JPH06508058A - adsorption matrix - 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] adsorption matrix ■, Background of the invention Technical field The present invention preferably uses salt-dependent adsorption chromatography (salt-dependent adsorption chromatography). liquid by ent adsorption chromatography) A novel thiophyl group for use in the isolation, purification and immobilization of proteins from thiophilic adsorption matrices, methods of making the same, and protein The present invention relates to a method for purifying white matter, preferably immunoglobulins.

Salt-dependent adsorption chromatography is used to detect high concentrations of salts, especially lyotropic ropic) (water-structure-forming) protein onto an adsorption matrix in the presence of a salt. Consists of combining. Specific binding of proteins is achieved by adjusting salt concentration The bound proteins are absorbed into the adsorbent matrix by reducing the salt concentration in the medium. eluted from the lix.

e.g. metal chelate matrices, hydrophobic matrices and thiophilics Salt-dependent adsorption matrices, including chemical matrices, are known. Specification In the book, the term “thiophilic adsorption matrix” means that the ligands A hydrophilic adsorption matrix containing vinyl sulfone groups attached to Concentrations of otrovic salts such as alkali sulfates and alkali phosphates Below is a matrix that can adsorb proteins such as serum proteins. this Particularly remarkable thiophilic adsorption is caused by the sulfur atom in the divinylsulfone group. It is mainly believed that the degree of thiophilicity is determined by the degree of thiophilicity attached to the vinyl sulfone group. to be strengthened or weakened by the given ligand .

Matrices exhibiting thiophilic adsorption can be Characterized by binding proteins. The difference lies in the thiophilic matrix. immunoglobulin from human serum is much stronger than albumin at a given salt concentration. This is reflected in the fact that the elements are combined. Binding albumin to a thiophilic matrix However, this is typically not necessary for binding immunoglobulins. Requires a higher concentration of salt than required. Hydrophobic matrix is immunoglobulin It is characterized by binding albumin more strongly than phosphorus.

Thiophilic adsorption matrices are especially suitable for nucleic acids, nucleotides, and serum. proteins, including immunoglobulins and enzymes, and other polypeptides. Used in the fractionation of biopolymers such as tides.

Two types of adsorption matrices with pronounced thiophilicity, the so-called T -Gel and nitrilophoric matrices are known It is being Furthermore, thioaromatic matrices that exhibit hydrophobicity are known.

prior art U.S. Patent No. 4,696.980 M-X-CH2CHz So, CH2CH2-3-Y (where M is polymer hydrophilic It is a network structure, and X is an O (oxygen), N (nitrogen) or S (sulfur) atom, Y is an optionally substituted alkyl, aryl or heteroaromatic group; do S is located two carbon atoms away from the sulfone group and belongs to the ligand. It is a sulfur atom.

Typical matrices belonging to this group include divinyl mercaptoethanol. by reacting with a sulfone-activated hydrophilic matrix (e.g. agarose). is generated.

First, the hydrophilic matrix M-OH is activated with divinylsulfone: M-OH+CHz=CH-5ot-CH=CH.

→M-0-CH, -CHl-3OI-CH=CH, then divinylsulfone Reacting the activated hydrophilic matrix with mercaptoethanol: M　OCHt　CHt　SO2CH=CH2+H3CHx　CHl　OH →M　OCHz　CHt-So! CHt CHl-3-CH, -CH, -OH The thiophilic effect is due to the fact that these matrices typically contain long alcohols as ligands. In contrast to known hydrophilic matrices with kill chains, albumin This entails not combining the components.

The above types of thiophilic adsorption matrices are similar to potassium sulfate. type IgG, I from serum and ascites fluid to which Bic salt was added at a concentration of 0.5M. Surprisingly, it has specific selectivity for gA and IgM immunoglobulins. It was discovered in detail. After immunoglobulin binding, the matrix was treated with 0.5M sulfur. Unbound contaminating protein can be removed by washing with acid potassium buffer.

Immunoglobulins are then prepared using low concentrations of sodium chloride, e.g. 0.1M sodium chloride. can be released by elution with a buffer containing a

This method requires relatively high Immune globulin is extracted from a fluid containing immunoglobulin at a concentration (approximately 1/- or more). It includes a rapid and convenient method of purification.

However, thiophilic adsorption matrices of the above type require strict It has many disadvantages associated with its chemical structure.

As evident from the above chemical structure of the thiophilic adsorption matrix, the sulfone Sulfur atoms located two carbon atoms apart after the group are included. in the past This sulfur atom is important for the very specific selectivity exhibited by the matrix. considered essential, and this is explained in European Patent No. 0.168.363. Book (Example 4) and Jerker Porath and Makonnen Bel Tibtech by ew, 1987, Vol. 5, pp. 225-229, especially It is clear from page 226 that if a sulfur atom is replaced with nitrogen or oxygen, It has been concluded that the ``thiophilic'' character would be much weaker if It has been reported that the series is tilted by S>N>>01Se. This teaching is Coupling to vinyl sulfone activated matrices can result in in the selection of possible and available substances that have the desired effect. This suggests strong limitations. In addition, polythiol compounds such as mercapto Ethanol is toxic and malodorous and therefore the activated matrix of the necessary safety measures to be taken when coupling the substance to Therefore, it is difficult to work with them. This means that, for example, activation The activator, divinyl sulfone, which is produced when Trix is formed early. This is especially true in situations of use where toxicity was not taken into account in advance. .

U.S. Pat. No. 3,897,467 has the structure=M-Y-X-R (wherein R is aliphatic or heteroaromatically substituted, both containing one nitrile group) is the basis, X is NQ, S or 0 (however, Q is H where n=0, !, 2 or 3 (CH2 ), and Y is preferably -CH, -CH, -5ot -CH , -CH, -) consisting of a hydrophilic polymer coupled with a ligand of A nitrilophoric adsorbent is described.

These adsorbents have properties comparable to those of the thiophilic matrices mentioned above. However, it does not necessarily contain the essential sulfur atom and two carbon atoms in the terminal direction from the sulfone group. No. Presumably, this is due to the presence of one or more nitrile groups in the ligand. It can be unique to the current situation.

Nitrilophoric adsorbents appear to have limited use; in aqueous solution, It is known that nitriles can be hydrolyzed, especially in basic or acidic solutions. This is true during and during use for the purification of immunoglobulins. Regeneration, sterilization and (typically at high or low pH and in an autoclave) Typically used for depyrogenization and pyrogenzation. This means that the adsorbent is unstable under certain conditions.

