WO2012001142A1 - Ligands d'affinité de liaison aux anticorps améliorée - Google Patents

Ligands d'affinité de liaison aux anticorps améliorée Download PDF

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Publication number
WO2012001142A1
WO2012001142A1 PCT/EP2011/061102 EP2011061102W WO2012001142A1 WO 2012001142 A1 WO2012001142 A1 WO 2012001142A1 EP 2011061102 W EP2011061102 W EP 2011061102W WO 2012001142 A1 WO2012001142 A1 WO 2012001142A1
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Prior art keywords
ligand
affinity
solid support
affinity resin
support material
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PCT/EP2011/061102
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English (en)
Inventor
Phaedria M. St. Hilaire
Per-Erik Gustavsson
Roice Michael
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Novo Nordisk A/S
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Publication of WO2012001142A1 publication Critical patent/WO2012001142A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • 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
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/29—Chiral phases
    • 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
    • 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
    • B01J20/3214—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
    • 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
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06086—Dipeptides with the first amino acid being basic
    • C07K5/06095—Arg-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to particularly useful affinity ligands covalently bound to a solid support material, such as a polymer matrix, and uses thereof in the purification and/or isolation of biomolecules, such as proteins, in particular antibodies, such as monoclonal antibodies.
  • Affinity chromatography enables selectively and reversibly adsorbing biological substances, such as monoclonal antibodies, to a complementary binding substance, such as an affinity ligand immobilised on a solid support material packed in an affinity column.
  • WO 2003/080655 Al discloses an immunoglobulin-binding protein and a matrix for affinity separation, which comprises an immunoglobulin-binding protein as ligand coupled to a solid support.
  • the resulting affinity resin exhibits improved stability towards aqueous base over prior art affinity resins comprising immunoglobulin-binding proteins.
  • a further improved stability towards aqueous base is desired.
  • the affinity resin according to WO 2003/080655 Al is relatively expensive to produce. Hence, there is a need for less costly affinity resins.
  • WO 2006/066598 discloses affinity resins comprising covalently immobilized affinity ligands comprising one or more hydrophobic functional group(s) and one or more cationic functional groups(s) for purification of antibodies.
  • affinity resins disclosed in WO 2006/066598 exhibit very beneficial properties, it is still desirable to develop even better affinity resins and processes for purification of e.g. antibodies.
  • FIGURE 1 Purification of antibody from CHO-cell supernatant using the Lll-EDA-Sepharose resin.
  • Figure 2 SEC-HPLC-analysis of the antibody feed and the elution peak from the purification in Figure 1.
  • Figure 3 Bioanalyzer-analysis of the elution peak in Figure 1.
  • Figure 4 Structures of reference ligands (a) Fl, (b) L13, and (c) TAAG.
  • affinity resins carrying a ligand of the formula I herein exhibit excellent properties with respect to an exceptionally high capacity and superior binding selectivity compared to structurally related ligands.
  • the present invention provides an affinity resin comprising a solid support material having covalently immobilized thereto, optionally via a linker, an affinity ligand of the Formula I:
  • the ligand of Formula I plays a crucial role for the functionality and beneficial properties of the affinity resin. It is envisaged that the ligand may either be used as the only ligand immobilised to the solid support material, or that the ligand may be used in combination with other ligand(s). In one embodiment, the ligand of Formula I constitutes the only ligand on the solid support material. It is also envisaged that a plurality of ligands, each of Formula I, may be used either alone or in combination with other ligand(s).
  • the integers X and Y which each independently may be 2, 3 or 4, determine the distance between the a-carbon of the two amino acids and the corresponding guanidino-functionality. In some embodiments one or both of X and Y are 3, in particular both of X and Y are 3.
  • immobilized ligand has the formula la
  • both arginines are in the L-configuration.
  • the ligand in fact a plurality of ligand molecules is covalently immobilised to a solid support material .
  • solid support materials are natural and synthetic polymers (e.g. polymer resins, such as cross- linked polymer resins), glasses, ceramics, metals, metal oxides, e.g. aluminium oxide and silicium oxide, and other inorganic oxides, etc. Molecules of the ligand may be covalently immobilised to the full or partial surface of such material and may further be immobilised within cavities or pores of such materials.
  • the solid support material comprises a polymer matrix, e.g. a cross- linked polymer matrix.
  • at least the surface of the solid support material carrying the ligand comprises hydrophilic moieties.
