EP0460026A1 - Hochleistungsaffinitätschromatographische säule mit nichtporösem, monodispersem, polymerischem packungsmaterial - Google Patents

Hochleistungsaffinitätschromatographische säule mit nichtporösem, monodispersem, polymerischem packungsmaterial

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Publication number
EP0460026A1
EP0460026A1 EP90903707A EP90903707A EP0460026A1 EP 0460026 A1 EP0460026 A1 EP 0460026A1 EP 90903707 A EP90903707 A EP 90903707A EP 90903707 A EP90903707 A EP 90903707A EP 0460026 A1 EP0460026 A1 EP 0460026A1
Authority
EP
European Patent Office
Prior art keywords
groups
poly
styrene
molar ratio
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP90903707A
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English (en)
French (fr)
Inventor
Marsha Denise Bale
Richard Calvin Sutton
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Eastman Kodak Co
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Eastman Kodak Co
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Publication date
Application filed by Eastman Kodak Co filed Critical Eastman Kodak Co
Publication of EP0460026A1 publication Critical patent/EP0460026A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • 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
    • 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
    • B01J20/3255—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
    • 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
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds

Definitions

  • HPAC high performance affinity chro atography
  • affinity chromatography has become a valuable tool for separating biological materials from aqueous media.
  • biologically active molecules such as small ligands, proteins, nucleic acids, enzymes, etc.
  • the basic principle of affinity chroma ⁇ tography involves immobilization of one of the components of the interacting system (e.g., the ligand) to an insoluble support which can then be used to selectively adsorb, in a chromatographic procedure, that component (e.g. an enzyme) of a fluid medium (e.g. aqueous solution) with which it can selectively interact thereby forming a complex of the two components.
  • a fluid medium e.g. aqueous solution
  • Elution of the desired component can then be achieved by any one of a number of procedures which result in disassociation of the complex.
  • the process may be used to isolate specific substances such as enzymes, hormones, specific proteins, inhibitors, antigens, antibodies, etc. on the basis of the biologic specific interactions with immobilized ligands .
  • High performance liquid affinity chroma ⁇ tography combines the remarkable specificity of affinity chromatography with the efficiency, sensitivity and speed of operation of high perfor ⁇ mance liquid chromatographic methods and apparatus.
  • Devices for carrying out high speed liquid chromatography and therefore high speed affinity chromatography have arranged in series along a path of flow, an eluant supply means comprising an eluant reservoir and a high pressure metering pump; a specimen material means comprising a specimen material injector; a separation device comprising a chromatographic column packed with packing material for the purpose of separating the biological material of interest; and a detector.
  • Procedures for carrying out high performance liquid affinity chromatography are described, for example, in Methods In Enzymology. vol. 104, pp. 212 et seq.
  • the procedure involves passing an aqueous sample comprising a biological material of interest through a chromatographic column.
  • the solution is passed through the column under pressure.
  • the column is packed with a support having an attached ligand.
  • the ligand has an affinity for a biological species in the sample.
  • the biological species binds to the ligand forming a ligand—biological species complex.
  • an eluant designed to dislodge the biological material from the ligand is passed through the column under pressure. The eluant breaks up the ligand-biological species complex thereby removing the biological species from the column.
  • the procedure can be carried out in a manner that allows the biological species of interest to pass through the column without binding while other materials from which the biological species is to be separated bind to the ligand.
  • the packing materials in prior art separa- tion devices used in high performance affinity chromatography are porous materials such as silica or porous polymeric particles. Such porous materials present several problems .
  • pores may be clogged by the biological species in the mixture to be separated. This decreases the efficiency of utilization of immobilized ligands and can lead to the requirement of higher pressure for maintenance of a constant flow rate.
  • the mechanical strength of porous packings is generally low enough to cause collapse of the bed at fairly low pressures ( ⁇ 1,000 psi for macroporous polymers and up to 4,000 psi for silica particles). This limitation also prevents use of concentrated samples. Thus it is generally necessary to dilute samples to accommodate porous particles. It would be expected that porous particles would provide greater surface area over non—porous particles. This, in practice, is not the case. Depending on the size of the pores, porous particles are relatively inefficient for use with large molecules of biological interest.
  • a useful ligand may be attached to a porous bead in one of the pores.
  • the biological species which it is desired to eliminate or separate from the liquid stream may be so large as not to be able to get down into the pores and reach the ligand.