The above thiophilic and nitrilophoric adsorbents can be used in a method consisting of the following steps: It has therefore been used for the purification of immunoglobulins from liquids: l) The liquid in which the immunoglobulin is to be purified is diluted with 0.5M, pH 7.6. Mix with a solution of potassium sulfate + 0.1M Tris/HCI and then typically The liquid is brought into contact with the adsorbent by passing the liquid through a column containing the adsorbent. 2) 0.5M potassium sulfate + 0.1M Tris/HCI! Unbound proteins are washed from the adsorbent using a buffer solution containing a washing step in which the 3) Low salt content, e.g. 0.05M sodium phosphate to grow pH 8.0 An eluate in which binding proteins, including immunoglobulins, are eluted using an existing buffer solution. Release process.

Analytical Biochem by Hutchens and Pora istry, Vol. 159 (1986), pp. 217-226, Immunization from serum This method is described as the general and best method for purifying globulin. Journal of Immunologica by Belew, M. et al. l Methods.

Volume 102, pp. 173-182 (1987) from ascites fluid and monochrome Purification of immunoglobulins from in vitro cell culture supernatants containing natural antibodies. We describe such a method in order to do so. As far as is known, adsorption and Immunoglobulins can be purified by using different pH and ionic strengths during rough washing. No instructions were given on how to purify it. A by Lihm and Heegaard Naval Biochemistry, Vol. 192, No. 64-6 9 (1991) to purify immunoglobulin from rabbit serum. Eel 0.5M potassium sulfate + 0.1M Tris/HCf (pH 7,6) describes the use of 0.75M ammonium sulfate at neutral pH.

In addition, other proteins other than immunoglobulins, such as flounder lectin and chicken Pushin (Analytical Bio by Huches and Porath) chemistry, Vol. 159, pp. 217-226 (1986)) and and serum albumin (Analytic by Lihme and Heegaard) alBiochemistry Vol. 192, pp. 64-69 (1991)) If the ionic strength is 0.5M potassium sulfate (1=1.5), then If the strength is increased beyond that it can be bound to a thiophilic adsorbent. It is known. Therefore, the binding of serum albumin is approximately 1.2M (1=3.6 ) is known to potentially occur at ammonium sulfate concentrations (Lihme et al. and Analytical Biochemist by Heegaard y Vol. 192, pp. 64-69 (+991)).

However, a drawback of that method is that in addition to immunoglobulins, e.g. Also contains significant amounts of contaminating proteins such as phosphorus and alpha-2-macroglobulin The eluate should be provided. Furthermore, the method is particularly effective at low concentrations of immunoglobulins. Fluid containing bulin, e.g. 0.01-0.1 μg of immunoglobulin per ml Binding immunoglobulins from cell culture supernatants typically containing have relatively low ability to do so.

These drawbacks limit the use of the separation principle and often lead to reduced yields. the need to combine the method with other supplementary purification steps, which increases costs. It necessarily involves sex.

2. Disclosure of the present invention The object of the invention is to provide a simple and inexpensive to produce and special Can be manufactured from divinyl sulfone activated matrix without safety measures The object of the present invention is to provide a new and different thiophilic adsorption matrix.

For this purpose, the divinyl sulfone group is is bonded to the hydrophilic polymer network via a nitrogen atom and further The hydrophilic polymer network structure is bonded to a vinyl sulfone group or a ligand. and the said ligand has the general formula (wherein, X is NQ (where N is nitrogen, Q is H(CH 2), (where n = 0.1.2 or 3) or 0 (oxygen)], and R is an optionally substituted aromatic ring system consisting of one or more rings or a heteroaromatic ring system, in which R does not contain a nitrile group. The present invention provides a thiophilic adsorption matrix characterized by Therefore, it is achieved.

The present invention provides that if the ligand has aromatic or heteroaromatic character and the divinyl sulfur The S coupling atom between the Hong group and the ligand is replaced by 0 or N If so, the thiophilicity is at least equal to that of the known thiophilic matrix. It was discovered that it is possible to provide a highly thiophilic adsorption matrix. It was done.

Therefore, adsorption according to the prior art (Porath & Belew, supra) Reduced thiophilicity of the matrix applies to the adsorption matrix of the present invention. It's surprising that you don't get hooked.

Particularly preferred ligands are selected from the substituents listed in claim 2 and are Particularly preferred are the ligands described in range 3. It doesn't cost anything In addition, these ligand precursors can be divinized via oxygen or nitrogen belonging to the ligand. Nylsulfone is easily attached to the vinyl sulfone of the activated polymer network. difference Furthermore, coupling bonds of these precursors generally do not require special safety measures. .

The concentration of the ligand can be varied. 50 per 1 of the lubricating matrix Ligand concentration of ~80 μmol, preferably 50-40 μmol, especially lO ~40 μmol degree is particularly preferred.

The hydrophilic network structure to which divinyl sulfone is bonded can be used as a particle, as a membrane, or as a It is contained in the membrane and contains agar, agarose, dextran, starch and sesame. polysaccharides such as lurose, and polyacrylamides, polyamides, and polyimides , polyesters, polyesters, polymeric vinyl compounds, and their substituted natural or synthetic organic polymers, respectively selected from synthetic organic polymers such as derivatives It can be your body. Agarose is particularly preferred.

It is easy and inexpensive to manufacture and must be manufactured under special safety measures. In addition to eliminating the need for strong acids, the novel thiophilic adsorption matrix of the present invention relatively stable in aqueous solution under conditions or strongly basic conditions and elevated temperatures. Ru.

Another object of the invention is to produce a thiophilic adsorption matrix according to the invention. The goal is to provide a method for These thiophilic adsorption matrices are The divinyl sulfone group is an ether oxygen atom, a thio-ether sulfur atom, or a nitrogen atom. comprising a hydrophilic polymer network bonded through a divinyl sulfonate The group is further covalently bonded to a ligand, said ligand having the general formula (wherein, X is NQ (where N is nitrogen and Q is H(CH,), (where n=0 ．． 1.2, or 3) or 0 (oxygen)], and R is one or more. an optionally substituted aromatic or heteroaromatic ring system consisting of more than one ring and R is a nitrile group, and the mold coalesced network is contacted with divinyl sulfone. The activated polymer network is then activated by Reacts with precursor. The ligand precursors are 2-hydroxypyridine and 4-hydroxypyridine. gin, xanthine, 4-methoxyphenol, l-hydroxybenzotriazo L, 4-aminobenzoic acid, 2-hydroxybenzyl alcohol, 2,4-dihyde Roxy-6-methylpyridine, 4-aminosalicylic acid, 2-aminothiazole, 2-aminopyridine, 2-aminopyrimidine, 2-hydroxypyrimidine, 4- Hydroxypyrimidine, imidazole, 3-amino-1,2,4-triazole , 4-hydroxybenzoic acid butyramide, 2-hydroxybenzhydroxamic acid , phenol and 4-chlorophenol.