  • the solid support material is typically in the form of beads (in particular substantially spherical beads), particles, a monolith, a filter, a membrane, a sheet, a plate such as a micro-array plate, a fibre, or a sensor, such as a cantelever, a surface plasmon resonance sensor, or a quartz crystal microbalance. Particularly preferred are substantially spherical beads.
  • the solid support materials have pores sufficiently wide for the target protein to diffuse through said pores and interact with ligand on the inner surface of the pores.
  • a monoclonal antibody with molar mass approx. 150 kDa an average pore diameter of 50-200 nm is preferred, such as approx. 100 nm.
  • suitable polymer resins are available, e.g. SepharoseTM, FractogelTM, CIMGELTM, Toyopearl.
  • the solid support material comprises a plurality of hydrophilic moieties.
  • the hydrophilic moieties can be polymer chains which, when cross-linked, form a cross-linked polymer matrix. Examples include e.g. polyethylene glycol moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
  • the solid support material is in the form of a polymer matrix which can be prepared from a variety of polymerisable monomers, including styrenes, acrylates and unsaturated chlorides, esters, acetates, amides and alcohols, including, but not limited to, polystyrene (including high density polystyrene latexes such as brominated polystyrene), polymethylmethacrylate and other polyacrylic acids, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane,
  • polystyrene including high density polystyrene latexes such as brominated polystyrene
  • polymethylmethacrylate and other polyacrylic acids polyacrylonitrile
  • polyacrylamide polyacrolein
  • polydimethylsiloxane polybutadiene
  • polyisoprene polyurethane
  • polyvinylacetate polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,
  • the beads are prepared from styrene monomers or PEG based macro-monomers.
  • the polymer is in preferred embodiments selected from the group consisting of polyethers, polyvinyls, polyacrylates, polymethacrylates, polyacylamides, polyurethanes, polyacrylamides, polystyrenes, polycarbonates, polyesters, polyamides, and combinations thereof.
  • Highly preferred surface and core moieties include cross-linked PEG moieties, polyamine moieties, polyvinylamine moieties, and polyol moieties.
  • the polymer matrix can also be selected from the group consisting of PS, POEPS, POEPOP, SPOCC, PEGA, CLEAR, Expansin, Polyamide, Jandagel, PS-BDODMA, PS-HDODA, PS-TTEGDA, PS-TEGDA, GDMA-PMMA, PS-TRPGDA, ArgoGel, Argopore resins, ULTRAMINE, cross-linked LUPAMINE, high capacity PEGA, Silica, Fractogel, Sephadex, Sepharose, Glass beads, cross-linked polyacrylates, and derivatives of the aforementioned; in particular, the polymer matrix is selected from the group consisting of SPOCC, PEGA, HYDRA, POEPOP, PEG-polyacrylate copolymers, polyether-polyamine copolymers, and cross-linked polyethylene di-amines.
  • the solid support material is selected from silica, cross-linked
  • polyacrylate cross-linked polymethacrylate, cross-linked polystyrene, cross-linked cellulose, and cross-linked agarose.
  • Resins useful for large-scale applications may be one of the above mentioned or other commercial resins such as SephadexTM, SepharoseTM, FractogelTM, CIMGELTM, Toyopearl, cross-linked agarose, and macroporous polystyrene or polyacrylate.
  • the solid support material may also be of a mainly inorganic nature, such as macroporous glass or clay minerals, or combinations of resins and and inorganics, such as Ceramic HyperDTM.
  • the ligand is associated to the surface of a sensor or an array plate and used to detect and/or quantify antibodies in a biological sample.
  • biological sample includes natural samples or samples obtained from industrial processes, e.g. recombinant processes, and include "body fluid", i.e. any liquid substance extracted, excreted, or secreted from an organism or tissue of an organism.
  • body fluid need not necessarily contain cells.
  • Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, urine, plasma, cerebral spinal fluid, tears, milk, sinovial fluid, and amniotic fluid.
  • a plurality of ligands are associated to the surface of an array plate and arranged in a plurality of spots, with each spot representing one ligand.
  • a functionalized array can be used to detect the presence of antibodies in a solution.
  • Such an array can be used for diagnostic applications to detect the presence of certain antibodies in a biological sample.
  • a plurality of ligands is associated to the binding surface of a cantilever sensor for detection and optionally quantification of antibodies.
  • a plurality of affinity ligands can be associated to a plurality of cantilevers with each cantilever
  • the ligand of Formula I is covalently bound to a solid support material, e.g . to cross-linked agarose, at a ligand density in the range of 17 - 1,700 ⁇ per gram dry affinity resin, such as 85 - 850 ⁇ per gram dry affinity resin, such as 170 - 425 ⁇ per gram dry affinity resin, such as 204 - 340 ⁇ per gram dry affinity resin .