  • immobilization of a large affinity ligand can only take advantage of a small portion of the total surface area since the ligand itself cannot penetrate the porous well. Thus, the efficiency of binding is diminished and the apparent surface area advantage of such porous particles becomes illusory.
  • the prior art porous particles are about 5—10 ⁇ m in size to avoid build up of back pressure and collapse of the bed.
  • the present invention provides a high performance affinity chromatography separation device comprising: A) a chromatographic column containing: B) a packing material; wherein the packing material is a plurality of non-porous, monodisperse polymeric particles having i) a particle size in the range of 0.01 to about 5 micrometers ( ⁇ m) and ii) surface reactive groups which are directly or indirectly reactive with free amino groups , sulfhydryl groups, carboxy groups or aldehyde groups of biological ligands.
  • the particles used as packing material are able to withstand greater pressures than porous beads.
  • the particles do not collapse under pressures at which porous particles of the prior art collapse. They have more effective surface area than the larger porous beads used of the prior art.
  • the particles are preferably 1 to 3 ⁇ m.
  • the present invention also provides a method of separating a biological species from an aqueous solution comprising the steps of:
  • the separation device of this invention includes a unique chromatographic column containing a bed of the above-described particles as packing materials.
  • the columns are generally made of stainless steel.
  • stainless steel No. 316 is often used. It is an alloy according to a U.S. standard defined by the American Institute for Standards in Industry (AISI). It is an alloy which shows an excellent resistance against corrosion.
  • the roughness of the tube of the inner wall is a specification governed by the chromatography. As a rule of thumb, the wall roughness—expressed as peak-to-peak value should be less than one-tenth of the means size of the particles packed in the column. This means that the wall roughness should be smaller than 0.5 ⁇ m, when 5 ⁇ m particles are packed in the column. Glass, polymer or gold—lined tubing is also used in chromatographic columns.
  • Tube diameters used for analytical columns range from 4 to 8 mm internal diameter for medium bore columns; 2 to 4 mm internal diameter for small bore columns ; and 1 to 2 mm internal diameter for micro bore columns.
  • the terms given to the diameter are indications currently used for the columns and for their chromatographic application.
  • the internal diameter of the column is a very important factor which has to be selected properly to match the application. Columns within the above ranges of internal diameter are commercially available. The columns are operated under high pressure. There are certain wall thicknesses required for safety reasons. The 0.25-inch tubing most often used has, with an internal diameter of 4.6 mm, a wall thickness of 0.9 mm which is sufficient.
  • Thicker wall tubing with the same internal diameter is offered by some manufacturers.
  • the length of the columns may be from 50 to 100 mm in length. Although larger or shorter lengths are also useful.
  • the column is packed with the chromatographic resin from a slurry.
  • One end of the column is fitted with a frit and end piece.
  • the other end is attached to a slurry packing device.
  • High pressures (often around 10,000 psi or 68,948 kPa) are applied to force the slurry into the column and pack it rapidly (within minutes).
  • Lower pressures up to 6,000 psi or 41,369 kPa) can also be used to pack the column more slowly (within 30-60 minutes).
  • the column is then dis- connected from the packing apparatus and fitted with an end piece.
  • HPLC equipment may be used for separations, generally at room temperature.
  • the flow rate is usually maintained at about 0.1-3 mL per minute for a standard 5 mm ID column.
  • a typical value is 1 mL per minute, which gives a pressure over a 5 cm column loaded with 2 ⁇ m particles of about 1,000-3,000 psi (6,900-21,000 kPa).
  • the packing material is made up of polymeric particles.
  • the particles are water-insoluble latex particles having a particle size in the range of from about 0.01 to about 5 ⁇ m, and preferably from about 0.1 to about 3 ⁇ m.
  • the particles are non—porous and monodisperse.
  • the particles also have surface reactive groups which are directly or indirectly reactive with nucleophilic free amino groups and sulfhydryl groups of biological ligands.
  • Such reactive groups are electrophilic and include: a) active halogen groups; b) activated 2-substituted ethylsulfonyl or activated vinylsulfonyl groups; c) reactive carboxyl groups; d) epoxy groups; e) isocyanate groups; f) aziridine groups; g) aldehyde groups; h) 2-substituted ethylcarbonyl groups; and i) succinimidoxycarbonyl groups.
  • Surface reactive groups a), b), c) and i) are preferred.
