US20240018184A1 - Method of Cleaning and/or Sanitizing a Separation Matrix - Google Patents

Method of Cleaning and/or Sanitizing a Separation Matrix Download PDF

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US20240018184A1
US20240018184A1 US18/354,413 US202318354413A US2024018184A1 US 20240018184 A1 US20240018184 A1 US 20240018184A1 US 202318354413 A US202318354413 A US 202318354413A US 2024018184 A1 US2024018184 A1 US 2024018184A1
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seq
zvar
separation matrix
immunoglobulin
binding
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US18/354,413
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Annika Forss
Gustav José Rodrigo
Tomas Bjorkman
Mats Ander
Jesper Ulf Hansson
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Cytiva Bioprocess R&D AB
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Cytiva Bioprocess R&D AB
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Priority claimed from GBGB1608229.9A external-priority patent/GB201608229D0/en
Priority claimed from US15/282,367 external-priority patent/US10654887B2/en
Priority claimed from US15/348,699 external-priority patent/US10703774B2/en
Application filed by Cytiva Bioprocess R&D AB filed Critical Cytiva Bioprocess R&D AB
Priority to US18/354,413 priority Critical patent/US20240018184A1/en
Assigned to CYTIVA BIOPROCESS R&D AB reassignment CYTIVA BIOPROCESS R&D AB CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: GE HEALTHCARE BIOPROCESS R&D AB
Assigned to GE HEALTHCARE BIOPROCESS R&D AB reassignment GE HEALTHCARE BIOPROCESS R&D AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HANSSON, JESPER ULF, ANDER, Mats, BJORKMAN, TOMAS, FORSS, ANNIKA, RODRIGO, GUSTAV JOSE
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    • 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
    • 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
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B01J20/267—Cross-linked 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
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on 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
    • B01J20/286—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
    • B01J20/30—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
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • 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
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography

Definitions

  • the present invention relates to a method of cleaning and/or sanitizing separation matrices that comprise multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.
  • the present invention further relates to a method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, as well as to use of a cleaning liquid comprising at least 70% by volume of an aqueous alkali metal hydroxide solution for the sanitization of a separation matrix.
  • Immunoglobulins represent the most prevalent biopharmaceutical products in either manufacture or development worldwide.
  • the high commercial demand for and hence value of this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective immunoglobulin manufacturing processes whilst controlling the associated costs.
  • Affinity chromatography is used in most cases, as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs).
  • mAbs monoclonal antibodies
  • pAbs polyclonal antibodies
  • a particularly interesting class of affinity reagents is proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent on the antigen-binding specificity of the antibody.
  • Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples such as but not limited to serum or plasma preparations or cell culture derived feed stocks.
  • An example of such a protein is staphylococcal protein A, containing domains capable of binding to the Fc and Fab portions of IgG immunoglobulins from different species. These domains are commonly denoted as the E-, D-, A-, B- and C-domains.
  • Staphylococcal protein A (SpA) based reagents have due to their high affinity and selectivity found a widespread use in the field of biotechnology, e.g. in affinity chromatography for capture and purification of antibodies as well as for detection or quantification.
  • SpA-based affinity medium probably is the most widely used affinity medium for isolation of monoclonal antibodies and their fragments from different samples including industrial cell culture supernatants.
  • various matrices comprising protein A-ligands are commercially available, for example, in the form of native protein A (e.g. Protein A SEPHAROSETM, GE Healthcare, Uppsala, Sweden) and also comprised of recombinant protein A (e.g. rProtein A SEPHAROSETM, GE Healthcare). More specifically, the genetic manipulation performed in the commercial recombinant protein A product is aimed at facilitating the attachment thereof to a support and at increasing the productivity of the ligand.
  • native protein A e.g. Protein A SEPHAROSETM,
  • Such contaminants can for example be non-eluted molecules adsorbed to the stationary phase or matrix in a chromatographic procedure, such as non-desired biomolecules or microorganisms, including for example proteins, carbohydrates, lipids, endotoxins, bacteria and viruses.
