? may vary1, depending on the number of layers of the swellable matrix and the specific properties used. It should be understood that the values may vary depending on the fixed matrix. typical Typically, α and α' are approximately 2-1/2°.

Lines D and Do represent the bottom 142 of the concentric distribution channel and the concentric collection channel, respectively. It represents a cross-sectional view of a plane including the bottom part 172 of the flannel, with respect to planes C and C''. and define angles β and β'. Therefore, for planes C and C′, it is The plane planes and Do, which form angles β and β′, respectively, are the depths of the channels gradually increasing. This defines the slope of channels 140 and 170. In the embodiment shown in FIG. In this case, β and β' are typically each about 5°. However, these All angles are mutually and absolutely variable.

Similarly, the radial distribution grooves 130 and/or the radial collection grooves 160 of the column Chromatography columns configured to increase volume from the inside to the periphery It is also within the scope of the present invention to do so. Such configurations can be found here No. 3,361,261, incorporated herein by reference.

As understood by those skilled in the art, the retention volume of a chromatography column is minimal. It is desirable to do so. With this in mind, the radially outwardly expanding chamber The optimal configuration is such that the distance d2 allows for maximum swelling of the swellable stationary phase. This is to ensure that there is no unused space left. In this way , the differential pressure at the periphery is minimized, and at the same time the retention volume is reduced to its lower limit. Ru. This housing configuration also allows for a single layer of matrix or no space between the layers. It is possible to use multiple layers of the matrix without intervening make it look like The radially outwardly expanding chamber cooperates with the stationary phase thus configured. to distribute the sample evenly across it.

To provide a chromatography media matrix that is consistent and easy to handle. at least one component used to form the perforated matrix. A self-bonding structural fiber having a long length is desirable. Such fibers are stationary phase , both in the wet "as-formed" state and in the final dry state. However, it provides sufficient structural integrity. Such structures are used during and for the purposes of processing. This makes it possible to treat the stationary phase as a sheet when used as Ru. Preferably, the sheet forming the chromatographic medium comprises an aqueous stream of fibers. Formed by vacuum filtering the slurry. The sheet can also be pressure filtered or Non-aqueous slurries can be felted. This sheet has excellent flow properties and It exhibits a uniform high degree of porosity and is substantially homogeneous. Generally, this media is thick Ranges from about 5 mils to about 150 mils (dry), but thicker or thinner Even if the sheet is thin, the sheet may be spirally wound or stacked as described above. If it is possible to form a column that can be operated in a similar manner, it can be used.

In constructing the chromatography column of the present invention, the the chromatographic medium has a uniform thickness over its entire length and width; It is also important that the media have a substantially uniform density throughout. medium It is preferred that the layer is substantially homogeneous with respect to itself, but It will be appreciated that for applications and materials, non-homogeneous structures can also be used. .

The solid stationary phase maintains a substantial pressure difference across the solid stationary phase when in use. Solid stationary phases are intended to perform separations by Have sufficient compressive strength to withstand deformation under applied loads is required. Such compressive strength exists not only in the medium itself, but also in the space. the carrier means (if present) and the chromatographic media or solid stationary phase It must also be present in the inner core that is accessed.

Because of the swellability of the media, as an element of the invention, the space between each layer of the media is There is cooperation between the support means and/or the walls of the chamber and the matrix. It takes Pacer means are not essential to this aspect of the invention and may be used in one embodiment. In some cases, chromatography columns are prepared without such spacer means. I can do it. In such columns, the self-supporting matrix of the invention is simply Consists of one continuous column medium. In columns where the medium does not undergo significant swelling Advantageously, spacer means are omitted. Spacer means medium conditioned This allows for expansion and increased distribution of the sample flowing through the stationary phase. swelling The spacer means provided between each layer of the chromatographic medium is This results in distributive movement of the sample as it passes through the solid stationary phase.

The spacer means uniformizes the thickness and density of the chromatographic medium during use. Functions to control. The spacer means may also be used to separate layers of the chromatographic medium. It can serve as a backing or support for. This last aspect is This is particularly useful.

Any spacer means used should be inert with respect to the chromatographic step. It is preferable that the structure be made of a material that is suitable for use. Inert means that the material is a solid stationary phase. This means that it does not adversely affect the functions of the

Referring to FIGS. 2 and 3, the spacer means comprises a mesh 22. Referring to FIGS.

Alternatively, if the column configuration is as shown in Figures 4 to 10. In this case, the spacer means 182 can consist of mesh or woven fabric and mesh. . The woven material can function to some extent to guide the sample flowing through the medium. , substantially uniformly distributing the sample axially and circumferentially through the medium. Metsu The material creates a spacing between the media and the flow due to the compression of this permeable media. It makes it possible to adjust the expansion of the medium in order to prevent "blockage" and assist in dispensing or guiding the sample flowing through it.

Menonyu material ranges from 1716 inches (1,6 mm) to 174 inches as a general guideline. It is an open type material with openings in the range of inches (6.4 m).

This selective spacing means, i.e. the thickness of the fabric and especially the mesh material, and the use The pore size of each used can be determined by conducting tests varying these factors. , which can be easily determined by a person skilled in the art. These spacers means Factors such as aperture and thickness may include, for example, swelling, wettability, thickness, chemical composition, etc. the type of medium used, the flow rate of the sample through the stationary phase, and e.g. the number of turns, the medium It depends largely on the surface area of the stationary phase, which depends on the thickness of the stationary phase, the diameter of the stationary phase, etc. Therefore, this It is very difficult to clearly identify these variables, and they are determined by trial and error. , or can be determined by more complex test methods to determine the optimal parameters. I have no choice but to say it.

The currently preferred mesh material is polypropylene C0NWED (TD- 620 grade).

The total width of the stationary phase according to the invention can be infinite, with the actual diameter limited by spatial constraints. limited only by practical considerations.

The overall column diameter or width can be increased without theoretical limits. Therefore, there is no limit to the size or material amount of the sample to be separated in the column bed. do not have. The diameter is therefore chosen to separate the desired amount of sample material to be produced. It is possible to increase it.

In operation, a sample is forced through a stationary phase and a chromatographic medium The body is separated into separate chromatographic fractions. selectively used The pacer induces and permits the flow of this sample as it moves through the column. In this way, the latent capabilities of the medium can be brought out and utilized in a better manner.

Referring to Figure 1, the sample is preferably introduced at the bottom of the column and solidified. flow to the outer surface of the body's stationary phase and then to the chromatographic medium and spacer. Flows radially inwardly through the layers of means into the channels 21 of the core tube 24. It entered and was washed away from the center. From what has been stated above, it can be seen that the radial flow It will be clear that the energy is circulated in the opposite direction.

Referring to FIG. 4, the sample is preferably introduced through the inlet 118 and the dispensing means 12 2 and secured by a radial distribution groove 132 and a concentric distribution channel 130. is substantially uniformly distributed over the surface of phase 116 and includes radial collection grooves 140 and and concentric collection channel 170 and exit via outlet 156.

The chromatography column of the present invention can be used to perform chromatography that is commonly performed using conventional columns. Separation by affinity chromatography and reversed-phase chromatography It can be used for any type of separation. Furthermore, the column of the present invention may also be found useful in areas where columns are impractical. .

The embodiments described above are based on the radial direction of the material through the chromatographic medium. only distributions are shown; however, non-radial distributions (e.g. It will be appreciated that linear or tangential distributions (such as linear or tangential distribution) may also be advantageously employed. cormorant.

The column of the invention can be used for separations in the analytical and preparative fields. Wear. The column can be coupled to all conventional types of chromatography equipment. how many Columns or solid stationary phase cartridges can be connected in series or in parallel. Wear. For large units, use the same or separate affinity columns for multiple columns. can accommodate medium for chromatography or reversed-phase chromatography. and they may be of the same or different lengths and/or diameters. example For example, Daly et al., U.S. Patent Application No. 611.6, filed May 18, 1984. See No. 62. The contents of this application are hereby incorporated by reference. and. The stationary phase described above can be passed through the column without compromising the adsorption capacity of the medium. It has been found that this has the unexpected effect of increasing flow. difference Furthermore, when performing protein and dye staining tests, the stationary phase of the present invention Increased pressure drop when compared to stationary phases not using spacer means as described in It has been found that this results in a uniform distribution of the sample flow through the stationary phase without the use of a stationary phase.

The stationary phase of the present invention reduces overall processing time and provides suitable ROM Has excellent binding ability when used with roughy media . This stationary phase can be used for standard type pumps, gravity feed, and syringes. The preferred mode of use is at pressures from 1 to 5 Qpsi, and even under vacuum. Vine is also used. The stationary phase of chromatography media ensures sterile conditions. Everything is contained in a container that is completely sealed. solid stationary phase Due to the fact that they are manufactured and assembled in a factory, each stationary phase is identical to the other. Unlike conventional columns, there is no deviation, and packaging expertise is required. Eliminates dependence on

Maximizes sample distribution and minimizes retention volume, decreasing from inside to periphery The column can be configured to maximize the utilization of the stationary phase by creating a pressure differential gradient that The useful life of the column is thereby substantially improved.