Another object of the invention is to provide a method for purifying proteins from liquids.

For this purpose, the thiophilic adsorption matrix according to the invention The liquid is a thiophyllic adsorption matrix. The protein is then contacted with a thiophilic adsorption matrix or liquid. This is achieved by providing a method for purifying proteins from liquids recovered from .

The proteins possible according to the invention include all proteins, especially immunoglobulins, albumin α-1-antitrypsin, orosomucoid, Gc-globulin and factors ■Serum proteins including streptavidin and β-galactosidase protein from the fermentation liquid, alkaline phosphatase from calf intestine, protein A and protein G.

Another object of the invention is to use known thiophilic and nitrilophoric adsorption matrices. by using the novel thiophilic matrix according to the present invention. extracting immunoglobulins from liquids, offering greater binding capacity than previously known methods. An object of the present invention is to provide a method of purification.

Its purpose is especially for types IgG+, IgG*A, IgG*s, TgGs In vitro cell culture containing IgM and IgH (murine) immunoglobulins Less than 1 immunoglobulin per ILl of fluid, including culture supernatant It is an object of the present invention to provide a method for purifying immunoglobulin from a liquid having a concentration of .

The purpose is to add a lyotropic buffer to a liquid and make the liquid absorb thiophilic. The thiophyllic adsorbent matrix is lyotropically Wash with buffer solution and dissolve the washed thiophyllic adsorption matrix. Elution occurs in a synergic liquid, and the synergic buffer in the liquid is 2.25 or more. Above, it preferably has an ionic strength of 2.25 to 4.5, particularly 3.0 to 4.0. This is achieved by providing a method for purifying immunoglobulins, characterized in that Ru.

An ionic strength of 2.25 or higher is better than the ability to achieve by known methods. Surprisingly, it was found that it produced quite a large capacity.

Another object of the invention is a method for obtaining purified immunoglobulins of greater purity than known methods. The goal is to provide the following.

This purpose is to ensure that the lyotropic buffer solution is below 2.25, preferably between 0 and 2.2. 5. A liquid paste, characterized in particular by having an ionic strength of 0.6 to 1.5. adding a Robic buffer and contacting the liquid with a thiophilic adsorption matrix; washing the thiophilic adsorption matrix with the lyotropic buffer solution; The thiophyllic adsorption matrix, which has been washed with This is achieved by providing a method for purifying immunoglobulins from a liquid.

Lower ionic strength does not allow this to result in smaller yields of immunoglobulin Surprisingly, it gives eluted immunoglobulins of greater purity. Do you get it.

In particularly preferred embodiments, the pH of the lyotropic buffer solution is 7.5 or higher. Below, preferably 2.5 to 7°5, particularly 3.0 to 6.5, particularly preferably 3.5 to 6°0, specifically 4.0 to 5.5.

In other preferred embodiments, the otorovic buffer in the liquid is about 2.25 or greater; preferably has an ionic strength of 2.25 to 4.5, especially 3.0 to 4.0; Offers both greater binding capacity and greater purity than more known methods .

Possible thiophilic adsorption matrices include the novel thiophilic matrices of the present invention. Also known as x.

Therefore, the thiophilic matrix has divinyl sulfone groups as ether oxygen sources. The thioether is attached to the polymer network via a sulfur or nitrogen atom. The divinyl sulfone group is further covalently bonded to the ligand, and the divinyl sulfone group is further covalently bonded to the ligand. Is it Iko? a) optionally substituted and linked via a sulfur atom to the divinylsulfone group; alkyl, aryl or heteroaromatic groups, b) bonded to the divinyl sulfone group via an oxygen atom or a nitrogen atom; is substituted with one or more rings, and the substituent is nitrile. consisting of an aromatic or heteroaromatic ring system containing no groups, and C) a nitrile group; or at least in a ring having one or more side groups containing a nitrile group. Divinyls with one nitrogen atom and via a sulfur atom, an oxygen atom or a nitrogen atom selected from aliphatic or heterocyclic, attached to a sulfone group; selected from the aforementioned divinylsulfone activated polymer networks.

Additionally, known thiophilic adsorption matrices and the novel thiophyllic adsorption matrices of the present invention Possible polymeric networks for the matrix can be formed as particles or as films. polysaccharides such as agar, agarose, dextran, starch and cellulose, especially agarose, polyacrylamide, polyamide, and Imides, polyesters, polyethers, polymeric vinyl compounds and their substitutions These are known polymer network structures such as derivatives of

For lyotropic buffers and lyotropic buffer solutions according to the invention Possible otrobic salts are sodium sulfate, potassium sulfate, ammonium sulfate, Known compounds such as sodium phosphate, potassium phosphate and ammonium phosphate Machine salt, or (sodium citrate, sodium tartrate, potassium citrate, alcohol Organic salts of polyhydric carboxylic acids such as potassium stone or mixtures thereof.

Liquids possible according to the invention include immunoglobulin-containing liquids, in particular blood, serum, dehydrated liquids. or a biological liquid such as a cell culture supernatant, especially a cell culture supernatant.

The immunoglobulins possible according to the invention include all immunoglobulins, especially type I gG+, I gG*A, 1 gGxs, IgG palm, IgA, IgM, IgD and and IgE immunoglobulins and especially murine and human immunoglobulins. yric adsorption matrix As mentioned, replacing the sulfur atom with, for example, oxygen or nitrogen gives Y and does not contain one or more nitrile groups. Thiophilic efficiency may occur if the ligand is an aromatic species or a heteroaromatic species. Surprisingly, we found that this is not the case if you have a family species. in contrast , for example, a matrix in which -3-R is replaced with a phenyl ring, i.e. structural formula : %formula% The matrix with the corresponding mercaptoethanol derivative according to the prior art It shows particularly strong thiophilic bonds, much stronger than can be achieved. I found out that Characteristic thiophilic phase of this phenyl-derivatized matrix The interaction is that human serum immunoglobulin G interacts more strongly with its matrix than albumin. It clearly shows itself in its union with Kusu. Mercaptoethanol Its strong thiophilic bonding of this matrix compared to its derivatives has been shown to be useful in human immunology. Globulin G binds more effectively at lower concentrations of otrovic salt in the sample. It clearly shows itself in that it is combined. This is due to the concentration of the ligand on the matrix. The effect is highly dependent on Trix requires the presence of lyotropic salts to bind effectively, but Even in the absence of otrovic salts, matrix beams with high ligand concentration Even binds immunoglobulins. This strong effect of immunoglobulins at high ligand concentrations A thiophilic bond may consist of, for example, ammonium sulfate or sulfate. In industrial applications where liquids containing large amounts of salts such as potassium present problems due to contamination. may be desirable and advantageous.

