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  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
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  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
  • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
  • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
  • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
  • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
  • B01J20/3253—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
  • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography

Definitions

• the invention concerns novel thiophilic adsorption ma ⁇ trices, preferably for use in the isolation, purification 10 and immobilization of proteins from a liquid by salt- dependent adsorption chromatography, a process for produc ⁇ ing them, and a process for purifying protein, preferably immunoglobulin.
• Salt-dependent adsorption chromatography comprises binding proteins to an adsorption matrix in the presence of high concentrations of salts, in particular lyotropic (water- structure forming) salts. Specific binding of proteins is achieved by adjusting the salt concentration, and the
• bound proteins are eluted from the adsorption matrix by reducing the salt concentration in the medium.
• Salt-dependent adsorption matrices are known, including e.g. metal chelate matrices, hydrophobic matrices and ma-
• thiophilic adsorption matrices means hydrophilic adsorption matrices comprising a vinyl sul ⁇ phone group to which a ligand is covalently bound, said matrices being capable of adsorbing proteins, such as
• Matrices exhibiting thiophilic adsorption are character ⁇ ized by binding proteins in another manner than hydropho ⁇ bic matrices. The differences are reflected i.a. in the fact that thiophilic matrices bind immunoglobulins from human serum much stronger than albumin at a given salt concentration. Albumin can be caused to bind to thiophilic matrices, but this typically requires a higher salt con ⁇ centration than is necessary for binding immunoglobulin. Hydrophobic matrices are characterized by binding albumin more strongly than immunoglobulin.
• Thiophilic adsorption matrices are used in particular in the fractionation of biopolymers, such as nucleic acids, nucleotides, and proteins, including serum proteins, immunoglobulins and enzymes, and other polypeptides.
• adsorption matrices having a distinct thio ⁇ philic nature are known, the so-called T-gels and the nitrilophoric matrices. Furthermore, thio-aromatic ma- trices having a hydrophobic nature are known.
• thiophilic adsorption matrices which consist of hydrophilic matrices having covalently coupled chemical structures (ligands) of the type:
• M is a polymeric hydrophilic network
• X is an 0 (oxygen), N (nitrogen) or S (sulphur) atom
• Y is an optionally substituted alkyl, aryl or hetero ⁇ aromatic group
• S is a sulphur atom positioned two carbon atoms away from the sulphone group and belonging to the ligand.
• a typical matrix belonging to this group is produced by reaction of mercapto ethanol with a divinyl sulphone acti ⁇ vated hydrophilic matrix (e.g. agarose):
• M-0-CH 2 -CH 2 -S0 2 -CH CH 2 + HS-CH 2 ⁇ CH 2 -OH - M-0-CH 2 -CH 2 -S0 2 -CH 2 -CH 2 -S-CH 2 -CH 2 -OH
• the thiophilic effect entails that these matrices do not bind albumin particularly effectively in contrast to known hydrophilic matrices which typically have long alkyl chains as ligands.
• Thiophilic adsorption matrices of the above-mentioned type have surprisingly been found to have particular selecti ⁇ vity to immunoglobulins of the type IgG, IgA and IgM from serum and ascites liquids to which lyotropic salts, such as potassium sulphate have been added in a concentration of 0.5 M. After binding of immunoglobulin the matrix can be washed with an 0.5 M potassium sulphate buffer to remove non-bound contaminating proteins. The immuno ⁇ globulin can then be released by elution with a buffer with a low concentration of sodium chloride, e.g. 0.1 M sodium chloride.
• This approach involves a rapid and convenient method of purifying immunoglobulins from liquids containing rela ⁇ tively high concentrations of immunoglobulin (above about 1 mg/ml), as is the case with e.g. serum and acites liquid.
• thiophilic adsorption matrices of the above- mentioned type have a plurality of drawbacks associated with the exact chemical structure of the ligand.
• a sulphur atom is included, positioned at a distance of two carbon atoms after a sulphone group.
• this sulphur atom was considered essential to the quite special selectivity exhibited by the matrices, which appears from the EP Patent Specification 0 168 363 (example 4) and moreover from Jerker Porath and Makonnen Belew, Tibtech, 1987, vol. 5, p.
• R is an aliphatic or heteromatic substituent com ⁇ prising at least one nitrile group
• Y is preferably -CH 2 -CH 2 -S0 2 -CH 2 -CH 2 -
• adsorbents have properties which are comparable with the thiophilic matrices described above, but they do not necessarily comprise the essential sulphur atom two carbon atoms terminally from the sulphone group. Presumably, this can be ascribed to the presence of one or more nitrile groups in the ligand.