These columns and disks are used to distribute the sample radially through the medium. It is particularly useful in that means are provided. Therefore, the sheet of media 'fP, in the form of a disc in the column of Figure 1, with or without spacer means; It can also be stacked without.

For the purpose of purifying fluid samples containing cells, e.g. fermentation media or blood, affinity Tangential flow of blood across the IJ wax is preferred.

“Tangential flow” or “tangential flow across a medium” means that a fluid sample is It is intended to be oriented across, but not through, the medium. Typical For a tangential flow arrangement, see Liberte, U.S. Pat. No. 4.551.435. (i985), the contents of which are incorporated herein by reference. I will include it. Blood contains various cells ranging in size from 1 to 20 μm and 400 flow through the medium due to the fact that it contains a variety of proteins. It is difficult to process blood due to this. As shown in Figure 14, When blood flows through matrix 201, blood cells 219 It tends to be seen on the surface.

Lysis of cell walls, e.g. erythrocytes, generally occurs in a very short time with 40,000 dyne/cff1 or more, or approximately 3.000dyne for a time of 1 second or more /co(with very high shear stress. The value of shear stress also depends on the surface area, )　IJ wax surface characteristics, adhesion of blood components to matrix, flow pattern depending on the coagulation mechanism induced by the specific matrix or wall, etc. Exists. Hemolysis, the escape of free hemoglobin from cells, is the destruction of red blood cells. This is a basic indicator.

Cell lysis can be reduced by tangential flow of blood across the surface of the matrix. Yes, the shear stress is less than 3.000 dyne/cffl.

When blood flows tangentially to a surface, there is a floating layer 4a next to the surface. Cell areas are formed. When the floating layer is lost, free hemoglobin leaks into the plasma. and glandular resistance increases. As shown in FIG. The tangential flow of blood across 01 allows cells 219 to pass through the medium. , while simultaneously allowing impurities to be adsorbed by the matrix. Improvements of the invention The well-defined matrix surface and separation device configuration, together with optimal flow conditions, It overcomes the problems of shear stress and cell lysis.

In general, cells prefer high-energy hydrophilic surfaces to lower-energy hydrophobic surfaces. Adheres more easily to surfaces. Platelets, which have an overall negative surface charge, are cationic For surfaces that have a positive overall charge, such as polyelectrolytes and certain metals, There is evidence that it sticks. However, the phase between platelets and positive polyelectrolyte surfaces Understanding interactions requires an understanding of surface adsorption to plasma proteins. Must be. Platelets are larger and fewer in number than the dissolved plasma components, It spreads much more slowly. Most platelets have already interacted with plasma proteins. There is a high probability that the layer will come into contact with the surface covered by the layer. Therefore, the port The interaction of the reamer surface with plasma proteins is more important than the interaction with platelets. It is a consideration. The reactivity of adsorbed protein molecules is Depending on the chemical nature of the surface being treated, this reactivity can cause blockages and blood flow. It affects the characteristics.

The most damaging points in the plasma flow system are the diameter of the tube and the steep drop in the direction of flow. It is a place of great change. Therefore, when configuring a blood purification system, It is necessary to reduce these factors. Also, blood flow against foreign surfaces It is also necessary to avoid or reduce direct collisions.

The flow of blood within a system is determined by the changing molecular interactions between its various components. It is accompanied by To maintain the stability of a chromatographic separation device, Minimize as much as possible any potential hindrances that may arise during the separation process. Should. The main parameters to consider are flow rate, pressure fluctuations and viscosity changes. be. Fortunately, the plasma in the blood is a lubricant that keeps the cells away from the walls of the device. It also functions as a diluent.

Another problem with blood flow within the device is the clumping of cells around the flow path. That is. Blood flow changes from a single inlet to multiple flow channels. As the flow flows into the expanded chamber, a region of stagnant or blocked flow with low shear forces is triggered. It occurs near the entrance of the channel, where platelets tend to adhere. localized blood In regions where hydrodynamics provides an appropriate combination of convective and diffusive transport, , platelets readily adhere to the surface of foreign materials. Other platelets coagulate on the attached platelets. to form aggregates. As the separation progresses, this aggregate coalesces and the final will block the entrance of the channel. This means that the progressive mass transport capacity It can be observed as an increase in resistance to blood flow, accompanied by a decrease. of platelets As the aggregates grow larger, red blood cells will eventually become trapped and adhere to them. As a result, the entire system becomes clogged.

In one preferred embodiment of the invention, at least two components in the sample are An apparatus for carrying out a chromatographic separation comprises (a) one or more sheets; , a corrugated sheet, or a tube-shaped modified polysaccharide material; (a) means for effecting a tangential flow of said sample across said modified polysaccharide material; There is.

The matrix material within the tangential flow separation device may be in sheet form.

For example, one or more sheets of matrix material can be placed at one end of the blood dispensing device. A parallel arrangement is possible with a stage and a blood collection means at the other end. The seat is It can be self-supporting or adhered to a rigid support member.

In this example, a plurality of housings with layers of sheet-like matrix material are used. Can be stacked. These layers are configured to receive the edges of the plate. The sample is separated by support means on the housing wall containing grooves or parallel to each other and parallel to the direction of sample flow to provide channels for tangential flow of The vines are separated by rows of filaments arranged in a vine. Dimensions of this filament and their spacing is high to minimize the pressure difference (Δp) between the inlet and outlet of the device It can be optimized for efficient separation. Thickness of filaments and between them The distance can be determined by one of ordinary skill in the art using routine experimentation. selectively, layer separated by two rows of filaments placed at an oblique angle to each other Can be done.

Advantageously, these two rows of filaments are fixed at their intersection.

Means for dispensing and collecting the sample are located inside the sides of the housing and one or more channels and nozzles on each side of the Contains grooves. Alternatively, the matrix may be constructed in the form of a corrugated sheet and the filament Channels can be created without using agents or nets.

In another embodiment, the matrix material can be configured as a hollow tube, through which so that the sample, e.g. blood, can flow tangentially to the surface of the matrix. Wear. The matrix forms a layer of uniform thickness on the inner wall of the impermeable tube is advantageous. This tube is then coiled within the housing and this Means are provided for introducing blood into the blood vessel and for collecting blood therefrom. choice Additionally, this housing also includes means for regulating the temperature of the coil, e.g. May include fans, thermostats, temperature controlled fluids, etc. See here No. 4,061,141 to Hyden et al., the contents of which are incorporated herein by reference. 977).

In a more preferred embodiment, the matrix is wound helically around a cylindrical core. in the form of one or more sheets. In this example, in the passing sample A separation device for chromatographic separation of at least two components of (1) A cylindrical core, (2) at least a first and second fixed layer wound around the cylindrical core; , there (a) the first phase comprises the modified polysaccharide polymer of the present invention; means for two phases to support and separate and provide channels tangentially to said core; Consisting of (3) means for dispensing a sample into said channel; and (4) collecting a sample from said channel. and (5) a cylindrical housing. It consists of

The device includes a third solid consisting of an impermeable sheet disposed between the first and second phases. It can further include constant phase. This impermeable sheet is suitable for blood and matrix It can be made of any impermeable material that is compatible with the Examples include, but are not limited to, vinylidene chloride (PVC), polyethylene, and others. It's not a thing.

means for providing support and separation and for providing channels tangential to the cylindrical core; are referred to here as meshes, nets, screens, etc. It can consist of two rows of filaments. These are spaced parallel to each other and a first row parallel to the axis of the cylindrical core, and a first row parallel to the axis of this cylindrical core; and a second row of orthogonal filaments. These two rows of filaments Advantageously, they are fixed at their intersection. These two columns are mutually orthogonal beveled if one row is perpendicular to the axis of the cylindrical core It can be arranged as follows. This type of screen or net is a cylindrical A series of fluid channels are provided tangential to the core of the shape.

The cross section of the individual filaments can be circular, square, rectangular or triangular. . The thickness and spacing of the filaments can be determined by experimenting with varying dimensions. Easily determined by the manufacturer. These factors, such as filament thickness and spacing, factors such as the type of medium used, e.g. swellability, wettability, thickness, chemical composition, etc. surface area of the sample stationary phase tangential to the stationary phase, e.g. number of turns, medium the thickness and other characteristics of the chromatographically processed material, e.g. blood or fermentation media. It is highly dependent on the type of sample. Clearly identifying these variables is very Although difficult, it may be determined by trial and error or more complex experimental methods.

Preferably, the first row of filaments is circular and 9.5 mil, and the filaments The second row is circular and 47 mils.

The first row of filaments is 49° to 90° to the second row of filaments. Vines placed at an angle. Most preferably, the first row is about 90° to the second row. placed at an angle of

The filament can be polyethylene, polypropylene, polystyrene, nylon or Advantageously made of elastomers or metals that are virtually undeformable but elastic . The material used deforms the adjacent matrix material layer or impermeable sheet. It can be easily separated, manufactured easily, and has no contact with the sample or matrix. It is something that cannot be affected by touch and deteriorate. Preferably filament The top is constructed of polypropylene.