  • the removal of such contaminants from the matrix is usually performed after a first elution of the desired product in order to regenerate the matrix before subsequent use.
  • Cleaning the matrix of contaminants usually involves a procedure known as cleaning-in-place (CIP), wherein agents capable of eluting contaminants from the stationary phase are used.
  • CIP cleaning-in-place
  • Inactivation of microorganisms usually involves a procedure known as sanitization-in-place (SIP) wherein agents capable of inactivating microorganisms are used. Some agents are capable of both cleaning and sanitizing the separation matrix, depending on the concentration of the agent and the contact time with the separation matrix.
  • SIP sanitization-in-place
  • Some agents are capable of both cleaning and sanitizing the separation matrix, depending on the concentration of the agent and the contact time with the separation matrix.
  • One such class of agents often used is alkaline solutions that are passed over said stationary phase.
  • the most extensively used cleaning and sanitizing agent for most separation matrices is NaOH, and the concentration thereof can range from 0.1 up to e.g. 1 M, depending on the degree and nature of contamination.
  • this strategy is associated with exposing the matrix to solutions with pH-values above 13.
  • affinity chromatography matrices containing proteinaceous affinity ligands such alkaline environment is a very harsh condition and consequent
  • the inventors of the present invention have recognised that alcohol solutions are suboptimal for the sanitization of affinity separation matrices. Alcohols are flammable, subject to regulation, and difficult to dispose of. Moreover, the inventors of the present invention have recognised that low concentration NaOH, such as 0.1 M NaOH, provides insufficient cleaning in order to allow the separation matrix to be used for the purification of a variety of different immunoglobulin products. Instead, the separation matrix is typically only used to purify separate batches of a single product in order to prevent carryover of impurities from one product to another. The inventors have recognised that concentrated aqueous alkali metal hydroxide solutions have a number of advantages as cleaning and storage solutions.
  • Concentrated alkali metal hydroxide solutions are bacteriostatic and can inactive most viruses, bacteria, yeasts, fungi and endotoxins. They are capable of effectively stripping the separation matrix of impurities to such a degree that the same separation matrix may be used to purify a variety of immunoglobulins. This facilitates the use of the separation matrix as a separation platform. Moreover, concentrated alkali metal hydroxide solutions are relatively cheap, easily disposed of, and removal from the separation matrix is simple to detect using pH and/or conductivity measurements.
  • the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine,
  • a highly alkali-stable separation matrix having a high dynamic binding capacity is obtained.
  • the inventors of the present invention have observed that such separation matrices are relatively stable upon impregnation with concentrated aqueous alkali metal hydroxide solution, and substantially retain dynamic binding capacity after contact with such concentrated alkali metal hydroxide solution. This means that such a separation matrices are suitable for cleaning and/or sanitization with concentrated alkali metal hydroxide solutions.
  • the immunoglobulin-binding alkali-stabilized Protein A domains may provide further enhancement of properties such as enhanced alkali stability.
  • the glutamine residue at position 1 of SEQ ID Nos: 51 or 52 may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position 35 of SEQ ID Nos: 51 or 52 may be mutated to an alanine.
  • the multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or nonamers.
  • the immunoglobulin binding capacity and alkali stability of the separation matrix may be increased.
  • the multimers of immunoglobulin-binding alkali-stabilized Protein A domains may each comprise a C-terminal cysteine residue for covalent coupling to the porous support.
  • the multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be coupled to the porous support via thioether links. This provides a robust, alkali-stable and well-proven method of attaching the ligands to the solid support.
  • the separation matrix may comprise at least 11 mg/ml, such as at least 15 mg/ml, of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support. This ensures a separation matrix with a good binding capacity.
  • the porous support may comprise cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml.
  • the porous support may for example be highly cross-linked agarose beads.
  • the aqueous alkali metal hydroxide solution used in the cleaning liquid may be sodium hydroxide solution, potassium hydroxide solution or a mixture thereof, preferably sodium hydroxide solution.