• Nitrilophoric adsorbents seem to have a limited use, it being known that nitriles can hydrolyze in aqueous solu ⁇ tions, in particular in basic or acid solutions, which means that the adsorbents are unstable under conditions which are typically used for regeneration, sterilisation and depyrogenization (typically at a high or low pH and autoclaving) between and during use for purification of immunoglobulins.
• the thiophilic and nitrilophoric adsorbents described above have been used for purification of immunoglobulin from a liquid according to a process comprising the following steps: 1) an adsorption step in which the liquid, from which immunoglobulin is to be purified, is admixed with a solution of 0.5 M potassium sulphate + 0.1 M Tris/HCl with pH 7.6, following which the liquid is contacted with the adsorbent, typically by passing the liquid through a column containing the adsorbent,
• Hutchens and Porath, Analytical Biochemistry, vol. 159, p. 217-226, 1986 describe the process as a general and op i- mal method of purifying immunoglobulins from serum
• Belew, M et al., Journal of Immunological Methods, vol. 102, p. 173-182, 1987 describes such a process for purify ⁇ ing immunoglobulin from ascites liquid and in vitro cell culture supernatants containing monoclonal antibodies.
• no processes have been taught for purify ⁇ ing immunoglobulins by using different pH and ion strength during adsorption and washing phase.
• Lihme and Heegaard, Analytical Biochemistry, vol. 192, p. 64-69, 1991 describe the use of 0.75 M ammonium sulphate at neutral pH for purifying immunoglobulins from rabbit serum instead of 0.5 M potassium sulphate + 0.1 M Tris/HCl pH 7.6, as described above.
• immunoglobu- lins e.g. lentillectin and trypsin, Hutchens and Porath
• a drawback of the method is the provision of an eluate which, in addition to immunoglobulins, also con ⁇ tains significant amounts of contaminating proteins, such as e.g. transferrin and ⁇ -2-macroglobulin.
• the process has a relatively low capacity for binding of immunoglobulins from liquids having a particularly low concentration of immunoglobulin, e.g. in vitro cell cul ⁇ ture supernatants which typically contain from 0.01 to 0.1 mg of immunoglobulin per ml.
• the object of the present invention is to provide new al ⁇ ternative thiophilic adsorption matrices which are simple and inexpensive to produce, and which can be produced from divinyl sulphone activated matrices without special safety measures.
• the invention provides a thiophilic nature of the adsorption matrix which is at least equal to the thiophilic nature of known thiophilic matrices, if the ligand has an aromatic or heteroaromatic nature and the S coupling atom between the divinyl sul- phone group and the ligand is replaced by 0 or N.
• Particularly preferred ligands are selected from the sub- stituents stated in claim 2, the ligands stated in claim 3 being particularly preferred.
• these ligand precursors are bound easily to the vinyl sulphone of a divinyl sulphone activated polymer network via oxygen or nitrogen belonging to the ligand. Furthermore, coupling of these precursors generally re ⁇ quires no special safety measures.
• the ligand concentration may vary. Particularly preferred is a ligand concentration between 5 and 80 ⁇ moles/ml, pre ⁇ ferably between 5 and 40 ⁇ moles/ml, especially between 10 and 40 ⁇ moles/ml of wet matrix.
• the hydrophilic network, to which the divinyl sulphone is bound may either be natural or synthetic organic polymers respectively selected from: polysaccharides, such as agar, agarose, dextran, starch and cellulose, and synthetic or- ganic polymers, such as polyacrylamide, polyamide, poly- imide, polyester, polyether and polymeric vinyl compounds, and substituted derivatives thereof either as particles, membranes or contained in membranes. Agarose is particu ⁇ larly preferred.
• novel thiophilic adsorption matrices of the invention are relatively stable in aqueous solutions under strongly acid or strongly basic conditions and at high temperatures.
• Another object of the present invention is to provide a process for producing thiophilic adsorption matrices according to the invention.
• the polymer network is activated by being contacted with divinyl sulphone, following which the activated polymer network reacts with a ligand precursor.
• the ligand precursor is selected from the group consisting of 2- hydroxypyridine, 4-hydroxypyridine, xanthine, 4-methoxy- phenol, 1-hydroxybenzotriazole, 4-aminobenzoic acid, 2- hydroxybenzylalcohol, 2,4-dihydroxy-6-methyl yridine, 4- aminosalicylic acid, 2-aminothiazole, 2-aminopyridine, 2- aminopyrimidine, 2-hydroxypyrimidine, 4-hydroxypyrimidine, imidazole, 3-amino-l,2,4-triazole, 4-hydroxybenzoic acid butyl amide, 2-hydroxybenzhydroxamic acid, phenol and 4- chlorophenol.