Additionally, the thiophilic matrix allows the use of nootropic salts in the sample. Depending on the concentration, it is only effective in binding and purifying immunoglobulins. They also contain other proteins such as other serum proteins other than immunoglobulins. It is noteworthy that it can be used to combine (Lihme & Analytical Biochemistry by Heegaard Vol. 192, p. 64, p. 3, p. 69 (1991)). In this case too, the It may be advantageous to use a lower concentration of salt in the pull.

Therefore, the coupling bonded phenate with divinylsulfone-activated agarose It is novel that Nol is thiophilic in the way it adsorbs proteins. This is surprising.

Contains an aromatic or heteroaromatic core with or without substituents In the study of many different ligands, if Y represents an aromatic or heteroaromatic species, If the sulfur atom is generally oxygen or nitrogen while maintaining thiophilicity, It has further been found that it can be replaced by The next substance is divinils Coupled to: 2-hydroxypyridine, 4-hydroxypyridine, xanthine, 4-methoxyphenol, l-hydroxy Cybenzotriazole, 4-aminobenzoic acid, 2-hydroxybenzyl alcohol 2,4-dihydroxy-6-methylpyrimidine, 4-aminosalicylic acid, 2 -aminothiazole, 2-aminopyridine, 2-aminopyrimidine, 2-hydro xypyrimidine, 4-hydroxypyrimidine, imidazole, 3-amino-1, 2,4-) Riazole, 4-hydroxybenzoic acid butyramide, 2-hydroxy Benzhydroxamic acid, phenol and 4-chlorophenol. They are all This provided an adsorption matrix with strong thiophilic properties.

Although the resulting adsorption matrix does not significantly bind albumin, They are all very effective and based on mercaptoethanol derivatives known type IgG, IgA and at least as effectively as the adsorption matrix. Binding of IgM and IgM immunoglobulins is common to the ligands studied.

In addition to strongly binding immunoglobulins, various adsorption matrices also bind other proteins. They were found to show small but significant differences in selectivity for white matter connections. Ta.

Therefore, for the purification of specific proteins, placement on aromatic or heteroaromatic cores is essential. It is important that substituents are chosen to provide the greatest possible potency and purity.

Therefore, routine testing will allow one skilled in the art to determine the most appropriate ligand for a particular protein. You can choose.

Furthermore, different isomers of the same substance differ in binding strength as well as in selectivity. It was found that there can be a large difference in the This means that 2-hydroxypyryly Coupling of dine (Example 1) and coupling of 4-hydroxypyridine (Example 1) 2), and 2-hydroxy pyridine derivatives are 4-hydroxy pyridine derivatives. Binds proteins much more than pyridine derivatives. they bind albumin Absent or weak albumin binding is common to both derivatives.

Purification of immunoglobulin As mentioned above, immunization with thiophilic or nitrilophoric matrices Known methods for purifying immunoglobulin contain very low concentrations of immunoglobulin (i.e. The ability to bind immunoglobulins from fluids with a under the conditions (0.5M potassium sulfate or 0.75M ammonium sulfate). The known method is limited as it is relatively low. much higher concentrations of immunoglobulins Low immunoglobulin concentrations are typical in contrast to serum and ascites fluid containing production of monoclonal antibodies by in vitro culture of hybridoma cells. This occurs in cell culture supernatants produced by

However, since (known thiophyllic and nitrilophorithic matrices) and the new thiophyllic matrix of the present invention) The ability of immunoglobulins to bind to adsorbent matrices with Increase the ionic strength (concentration) of known lyotropic salts in the liquid being purified. Surprisingly, it was found that it increases rapidly as the temperature increases.

Therefore, binding of monoclonal antibodies from in vitro cell culture supernatants The ionic strength of the liquid during the combination treatment is 2.25 to 3.0 (ammonium sulfate in the liquid was found to be enhanced by increasing the concentration of 0.75 M-IM). Ta. This increased binding strength corresponds to an overall binding capacity of 300-1000%. causes an increase in 0.5M potassium sulfate (ionic strength = 1..5) and IM When compared to ammonium sulfate, it offers a correspondingly large increase in binding capacity. provided. Potassium sulfate exhibits an increased solubility of about 0.6M and therefore the required It should be noted that the ionic strength cannot be reduced.

Increasing the ionic strength with ammonium sulfate to 3.8 and 4.0 binds The lower the concentration of immunoglobulin, providing an additional increase in strength, There is a rule that higher ionic strength is required to maintain binding capacity. Ru. The ionic strength of the lyotropic salts that are therefore desirable for use. There is no upper limit.

However, depending on the immunoglobulin concentration in the fluid, a minimum ionic strength of 2.4 It is always preferable to have a degree and 0. O1■/- to 1■/- immunoglobulin In a typical case using an in vitro cell culture supernatant having a concentration of 2. ．． It is preferred to use an ionic strength of 7 to 4.5. 3.0 for normal use It is preferable to use an ionic strength of ˜4.0.

Binding immunoglobulins to thiophilic and nitrilophoric adsorption matrices Otrobic (water structure-forming) salt groups that can contribute to Belongs to the loop. Examples of such are typically sodium as the counterion, Contains sulfate or phosphate ions along with potassium or ammonium ions It's salt.

Furthermore, certain organic ions, such as polyvalent anions of organic polycarboxylic acids (e.g. citrate or tartrate ions) also have lyotropic activity. but while increasing the ionic strength above that used in the prior art. The provision of increased binding capacity by ammonium sulfate is not limited to ammonium sulfate; The increased binding capacity that can be achieved is due to all inorganic and otrobic properties. It has been found in accordance with the present invention that the present invention is applicable to organic salts and organic salts.

Immunoglobulins by thiophilic or nitrilophoric adsorption matrices Other mentioned drawbacks of known methods for purifying eluted immunoglobulin (products of purification) or other proteins, such as transferrin and α-2-mer. It is highly contaminated with cloglobulin.

This contamination occurs when the pH is below 7.5 after immunoglobulin adsorption. The ionic strength in the solution causes immunoglobulin to adsorb onto the adsorption matrix. of the oliotrovic salt present in such an amount that it is lower than the ionic strength of the solution used for This can be greatly prevented by flushing the adsorption matrix with a solution. Surprisingly, it turned out that it was possible to do this.