  • the affinity resin is swollen in deionized water at 25 °C, and the ligand density is in the range 1 - 100 ⁇ per gram wet affinity resin, such as 5 - 50 ⁇ per gram wet affinity resin, such as 10 - 25 ⁇ per gram wet affinity resin, such as 12 - 20 ⁇ per gram wet affinity resin .
  • Linkers
  • the above-mentioned ligand is covalently immobilized to a solid support material, possibly through a linker.
  • the ligand is covalently attached to a linker which again is covalently attached to the polymer matrix.
  • General techniques for linking of affinity ligands to solid support materials can be found in Hermanson, Krishna Mallia and Smith, Immobilized Affinity Ligand Techniques", Academic Press, 1992.
  • the linker forms a strong and durable bond between the ligand and the solid support material .
  • This is particularly important, when the solid support material of the present invention is to be used for repeated purification of monoclonal antibodies or fragments thereof.
  • lin kers can be selectively cleavable. This can be useful when the solid support is to be used for analytical purposes.
  • the ligand is attached to the solid support through a linker having a length of less than 50 A, such as a length of from 3 to 30 A, for example a length of from 3 to 20 A, such as a length of from 3 to 10 A.
  • the linker is attached to the affinity ligand via the C-terminal carboxylic acid group.
  • the linker may consist of one or of a plurality of covalently linked subunits, e.g . such that the subunits are selected from identical and non-identical linker subunits.
  • the linker comprises 3-50identical or non-identical, covalently linked subunits.
  • linker includes or consists of subunits selected from the group consisting of glycine (Gly), alanine (Ala), 3-aminopropionic acid, 4-aminobutanoic acid, and 4-hydroxymethyl-benzoic acid (HMBA).
  • Gly glycine
  • Al alanine
  • HMBA 4-hydroxymethyl-benzoic acid
  • the linker includes or consists of subunits selected from the group consisting of polydisperse polyethylene glycol; monodisperse polyethylene glycol, such as ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, and heptaethylene glycol; an amino acid; a dipeptide; a tripeptide; a tetrapeptide; a pentapeptide; a hexapeptide; a heptapeptide; octapeptide; a nonapeptide; a decapeptide; a polyalanine; and a polyglycine, including any combination thereof.
  • polydisperse polyethylene glycol monodisperse polyethylene glycol, such as ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, and heptaethylene glycol
  • an amino acid a dipeptid
  • the linker includes or consists of one or more subunits selected from the group consisting of: -(Gly) n -, -0-(CH 2 ) n -0-, -NH-(CH 2 ) n -NH-, -NH-(CH 2 ) n - S-, -(0-CH 2 CH 2 ) n - / and -S-, wherein each n is an integer of 1 to 10.
  • Examples of such linker subunits are -Gly-, -ethylenediamine-, -cysteamine-, etc.
  • the present invention also relates to the ligand per se. i.e. a compound of the Formula II
  • both of the arginines are in the L-configuration.
  • such a compound may have interesting therapeutic, diagnostic and analytical applications, e.g. as a drug binding to Fc for use in e.g. in inflammatory diseases, as a side chain covalently bound to a pharmaceutical protein with the aim of increasing the half life of said protein, immobilised on a surface of a sensor used to detect biomolecules such as IgG, and covalently bound to a marker, such as a fluorescent marker, to detect biomolecules such as IgG.
  • the present invention further relates to a ligand-binding partner conjugate, wherein the ligand of Formula II is as defined hereinabove, e.g. Z represents the binding partner, optionally attached via a linker (as above).
  • the present invention also relates to the use of the affinity resin defined herein in a method for the isolation of a protein, e.g. an antibody, serum albumin or human growth hormone, or a derivative thereof.
  • the method comprises the steps of: a) providing an affinity resin comprising a solid support material having covalently immobilized thereon an affinity ligand, as defined herein, b) contacting said affinity resin with said sample comprising said protein or derivative thereof under conditions which allows said protein or derivative thereof to become bound to said affinity resin, and c) separating the affinity ligand from said sample.
  • step a the affinity resin is provided, e.g. in accordance with the description provided in the present application, in particular the examples.
  • steps b) and c) the handling of the solid support material, the procedure for contacting the affinity resin with the sample and the following separation step, etc. essentially follows that of conventional affinity chromatographic techniques.
  • the protein is an antibody, e.g. a polyclonal antibody or a monoclonal antibody.