  • Sodium hydroxide solution is relatively cheap, readily available and widely accepted for use as a cleaning and sanitization solution.
  • the aqueous alkali metal hydroxide solution may have a molarity of from 500 mM to 5 M, such as from 1 M to 2 M. This ensures a solution with good bactericidal properties.
  • the cleaning liquid may in some instances further comprise a C2-C7 alcohol, such as ethanol, isopropanol or benzyl alcohol.
  • a cleaning liquid combining an alcohol and an alkali metal hydroxide may be more effective in inactivating certain microorganisms, such as some spore-forming bacteria.
  • the cleaning liquid may comprise at least 70% by volume aqueous alkali metal hydroxide solution, such as at least 90% by volume aqueous alkali metal hydroxide solution, preferably at least 99% by volume aqueous alkali metal hydroxide solution.
  • the cleaning liquid may consist of, or consist essentially of, aqueous alkali metal hydroxide solution.
  • the predetermined contact time may be a time sufficient to provide a 6-log 10 reduction in endotoxin concentration and/or microorganism concentration in the separation matrix.
  • the predetermined contact time may be from 10 minutes to 50 hours, such as from 30 minutes to 24 hours, or such as from 1 hour to 12 hours. Such contact times are typically sufficient to inactivate a wide range of microorganisms.
  • Steps a)-c) of the method above may be repeated at least 10 times, such as at least 50 times or 50-200 times.
  • the separation matrix may be reused many times while still retaining sufficient capacity to achieve an acceptable purification.
  • the object of the invention is further achieved by a method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, as defined by the appended claims.
  • the method comprises the steps of purifying a first immunoglobulin and cleaning and sanitizing the separation matrix by performing the steps a)-c) of the method of cleaning and/or sanitizing a separation matrix as described above.
  • the method further comprises the step of d) purifying a mixture comprising a second immunoglobulin using the separation matrix, wherein the second immunoglobulin is different from the first immunoglobulin.
  • the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.
  • SpA Staphylococcus Protein A
  • antibody and “immunoglobulin” are used interchangeably herein, and are understood to include also fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.
  • an “Fc-binding polypeptide”, “Fc-binding domain” and “Fc-binding protein” mean a polypeptide, domain or protein respectively, capable of binding to the crystallisable part (Fc) of an antibody and includes e.g. Protein A and Protein G, or any fragment or fusion protein thereof that has maintained said binding property.
  • linker herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.
  • spacer herein means an element connecting a polypeptide or a polypeptide multimer to a support.
  • % identity with respect to comparisons of amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLASTTM) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410. A web-based software for this is freely available from the US National Library of Medicine.
  • BLASTTM Basic Local Alignment Tool
  • blastp protein-protein BLAST
  • the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • FIG. 1 shows an alignment of the Fc-binding domains as defined by SEQ ID NOs: 1-7 and 51-52.
  • FIG. 2 shows results from Example 2 for the alkali stability of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to an SPR biosensor chip.
  • FIG. 3 shows results from Example 4 for the alkali stability (0.5 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to agarose beads.
  • FIG. 4 shows results from Example 4 for the alkali stability (1.0 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to agarose beads.
  • FIG. 5 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO: 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qb10%, 6 min residence time) vs. incubation time in 1 M NaOH.
  • FIG. 6 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO: 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qb10%, 6 min residence time) after 31 h incubation in 1 M NaOH vs. the ligand content of the prototypes.
  • FIG. 7 shows results from a pH gradient elution of polyclonal human IgG a) from the reference matrix MabSelect SuRe LX and b) a matrix according to the invention.
  • FIG. 8 shows analyses of the IgG1, IgG2 and IgG4 components in fractions from the chromatograms of FIG. 7 .
  • the first bar blue
  • the second (red) IgG 4 the third (green) IgG 2.