  • the polymer particles can have surface reactive nucleophilic groups such as amines, sulfhydryl, etc.
  • the polymers employed to form the particles of this invention conform to the general structure: T fA-> 0 fB p fD q wherein —A- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers, -B- represents recurring units derived from one or more ethylenically unsaturated monomers having the requisite reactive groups which will directly or indirectly react with the free amine or sulfhydryl groups of a ligand with biological specificity as described herein, and —D— represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by -A- or -B-.
  • o is from 0 to 99.9 mole percent
  • p is from about 0.1 to 100 mole percent
  • q is from 0 to about 20 mole percent.
  • o is from about 45 to about 99 mole percent
  • p is from 1 to about 50 mole percent
  • q is from 0 to about 10 mole percent.
  • the —A— recurring units are derived from one or more hydrophobic ethylenically unsaturated monomers. Such monomers are insoluble in water.
  • Representative hydrophobic monomers include, but are not limited to, styrene and styrene derivatives (for example, vinyltoluene, 2,5-dimethylstyrene, 4—£—butylstyrene and 2—chlorostyrene), acrylic and methacrylic acid esters (for example, n—butyl acrylate, propyl methacrylate, methyl acrylate, ethyl methacrylate, 2—ethylhexyl methacrylate, N—phenyl- acrylamide and methyl methacrylate), acrylonitrile and vinyl acetate.
  • the polymer can be crosslinked, if desired, in any suitable fashion.
  • One method is to incor ⁇ porate a small amount, that is up to about 15 mole percent, and preferably from about 0.3 to about 5 mole percent, of a monomer having two or more ethylenically unsaturated polymerizable groups. These monomers are included among the hydrophobic monomers from which A is derived. Representative monomers are described in Research Disclosure.
  • Particularly useful monomers from which -A- is derived are styrene, vinyltoluene, ethylene dimethacrylate, butyl acrylate, divinylbenzene, 2-ethylhexyl methacrylate and methyl methacrylate.
  • the —B- recurring units compromise an appended reactive group that readily reacts with an amine or sulfhydryl group with or without the use of an intermediate crosslinking agent.
  • the B groups can therefore be derived from any monomer containing such reactive groups.
  • Preferred monomers are those comprising appended reactive groups a) to i) mentioned hereinbefore.
  • One preferred class of monomers which provide the requisite reactive groups are those comprising an active halogen atom which readily reacts with amine and sulfhydryl groups.
  • monomers having an active halogen atom include vinyl chloroacetate, vinyl bromoacetate, haloalkylated vinyl aromatics (for example, chloromethylstyrene or bromomethylstyrene) , haloalkyl acrylic or methacrylic esters (for example, chloroethyl methacrylate, 3—chloro-2-hydroxypropy1 methacrylate and 3-chloropropyl acrylate) and others known to one skilled in the art.
  • the haloalkylated vinyl aromatics for example those having active haloalkyl groups of 1 to 3 carbon atoms, are preferred when the active halogen atom is used as the reactive group. Chloromethylstyrene is very useful.
  • monomers having active halogen atoms exhibit many advantages
  • monomers having activated 2-substituted ethylsulfonyl and vinyl- sulfonyl groups possess additional advantages in that proteins can be attached to the polymers under milder -10- conditions and require less process control during manufacture. This renders manufacture more efficient and less costly.
  • a number of representative monomers having the latter groups are known in the art, including those disclosed in U.S. Patents 4,161,407 (issued July 17, 1979 to Campbell) and 4,548,870 (issued October 22, 1985 to Ogawa et al).
  • Preferred activated 2-substituted ethyl- sulfonyl and vinylsulfonyl monomers can be represented by the formula (II):
  • R is hydrogen or substituted or unsubstituted alkyl (generally of 1 to 6 carbon atoms, such as methyl, ethyl, isopropyl or hexyl.
  • R is hydrogen or methyl.
  • a base such as halo, acetoxy, alkylsulfonyloxy such as methylsulfonyloxy, aryl- sulfonyloxy such as r>-tolylsulfonyloxy, trialkyl- ammonio, for example, a trimethylammonio salt or pyridinio salt).
  • R is hydrogen, substituted or unsubstituted alkyl (generally of 1 to 6 carbon atoms as defined for R), or substituted or unsubstituted aryl (generally of 6 to 12 nuclear carbon atoms, such as phenyl, naphthyl, xylyl or tolyl).