The means for collecting the sample consists of one or more grooves along the length of the cylindrical core. Ru. These grooves are tangential flow channels and nozzles located at the end of the cylindrical housing. It communicates with Le. The means for dispensing the sample are distributed along the length of the cylindrical housing. Consisting of one or more grooves, these are connected flow channels and cylindrical housings. It communicates with the provided nozzle.

Of course, the means for collecting the sample can also be used as the means for dispensing the sample; It should be understood that any means used can be used as a means for collecting samples.

As shown in FIG. 17, the apparatus includes two layers of matrix material 201 per turn. consisting of two sheets, thus the channel has a layer of matrix on both sides. become. As shown in Figure 15, these two sheets contain two different ligands. nothing. For example, one sheet 220 of the matrix may preferentially contain bacteria 221 or lipids. The second sheet 222 of the matrix is a material that selectively adsorbs heparin. including.

! In one embodiment shown in FIG. The hydrodynamic ram is a helically wound chromatography system around a cylindrical core 200. Two layers of media 201, a spirally wound impermeable film 202, and a spirally wound impermeable film 202. It consists of a flow guiding web 203 wound into a ring. Housing 207 and exit cap Pool 208 houses this spirally wound device. Is this spirally wound? In the media, the impermeable film and web 203 are attached to each end 209 as described above. Sealed by thermoplastic welding using technology. Outlet cap 208 and The housings are held together by welds 210. This housing also , a protective cap 213 containing protected inlet and outlet means 211 and 212.

Referring to the embodiment shown in FIG. 11, one of the chromatography media 201 layers, an impermeable sheet 202 and a flow guiding web 203 form a cylindrical core 20. It is wrapped around 0. The sample is chromatographed in channel 205. a cylindrical core that flows tangentially to the medium and communicates with these channels 205; 200 are collected by a series of grooves 214. The thickness of this media web and layer is 20 4 to obtain optimal flow rate, chromatographic efficiency and cell stability. It is adjustable.

Referring to FIG. 13, a tangential flow filter with one layer of chromatography media 201 is shown. -) IJ Edge places this medium 201 and impermeable film 202 on web 203. is prepared by placing one end 21 of the web 203 on the 5 is not covered with medium 201 or film 202. Opposite end 21 of the media 6 has an additional layer of web 203 thereon and extends beyond the layer of media 201. ing. This medium 201, film 202 and web 203 are then shaped into a cylindrical shape. It is wound around the core 200.

FIG. 16 shows an impermeable sheet 202 and a web 203 as a flow guide. , a layer of PIIMA 216, a highly hydrophilic layer that facilitates protein transfer. and a tangential flow having a BOA or pyrogen or bacteria adsorption layer 217. Figure 3 shows a top plan view of the cartridge. The sample is injected into the blood inlet 218. channel 205 defined by the web.

use For affinity chromatography of the present invention, for reversed phase chromatography, Or bioactive materials can be analyzed by affinity or reversed phase chromatography well known in the art. for any treatment by - or as a support for bioreactors. Available for use.

The material obtained in the present invention has unique properties not found in materials used so far. have. For example, modified polysaccharides such as cellulose, other polysaccharides such as microcrystalline cellulose, etc. The two-component system formed by mixing with the seed polysaccharide and forming a fibrous sheet is It has the advantage of not containing silica material, which normally exhibits non-specific adsorption to proteins. do. Lack of substantial swelling or adjusting the many variables present within the system The highly tunable degree of swelling, which can be easily controlled by Lulose can be replaced by other mechanical reinforcements, manufacturing costs are low, and Equipped with a high degree of exchange or fixation ability.

This will in any case depend on various conditions, including the ratio of comonomers (a) and (b), etc. It can be changed by adjusting the conditions.

Three components formed from modified polysaccharide, modified or unmodified fine particles, and unmodified fibers A system has the ability and rigidity that can be obtained by changing the many variables that exist within the system. and has the advantage of potentially maximizing swelling. The flow rate is By varying the ratio of organic components to clause is possible. In addition, this type of system is different from conventional technology that uses unmodified cellulose. system, it exhibits the advantage that refined pulp is not required in many cases. this is , a polymer bound to a polysaccharide cross-links microparticles to the fiber surface in purified pulp. This is because it functions just as well.

The polymeric component in this system of the invention also functions as a binding resin and therefore Addition of resin to the slurry can be omitted if desired.

In general, conventional techniques use materials with a large surface area to bond the maximum number of chemical groups. However, the material of the present invention has polyfunctional groups in each polysaccharide molecule. It provides a means to combine the These functional groups act as scaffolds for fixation. Preparation should not be limited to substances with large surface areas, as long as they are made available. That's no longer the case.

In protein separation and purification, the main factors to be avoided are This is damage to. In the present invention, this means that only a limited amount of organic polymer This can be avoided by using biocompatible materials such as polysaccharides with Ru. This material is swellable and has minimal denaturation of proteins. biological content Non-specific adsorption of molecules is reduced, but this is due to the absence of acrylic and sugar polymers. This is because they have extremely few such properties and are inherently hydrophilic.

For samples containing cells, such as blood or fermentation media, the sample may cartridges configured to flow tangentially rather than through the Cell lysis can be avoided by passing the sample through the tube. Thus the tangential flow The cartridge can be used to separate or purify components of blood or fermentation media.

For example, Ma) IJ Fuchs, chemical or biological methods used to separate components in a sample. with an extended aldehyde group on the spacer arm to bind the Gantt Consists of cellulose-acrylate composite polymer. creatures like this Chemical ligands include, for example, the commonly used coagulant citric acid-phosphoric acid. - Removal of dextrose (CPD) or heparin from the blood if possible Contains an anticoagulant-adsorbing ligand that binds to the anticoagulant. In particular, three ligands, namely ligand A (low molecular weight amino compound containing hexamethylene diamine), Ligand B (prota amine) and Ligand C (high molecular weight amino polymer containing polyethyleneimine) have been found to be particularly useful for the adsorption of anticoagulants. Lithium containing amino groups The gun is attached to the matrix material as described above.

Another area for design flexibility and control is the ability to carry a variety of anchoring groups. The length of the acrylic polymer is adjustable.

Possibility of changing polymer length due to steric hindrance resulting in solute or ligand accessibility It not only eliminates the oxidation (but also minimizes the leakage of the ligand from the matrix).

To avoid the possibility of internal hydrophilic interactions such as folding, The "me" should not be too long. Approximately 5 to 20 molecules are required to attach the bioligand. A child "arm" is generally ideal.

By using affinity ligands or anchoring groups for well-known scaffolds for biomolecules. This substance is similar to commercially available substances such as 5ephadex@ and 5epharose■. Can incorporate any of the synthetic methods developed in the prior art It is.

Finally, this ma) IJ Fuchs is chemically and physically stable, free from salts and eluents. There is minimal change in dimensional stability upon contact with.

Example Having thus far described the present invention generally, the present invention will now be described with reference to specific embodiments. can be better understood by They are included here for illustrative purposes only. and unless otherwise specified, are not intended to limit the invention. It's not something you can do.

Example 1 Preparation of affinity chromatography system Component A: Cellulose fiber Component B nigericidyl methacrylate Component C: monosaccharide or disaccharide Step I Coupling of C to B Component B (10%) Polymerization using redox catalyst 10% B and 90% component BC (90%) BC copolymer 1st stage 3. The above With the excess amount of catalyst left in bonding step 2 to the components of the polymer, The epoxy groups of component B of the above copolymer are separated by increasing the temperature to 90°C. Can be bound to lurose. While component C is 1,000, component The chemical reaction is exactly the same as in step 1, except that he is polymeric.

90% of the available epoxy groups react with component C under acid or base catalysis. Component C is added to the polymer in the appropriate molar ratio. The remaining 10% is after Leave the cellulose available for binding onto the surface.

Cotton linter fiber 36 g methacrylic acid (or β-carbon Ruboxy 90ml ethyl acrylate) Glycidyl methacrylate 91Triton X-1002, 0g Sodium lauryl sulfate 1.5g ammonium persulfate 3.6g Sodium thiosulfate 3.6g Butylamine　　　　　　　　　1.0+al Examples 3 and 4 are Concerning the formation of a ligand matrix. Example 5 shows that the benzamidine ligand Concerning binding.

Example 3 Preparation of cellulose-GA preligand affinity matrix Purified cellulose 5.0g glycidyl acrylate-1-10,01111Et hoquad C/25 0.5 m11.0M l IC116, 67ml D. 1. HJ 250 l111 method Cellulose 250...1 glue, 1. Minutes at 80°C with stirring in H2O The glycidyl acrylate was added to the reaction vessel. Maintain temperature and continue stirring. While continuing, APS, STS and IIcI were added and the reaction was allowed to run for 1 hour.

The covalently bound cellulose-GA pre-1 Nogando matrix was removed and 7×2 liter of deionized water and subsequent treatment (as in Example 5 below). (such as conversion to Affinity 9 Gantt).