• Another object of the invention is to provide a process for purifying protein from a liquid.
• This object is achieved by providing a process for puri ⁇ fying protein from a liquid, wherein the liquid is con ⁇ tacted with a thiophilic adsorption matrix, and the pro- tein is then recovered from either the thiophilic adsorp ⁇ tion matrix or from the liquid, characterized in that the thiophilic adsorption matrix is a thiophilic adsorption matrix according to the invention.
• Possible proteins according to the invention are all pro ⁇ teins, in particular serum proteins, including immunoglo ⁇ bulins, albumin, ⁇ -1-antitrypsin, orosomucoid, Gc-globu- lin, and factor VIII, proteins from fermented liquids, including streptavidin and 0-galactosidase, alkaline phosphatase from calf intestines, protein A and protein G.
• serum proteins including immunoglo ⁇ bulins, albumin, ⁇ -1-antitrypsin, orosomucoid, Gc-globu- lin, and factor VIII
• proteins from fermented liquids including streptavidin and 0-galactosidase, alkaline phosphatase from calf intestines, protein A and protein G.
• Another object of the invention is to provide a process for purifying immunoglobulin from a liquid which gives a greater binding capacity than heretofore known methods by using both known thiophilic and nitrilophoric adsorption matrices and novel thiophilic matrices according to the invention.
• the object is particularly to provide a process for purifying immunoglobulins from a liquid with a concen ⁇ tration of than less than 1 mg of immunoglobulin per ml of liquid, including in vitro cell culture supernatants con ⁇ taining (murine) immunoglobulins of the type IgG_, IgG.-,-. IgG 2B , IgG 3 , IgM and IgE.
• This object is achieved by providing a process for purify ⁇ ing immunoglobulin, comprising adding a lyotropic buffer to the liquid, contacting the liquid with a thiophilic adsorption matrix, washing the thiophilic adsorption ma ⁇ trix with a lyotropic buffer solution, and eluting the washed thiophilic adsorption matrix with an elution li ⁇ quid, characterized in that the lyotropic buffer in the liquid has an ion strength above 2.25, preferably between 2.25 and 4.5, in particular between 3.0 and 4.0.
• Another object of the invention is to provide a process giving a greater purity of the purified immunoglobulin than the known processes.
• This object is achieved by providing a process for purify ⁇ ing immunoglobulin from a liquid, comprising adding a lyo ⁇ tropic buffer to the liquid, contacting the liquid with a thiophilic adsorption matrix, washing the thiophilic ad- sorption matrix with a lyotropic buffer solution, and eluting the washed thiophilic adsorption matrix with an elution liquid, characterized in that the lyotropic buffer solution has an ion strength below 2.25, preferably be ⁇ tween 0 and 2.25, in particular between 0.6 and 1.5.
• pH in the lyotropic buffer solution is below 7.5, preferably between 2.5 and 7.5, in particular between 3.0 and 6.5, particularly pre ⁇ ferred being 3.5 to 6.0, especially 4.0 to 5.5.
• the lyotropic buffer in the liquid has an ion strength above 2.25, preferably between 2.25 and 4.5, in particular between 3.0 and 4.0, thereby providing both a greater binding capacity and a greater purity than in the known methods.
• the thiophilic matrix is selected from divinyl sulphone acti- vated polymer network, to which the divinyl sulphone groups are bound via an ether oxygen atom, a thioether sulphur atom or a nitrogen atom, the divinyl sulphone groups being moreover covalently bound to a ligand selected from:
• an aromatic or heteroaromatic ring system which is optionally substituted, consisting of one or more rings whose substituents do not comprise nitile groups, and which is bound to a divinyl sulphone group via an oxygen atom or a nitrogen atom, and
• a possible polymeric network for the known and novel thiophilic matrices of the invention is known poly ⁇ mer networks, such as polysaccharides, e.g.
• agar agarose, dextran, starch and cellulose, in particular agarose, polyacrylamide, polyamide, polyimide, polyester, poly- ether, polymeric vinyl compounds and substituted deri ⁇ vatives thereof either as particles, membranes or con ⁇ tained in membranes.