Analytical Bioc by Hutchens and Porath hemistry Vol. 159, pp. 217-226 (1986) However, it is stated that the binding of immunoglobulin to the adsorbent is pH dependent. However, in connection with the flushing method after adsorption of immunoglobulin, the use of this pH dependence has been proposed. Previously, it should be possible to increase the purity of the eluted immunoglobulin. has not been reported, disclosed, or suggested.

The methods of the invention include, for example, monoclonal in vitro cell production according to the following specific method. It can be used in the purification of immunoglobulins from culture supernatants. So The method is: Final concentration of 1.0 M ammonium sulfate and 0.05 M sodium acetate, p The cell culture supernatant was treated with ammonium sulfate and sodium acetate until H5.2. and then typically mix that liquid into a column containing an adsorption matrix. contacting the liquid with the adsorption matrix by passing the liquid through Buffer solution consisting of 0.3M ammonium sulfate + 0.05M sodium acetate (pH 5.2) to wash unbound and bound contaminating proteins from the matrix. out, and Low salt content, e.g. 0.05M Tris/HCI! Buffer (1) H9 with , 0) to elute binding proteins including immunoglobulins. Consists of things.

The method of the invention also allows for the purification of immunoglobulins from other fluids, such as serum or ascitic fluid. However, when it comes to otrovic salts, The ionic strength of the flushing buffer and the pH value of the flushing buffer are It is necessary to adjust each.

The results show that immunoglobulin binding can be achieved without significantly reducing the binding capacity of the adsorption matrix. There will be increased purity of Brin. Similarly, each of ionic strength and pH value It is desirable that the regulation of IgG+, IgG**, IgG), IgM or I depending on whether gE is included).

The preferred ionic strength of the flushing buffer will depend on the specific application and any contamination present. However, it is typically between 0 and 2.25. in most cases The most preferred range is 0.6-1.5. In the same way, flushing buffer The preferred pH value depends on the particular application, but typically ranges from pH 2.5 to The pH is 7.5. Stability of immunoglobulin during flushing process and how A more preferred range for the efficiency of the method is pH 3.0 to pH 6.5, but the most preferred range is pH 3.0 to pH 6.5. A preferred range would be pH 3.5 to pH 6.0. A pH of 4.0 to 5.5 is particularly preferred. Delicious.

The pH value of the elution buffer is preferably 7.0 or higher, but may also be lower. melt Separation also changes the dielectric constant of the buffer solution, for example by adding ethylene glycol. This can be achieved by The choice of elution method depends on how the immunoglobulin binds. The method and subsequent cleaning of contaminants are generally carried out independently.

Preparation of 2-hydroxypyridine-derivatized divinylsulfone-activated agarose l) Contains approximately 40 micromoles of vinyl sulfone groups per l of gel excluding water. divinylsulfone-activated agarose (Kem-En-Tec, Denmark) (Mini Leak High) by suction filter with 2 to 3 volumes of ion exchange water. Washed above. Drain the gel with a little suction and drain the wet gel We weighed 10g of the following.

2) 0.5g of 2-hydroxypyridine to 10-0°IM dipotassium phosphate Dissolved in 10 ml of buffer and titrated with sodium hydroxide to pH 11.0. I made it.

Sodium boronate is added and the resulting solution is divinylsulfone activated agar. Mixed with loin. The gel was incubated with the solution for 18 hours at room temperature.

3) Next the gel was mixed with 50% ethanol in water, 25% ethanol in water and finally pure water. Washed thoroughly.

The resulting matrix is about 40 microns per liter of drained wet matrix. It contained 2-hydroxypyridine of 2-hydroxypyridine.

Example 2 Preparation of 4-hydroxypyridine-derivatized divinylsulfone-activated agarose The procedure was the same as that described in Example 1, but with 2-hydroxypyri 4-hydroxypyridine was used instead of gin.

The resulting matrix is about 40 microns per liter of drained wet matrix. It contained romol of 4-hydroxypyridine.

Example 3 Production of 4-methoxyphenol-derivatized divinylsulfone-activated agarose The procedure was the same as that described in Example 1, but with 2-hydroxypyri 4-methoxyphenol was used instead of gin.

The resulting matrix has approximately 40 RI per 11 RI of the drained wet matrix. Contained micromoles of 4-methoxyphenol.

Example 4 Production of 4-aminobenzoic acid-derivatized divinylsulfone-activated agarose The procedure was the same as that described in Example 1 but with 2-hydroxypyridine. 4-aminobenzoic acid was used instead.

The resulting matrix is about 40 microns per liter of drained wet matrix. Contains romol of 4-aminobenzoic acid.

Example 5 Production of phenol-derivatized divinylsulfone-activated agarose The procedure was the same as that described in Example 1, but with 2-hydroxypyri Phenol was used instead of gin.

The resulting matrix is about 40 microns per 7' of drained wet matrix. Contains a large amount of phenol.

Example 6 Preparation of divinylsulfone-activated agarose coupled with various ligands Construction To create divinylsulfone-activated agarose derivatized with the following ligand precursors: The method described in Example 1 was used for: instead of 2-hydroxypyridine , xanthine, l-hydroxybenzotriazole, 2-hydroxybenzyl Alcohol, 2,4-dihydroxy-6-methylpyrimidine, 4-aminosalici acid, 2-aminothiazole, 2-aminopyridine, 2-aminopyrimidine, 2 -Hydroxypyrimidine, 4-hydroxypyrimidine, imidazole, 3-amidine Nor-1,2,4-triazole, 2-hydroxybenzhydroxamic acid and 4 -Chlorophenol.

All compounds described are commercially available.

The resulting matrix contained approximately 30-40 micromoles of ligand.

Example 7 of phenol-derivatized divinylsulfone-activated agarose with moderate concentration of ligands. manufacturing The procedure was the same as that described in Example 1 but with 1 nL of wet matrix. Divinylsulfone activated containing 20 micromoles of vinylsulfone groups per liter Agarose (Mini Leak Medium manufactured by Kem-En-Tec) and phenol was used in place of 2-hydroxypyridine.

The resulting matrix has a concentration of about 20 per ml of drained wet matrix. Contained micromoles of phenol.

Example 8 of phenol-derivatized divinylsulfone-activated agarose with low concentration of ligands. manufacturing The procedure was the same as that described in Example 1 but with a drained damp mask. divinyl containing about 5 micromoles of vinyl sulfone groups per 1- Sulfone-activated agarose (Mini Leak L manufactured by Kem-En-Tea) ow) and phenol was used instead of 2-hydroxypyridine.

The resulting matrix is about 5 microns per liter of drained wet matrix. Contains moles of phenol.