  • FIG. 9 shows results from accelerated alkali stability measurements with 1 M NaOH incubation for the reference matrix MabSelect SuRe LX (MSS LX) and a matrix according to the invention.
  • the stability is expressed as the percentage of the 10% breakthrough capacity remaining after incubation.
  • FIG. 10 shows results from repeated CIP cycles using 2 M NaOH as the cleaning liquid for the reference matrix MabSelect SuRe (MSS) and a separation matrix according to the invention (Inv. Ex).
  • the capacity of each separation matrix relative to initial capacity is shown as a function of cycle number.
  • FIG. 11 shows results from incubation with 1 M NaOH the reference matrix MabSelect SuRe LX (MSS LX) and a separation matrix according to the invention (Inv. Ex). The relative fluorescence as a function of time due to binding of labelled hIgG is shown for each of the separation matrices incubated for set periods of 0, 16 and 32 hours.
  • FIG. 12 shows results from incubation with 1 M NaOH the reference matrix MabSelect SuRe LX (MSS LX) and a separation matrix according to the invention (Inv. Ex). The relative fluorescence as a function of time due to binding of labelled hIgG is shown for each of the separation matrices incubated for set periods of up to 32 hours.
  • FIG. 13 shows results from the inactivation of B. subtilis spores using cleaning fluids comprising 1 M NaOH.
  • One aspect of the present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.
  • the immunoglobulin-binding alkali-stabilized Protein A domains of the invention also termed herein as “the polypeptide”, comprise, consist essentially of, or consist of mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.
  • SpA Staphylococcus Protein A
  • SEQ ID NO: 51 (truncated Zvar) QQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SANLLAEAKK LNDAQ
  • SEQ ID NO 52 (truncated C domain) QQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV
  • Such immunoglobulin-binding alkali-stabilized Protein A domains may comprise, consist essentially of, or consist of mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90%, at least 95% or at least 98% identity to, SEQ ID NO: 1 (E-domain), SEQ ID NO: 2 (D-domain), SEQ ID NO: 3 (A-domain), SEQ ID NO: 22 (variant A-domain), SEQ ID NO: 4 (B-domain), SEQ ID NO: 5 (C-domain), SEQ ID NO: 6 (Protein Z), SEQ ID NO: 7 (Zvar), SEQ ID NO: 51 (Zvar without the linker region amino acids 1-8 and 56-58) or SEQ ID NO: 52 (C-domain without the linker region amino acids 1-8 and 56-58) as illustrated in FIG.
  • SpA Staphylococcus Protein A
  • the parental, i.e. non-engineered, Staphylococcus Protein A comprises five Fc-dining domains termed domain E (SEQ ID NO: 1), D (SEQ ID NO: 2), A (SEQ ID NO: 3), B (SEQ ID NO: 4) and C (SEQ ID NO: 5).
  • Protein Z (SEQ ID NO: 6) is a mutated B-domain as disclosed in U.S. Pat. No. 5,143,844, hereby incorporated by reference in its entirety.
  • SEQ ID NO: 7 denotes a further mutated variant of Protein Z, here called Zvar, with the mutations N3A, N6D, N23T.
  • SEQ ID NO: 22 (not shown in FIG. 1 ) is a natural variant of the A-domain in Protein A from Staphylococcus aureus strain N315, having an A46S mutation, using the position terminology of FIG. 1 .
  • SEQ ID NO: 51 is Zvar (SEQ ID NO: 7) without the linker region amino acids at positions 1-8 and 56-58.
  • SEQ ID NO: 52 is the C-domain of protein A without the linker region amino acids 1-8 and 56-58) as illustrated in FIG. 1 .
  • polypeptide can also be described as an Fc- or immunoglobulin-binding polypeptide, or alternatively as an Fc- or immunoglobulin-binding polypeptide unit.
  • the immunoglobulin-binding alkali-stabilized Protein A domains may comprise, consist essentially of, or consist of a sequence as defined by, or having at least 90%, at least 95% or at least 98% identity to SEQ ID NO 53.