  • an antibody e.g. a polyclonal antibody or a monoclonal antibody.
  • Sepharose 6FF resin supplied by GE Healthcare
  • Sepharose 6FF resin was activated by modification of literature procedure (Ref: J. A. Scoble, R. Scopes; J. Chromatography A 752, 1996, 67-76). The degree of activation was approximately 25 ⁇ /g.
  • EDA Ethylenediamine activated Sepharose 6FF resin
  • the epoxy activated Sepharose 6FF resin (100 ml_) was washed with water. Ethylenediamine (EDA) solution 40 - 80% EDA in water) was taken in 1 L RB flask. The epoxy activated Sepharose 6FF resin was added to the EDA solution and stirred for overnight at room temperature. The resin was washed with water, 50% EtOH/water, EtOH, 50% EtOH/water, and water. The amino loading was typically 18 -23 ⁇ mol/mL.
  • EDA Ethylenediamine
  • Cysteamine activated Sepharose 6FF resin (Sepharose 6FF-Cysteamine)
  • the epoxy activated Sepharose 6FF resin (100 ml_) was washed with water and was transferred to a 1 RB flask.
  • the resin was washed with water, 50% EtOH/water, EtOH, 50% EtOH/water, and water.
  • the amino loading was typically 18 - 20 ⁇ /nnL.
  • the epoxy activated cellulose resin (100 ml_) was washed with water and was transferred to a 1 L RB flask. Ammonia solution (25%) was added to the resin and stirred overnight at room temperature. The resin was washed with water, 50% EtOH/water, EtOH, 50% EtOH/water and water. The amino loading was typically 90 - 100 ⁇ /nnL. D. Coupling of Ligand to resin
  • the resin (100 mL) was sequentially washed with 25% EtOH/water, 50% EtOH/water, 75% EtOH/water, EtOH, 25% NMP/EtOH, 50% NMP/EtOH, 75% NMP/EtOH, and NMP.
  • the ligand (3 equiv) was dissolved in NMP/DMSO (2: 1, 50 mL) and was activated with EDC (l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide) (3 equiv), HOAt (1-hydroxybenzotriazole) (3 equiv) and DIPEA (N,N'-diisopropylethylamine) (4 equiv).
  • the reaction mixture was stirred for 5 min and was added to an EDA (ethylenediamine)/Cysteamine functional resin and shaken for 6 hours at room temperature.
  • the resin was filtered off and was sequentially washed with NMP, 75% NMP/EtOH, 50% NMP/EtOH, 25% NMP/EtOH, EtOH, 75% EtOH/water, 50% EtOH/water, 25% EtOH/water, water and 20% EtOH/water.
  • the ligand loading ranged from 10 - 40 ⁇ /nnL depending on resin.
  • the resin was packed in a Tricorn 5/50 column (GE Healthcare) to a bed volume of 1 mL (5.1 cm bed height).
  • the column was coupled to the AKTAlOOexplorer system and equilibrated with 5 mL of equilibration buffer (50 mM sodium phosphate, pH 7, 0.1 M NaCI).
  • the antibody feed was loaded to the column by the super-loop followed by washing with 15 mL of wash buffer (50 mM sodium phosphate, pH 7, 0.1 M NaCI).
  • Adsorbed antibody to the column was then eluted by 15 mL of elution buffer (e.g. 10 mM Na-Formate, pH 3.6, 100 mM NaCI).
  • the column was regenerated using a cleaning in place step with 5 mL of CIP- solvent (e.g. 1 M sodium hydroxide solution) followed by a re-equilibration step with 10 mL of equilibration buffer (50 mM sodium phosphate, pH 7, 0.1 M NaCI (not shown in figure 1)).
  • CIP- solvent e.g. 1 M sodium hydroxide solution
  • equilibration buffer 50 mM sodium phosphate, pH 7, 0.1 M NaCI (not shown in figure 1)
  • the flow rate for the equilibration, load, wash and elution step was 0.33 mL/min (100 cm/h) and the flow rate for the CIP and re-equilibration step was 0.5 mL/min.
  • Eluted fractions were adjusted to pH 7 by 0.5 M Na 2 HP0 4 if necessary and then analysed by HPLC and Bioanalyzer.
  • FIG. 1 A typical chromatogram is shown in Figure 1.