” Known mercaptoethanol-derivatized divinylsulfone-activated agaro for comparison manufacturing of l) Approximately 40 micromoles of vinyl sulfone groups per l of drained wet gel. containing divinylsulfone-activated agarose (Kem-εn-Te from Denmark) Aspirate Mini (LeakHigh) made by company C with 2 to 3 volumes of ion-exchanged water. Washed on filter. Drain the gel with slight suction and 10 g of the gel was weighed.

2) 5% vol/vol mercaptoethanol in water to pH 9 with sodium hydroxide , 5 and the resulting solution was mixed with the gel.

The gel was incubated with the solution for 18 hours at room temperature.

3) Next, add the gel to 50% ethanol in water, 25% ethanol in water, and finally pure water. Washed thoroughly.

The resulting matrix has a concentration of about 40 per Irn of the drained wet matrix. Contained micromoles of mercaptoethanol.

Example 10 Purification of immunoglobulins from human serum according to the prior art according to Examples 1-4 and Example 9 The prepared matrices contain immunoglobulins from human serum according to the following known methods. (Anal ytical Biochemistry Vol. 192, pp. 64-69 (19 91)): 1) Three human sera were diluted with 1.5M (NH,'), 304 to 1 l.

2) Transfer the sample to 3 g of gel (2-hydroxypyridine derivatized, 4-hydroxypyridine derivatization, 4-methoxyphenol derivatization, 4-aminoamine Divinyl sulfone activation method for zoic acid derivatization and mercaptoethanol derivatization. (Galose) packed column. Each column is preset to 0.7 It was equilibrated with 5M (NH,)ISO.

3) After applying the sample, leave the column at 0 until unbound protein is washed out of the column. ．． Washed with 75M (NH4)*SO4 buffer.

4) Bound proteins including immunoglobulins were eluted with 0.1M NaCl.

Expression of the bound and then eluted amount of protein from each matrix and The EU quantity of the eluate is calculated by the following formula: (E2.. of eluate)・(volume of eluate)=EU(-1 The qualitative composition of the eluate was determined by cross-immunoelectrophoresis. It was done. Result is: Therefore, the matrix of the present invention and the known matrix (mercaptoethanol) bound comparable amounts of total protein and substantially the same amount of immunoglobulin.

There is no matrix that binds albumin. 4-hydroxypyrimidine matrix The smaller amount of total protein eluted from the immunoglobulin matrix than from other matrices This reflects the high selectivity for Brin. Therefore the position and totality of substituents Therefore, the precise structure of the ligand has a decisive influence on the selectivity of the matrix. In conclusion, phenol derivatization with various ligand concentrations according to the present invention Determination of known proteins from human serum using divinylsulfone-activated agarose purification As the effect of ligand concentration on protein fractionation was described in Examples 5.7 and 8. Three types of phenol-derivatized divinyl sulfone-activated matrices prepared from was investigated.

The raw material is human serum and the conditions during purification are similar to those of most serum proteins. (i.e., for selective binding of immunoglobulins) A higher concentration of ammonium sulfate was used than that used for l) human serum in 3. Diluted l:1 with 0 M (NH,), So, and 30 min at 4000 revolutions/min. Centrifuged. The precipitate was discarded, while the supernatant was used for purification.

2) about 40 each per ml of matrix (from examples 5.7 and 8) micromoles, about 20 micromoles and about 5 micromoles of phenol. Transfer the supernatant to three columns packed with lomi' of phenol-derivatized agarose. The solution was applied.

3) After application of the sample, 0. OIM, HPO, and l.

Flush the column with 5M (NH,)2So, buffer (pH 7.2). Ta.

4) The matrix was mixed with 0.01M KtHPO4 and 1. 5M (NH,)2S o, (pH 7,2) to O, OIM, HPO4 and 0.25M NaCl!　 (pH 7.2).

The eluate in the fractions was collected and a molten rocket immunosorbent was used for qualitative determination of protein content. It was analyzed by electrophoresis.

The results show that the adsorption magenta with the highest content of phenyl groups (40 micromol/-) The matrix that binds the protein most strongly, i.e. the protein binds the two other adsorption matrices. generally eluted at lower ionic strengths compared to Furthermore, this matric Also known thiophyllic matrices such as mercaptoethanol derivatives It binds proteins much more strongly than immunoglobulin G or 40% ethylene glycol. immunoglobulins so strongly that they could only be liberated by subsequent elution using Phosphorus was combined.

However, albumin binds to the matrix much weaker than immunoglobulins. and much weaker than known hydrophobic matrices such as Octyl-Sepharose. The bonded points clearly showed thiophilicity itself.

An adsorption matrix with a content of about 20 micromoles of phenyl groups per me of wet gel. Trix has a higher ligand concentration (approximately 40-60 micromol/-) corresponds approximately to the pattern achieved using the thiophilic matrix of The binding pattern was shown.

Adsorption matrix with a phenyl group content of approximately 5 micromoles per l Somewhat weakly bound proteins, but still resistant to immunoglobulin binding. showed priority.

All matrices bound immunoglobulin much more strongly than albumin. .

Example 12 Binding capacity for immunoglobulins from in vitro cell culture supernatants (method comparison) Method ■ (according to prior art) l) Final concentration of potassium sulfate is 0.5M and tris-hydroxymethylamic acid 50 Mg per lrd until the final concentration of methane (Tris) is 0.1 Contains monoclonal immunoglobulin G1 and 10% volume/volume fetal bovine serum. 3001R1 of in vitro hybridoma cell culture supernatant with potassium sulfate. and Tris, and the pH was adjusted to 7.6 with hydrochloric acid.

2) 2-Hydroxypyridine-derivatized divinyls as prepared in Example 1 The supernatant was passed through a column filled with 51 nl of fluorine-activated agarose. . The effluent from the column is collected into fractions, which are separated by immunodiffusion into murine immunoglobules. were analyzed for their content of phosphorus.

Method ■ (according to the present invention) The test was carried out similarly to method I, but with the addition of potassium sulfate in section 1). ammonium sulfate is replaced with 0.8M ammonium sulfate, and the pH is kept constant at 7.6. be done.

Method■ The test was carried out similarly to method ■, where the concentration of ammonium sulfate was simply increased to 1.0M. It will be done.

Method■ The test was carried out similarly to method ■, where the concentration of ammonium sulfate was simply increased to 1.2M. It will be done.

The results show that the effluent concentration of murine immunoglobulin is 50% of the starting concentration (“50% saturation”). (defined as ) before passing through a column with an adsorption matrix Expressed as the number of - of cell culture supernatant.

Method 1 20 Method II 30 Method ■ 250 Method IV >300 As can be seen from Table I, the increase in medium ionic strength is results in a strong increase in binding capacity for immunoglobulins.