  • R is -CH 2 CH 2 .
  • This group which is an activated 2-substituted ethyl group, can be substituted with any group which does not impair the displacement of the leaving group X.
  • L is a linking group which can be a substituted or unsubstituted alkylene generally having 1 to 20 carbon and hetero atoms in the backbone.
  • alkylene is meant to include alkylene groups interrupted or terminated with oxy, thio, -NR 3- [wherein R3 is hydrogen, substituted or unsubstituted alkyl of 1 to 6 carbon atoms (such as methyl, chloromethyl or 2—hydroxy- ethyl) or substituted or unsubstituted aryl of 6 to
  • alkylene groups include methylene, ethylene, isobutylene, hexamethylene, carbonyloxyethoxy- carbonyl, methylenebis(iminocarbonyl), carbonyloxy- dodecylenecarbonyloxyethylene, carbonyliminom- ethyleneiminocarbonyliminoethylene, carbonylimino - ethyleneiminocarbonylethylene and other groups described or suggested by U.S. Patents 4,161,407 and 4,548,870, noted above.
  • L can also be substituted or unsubstituted arylene generally having 6 to 12 nuclear carbon atoms .
  • Representative arylene groups include phenylene, tolylene, naphthylene and others noted in the patents mentioned above.
  • divalent groups which are combinations of one or more of each of the alkylene and arylene groups defined above (for example, arylenealkylene, alkylenearylenealkylene and others readily determined by one of ordinary skill in the art).
  • L is substituted or unsubstituted phenylenealkylene, phenylenealkylene substituted with one or more alkyl groups (as defined for R), alkoxy groups (generally of 1 to 6 carbon atoms, for example, methoxy, propoxy or butoxy) or halo groups, or carbonyliminomethyleneiminocarbonylethylene.
  • alkyl groups as defined for R
  • alkoxy groups generally of 1 to 6 carbon atoms, for example, methoxy, propoxy or butoxy
  • halo groups generally of 1 to 6 carbon atoms, for example, methoxy, propoxy or butoxy
  • Representative 2-substituted ethylsulfonyl and vinyl sulfonyl monomers from which B can be derived include E & £-(2—chloroethylsulfonyl— methyl)styrene, m.
  • the first monomer is preferred.
  • Another preferred reactive group that can be appended to form recurring units B is the carboxyl group.
  • Carboxyl groups can be added to the particles by incorporating monomers containing such groups as, for example, acrylic acid, methacrylic acid, itaconic acid, 2-carboxyethyl acrylate, fumaric acid, maleic acid, 2-carboxyethyl methacrylate carboxymethylstryene, methacrylamidohexanoic acid, N-(2-carboxy-l,l-dimethylethyl)acylamide, and the like), or by further chemical reaction of a polymer having other reactive groups which can be converted to carboxyl groups (for example, by hydrolysis of anhydrides, such as maleic anhydride, or by oxidation of surface methylol or aldehyde end groups).
  • monomers containing such groups as, for example, acrylic acid, methacrylic acid, itaconic acid, 2-carboxyethyl acrylate, fumaric acid, maleic acid, 2-carboxyethyl methacrylate carboxymethylstryene,
  • auxiliary crosslinking agent is used to covalently attach proteins, e.g., antigens, antibodies, haptens, etc. via the carboxyl groups since the carboxy groups alone react too slowly with amine and sulfhydryl groups for most practical purposes.
  • proteins e.g., antigens, antibodies, haptens, etc.
  • auxiliary crosslinking agents are the well—known carbodiimides , e.g., l-cyclohexyl-3-[2-morpholinyl-(4)-ethyl]carbodiimide metho-p-toluenesulfonate, which have been used for crosslinking gelatin in photographic gelatin layers and for making diagnostic reagents as described in U.S. Patent 4,181,636.
  • auxiliary crosslinking agents includes the carbamoylonium salts such as are described in U.S. Patent 4,421,847 (issued December 20, 1983 to Jung et al).
  • Representative carbamoylonium compounds include l-(4-morpholinocarbonyl)-4-(2-sulfoethyl)pyridinium hydroxide, inner salt, and l-(4-morpholino— carbonyDpyridinium chloride.