Purified cellulose 5.0g glycidyl methacrylate 12.5111 Cellulose in n, i, H2O with stirring The mixture was dispersed and heated to 80° C. with stirring. Add 9 Glyshiji J Remetaku to the reaction container. Salt, APS and STS were added and the reaction was run for 1 hour. Terminate the reaction and The bound GMA-cellulose matrix was removed and placed in a 5 x 1.8 liter Washing with I, HJ and subsequent treatment (such as in Example 5 below) (conversion into a ligand): This was saved.

Example 5 GMA-cellulose matte of Example 4 The lix was washed with 5 volumes of deionized water. Treated with 1.5% aqueous solution of Na1L Then, 5 volumes of Na1On solution were circulated through the matrix for 1-2 hours at room temperature.

As a result, the epoxy groups were converted to aldehyde groups by the Na1Oi solution. The bath was washed with 10 volumes of deionized water at 25°C to a pH of 7.8. Equilibrated with 01M phosphate buffer. The matrix to which this aldehyde is attached Benzamidine at a concentration of 20 mg/nI was added to a concentration of approximately 1 mg/ml through NaCNBtl, was circulated overnight at 4° C. at a flow rate of 2 cc/min.

Alternatively, for benzamidine binding, add the above benzamidine solution at room temperature for 8 hours. It can also be carried out at room temperature by circulating it for a while. After binding, phosphate buffer at pH 7.8 Unbound proteins were removed from the matrix by washing with buffer solution.

The remaining aldehyde groups were removed from glycine at pH 6.5 in the presence of NaCNBH, The ethyl ester was circulated for 4 hours to inactivate it. This glycine ethyl ester is made by dissolving 1g of glycine ethyl hydrochloride in 100cc of deionized water. The pl+ was adjusted to 6.5 by adding sodium hydroxide solution.

Example 6 Media of the invention containing hydrophobic groups (a) Treatment Poly(n-octyl acrylate)-g-cellulone reagent amount Refined pulp (+260) 20 gn-octyla Acrylate 50ml glycidyl methacrylate 5g ammonium persulfate 2g thio Sodium sulfate 2g1, refined pulp (+260) was thoroughly dispersed in water in 30 3 liter round bottom flasks.

2. Thoroughly mix n-octyl acrylate and glycidyl methacrylate. and poured into the reaction vessel.

3. After injecting the monomer into the reaction vessel and thoroughly mixing the reaction mixture, add persulfuric acid The ammonium solution and sodium thiosulfate solution were charged into the reaction vessel at room temperature.

4. The reaction mixture was stirred vigorously and the reaction temperature was raised to 82°C within 15 minutes. .

5. Stirring was continued for one hour in the temperature range of 80-85°C.

6. After cooling the reaction mixture, the product was thoroughly washed with water.

Refined pulp (+260) 5. Og glycidyl methacrylate 12.5ml ammonium persulfate 0. 5g Sodium thiosulfate 0.5g Sodium iminodiacetate 2g1, purification valve (+260) in 30 reaction vessels 800ml of water (make sure it is thoroughly dispersed).

2. Inject glycidyl methacrylate into the reaction container (inject it into the reaction container) and pour the reaction mixture thoroughly. After mixing, add ammonium persulfate and sodium thiosulfate to a reaction vessel at room temperature. Filled.

3. Stir the reaction mixture vigorously and raise the reaction temperature to 80℃ within 15 minutes. I set it.

4. Stirring was continued for 1 hour at a temperature range of 80-85°C.

5. Cool the reaction mixture to 60°C, then add 1 noum of sodium iminodiacetate to the reaction volume in G). Filled into the container. The reaction was continued for an additional 26 hours.

6. Cool the reaction mixture, filter and wash the product.

Example 8 Hyoji 3rd period Hyakuhiki prostitution attack Is it possible to separate Protein A from Staphylococcus A? , (thin n-wall, tangy texture) . Its relative 11-11 reactivity towards the PC region of IgG (thus, the protein Contain A is a powerful biochemical for the selective removal of IgG from normal or immune blood. It becomes a body ligand and becomes 0. The immobilization of protein A on a solid support is To isolate whole or individual subclasses of gG, to separate cells, or as a means of immunotherapy was studied to remove immune complexes from plasma.

(a) Materials and methods Recombinant protein A is used as highly purified natural protein A.

Recombinant protein A has the following physicochemical properties, Repli Purchased from gen. Purity is TSK-3000HPL analyzed with 214n111 It is 98% or more due to C, and 94% or more at 280 nm. 5OS-PAGE Therefore, there is a single main band at a molecular weight of 45.000Mw + 1.000. generated.

It has an extinction coefficient of 0.1% of 0.16+0.02 at 270nm. have IgG binding activity was determined by immobilized IgG binding activity when analyzed by the Lowry method. According to the total protein percent bound to the HiI-1gG column, more than 95% It was above.

Structural studies performed on recombinant protein A by Repligen According to the DNA sequence originally described, four homologous FC-binding domains D , A, B, and C positions.

b) Preparation of medium Composite media prepared according to Example 4 prepared by grafting onto cellulose The cellulose acts as a solid support and the GIAA or GA polymer Provided chemical functionality for bonding. Then two types of affinity matrices A sample was prepared. Type A carries an amine group and is suitable for protein binding. Glutaraldehyde was used. G type carries a hydroxyl group and is a protein Requires oxidation by peroxide to form aldehydes as a means of bonding Was.

Protein A binding Covalently bonding bovine san to 1,6-diamino to the glycidyl group of the matrix By doing so, amino groups were introduced into the matrix. 0. using IN hydrochloric acid The total number of amino groups titratable by potentiometric titration is 1 It was about 1.8 meq per g. The matrix carrying amino groups is PI (7,3) as a sexualization buffer equilibrated with 0.1 M sodium phosphate; followed by 1.0 mg/+n I of sodium cyanoborohydride. and 10% glutaraldehyde in the same buffer for 6 hours at room temperature. Elution Activate until the solution shows the absence of glutaraldehyde in the Schick reagent solution. Remove excess glutaraldehyde by washing the matrix with sexing buffer. there was. MaI-IJ socks carry an activated aldehyde group with the following shape, amino The preparation is now complete for protein A binding via the group.

A solution of protein A was prepared in lO+ng in 0.1 M sodium phosphate at pH 7.3. /m+ and the presence of (1,0 mg/ml) sodium cyanoborohydride. The mixture was circulated for 16 hours at room temperature. Unbound ligand is removed in the eluate. The matrix was removed by washing with buffer until no traces were detected. . Excess amount of active groups was removed using 0.1M sodium phosphate at pH 6.5 as blocking buffer. Inactivated by circulating 1% hydrochloric acid glycine ethyl ester in aluminum for 4 hours. It became sexualized. The matrix was then diluted with 0.25M chloride) washing with the desired buffer, followed by the equilibration buffer used in the affinity imparting step. The amount of ligand bound to the cartridge by Lowry protein analysis is performed and this value is It was determined by subtracting it from the amount of Tin A.

IgG purification procedure Cartridge with 50mM NaP at pH 7.6 plus 25011IM sodium chloride. The edge was balanced. Human gamma globulin in the above buffer width of about 2.5 mg/ml <IIGG) solution (SigmaG-4386> by - times of passage or circulation, Flowed through the cartridge. Next, unbound gamma globulin is matrixed. The mixture was washed out using the buffer described above. The bound gamma globulin is p The matrix was eluted with H2,3 in 0.2 MGIy-HCI buffer. Conclusion The amount of combined 116G was determined by 0D28° of the eluate. Then M) I Re-equilibrate with J Sox PH7-6 buffer and ready to bind another sample. Ta.

(6) Spacer arm length and 1 (to change the length of the GG coupling spacer arm) The effects of hexamethylene diamine (21 people, sample Nαl-3), ethylene Epoxy with diamine (16 people, sample Nα4-6) and periodate (6,0 Human, sample Nα7-9) was used to conjugate protein A, and the bound protein A measure the amount of IIG, bind HGG, measure the amount of bound HGG, and This was investigated by measuring the G-protein binding ratio. The results are shown in Table■ .

Example 9 Using glutaraldehyde as the linking group, follow the procedure for type A (amino group) media. Protein A was immobilized on the matrices of Examples 4.5 and 8 by The ability to bind HGG was investigated. The results are shown in Table ■.

Effects of type of medium and type of binding method on the action of immobilized biomolecules To demonstrate the effect of forming aldehyde groups via periodate oxidation, Goat anti-116G was immobilized on a G-type matrix by Shown in V.

cloud Example 1O (1) Prepared according to Examples 1.4 and 8 in the form of a radial flow cartridge. The matrix was treated with 150mM NaP and 0.25M sodium chloride at pH 6.2. and equilibrated.

(2) Circulating 10% glutaraldehyde solution (in the above buffer) for 5 hours (133+nl).

(3) The cartridge was washed with the above buffer.

(4) 6.4 g r’AB and 6.4 g STI in each cartridge. 640 ml of the above buffer + 0.2% NaC to be circulated overnight through a NBHa).

(5) Using the above buffer solution and 0.2MG1y-HCI at pH 2 and 3 alternately, The cartridge was thoroughly cleaned.