• Possible lyotropic salts for the lyotropic buffer and the lyotropic buffer solution according to the invention are known inorganic salts, such as sodium sulphate, potassium sulphate, ammonium sulphate, sodium phosphate, potassium phosphate and ammonium phosphate, or organic salts of polyvalent carboxylic acids, such as sodium citrate, so ⁇ dium tartrate, potassium citrate, potassium tartrate, or mixtures thereof.
• Possible liquids according to the invention are immunoglo- bulin-containing liquids, in particular biological li ⁇ quids, such as blood, serum, ascites liquid or cell cul ⁇ ture supernatants, in particular cell culture super- natants.
• Possible immunoglobulins according to the invention are all immunoglobulins, in particular immunoglobulins of the type IgG. ⁇ IgG 2A , IgG 2B , IgGg, IgA, IgM, IgD and IgE and in particular murine and human immunoglobulins.
• the thiophilic matrices are not only useful for binding and purifying im ⁇ vunoglobu- lins, but they can also be used for binding other pro ⁇ teins, such as other serum proteins than immunoglobulins, depending upon the used concentration of lyotropic salts in the sample (Lihme & Heegaard, Analytical Biochemistry, vol. 192, p. 64-69, 1991). In this case too it will be advantageous with a smaller concentration of salts in the sample.
• the known process for purifying immunoglo ⁇ bulins by means of thiophilic or nitrilophoric matrices is restricted i.a. because the binding capacity for immuno ⁇ globulin from liquids with very low concentrations of immunoglobulin (i.e. below about 1 mg/ml) is relatively low under known binding conditions (0.5 M potassium sul ⁇ phate or 0.75 M ammonium sulphate).
• the low immunoglobulin concentration typically occurs with the cell culture supernatants resulting from the production of monoclonal antibodies by in vitro cultivation of hybridoma cells, in contrast to serum and ascites liquids which contain a much higher concentration of immunoglobulin.
• the binding capacity for immunoglobulin to adsorption matrices having a thiophilic nature increases steeply with increasing ion strength (concentration) of the known lyotropic salts in the liquid from which the immunoglobulin is to be purified.
• the salts capable of contributing to binding immunoglobu ⁇ lin to thiophilic and nitrilophoric adsorption matrices belong to the group of lyotropic (water structure forming) salts. Examples of such are salts containing sulphate or phosphate ions typically with sodium, potassium or ammo- nium ions as counter ions. Furthermore, some organic ions also have lyotropic activity, e.g. the multivalent anions of organic polyvalent carboxylic acids (e.g. citrate or tartrate ions).
• provision of an increased binding ca- pacity by increasing the ion strength over the ion strength used in the prior art is not limited to ammonium sulphate, but that the increased binding capacity achiev ⁇ able applies to all inorganic and organic salts having lyotropic properties.
• the process of the invention may e.g. be used in the puri ⁇ fication of immunoglobulin from monoclonal in vitro cell culture supernatants according to the following specific procedure, comprising:
• eluting the bound proteins, including immunoglobulins with a buffer solution, which has a low salt content, e.g. 0.05 M Tris/HCl, pH 9.0.
• the process of the invention may also be used for purify- ing immunoglobulins from other liquids, e.g. serum or ascites liquid, but then requires individual adjustment of the ion strength of the flushing buffer with respect to the lyotropic salt and the pH value of the flushing buffer.
• the result will be an increased purity of the immunoglobulin, without the binding capacity of the ad ⁇ sorption matrix being significantly diminished.
• individual adjustment of ion strength and pH value will be desirable depending upon the type of the immunoglobulin (for murine antibodies depending upon whether IgG-, IgG 2A - IgG 2B , I G , IgM or IgE is involved).
• the preferred ion strength of the flushing buffer depends upon the specific application and the present contamina ⁇ tions, but is typically between 0 and 2.25. In most cases the most preferred range is between 0.6 and 1.5.
• the preferred pH value of the flushing buffer depends upon the specific application, but is typically between pH 2.5 and pH 7.5. Owing to the stability of the immunoglobulin during the flushing procedure and the efficiency of the process, a more preferred range will be between pH 3.0 and pH 6.5, while the most preferred range will be between pH 3.5 and pH 6.0. It is particularly pre ⁇ ferred that pH is between 4.0 and 5.5.
• the pH value of the elution buffer is preferably above 7.0, but may also be lower. Elution can also be performed by changing the dielectric!ty constant of the buffer, e.g. by addition of ethylene glycol.
• the selection of elution method is generally independent upon the process for bind ⁇ ing the immunoglobulin and subsequent washing of contami- nations.
• the resulting matrix contained about 40 micromoles of 2- hydroxypyrine per ml of wet drained matrix.