  • monomers which can be incorporated in the polymers to provide the requisite reactive groups include monomers containing epoxy groups (such as glycidyl acrylate, glycidyl methacrylate, vinyl glycidyl ether or methallyl glycidyl ether), monomers containing isocyanate groups (such as isocyanatoethyl acrylate, isocyanatoethyl methacrylate, or ⁇ , ⁇ -dimethylmetaisopropenylbenzyl isocyanate), monomers containing an aziridine group [such as vinylcarbamoyl aziridine, N-methacryloylaziridine, N-acryloylaziridine and 2—(l-aziridinyl)ethyl acrylate], monomers containing aldehyde groups (such as vinyl benzaldehyde or acrolein) or 2-substituted ethylcarbonyl containing monomers (such as 2—
  • D represents recurring units derived from one or more ethylenically unsaturated monomers other than those represented by A or B.
  • Such monomers can have ionic or other hydrophilic groups which add dispersion stability to the resulting particles in aqueous solution or influence the biological activity of an immobilized ligand.
  • Useful ionic monomers include, but are not limited to, sodium 2-acryl- amido-2-methylpropanesulfonate, sodium 3-acryloyloxy- propanesulfonate, sodium acrylate, sodium methacry ⁇ late, and sodium styrenesulfonate, as well as other known sulfonates, sulfates, carboxylates, their salts or anhydrides, and useful nonionic polar monomers include 2—hydroxyethyl acrylate, 2,3-dihydroxypropyl acrylate, acrylamide, 2-hydroxyethyl methacrylate, N-isopropylacrylamide, 2-hydroxypropyl methacrylate, acrylonitrile and N-isobutoxymethyl acrylamide.
  • Preferred monomers are sodium 2—acrylamide—2-__ethyl- propanesulfonate, sodium acrylate, sodium 3-acryl- oyloxypropanesulfonate, sodium methacrylate, 2-hydroxyethyl acrylate, 2,3-dihydroxypropyl acrylate, acrylamide, N-isopropylacrylamide and acrylonitrile.
  • the polymer particles of this invention can be homogeneous particles being composed of the same polymer throughout, or they can be particles composed of more than one polymer such as graft copolymers as described, for example, in U.S. Patent 3,700,609 (issued October 24, 1972 to Tregear et al) and core—shell polymers described for example in U.S. Patent 4,401,765 (issued August 30, 1983 to Craig et al).
  • graft copolymers as described, for example, in U.S. Patent 3,700,609 (issued October 24, 1972 to Tregear et al) and core—shell polymers described for example in U.S. Patent 4,401,765 (issued August 30, 1983 to Craig et al).
  • a polymer particle can be prepared from cheap monomers, or monomers that regulate buoyancy, then polymerization is continued to add a shell of a different polymer having the requisite surface groups.
  • the polymeric particles can be prepared using any suitable polymerization technique, including emulsion (including batch, semi—continuous and continuous) and suspension polymerization techniques, graft copolymerization, and others known to one skilled in the polymer chemistry art.
  • Emulsion polymerization is preferred as it can be used to provide generally smaller particles without the use of surfactants or e ulsifiers as described for example in U.S. Patent 4,415,700 (noted above) and Research Disclosure publication 15963 (July, 1977). Research Disclosure is a publication available from Kenneth Mason Publications, Ltd., The Old Harbourmaster's, 8 North Street, Emsworth, Hampshire P010 7DD, England.
  • Staged emulsion polymerization can be used to provide a core—shell polymer composed of two different polymers.
  • Emulsion polymerization of the core is carried to substantial completion by continuously adding reactants to a reaction vessel under standard conditions. Monomers and catalysts needed to make the shell polymer are then continuously added to the vessel containing the latex of the core polymer. In this manner, the shell has a definite known composition rather than being a mixture of core and shell monomers .
  • Representative polymers useful in this invention include the following: poly(E & p_—chloro ⁇ methylstyrene) , poly(styrene-£•___—___. & _>—chloromethyl- styrene-co-2-hydroxyethyl acrylate) (67:30:3 molar ratio), poly(styrene-___-m & p-chloroethylsulfonyl- methylstyrene) (95.5:4.5 molar ratio), poly ⁇ styrene-___-N-[m &.
  • Any reactive amine- or sulfhydryl-containing ligand can be attached to polymeric particles according to the present invention as long as that ligand contains a reactive amine or sulfhydryl group, respectively which will react with the reactive groups on the polymer or with the intermediate formed by the reaction of a carbodiimide or a carbamoylonium compound with carboxyl groups on the particles in the case which the polymer has reactive carboxyl groups .