(6) The cartridge was inactivated with 1% glycine ethyl ester.

et al.) Test method: (1) Trypsin solution in 50mM NaP at pH 7.6 (Porcine Pa ncrease trypsin, Sigma) 41 and 250mM sodium chloride Buffer was flowed through each cartridge at two different flow rates. .

Fractions were collected.

(2) The cartridge was eluted with 0.2MGIy-11cI at pH 2 and 3.

(3) Protein was detected by 0021°.

(4) Trypsin activity was determined by analysis using Kabi's colorimetric substrate S-2302. sex was detected.

One unit of trypsin activity = 1. O/+nl/min 00. . .

(5) The results are shown in Table VI (PAB) and Table ■ (PAR and STI).

Example 11 Immobilization of glucoamylase enzyme on affinity cartridges for processing beverages Immobilization of glucoamylase is essential for the industrial production of fructose syrup from starch. It has already been applied to construction. The preparation method used for large-scale production of glucose is Covalent attachment of enzymes to porous inorganic materials containing mino groups by glutaraldehyde obtained by combining However, when removing glucoamylase from beer, In some cases, inorganic materials such as silica affect the beer's texture and color. That's called This is because silica ionizes as silicic acid on solid surfaces at low pH. Ru. Cellulose-based substrates are inert and do not ionize, so they are suitable for purifying and separating beverages. It stands out as more preferable.

Glue purchased from Sigma Chea+1ca1, St. Louis, Missouri. Coamylase (α-1,4-glucan glucohydrolase, EC: 3.2.1 ．． 3) is obtained from a strain of Aspergillus oryzae. This enzyme is It has a molecular weight between 48,000 and 80,000, and the subunit structure usually has Not worth it. The carbohydrate content of this enzyme ranges from 3% to 30%, mainly man Contains glucose, galactose, and in some cases glucosamines Also present are xylose and xylose. The structure of carbohydrates is serine and threonine water Monosaccharides, trisaccharides, trisaccharides and Exists as a tetrasaccharide unit. The isoelectric point is between 3.9 and 5.5, and the optimum pH is 5.0 to 5.5.

Measurement of enzyme activity Enzyme activity determines the amount of glucose released from starch by gel coalase. It was measured by measuring dynamically versus time.

Glucose production was performed using a SigIIIa analyzer purchased from Chemica1. (StockNa 15-10) and Sigma's technical booklet Co, 15 It was measured using the one described in LIV. Fluctuations in glucose production are caused by Cary As the change in optical density per minute at 340 nm by a 210 spectrophotometer It was measured.

Glucose analysis principle This analysis involves the conversion of glucose by ATP in the presence of hexonase. Conversion to su-6-phosphate and subsequent conversion of NADP to NAIIPH It is based on reduction. NADPII shows high absorption at 340na+ A , 4°) Since NAIIP shows no absorption at this wavelength, this reaction is caused by Al1 This can be tracked by measuring the increase in °. A34° due to the formation of NADPH The increase in is directly proportional to the amount of glucose present.

One unit is obtained from starch at pH 4.5 at 55°C. On+g glucose Defined as liberating gas.

Immobilization of glucoamylase on A-type affinity matrix Type A matrices prepared according to Examples 4 and 8 were activated by glutaraldehyde, thus making it suitable for immobilizing enzymes. This is an amino group matrix of a 12-atom spacer.

(a) Connection method 1. The matrix was washed with 50 ml of deionized water.

2. At room temperature, 0.05M phosphate buffer with pH 7.5 + 0.025M sodium chloride Equilibrate with 50 ml of water.

3. Circulating 50+ul of 1.0% glutaraldehyde in buffer at room temperature. It was activated by

4. Remove excess glutaraldehyde by washing twice with the above buffer. did.

5. In the presence of Na 4' (usually using 1 mg/ml NaBH+), Ligand (10 mg/ml in the same buffer) was bound overnight at room temperature.

1g of glycine hydrochloride ethyl ester was dissolved in 100ml of phosphate buffer. . The final pH was adjusted to 6.5. Then add NaB11 to bring its concentration to l. It was set as mg/a+1.

6. Unbound glue gunt was washed out with the same buffer until baseline was obtained.

7. Glycine hydrochloride at pH 6.5 in the presence of NaB114 for about 4 hours at room temperature Excess active groups were inactivated by circulating the ethyl ester.

8. Wash out excess reagent with the same buffer.

9. The amount of nitrogen bound to the column and gas was determined by nitrogen analysis.

The activity of glucoamylase samples was measured using a 1% solution of. At time 0 , 0.25 ml of sample was added to this substrate 11 in a 55° C. bath. 1 After 0 minutes, 1.25 ml of 0.2M potassium phosphate buffer p) 17.5 was added. . A 40 μm aliquot of this solution was then reconstituted with Sigo + a glucose fraction. Add analysis Nα15-UV 3.0+ol and record A34° after 5 minutes of incubation. Ta. Subtract an appropriate blank value from the absorbance reading to obtain As4°, and use this A34 The activity unit of the sample was calculated from ゜.

(C) Enzyme activity test method 1. Approximately 1 g of the matrix prepared as described above was mixed with a phi of 0.2 Suspended in 101 M sodium acetate.

-2, the suspension was vortexed and then centrifuged at 2600 rpm for 10 minutes.

3. The supernatant was decanted.

4. Add 0.2g of pellets to 1.0% amy-pectin1. In addition to Oal+, the above Enzyme activity in the matrix was measured by the method described above.

Table VIII O, 25m1 1. Oml 1.25 mI O, OPHM Binding of protamine to A medium. 10m1 (media 0.5g) and 100m1 (medium 5g) size radial flow cartridge configuration.

PIIMA is a polyhydroxy methacrylate, which is produced by acid hydrolysis of GMA. It was prepared by converting the glycidyl group to a hydroxyl group as described above. Li The conditions for activating PHMA for Gund binding are as follows.

a. Car) Wash the IJ with 2β water, 1.5% Na1O, (aqueous solution) Wash with activated C1 water for 60 minutes. d. Equilibrate the cartridge with 0.1M borate buffer (pH 8.2) e. Above Add 42 ml of a 10 mg/ml protamine solution in buffer (Sigma NaP -4005 protamine, free base derived from salmon sperm grade IV) 15m1Z 4 hours circulation in minutes f, Wash the cartridge with O, LM borate buffer at pH 8.2 (the washing solution should not bind (save for determination of the amount of protamine) g, pl + 8.2 g, 15% Min solution (ethanolamine or diethylaminopropylamine) 100ml was added with 4 x 500 g of Na0)1a and circulated at 15 mf/min for approximately 20 hours. Wash the cartridge with the following solution 11 (1) 0.1M borate buffer, pH 8 , 2 (2) 0.15M sodium chloride, pH 2,7 (pal lowered with hydrochloric acid) ( 3) 0.05M NaP + 0.25M NaCl, pH 7,6 (4) 0.2M Glycine-HCl 11112.3i, 0.02M NaP + 0.15M NaC Equilibrate with l5 pH 7.0 buffer [10ml size cartridge] ', the same binding procedure scaled down approximately 50 times [combination efficiency 80-90%] 2. Test method a, 10ml cartridge 501 of plasma bound with protamine and 20 units/ml Heparin (Sigma Heparindoor 005, activity 15, derived from pig intestinal mucosa) 0-18011sPK1 unit/mg) was flowed through the column at a flow rate of 1 ml/min. Ta.

b. 100ml cartridge 10100O plasma and 50 units of protamine bound Heparin was flowed through the cartridge at a flow rate of 15+ml/min.

3. Analysis method a, Michael D., Klein et al., “Colorimetric fraction of chemical heparin in plasma” This analysis utilizes the color change of Azur A when heparin is added. . It is useful for 0 to 10 units/ml and is useful for heparin anticoagulants. independent of quality activity. This analysis is performed on plasma, whole blood, and buffered physiological Can be used for saline solutions. Mix the dye and sample together and then immediately add 620 na Read with a spectrophotometer at +.

Controls include samples with known concentrations of heparin and samples without heparin. be.

b. Coagulation procedure for quantitative analysis of heparin in plasma, kit #8 from Sigma. 7B Test principle: Activated factor X (Xa) is introduced into plasma in the presence of traces of heparin. Neutralize rapidly with inhibitors. Plasma inhibitors and factor Xa in the presence of heparin At a particular time in the first step of the reaction, the amount of factor Xa neutralized is Proportional to the concentration of heparin present.