• the resulting matrix contained about 40 micromoles of 4- hydroxypyrine per ml of wet drained matrix.
• the resulting matrix contained about 40 micromoles of 4- methoxyphenol per ml of wet drained matrix.
• the resulting matrix contained about 40 micromoles of 4- aminobenzoic acid per ml of wet drained matrix.
• the resulting matrix contained about 40 micromoles of phe ⁇ nol per ml of wet drained matrix.
• the resulting matrices contained about 30-40 micromoles of ligands.
• the resulting matrix contained about 20 micromoles of phe ⁇ nol per ml of wet drained matrix.
• the resulting matrix contained about 5 micromoles of phe- nol per ml of wet drained matrix.
• the gel was incubated with the solution for 18 hours at room temperature.
• the resulting matrix contained about 40 micromoles of mer ⁇ capto ethanol per ml of wet drained matrix.
• the matrices which were produced according to examples 1- 4 and example 8, were used for purifying immunoglobulins from human serum according to the following known process (Lihme & Heegaard, Analytical Biochemistry, vol. 192, p. 64-69, 1991):
• the EU figure of the eluate was calculated by means of the following formula:
• the matrices of the invention and the known matrix thus bound comparable amounts of total protein and substantially the same amounts of immunoglobu ⁇ lin. None of the matrices bound albumin. The smaller amount of total protein eluted from the 4-hydroxypyridine matrix reflects a higher selectivity for immunoglobulin than the other matrices. It can therefore be concluded that the position of the substituent and on the whole the fine structure of the ligand have a decisive influence on the selectivity of the matrix.
• the raw material was human serum, and purification was performed so that the conditions during application pro- mote binding of most serum proteins, (i.e. a higher ammo ⁇ nium sulphate concentration was used than the one used for selective binding of immunoglobulins) .
• the matrices were eluted with a gradient from 0.01 M K 2 HP0 4 , 1.5 M (NH 4 ) 2 S0 4 pH 7.2 to 0.01 M K 2 HP0 4 , 0.25 M NaCl pH 7.2.
• the eluates were collected in fractions and analyzed by fused rocket immunoelectrophoresis for qualitative deter ⁇ mination of the protein content.
• the adsorption matrix with the highest content of phenyl groups (40 micromoles/ml) bound the proteins most strongly, i.e. the proteins were gene ⁇ rally eluted at a lower ion strength compared with the two other adsorption matrices.
• this matrix also bound the proteins more strongly than known thiophilic matrices, such as the mercapto ethanol derivative, the immunoglobulin G bound so strongly that it could only be liberated by a subsequent elution with 40% ethylene gly- col.
• the thiophilic nature showed itself clearly in that albumin bound much more weakly to the matrix than immunoglobulin and much more weakly than the known hydro- phobic matrices, such as octyl-Sepharose.
• the adsorption matrix with a content of about 20 micro ⁇ moles of phenyl groups per ml of wet gel, exhibited a binding pattern which corresponds closely to the pattern achieved with known thiophilic matrices with a higher ligand concentration (about 40-60 micromoles/ml).
• the adsorption matrix with a content of about 5 micro ⁇ moles of phenyl groups per ml, bound the proteins rather weakly, but still exhibited preference to binding of immunoglobulins.
• the test is performed like process I, but the potassium sulphate added in item 1 is replaced by 0.8 M ammonium sulphate, pH being kept constant at 7.6.
• the test is performed like process II, the ammonium sul- phate concentration being merely increased to 1.0 M.
• the test is performed like process II, the ammonium sul- phate concentration being merely increased to 1.2 M.
• results are expressed as number of ml cell culture supernatant passing the column with the adsorption matrix before the effluent concentration of murine immunoglobulin is 50% of the start concentration (defined as "50% satura ⁇ tion” ).
• Process III >300 Process IV >300
• an increase in the ion strength also has a strong positive effect on the binding capacity when us ⁇ ing sodium sulphate instead of ammonium sulphate.
• the eluate was collected in one fraction and analyzed for purity by means of sodiumdodecyl polyacrylamide electrophoresis followed by electronic scanning.
• the yield in mg of murine immunoglobulin G. is determined by quantitative rocket immunoelectrophoresis.
• the purity is the immunoglobulin in % of the total amount of protein in the sample.
• the test was performed like process I, the washing buffer in item 3 being merely replaced by 0.3 M ammonium sulphate + 0.05 M sodium acetate, pH 5.2.
• Process I 11.5 about 30 Process II 11.0 90 Process III ⁇ 0.5 95

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