  • Polymers having reactive groups that readily react directly with the amine or sulfhydryl groups on the ligands are simply mixed with the ligands, in an appropriate buffer if necessary, and allowed to react.
  • the attachment of the ligand to carboxyl group-containing polymer particles is carried out in two steps, the first of which involves contacting an aqueous suspension of the polymeric particles with a carbodiimide or a carbamoylonium compound to produce reactive intermediate polymer particles having intermediate reactive groups in place of the carboxyl groups.
  • This step is carried out at a suitable pH using suitable acids or buffers to provide the desired pH.
  • the pH is less than 6, but this is not critical as long as the reaction can proceed. More likely, the pH is between about 3.5 and about 6.
  • the molar ratio of carbodiimide or carbamoylonium compound to the carboxyl groups on the surface of the particles is from about 1:100 to about 10:1, and preferably from about 1:10 to about 2:1.
  • the reactive intermediate formed in the first step is contacted with a reactive amine- or sulfhydryl—group containing ligand.
  • a covalent linkage is thereby formed between the particles and the reactive compound.
  • the weight ratio of the reactive compound to the polymeric particles is generally from about 1:1000 to about 1:1, and preferably from about 1:100 to about 1:10.
  • Biologicc.l ligands of interest having the requisite free amino or sulfhydryl .group include: a ) Protein A which has an affinity for the Fc portion of IgG antibodies. b) Avidin or avidin complexes which have an affinity for biotin or biotin complexes.
  • Avidin and biotin derivatives which can be used to prepare the reagents of this invention include streptavidin, succinylated avidin, monomeric avidin, biocytin (that is, biotin- ⁇ -N-lysine) , biocytin hydrazide, amine or sulfhydryl derivatives of 2-iminobiotin and biotinyl- ⁇ -aminocaproic acid hydrazide, biotin derivatives, such as biotin-N-hydroxy- succinimide ester, biotinyl- ⁇ -aminocaproic acid— —hydroxysuccinimide ester, sulfosuccinimidyl 6-(biotin amido)hexanoate, N-hydroxysuccinimide- iminobiotin, biotinbromoacetylhydrazide, p—diazo- benzoyl biocytin and 3-(N-maleimidopropionyl)bioc
  • Macromolecules that have specificity for particular classes of biological molecules such as Concavalin A which has specificity for certain sugars and sugar—containing macromolecules; heparin which has affinity for coagulation factors, lipoproteins, plasma proteins, etc..
  • Small molecules that have specialized affinity for classes of biological molecules such as the dye Cibarcon® Blue F3G—A and other protein specific hydrophobic dyes that have specificity for albumen, enzymes requiring adenlyl-containing cofactors, coagulation factors and interferron.
  • a general procedure for preparing the packing material from the polymer particles includes covalently attaching the selected ligand to the particles using generally known reactions.
  • the ligand can be directly attached to the particles.
  • the polymer particles are mixed with the ligand in an aqueous buffered solution (pH generally from about 7 to about 10) and a concentration of from about 0.1 to about 40 weight percent polymer particles (preferably from about 0.1 to about 10 weight percent).
  • the amount of ligand is at a ratio to polymer of from about 0.1:1000 to about 1:10, and preferably from about 1:100 to about 1:10.
  • Mixing is carried out at a temperature in the range of from about 5 to about 50°C, and preferably at from about 5 to about 40 ⁇ C, for from about 0.5 to about 48 hours. Any suitable buffer can be used.
  • the pendant reactive groups on the outer surface must be modified or activated in order to cause covalent attachment of the ligand.
  • carboxyl groups must be activated using known carbodiimide or carbamoylonium chemistry, described supra.
  • an epoxy group on the outer surface can be hydrolyzed to form a diol compound capable of reacting with cyanogen bromide which can act as a coupling agent for amine groups in the immunological species.
  • Aldehydes can react directly with amines to form a Schiff's base which can be subsequently reduced to form a covalent link.
  • the aldehyde can be oxidized to an acid and chemistry identified above for carboxyl groups can be used to form an amide linkage.
  • HPAC Chromatography
  • HPAC High Performance Affinity Chromatography
  • the flow rate was reduced to 1.5 mL/minute for 6 minutes and then reduced to 1.4 mL/minute for 25 minutes .