Table IX 4. Results: PIIMA 77 units 128 units - Answer 1-1 2.98 mg of protamine bound; the inactivating agent used was diethylaminoprotamine. Pyramine $2 - 12.86 mg of protamine bound; inactivating agent used was ethanol amine B. Removal of heparin from plasma with PHMA + protamine, 100 m IPIIMA + Fo 9 Mi 7 $1 42.194 units 12 0 units P) IMA + Pro 9 Mi 7! :2 24.490 units 68 ．． #2-ethanolamine inactivated with 6 units #l-diethylaminopropylamine inactivated by B. PHMA + Promin with heavy length of ``Shoka'', grip 11PH MA+7tl Mini/$1 42.194 units 120 units PHM A+B o9 Mi712 24,490 units 68.6 units 11-Jie Inactivated with thylaminopropylamine I2-Inactivated with ethanolamine 1. Materials made by Kosara (all buffer solutions/materials are pyrogen-free water and pyrogen-free Prepared using a glass container. ) PHMA medium in a 10d column a. Wash with 20 d of water S, 1.5χ Na1O, aqueous solution passed at 1 m/min for 30 minutesc, 20a Wash with water from d. d, Equilibrate with O, 1M sodium phosphate buffer at pH’y, s. e. The above buffer. Circulate 6d of sulfuric acid polymyxin B solution of 5■/I11 in liquid overnight (0,571 minutes) f, Wash out unbound polymyxin B sulfate with 0.1M phosphate buffer (unbound (Save for measurement of quantity) g, then equilibrate the column with p)I a, o 0.1M borate buffer, 1% ethanol solution while adding 4×25 μ of NaBH4 60 min apart. Circulate 20m of min solution i, p) I 7.5 0. IM NaP buffer and 0.15M at pH 2,7 Wash the column alternately with hydrochloric acid ±0.25M sodium chloride solution (2 (ld, each 2 times per solution) j, pu 7.5 0. Equilibrate the column with IM NaP buffer (PHM^) For the 250d cartridge (12.5g of media), scale up 75 times. same process] This 10m column is a small column with a diameter of 10am.

2. Invasion of mice a, 0.02M NaP + p) 17.0 of 0.15M sodium chloride + 10 xrg/d E, coli LPS (Sigma's E, co 055:8 Ribopolysaccharide Hidden L-2880 derived from 5-phenolic extract was attached to the IOd cartridge. For the 250d cartridge, the flow rate was 2 M/min.

b. Same as a. except that 20tIg/rail of BSA was added to the buffer solution. character.

3. Level of sacrifice Test method used: Whittaker bioproduct QCL100O dye quantitative LA L analysis Principle of this test method The endotoxin of Durham-negative bacteria is Lfulus Amebocyte Lysate. (LAL) catalyzes the activation of the proenzyme. The initial rate of activation is Measured by the concentration of toxin. The activated enzyme generates p-nitrogen from a colorless substrate. After catalyzing the splitting of troaniline (ρNA) and stopping the reaction with acetic acid, spectroscopic Yellow color is measured at 405 nts.

After experimenting with standards, the correlation between absorbance and endotoxin concentration was 10- It is linear in the range of 200 pg/yd.

Application of this test method Dilute the starting material with sterile physiological saline to an appropriate concentration range, analyze and draw a curve. A similarly diluted unknown sample is analyzed against this curve. This unknown test The pgld value obtained for the sample multiplied by the dilution factor is the endotoxin value in the unknown sample. is equal to the concentration of

4, u a, binding efficiency biactivation P) 60 μ of polymyxin B sulfate per 1 g of IMA gives 26-31 .mu./g can be combined.

b. LPS binding ability (everything depends on the concentration of LPS) test method In A, 10-Cartridge (700-1000) ■/g medium 33.5 μg/■ Sulfate polymyxin B 250m cartridge 43.7Mg/af PMBS 530μg/g medium In test method B, 1OH1 cartridge 26.5Mg/■PMBS250ad cartridge 3 2.1μg/■Pl’lBS Urasen ± once ″ Blow-in A and Blow-in G Fill-immobilized recombinant Using protein A and protein G as reference proteins, affinity separation media An example of the function is given. Protein A is a well-known protein derived from staphylococci. The Fc5l region of immunoglobulin interacts with immunoglobulin G. proty Uses of Contain A include detection and purification of antibodies and immune complexes, immunoglobulin flux. certain pathogens through large-scale production of antibodies, monoclonal antibodies, and extracorporeal plasma filtration. These include potential treatments for cancer and autoimmune diseases.

Protein G is an immunoglobulin-binding bacterial cell isolated from the G group of streptococci. It is a cell wall protein. It was recently cloned and has a cell wall system. and thus in sufficient quantities to fully explore its properties. available. Is protein G Fc of immunoglobulin? join to if area Similar to protein A in that it It has been reported to be a binding reagent. This is because protein G is protein A subclass of animal IgG and human IgG that has little or no interaction with A. This is because it combines . The ■type receptor (protein G) is the ■type Fc receptor. Protein A is thought to have broader immunological uses than protein A. Ru.

1. Square and standing materials Recombinant protein A was purchased from Repligen (Cambridge, MA). Purchased from J). Protein G (type GXII@) is available from Genex (Merry Gaithersburg, Rand). Both proteins are shown in Table X and XI. The affinity support used was 1 activated to have an aldehyde group at the end of the 8 atom long spacer arm. . This support with extended arms immobilizes macromolecules and makes it possible to immobilize giant molecules of similar size. To avoid steric problems that sometimes occur when used to purify large molecules. chosen. All standard chemicals used during the immobilization or evaluation steps were It is of reagent grade and purchased from a regular laboratory supply supplier.

Purified bovine and human 1-core G fractions are manufactured by Stg*a Chemica1 Co., Ltd. (Mizu Purchased from St. Louis, California).

Human serum was obtained from the American Red Cross (Farmington, CT) and It was partially purified by exchange. Polyclonal rabbit anti-human albumin serum, and goat antiserum against human) IgG from Cone BioProducts. (Seguin, Texas), and also ion-exchanged and partially ion-exchanged it before use. Purified. In one experiment, both affinity purified goat anti-human IgG were used. . During binding to the affinity support and testing, to obtain the required flow rate, A screw pump (Ratnin) was used. online to observe dynamic events. A UV monitor (Gilson) was used. Protein measurement at 0.0. . .

It was verified by the Lowry method.

2. Examination of Keiraishi There have been few reports regarding experiments or the optimal binding conditions for Gantt. Because of this, two sets of experiments were conducted. For the first set, as reported in Table X, Both ligands were attached to aminophilic supports via glutaraldehyde.

In this case, the ligand was stabilized using sodium borohydride as a reducing agent. . Comparative tests using various polyclonal 1gG Consistently demonstrated high elution capacity.

The results shown in Table XI highlight several additional points.

(1) Regarding protein A and protein G, trimethylamine borane is also Uke is a reducing agent that can be added. (2) The novel matrix of the present invention can be easily scaled up. By increasing the size of one column and the amount of bound ligand by 2.5 times, which The column also yields an elution amount of TgG that is close to a complete scale-up. (3 ) The relatively weak interaction of bovine species with proteins is consistent with published literature. I will.

Overall, a weak interaction with protein A of especially goat and bovine IgG was observed. The data are consistent with the published literature, but are not fixed. All of the ligands obtained were recombinant in nature and therefore the results cannot be interpreted as a result of cloning. It should be noted that this may vary depending on the origin and consistency of the product. all Total protein recovery in all experiments was in the range of 91-100%, which This indicates that unrecoverable protein loss to the matrix is not a factor. is suggesting.

Comparison of protein A column and protein G column: Reducing sodium borohydride Interaction with various polyclonal 1gG as base agent Human IgG (pure) 17.52 3.5 19.81 4.52 Human RGC (Serum> 16.85 3.37 16.85 3.76 Rabbit Al5A (antiserum) 10.16 2.03 16.54 3.78 Yagi Hakko gG (affinity purified product) 6.88 1.38 19.44 4.44 Binding and experimental conditions A type matrix (1,6-diamithexane bonded matrix) A laboratory column containing 1 g of was used. The column was buffered with 0.1M borate buffer at pH 8.2. It was activated with 0.25% glutaraldehyde in the solution. excess glutaraldehyde After washing the column to ensure that there is no residue, add Gantt's solution (5 μ/d) in the same buffer. It was circulated for 4 hours. Reduce the column with sodium borohydride (NaBH) , blocked with 1% hydrochloric acid glycine ethyl ester, pH 8.2. this block and reduction steps were allowed to proceed overnight for convenience. Buffer (0.05M solution, pH 7.6) sodium chloride and 0.25 M sodium chloride) and 0.2 M glycine-hydrochloric acid ( After several alternating washes with pi(2,3) 0,15M acidified to pH 2,0. Wash the column with sodium chloride, then 0.25M sodium chloride and 0.0 Equilibrate with 5M sodium phosphate pH 7.6 buffer followed by IgG fraction. It passed through Shigon. The flow rate was 2 d/min throughout. Maximize contact time All IgG solutions were circulated for 1 hour to ensure stability. The elution from the rgc column is , using glycine-hydrochloric acid at pH 2 and 3.

According to the Lowry method, the protein A column has an efficiency of 100χ. and contained 5 μm of immobilized ligand. On the other hand, protein G The column bound with 88% efficiency and contained 4.4μ of immobilized ligand. Ta. To keep the experiment constant, the solution used in the experiment was a total of 50+ag on each column. tJNvI (by dilution) to provide (±5+++g) of IgG, Protein A and Protein G columns avoid possible variations due to starting samples. Therefore, the experiments were conducted simultaneously using the same pool of experimental samples. The above ratio is the eluted The interaction ratio between total IgG and immobilized ligand (i.e. 1 gG eluted/ligan bound) Defined as

l-■ Comparison of protein A column and protein A column Interaction with various polyclonal 1gG as base agent Human RGC (pure) 43.99 3.55 46.66 4.42 Human IgG (Serum) 39.03 3.15 40.05 3.79 Bovine IgG (pure) 25.19 2.03 44.44 4.20 Binding and experimental conditions Using a slightly larger column containing 2.5 g of the amino-type matrix of Table X, the present invention The scale-up efficiency of the new matrix was experimented.