  • Figure 1 In the application step of the purification process ( Figure 1), 1 mL fractions were collected so the 10% dynamic breakthrough profile of the antibody could be determined by HPLC- analysis. Analysis of the purity of the eluted antibody and the high selectivity of the affinity of the resin is demonstrated in Figure 2, which shows the HPLC analysis of the antibody feed (before purification) and the elution peak (after purification). Purity was also determined by Bioanalyzer (Figure 3). Table 1 also shows the results for purification of an antibody of IgG subtype 4 on various affinity resins. The 10% dynamic binding capacity (DBC) values ranged between 15 and 25 mg/mL, and eluted antibody was obtained in high purity (around 90%) and with high recovery values (between 80 and 90%). Table 1 : Results for the purification of IgG4 G using ligands Ll l and L13 attached to different base resins and with elution at pH 3.6.
  • DPC 10% dynamic binding capacity
  • Ll l is a particularly good ligand for selective binding and de-binding of antibodies.
  • the optimal pH for loading varies somewhat for individual monoclonal antibodies, however, most immunoglobulins bind well to the Lll-resin when applied at pH 6-7.
  • the feed stock pH is adjusted to match that of the equilibration buffer. Suggested buffers: 50 mM Sodium Phosphate augmented with 0.1 M NaCI, pH 7; 50 mM Sodium Phosphate augmented with 0.1 M NaCI, pH 6.
  • Washing is monitored until the UV absorbance is back to low levels. Washing can be carried out with the same buffers for used for equilibration.
  • Bound antibody is eluted by pH change. Elution occurs in the pH range of 5 to 3, with somewhat lower yields at higher pH. Recommended elution conditions for minimal HCP is pH 4.6, while elution for a maximized yield occurs with the use of pH 3.6. Cleaning in Place (CIP)
  • Residual material bound to the column can be cleaned using 5 column volumes of 0.5 to 1 M NaOH at a flow velocity of 150 cm/h.
  • the resin may be sanitized by treatment with 0.5 to 1 M NaOH for 1 hour.
  • the different antibodies tested spanned over different subclasses of IgG such as IgG4, IgGl and IgG2 as well as several different constructs of IgG4. All of the tested antibodies could be purified by the Lll-EDA-Sepharose resin.

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Abstract

La présente invention concerne des ligands particulièrement utiles, liés de façon covalente à un matériau de support solide, tel qu'une matrice polymère, et leur utilisation dans la purification et/ou l'isolement de biomolécules, telles que des protéines, en particulier des anticorps, par exemple des anticorps monoclonaux. Les ligands incluent des composés de formule (3,5-di-tert-butyl-4-hydroxy-benzoyl)-Arg-Arg-.
PCT/EP2011/061102 2010-07-02 2011-07-01 Ligands d'affinité de liaison aux anticorps améliorée WO2012001142A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105377875A (zh) * 2013-05-31 2016-03-02 斯蒂法诺·梅内加蒂 用于纯化抗体或抗体片段的拟肽亲和配体
US10914659B2 (en) 2013-09-30 2021-02-09 3M Innovative Properties Company Guanidine-functionalized metal silicate particles and methods of making and using such particles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080655A1 (fr) 2002-03-25 2003-10-02 Amersham Biosciences Ab Proteine de liaison a l'immunoglobuline mutee
WO2006066598A2 (fr) 2004-12-23 2006-06-29 Novo Nordisk A/S Ligands affinitaires se liant a des anticorps

Patent Citations (2)

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WO2003080655A1 (fr) 2002-03-25 2003-10-02 Amersham Biosciences Ab Proteine de liaison a l'immunoglobuline mutee
WO2006066598A2 (fr) 2004-12-23 2006-06-29 Novo Nordisk A/S Ligands affinitaires se liant a des anticorps

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J.A. SCOBLE, R. SCOPES, J. CHROMATOGRAPHY A, vol. 752, 1996, pages 67 - 76
VERSAMATRIX A/S: "Antibody Ligands - Patent Application Number PA 2004 02010", EUROPEAN PATENT REGISTER, 21 July 2006 (2006-07-21), pages 1 - 102, XP055007901, Retrieved from the Internet <URL:https://register.epo.org/espacenet/application?documentId=EKQ6KY1G4246FI4&number=EP05822928&lng=en&npl=false> [retrieved on 20110923] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105377875A (zh) * 2013-05-31 2016-03-02 斯蒂法诺·梅内加蒂 用于纯化抗体或抗体片段的拟肽亲和配体
CN105377875B (zh) * 2013-05-31 2019-11-26 斯蒂法诺·梅内加蒂 用于纯化抗体或抗体片段的拟肽亲和配体
US10914659B2 (en) 2013-09-30 2021-02-09 3M Innovative Properties Company Guanidine-functionalized metal silicate particles and methods of making and using such particles

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