Example 13 The test described in Example 12 was repeated and the adsorption matrix or was simply replaced by the mercap I ethanol derivative.

Method ■ 230 Method IV >300 As can be seen, the increase in ionic strength also increases with known thiophilic adsorption matrices. It had a strong positive effect on the binding ability of x.

Example 14 The test described in Example 12 was repeated and ammonium sulfate or simply sulfuric acid Replaced by l.rium.

Table■ Method 1 30 Method n 40 Method III >300 Method IV >300 As can be seen, when using sodium sulfate instead of ammonium sulfate, the ion The increase in strength also has a strong positive effect on binding capacity.

Example 15 Purity of immunoglobulins isolated from in vitro cell culture supernatants (comparison of methods) ) Method I (known washing) 1) The final concentration of ammonium sulfate is 1. OM and tris-hydroxymethyla 50 Mg per l until the final concentration of minomethane (Tris) is 0.1 M. Murine monoclonal immunoglobulin (G) and 10% vol/vol fetal bovine blood 300% of in vitro hybridoma cell culture supernatant containing supernatant was diluted with ammonium sulfate. Monium and Tris were mixed and the pH was adjusted to 7.6 with hydrochloric acid.

2) 2-Hydroxypyridine derivatized divinyls as prepared according to Example 1 The supernatant was passed through a 5-filled column of sulfone-activated agarose.

3) 1.0M ammonium sulfate + 0.01M Tris/HCI! (pH The column was washed with approximately 50 ml of 7,6).

4) Release the binding protein with 0.05M Tris/HCR (pH 9,0') and eluted.

The eluate was collected in one fraction and subjected to sodium dodecyl polyacrylamide electrophoresis. The samples were analyzed for purity by dynamic and then electronic scanning. Mouse immunoglobulin G1 The total yield is determined by quantitative rocket immunoelectrophoresis. Purity is sample The immunoglobulin test for the total amount of protein in % is carried out in the same manner as method I and described in section 3). The washing buffer was 0.3M ammonium sulfate + 0.05M sodium acetate. (pH 5,2). Washing buffer or 0.3M ammonium sulfate + 0.1M Tris/ It was simply replaced by HCl (pH7,6).

Yield (■) Purity (%) Method I 11.5 approx. 30 Method II 11.0 Approx. 90 Method I [I < 0. 5 Approximately 95 Reductions in ionic strength and pH in the flushing buffer maintain high performance. The results show that the purity of the eluted immunoglobulin is increased while maintaining the purity of the eluted immunoglobulin.

High purity can be obtained if the pH is not reduced at the same time as the ionic strength. Oak yield is very small.

Example 16 A comparative test of the method was carried out analogously to Example 15, in which the adsorption matrix was prepared according to Example 9. was simply replaced by the synthesized mercaptoethanol derivative.

The results correspond completely to those in Example 15, indicating that the method of the invention also Increasing the purity of eluted TgG in Ohiri Tsukumatori Socus.

international search report international search report Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), 0A (BF , BJ, CF, CG, CI, CM, GA, GN, ML, MR, SN, TD, TG. ), ATDK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, MN, MW, NL, NO, PL, RO, RU, SD, SE, US

Claims (18)