The column was activated and bound the ligand as described for Table X. this In the experiment, trimethylamine borane was used as a reducing agent. This reagent is was dissolved in IM acetic acid and then diluted with glycine ethyl hydrochloride (final concentration 1 In combination with 65χ, pi(6,0), unbound sites were blocked. prevention and return The original lasted one night. The column was washed, equilibrated, and run as described above.

Protein A columns have an efficiency of 99%, as determined by the Lowry method. and in this case contained 12.4 μ of immobilized ligand. Pro for this The Ting G column bound with 97% efficiency and 10.6μ of immobilized ligand. It contained. To keep the experiment consistent, the total amount of solution used in the experiment was added to each column. The starting sample was adjusted (by dilution) to provide 125 ■ (±5 ■) IgG. The Protein A and Protein G columns were The above ratios as in Table is eluted IgG/bound ligand.

The matrix composition and attachment method are compatible with either ligand.

Based on the results obtained, it appears that bacterial Fc receptors are not important in the first It is linked to the solid support through the amino site, and connects to the FceJl region of immunoglobulin. This leaves the Fc receptor site accessible for interaction. Ru. Column containing 2.5 times more matrix and immobilized ligand The arithmetic scale-up observed when experimenting with human IgG The same applies to devices with a flow rate in the range of 100-1,000 d/min. This shows that it can be manufactured using

Further support for matrix scale-up potential is provided by novel geometries. was obtained when human IgG was bound to this matrix. This support is It differs in that it has been preactivated to the aldehyde form.

(Pre-activation involves diluting the glycidyl groups of cellulose-bound GhA with diluted perchloric acid. by reacting with a weak acid such as to form the dihydroxy form, i.e. PHMA. achieved. This PHMA is then reacted with periodate to form the aldehyde It takes shape. ) Experimental results show flow rates of 2d/min, 54/min and 125i/min. For devices that can be flushed, 10■, 80■ and 3,000■ glue gant fixation Establishment ability was introduced. Maximum affinity filter is 3g or more from goat anti-human serum. The upper IgG could be separated.

Urasengo-” Rena Proin A 13.8g i-matrix Constructed in a tangential flow cartridge type with 250 id containing hexamethylene diamine. Protein A was immobilized on the induced matrix. This cartridge first Equilibrate with 0.1M borate buffer (approx. 2000m) at pH 8.2, then , 2 of 10++g/d protein AI solution 7d and passed through the cartridge. Circulated overnight (21 hours). ? The JL amount is approximately 100 for the first 2.5 hours. d/min, 50d/min for the next 1.5 hours, and 25d/min for the remaining hours. Met.

This cartridge then added glycine ethyl at pH 8.2 to the already existing solution. Add 5 m of a 26% solution of ester to a final concentration of about 1% (dead volume is about 1 30d).

Add 2 parts (100 μl) of sodium borohydride 20 minutes apart from each other. , the solution was circulated for an additional 4 hours, then the cartridge was washed with pH 8.2 buffer. The eluate was saved as a control for washing and protein measurement.

Pass 500 d of 1% glycine ethyl ester solution of pi (8,2) through the cartridge. The column was inactivated by circulating the sodium borohydride solution. (4 x 250 µm each) were added at 30 minute intervals. After about 1-1 portions and 2 hours, The solution was circulated overnight at 4 °C and then 4 parts sodium borohydride (400 (2) was added at intervals of 45 minutes) and the solution was circulated at room temperature for about 4 hours.

The column was then washed with the following solutions:

= O, 1M boric acid solution at pH 8.2 680--0.2VI glycine at pH 2.3 - Hydrochloric acid solution 1140d - 0.1M boric acid solution at pH 8,2 450d - pH 2,3 0.2M glycine-hydrochloric acid solution 1140d - pH 6.5 buffer 450m -0.2M glycine-hydrochloric acid solution 570d-p) I6.5 buffer solution 570d 70 in the matrix, based on Lowry's color test, as described above. protein A was bound to it. The graph of pressure difference (Δp) convection 11 (-7 minutes) is This is shown in Figure 26.

The tangential flow cartridge was then tested for HTG purification. Three different experiments The test was carried out at a flow rate of HrG pH 7.6 containing 0.25M sodium chloride 25.100 and 140 d The color was circulated for 60 minutes at a flow rate of The capacity of the system is 50+d/min (194■) at a flow of 100d/min (208■). ) and 25 id/min (195 ■).

Table X11 50 747 1920 194 1500 2.18 93 (5mg/m1) 1.00 683 2000 208 1700 2.9 7 95 (5B/m1) 25 683 2160 195 1700 2.78 95 (5mg/ml) In another example, undiluted plasma (7kl) at pH 7.8 was pumped at 75ml/min for 60 minutes. circulated. As shown in Table XTIr, 98% recovery of TgG was observed. It was.

Table XIII (0, [1,650) In the third experiment, IgG in hemolyzed blood (blood stored at 4°C for 2 months) was purified. Manufactured. The tangential flow cartridge was first washed with a 0.3M sodium chloride solution at pH 7.8. Equilibrate, then empty the cartridge and lyse at pH 6.76 (unadjusted). Refilled with blood (50d). This blood is circulated at 75 m/min for 60 minutes. As shown in Table XIV, the amount of eluted IgG was ,. It is 486.4■ based on 0Dbs. Based on the Lowry method of 20 It was 3IIIg. Is the purity of IgG greater than 95% as shown in Figure 27? It was. ″A summary of the experimental results is shown in Table XIV.

Boar! Two masters ・-The 1 gun of the heparin that is in charge of ゛)゛ The experimental medium was placed in test tubes. Buffered saline, human plasma and blood The whole was mixed with lO or 20 units/I11 of heparin (heparin from porcine intestinal mucosa, Sigma) I-7005, activity 150-180 USPKI U (unit) /■). The test fluid is left in contact with the medium for 30 minutes, The samples were then centrifuged at low speed for approximately 5 minutes, just until the media pelleted.

Color analysis of heparin in plasma published by Michael D. Klein Law Anal tical Bioches+1str 124:59-64 ( (1982) (see Example 13).

Graph showing the relationship between surface area and % heparin removal for three affinity ligands. (vehicle 0.1g/10cffl, contact time 1 hour, blood volume 10d heparin concentration 20 units, citrate concentration 2.9 μ/d) is shown in FIG.

As shown in Figure 19, Ligand C is a high molecular weight polymer (polyethylene). (imine ligand) was the strongest heparin adsorbent. For 250d medium By contacting 10 d of blood containing 20 units/I11, 100 Heparin removal of χ is achievable. In addition, 29 ■ quinnes present in the blood It was also found that approximately 20% of the acid salt was adsorbed by the same medium.

Next, the effect of contact time was measured. The results are shown in Figure 20, where In different cases, using ligand A (hexamethylene diamine) and protamine. The % removal of heparin is shown relative to the time interval.

This experiment consisted of 7CIX7.5CI11 or 52.5 in a 150 d blood bag. This was done by laminating CD-sized pieces of tzetaphilic paper. . With Ligand A, 680 units of heparin (452 x 150 i x 1 0 units/d) was found to be removed per cii of paper. This corresponds to the ability of the medium to remove 13 units of heparin.

This result is shown in Figure 20, where 90% of the adsorption sites for heparin binding It has been shown that approximately 1 hour of contact time is required for use.

Next, we measured the effect of protamine on the amount of heparin removed from the blood. 0 various concentrations of glue Gant (0-200 ■ protamine/g of medium), 20 units/d 10 IIN of blood with 50% heparin.

The samples were allowed to react for 1 hour and the amount of bound heparin was then determined as described above. Ta. For two sheets of matrix material with surface areas of 12d and 6cia. A graph showing the amount of heparin bound to the matrix is shown in FIG.

Backyard ■-” The tangential flow cartridge is a 250D size cartridge shown in Figure 11. It was constructed based on the following. Unilabine first so that the medium does not obscure the core. The material is wrapped twice around the core, then the media and the impermeable film are wrapped around the core. Roll together with the rolling material until the diameter is 2.600 in (66,04 am). there was. Cut the media and film and cut them into 4 inch (101,6 m) pieces of sea urchin rubbing. ) pieces 2 in (50,8++ m +) Inserted. Finally this subassembly is held together by elastic bands and polypropylene Cap with a lens end cap.

A 250 d size cartridge (housing volume) has a 14.4 g matrix. The total surface area was 180 cd.