  • the column was pretreated with the binding and elution solvents (mobile phases) which were used in the intended separation.
  • the column was equilibrated with a solution containing a) 1.5 M glycine and 2.0 M-NaCl (pH 8.9); then with a solution containing b) 0.1 M sodium citrate (pH 3); and then with a solution containing c) 1.5 M glycine and 2.0 M NaCl (pH 8.9).
  • the column was then treated with 1 mL of 1% BSA; and finally with 0.1 M sodium citrate (pH 3) until the baseline of absorbance at 280 nm returned. All flow rates were 0.5 mL/minute.
  • the column was stored at 4 ⁇ C in PBS containing 0.1 mM sodium azide.
  • Example 2 Separation of a Mouse I G 2A from Ascites Fluid
  • a sample of reconstituted ascites fluid (Sigma)(0.05 ml) was applied to the column prepared in Example IB at 1 mL/minute following equilibration of the column with the 1.5 M glycine and 2 M NaCl buffer (pH 8.9).
  • the non-binding material was eluted with the same buffer.
  • Elution of the mouse IgG 2A was with 0.1 M sodium citrate, pH 3.0.
  • Fractions of 1 mL each were collected into tubes containing 0.5 mL 0.2 M Tris pH 8.0.
  • Column fractions were quantified using an ELISA for mouse IgG 2A , and the results are shown in Table III. It is clear that all of the IgG 2A applied to the column is being recovered with ⁇ 1% of it coming through with the non—binding fraction.
  • This column was prepared in situ.
  • a quantity (14 ml at 8.0% solids) of 2 ⁇ m poly(styrene-co-2-chloroethylsulfonylmethylstyrene) non-porous beads (molar ratio 95.5/4.5) was charged into a Micrometrics preparative slurry packer fitted with a precolumn and packed into a 4.6 x 50 mm column by pumping water into the packer at 0.8 mL/minute.
  • the column was disconnected from the packer, and an end fitting was added. Determination of the solids of the unpacked latex showed that a total of 0.54 g of the non—porous beads were packed into the column.
  • Lysine ligand was coupled covalently to the non-porous beads by pumping lysine at 1 mM in 0.1 M EPPS, pH 8.5 through the column at 0.3 mL/minute for 15 hours. Aliquots of bovine serum albumin (1 mg in 1 mL) were injected into the column at 0.5 mL/minute until the entire protein sample added was eluted (elution buffer 0.1 M EPPS, pH 8.5). A total of 6 aliquots was injected ; 0.4 mg of bovine serum albumin was retained by the column. The column was allowed to incubate at room temperature overnight to allow coupling of bound bovine serum albumin to the resin.
EP90903707A 1989-02-21 1990-02-13 Hochleistungsaffinitätschromatographische säule mit nichtporösem, monodispersem, polymerischem packungsmaterial Withdrawn EP0460026A1 (de)

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US7371852B2 (en) * 2003-01-22 2008-05-13 Serenex, Inc. Alkyl-linked nucleotide compositions
CA2687930C (en) * 2007-05-25 2016-05-17 Merck Patent Gesellschaft Mit Beschraenkter Haftung Graft copolymers for cation exchange chromatography
US20130171658A1 (en) * 2010-09-17 2013-07-04 Prozyme, Inc. Isolation and deglycosylation of glycoproteins
DE102013020400A1 (de) * 2013-12-10 2015-06-11 Merck Patent Gmbh Reinigungsvorrichtung
JP2015114272A (ja) * 2013-12-13 2015-06-22 東ソー株式会社 重合性モノマー及びその用途
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US4161407A (en) * 1977-10-06 1979-07-17 Eastman Kodak Company Crosslinkable polymers having vinylsulfonyl groups or styrylsulfonyl groups and their use as hardeners for gelatin
DE3130924A1 (de) * 1981-08-05 1983-02-17 Röhm GmbH, 6100 Darmstadt Oberflaechenreiche systeme zur fixierung von nucleophile gruppen enthaltenden substraten
SE470261B (sv) * 1984-05-17 1993-12-20 Jerker Porath Adsorbent för separation och immobilisering av proteiner, sätt att framställa ett adsorbent, samt dess användning för biopolymer fraktionering
DE3609021A1 (de) * 1985-03-25 1986-09-25 Bio-Rad Laboratories, Inc., Richmond, Calif. Chromatographisches trennmedium fuer die schnelle analyse von kleinen proben

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