[Claims] 1. General formula for particle distribution X-R (wherein, X is NQ [where N is nitrogen, and Q is H(CH2)n (where n= 0, 1, 2 or 3) or O (oxygen)], and R is one or more an optionally substituted aromatic or heteroaromatic ring system consisting of and R is a group (not containing a nitrile group) characterized by Divinyl sulfone group is ether oxygen atom, thioether sulfur atom or nitrogen atom and the divinyl sulfone group is bonded to the hydrophilic polymer network via further covalently bonded to the distributed particles, the hydrophilic polymer network structure Thiophilic adsorption matrix. 2. Alkyl such as methyl, ethyl, propyl and butyl; methoxy, ethyl Alkoxy such as xy, propyloxy, butyloxy; -(CH2)nCO Carboxy such as OH (where n=0, 1, 2 or 3); -(CH2)2C Carboxamides such as ONH2 (where n=0, 1, 2 or 3); -(CH2)nCONHOH (where n=0, 1, 2 or 3) cyhydroxyamide; halogens such as -F, -Cl, -Br and -I; b (-NO2); sulfonic acid (-SO2H); hydroxy (-OH); -(CH 2) Alcohol such as nOH (where n=1, 2 or 3); -(CH2)n R is a substituent selected from amines such as NH2 (where n=1, 2 or 3); The thiophilic adsorption according to claim 1, characterized in that matrix. 3.2-hydroxypyridine, 4-hydroxypyridine, xanthine, 4-meth xyphenol, 1-hydroxybenzotriazole, 4-aminobenzoic acid, 2 -Hydroxybenzyl alcohol, 2,4-dihydroxy-6-methylpyrimidine 4-aminosalicylic acid, 2-aminothiazole, 2-aminopyridine, 2- Aminopyrimidine, 2-hydroxypyrimidine, 4-hydroxypyrimidine, i Midazole, 3-amino-1,2,4-triazole, 4-hydroxybenzoic acid Butyramide, 2-hydroxybenzhydroxamic acid, phenol and 4-hydroxybenzhydroxamic acid A hydrophilic polymer network structure is formed using a particle distribution precursor selected from the group consisting of lolophenol. Coupling of the particles is achieved by reacting with the divinyl sulfone group of Thiophilic adsorption according to claims 1 and 2, characterized in that matrix. 4. The concentration of the particles is preferably 5 to 80 micromoles per ml of wet matrix. or 5 to 40 micromoles per ml, especially per ml of wet matrix. Claims 1 to 3, characterized in that the amount is 10 to 40 micromoles. thiophilic adsorption matrix. 5. The polymer network structure can be used as particles, as a membrane, or contained in a membrane to form a polysaccharide. preferably agar, agarose, dextran, starch and cellulose. , especially agarose, and synthetic organic polymers, preferably polyacrylamide, polymers, etc. Lyamide, polyimide, polyester, polyether, polymeric vinyl compounds and and substituted derivatives thereof. Thiophilic adsorption matrix according to paragraph 4. 6. The divinyl sulfone group is an ether oxygen atom, a thioether sulfur atom, or a nitrogen atom. is bonded to the hydrophilic polymer network through atoms and the divinyl sulfur The phon group is further covalently bonded to the particle distribution, and the particle distribution has the general formula X-R (wherein, X is NQ [where N is nitrogen and Q is H(CH2)n (where n=0 , 1, 2 or 3) or O (oxygen)], and R is one or more an optionally substituted aromatic or heteroaromatic ring system consisting of and R is a group (does not contain a nitrile group), the hydrophilic polymer network structure In the method for producing a thiophilic adsorption matrix, the polymer network structure Activate the polymer network by contacting it with divinyl sulfone; and then reacting the activated polymer network structure with a particle distribution precursor. Method. 7. The particle distribution precursor is the following substance: 2-hydroxypyridine, 4-hydroxypyridine xanthine, 4-methoxyphenol, 1-hydroxybenzotriazole , 2-aminobenzoic acid, 2-hydroxybenzyl alcohol, 2,4-dihydro xy-6-methylpyrimidine, p-aminosalicylic acid, 2-aminothiazole, 2-aminopyridine, 2-aminopyrimidine, 2-hydroxypyrimidine, 4- Hydroxypyrimidine, imidazole, 3-amino-1,2,4-triazole , 4-hydroxybenzoic acid butyramide, 2-hydroxybenzhydroxamic acid , phenol and 4-chlorophenol. The method described in scope item 6. 8. The thiophilic adsorption matrix has divinyl sulfone groups as ether zymogens. into the hydrophilic polymer network via a thioether sulfur atom, or a nitrogen atom. and the divinyl sulfone group is further bonded to the particle distribution, The particle distribution has the general formula X-R (wherein, X is NQ [where N is nitrogen and Q is H(CH2)n (where n=0 , 1, 2 or 3) or O (oxygen)], and R is one or more an optionally substituted aromatic or heteroaromatic ring system consisting of and R is a group (does not contain a nitrile group), the hydrophilic polymer network structure contacting the liquid with the thiophilic adsorption matrix, characterized in that the liquid is and then protein from the thiophilic adsorption matrix or from the liquid. A method for purifying proteins from liquids. 9. Alkyl such as methyl, ethyl, propyl and butyl; methoxy, ethyl Alkoxy such as xy, propyloxy, butyloxy; -carboxy such as (CH2)nCOOH (where n=0, 1, 2 or 3); - Carboxylic acid such as (CH2)nCONH2 (where n=0, 1, 2 or 3) Amide; such as -(CH2)nCONHOH (where n=0, 1, 2 or 3) Carboxyhydroxyamide; halogens such as -F, -cl, -Br and -I Nitro (-NO2); Sulfonic acid (-SO2H); Hydroxy (-OH); - Alcohols such as (CH2)nOH (where n=1, 2 or 3); selected from amines such as -(CH2)nNH2 (where n=1, 2 or 3) 9. The method according to claim 8, wherein R is substituted with a substituent. 10.2-hydroxypyridine, 4-hydroxypyridine, xanthine, 4-methane Toxyphenol, 1-hydroxybenzotriazole, 4-aminobenzoic acid, 2-hydroxybenzyl alcohol, 2,4-dihydroxy-6-methylpyrimi gin, 4-aminosalicylic acid, 2-aminothiazole, 2-aminopyridine, 2 -aminopyrimidine, 2-hydroxypyrimidine, 4-hydroxypyrimidine, Imidazole, 3-amino-1,2,4-triazole, 4-hydroxybenzoin acid butyramide, 2-hydroxybenzhydroxamic acid, phenol or 4- A particle distribution precursor selected from the group consisting of chlorophenols is formed into a hydrophilic polymer network structure. The particles are coupled by reacting with the divinyl sulfone group of the body. 10. A method according to claims 8 and 9, characterized in that: 11.3 The lyotropic buffer in the liquid is 2.25 or more, preferably 2.25 Lyot, characterized by having an ionic strength of ~4.5, in particular 3.0-4.0. Adding a tropic buffer to a liquid and contacting the liquid with a thiophilic adsorption matrix and wash the thiophilic adsorption matrix with lyotropic buffer solution. The washed thiophilic adsorption matrix is then eluted with an eluent. A method for purifying immunoglobulin from a liquid, comprising: 12. The lyotropic buffer solution is 2.25 or less, preferably 0 to 2.25. Lyotropic to liquid, characterized by having an ionic strength of 0.6 to 1.5. add a thiophyllic adsorption matrix and contact the liquid with the thiophyllic adsorption matrix; Wash the ophilic adsorption matrix with a lyotropic buffer solution and then Consists of eluting the washed thiophilic adsorption matrix with an eluent , a method for purifying immunoglobulins from liquids. 13. pH of the lyotropic buffer solution is 7.5 or less, preferably 2.5 to 7.5 , particularly 3.0 to 6.0, particularly preferably 3.5 to 6.0, particularly 4.0 to 5.5. 13. The method according to claim 12, characterized in that: 14. The lyotropic buffer in the liquid is 2.25 or more, preferably 2.25 to 4. 5, in particular having an ionic strength of 3.0 to 4.0. The method according to paragraphs 2 and 13. 15. The thiophilic adsorption matrix has divinyl sulfone groups attached to ether oxygen. attached to the polymer network via a thioether sulfur atom or a nitrogen atom. and the divinyl sulfone group is further covalently bonded to the particle distribution. , the particle distribution is a) optionally substituted and linked via a sulfur atom to the divinylsulfone group; alkyl, aryl, or heteroaromatic groups, b) bonded to divinyl sulfone via an oxygen or nitrogen atom; is substituted with one or more rings, and the substituent is a nitrile group. and c) a nitrile group. or a nitrile group. divinyl sulfate via a sulfur atom, an oxygen atom or a nitrogen atom. an alicyclic or heterocyclic group attached to a phon group; selected from selected from the divinyl sulfone activated polymer network structure, The method according to claims 11 to 14. 16. The polymer network structure can be used as particles, as a membrane, or contained in a membrane to produce polysaccharides. , preferably agar, agarose, dextran, starch and cellulose, especially agarose, and a synthetic organic polymer, preferably polyacrylamide, polyamide polyimides, polyimides, polyesters, polyethers, polymeric vinyl compounds and Claim 15, characterized in that the compound is selected from substituted derivatives thereof. How to put it on. 17. Lyotropic buffers and lyotropic buffer solutions are sodium sulfate , potassium sulfate, ammonium sulfate, sodium phosphate, potassium phosphate and Lyotropic salts selected from ammonium phosphate or sodium citrate , polyhydric carbones like sodium tartrate, potassium citrate, potassium tartrate Claim 11, characterized in that it contains an organic salt of an acid or a mixture thereof. ~The method according to item 16. 18. The liquid is a biological fluid, preferably blood, serum, ascites fluid or cell culture supernatant. Claims 11 to 1, characterized in that the cell culture fluid is a cell culture supernatant, especially a cell culture supernatant. The method described in Section 6.