The tangential flow cartridge was completely filled with saline at 50 d/min. 1 unit 10U/- of heparin is given to the blood of the patient and administered through this pre-filled cartridge. The filtered blood was delivered at a flow rate of 12 d/min (Δp = 1.5 psi). For every d fraction, the last 10 d fractions were collected. Heparin concentration in each fraction The intensity was analyzed by color analysis and plotted as shown in FIG. Figure 23 shows The figure shows that approximately 70% of heparin was removed. In another experiment (de (data not shown) 2 units of blood were passed through a -800d size cartridge. 80% of the heparin was removed by washing.

Then for blood and saline, at a flow rate between 0 and 160 d/min. The pressure difference across the tangential flow cartridge was measured.

The results shown in Figure 22 show that for a medium with a length of 6'11'', the increase in flow rate As a result, the pressure difference for blood is more steep than that for saline. It shows that it will increase rapidly.

In Table xv, various volumes of filtration for a 250d tangential flow cartridge are shown. Blood component analysis for blood is shown. Table XVI shows the 800IIi size Blood components for various volumes of blood when using tangential flow cartridges shows an analysis of

1 aircraft 4 to 8 Rent (Shishi) 41 and a half 4t? vl’l (cm2) Kibansamu, Gin PA (min・) Rikkapanto”Jl　　(m97’t:+’I Me7 (Islands)/q-+Z<yp, ) Bruise i (ml 7ml 1n) Blood 5 years old ≧ 1 year old ≧ 1 year old 1 year old (ml) International investigation report

Claims (1)

[Claims] 1 (1) a water-insoluble polysaccharide covalently bonded to a synthetic polymer; and (2) a polysaccharide covalently bonded to a synthetic polymer; Ma is (a) a polymer having an epoxy group that can be directly covalently bonded to the polysaccharide; (b) one or more polymerizable compounds, (i) said synthetic polymer for affinity-claimed ligands or biologically active molecules; a chemical group capable of forming a covalent bond, or (ii) A modified polyester made of a material having a hydrophobic chemical group. sugar substance. 2. The material of claim 1, wherein the synthetic polymer is a homopolymer. 3 The synthetic polymer is glycidyl acrylate or glycidyl methacrylate 3. The material of claim 2, which is a homopolymer of. 4. The material of claim 1, wherein the synthetic polymer is a copolymer. 5. The material of claim 1, wherein the polysaccharide is cellulose. 6. Said chemical group capable of forming a covalent bond reacts with an affinity ligand. 2. The substance of claim 1. 7. Causes covalent bonding of the synthetic polymer to the affinity ligand. wherein said chemical group capable of decoding is selected from the group consisting of primary amines and aldehydes; Substance of requirement 1. 8 The affinity ligand is an enzyme, a nucleic acid, an antigen, an antibody, a carbohydrate, a lectin, an enzyme cofactor. 7. The substance of claim 6, which is an enzyme inhibitor or a binding protein. 9 The ligand is benzamidine, protein A, protein G, polymixi 9. The substance of claim 8, selected from the group consisting of protein B, protamine and heparin. 10. A self-supporting cellulosic fibrous matrix comprising the material of claim 6. 11 Improved type consisting of using the substance of claim 1 as an insoluble ligand support Affinity chromatography method. 12 A method of carrying out a chemical reaction using an insolubilized biologically active molecule, the method comprising: , wherein the insoluble support for said molecule is the material of claim 1. 13. Claim wherein said material has the form of a self-supporting cellulosic fibrous matrix. Any one method of item 11 or 12. 14. A method for preparing the modified polysaccharide substance of claim 1, comprising: (1) the polysaccharide The compound (a) having an epoxy group capable of reacting with the hydroxyl group of the compound (b) and covalent bonding of the compound (a) to the polysaccharide in the presence of the polysaccharide. polymerization under insufficient temperature conditions, thereby forming synthetic polymers (a) and (5). (2) combining the polysaccharide with the chemical group of compound (a) of the synthetic polymer; A method comprising reacting under conditions sufficient to produce a covalent bond. 15 Chromatography of at least two components of the sample flowing through the column A chromatography column for separating housing and at least one solid stationary phase within said housing, said chromatography As a chromatographic means of dispersing a sample through a stationary phase that has functional groups. and a matrix that is useful for means for collecting the sample after the sample has flowed through the stationary phase; A chromatography column in which the trix comprises the substance of claim 1. 16 Chromatography of at least two components of the sample flowing through the column A chromatography column for separating (1) a housing, the housing comprising: (a) an inlet housing member; and (b) an outlet housing member, the inlet housing member and the outlet housing member; the ging member defines a radially outwardly extending stationary phase chamber; (2) the stationary phase in the stationary phase chamber expanding outward in the radial direction; A matrix that has chromatographic functionality and is effective for chromatographic separations. said stationary phase and said stationary phase chamber extending radially outward cooperate with each other. to provide a substantially uniform radial distribution of the sample across the stationary phase; A column in which the matrix comprises the substance of claim 1. 17 Chromatography of at least two components of the sample flowing through the column A chromatography column for separating (1) a housing, the housing comprising: (a) an inlet housing member; and (b) an outlet housing member, the inlet housing member and the outlet housing member; the mounting member defines a stationary phase chamber; (2) the stationary phase in the stationary phase chamber expanding outward in the radial direction; A matrix that has chromatographic functionality and is effective for chromatographic separations. The stationary phase chamber and the stationary phase cooperate to transfer the sample through the stationary phase. a column distributed across the column, said matrix comprising the material of claim 1; Mu. 18 Chromatography of at least two components of the sample flowing through the column A chromatography column for separating (1) a housing, the housing comprising: (a) an inlet housing member; and (b) an outlet housing member, the inlet housing member and the outlet housing member; the ging member defines a radially outwardly extending stationary phase chamber; (2) a stationary phase in the chamber that expands radially outward; A matrix that has chromatographic functionality and is effective for chromatographic separations. including sous, The stationary phase chamber and the stationary phase cooperate to distribute a sample across the stationary phase. 2. A column comprising: a matrix comprising a substance according to claim 1; 19 A method for chromatographic separation of at least two components of a sample. contacting said sample with a self-supporting fibrous matrix containing a modified polysaccharide material. The modified polysaccharide substance (1) binds to the water covalently bonded to the synthetic polymer. an insoluble polysaccharide, and (2) the synthetic polymer, (a) a polymer having an epoxy group that can be directly covalently bonded to the polysaccharide; (b) one or more polymerizable compounds, (i) said synthetic polymer for affinity-claimed ligands or biologically active molecules; a chemical group capable of forming a covalent bond, or (ii) made of a material having a hydrophobic chemical group; 20. Claim wherein said contact consists of a radial flow of said sample through said matrix. The method of item 19. 21. Claim wherein said contact consists of a tangential flow of said sample across said matrix. 19 methods. 22. Clamp for chromatographic separation of at least two components of a sample. A chromatography device comprising: (A) a modified polysaccharide material comprising: (1) a water-insoluble polysaccharide covalently bonded to a synthetic polymer; and (2) the synthetic polymer. but, (a) a polymer having an epoxy group that can be directly covalently bonded to the polysaccharide; (b) one or more polymerizable compounds, (i) said synthetic polymer for affinity-claimed ligands or biologically active molecules; a chemical group capable of forming a covalent bond, or (ii) being made from something with a hydrophobic chemical group; , and (B) a procedure for performing a tangential wave of the sample across the modified polysaccharide material. A device consisting of steps. 23. The claim that the modified polysaccharide substance is in the form of a sheet, a corrugated sheet or a tube. The device of item 22. 24 Clamp for chromatographic separation of at least two components of a sample A chromatography device comprising: (1) a cylindrical core; (2) at least first and second stationary phases wound around the cylindrical core; Consisting of (a) said first phase comprises a modified polysaccharide substance, said substance being combined with (i) a synthetic polymer; (ii) the synthetic polymer comprises a covalently bonded water-insoluble polysaccharide; (1) Polymer having an epoxy group that can be directly covalently bonded to the polysaccharide (2) one or more polymerizable compounds, (a) said synthetic polymer for affinity-claimed ligands or biologically active molecules; a chemical group capable of forming a covalent bond, or (b) consists of a substance having a hydrophobic chemical group, and the first phase is in the form of a sheet; (b) the second phase provides support and separation and is tangential to said cylindrical core; (3) dispensing a sample into said channel; (4) means for collecting a sample from said channel; and (5) a cylindrical tube. A device consisting of Uzing. 25 further comprising a nozzle disposed at an end of the cylindrical housing, the tip Means for collecting a sample from the channel extends forward along the axial length of the cylindrical core. one or more grooves in fluid communication with the channel and the nozzle; 25. The apparatus of claim 24. 26 further comprising a nozzle disposed at an end of the cylindrical housing, the tip means for dispensing sample into the channel along the axial length of the cylindrical core; a channel and one or more grooves in fluid communication with the nozzle; The device according to claim 24. 27. Said means for supporting and separating and providing channels may (1) a first row spaced in parallel and perpendicular to the axis of the cylindrical core; (2) a second row spaced parallel to each other and arranged at an angle to said first row; row of 25. The device of claim 24, comprising two rows of filaments consisting of. 28. Said first column and said second column are fixed at the point where they intersect, The device according to claim 27.