  • amino acid residues in SEQ ID NO: 53 may individually of each other be:
  • amino acid residues may individually of each other be:
  • the N11 (X 2 ) mutation may be the only mutation or the polypeptide may also comprise further mutations, such as substitutions in at least one of the positions corresponding to positions 3, 6, 9, 10, 15, 18, 23, 28, 29, 32, 33, 36, 37, 40, 42, 43, 44, 47, 50, 51, 55 and 57 in SEQ ID NOs: 4-7.
  • the original amino acid residue may e.g. be substituted with an amino acid which is not asparagine, proline or cysteine.
  • the original amino acid residue may e.g.
  • amino acid residues may be deleted, e.g. from positions 1-6 and/or from positions 56-58.
  • the amino acid residue at the position corresponding to position 9 in SEQ ID NOs: 4-7 is an amino acid other than glutamine, asparagine, proline or cysteine, such as an alanine or it can be deleted.
  • the combination of the mutations at positions 9 and 11 provides particularly good alkali stability, as shown by the examples.
  • the amino acid residue at position 9 is an alanine and the amino acid residue at position 11 is a lysine or glutamic acid, such as a lysine. Mutations at position 9 are also discussed in copending application PCT/SE2014/050872, which is hereby incorporated by reference in its entirety.
  • the amino acid residue at the position corresponding to position 50 in SEQ ID NOs: 4-7 (X 13 ) is an arginine or a glutamic acid.
  • the amino acid residue at the position corresponding to position 3 in SEQ ID NOs: 4-7 is an alanine and/or the amino acid residue at the position corresponding to position 6 in SEQ ID NOs: 4-7 is an aspartic acid.
  • One of the amino acid residues at positions 3 and 6 may be an asparagine and in an alternative embodiment both amino acid residues at positions 3 and 6 may be asparagines.
  • the amino acid residue at the position corresponding to position 43 in SEQ ID NOs: 4-7 is an alanine or a glutamic acid, such as an alanine or it can be deleted.
  • the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acid respectively, while the amino acid residue at position 43 is alanine or glutamic acid.
  • amino acid residue at the position corresponding to position 28 in SEQ ID Nos: 4-7 (X 5 ) is an alanine or an asparagine, such as an alanine.
  • the amino acid residue at the position corresponding to position 40 in SEQ ID NOs: 4-7 is selected from the group consisting of asparagine, alanine, glutamic acid and valine, or from the group consisting of glutamic acid and valine, or valine, or it can be deleted.
  • the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and glutamic acid respectively, while the amino acid residue at position 40 is valine.
  • the amino acid residue at position 43 may then be alanine or glutamic acid.
  • amino acid residue at the position corresponding to position 42 in SEQ ID NO: 4-7 (X 10 ) is an alanine, lysine or arginine or it can be deleted.
  • amino acid residue at the position corresponding to position 18 in SEQ ID NO: 4-7 (X 3 ) is a lysine or a histidine, such as a lysine.
  • amino acid residue at the position corresponding to position 33 in SEQ ID NO: 4-7 (X 7 ) is a lysine or a serine, such as a lysine.
  • amino acid residue at the position corresponding to position 37 in SEQ ID NO: 4-7 (X 8 ) is a glutamic acid or an aspartic acid, such as a glutamic acid.
  • amino acid residue at the position corresponding to position 51 in SEQ ID NO: 4-7 (X 14 ) is a tyrosine or a leucine, such as a tyrosine.
  • the amino acid residue at the position corresponding to position 44 in SEQ ID NO: 4-7 is a leucine or an isoleucine.
  • the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acid respectively, while the amino acid residue at position 44 is isoleucine.
  • the amino acid residue at position 43 may then be alanine or glutamic acid.
  • the amino acid residues at the positions corresponding to positions 1, 2, 3 and 4 or to positions 3, 4, 5 and 6 in SEQ ID NOs: 4-7 have been deleted.
  • the parental polypeptide is the C domain of Protein A (SEQ ID NO: 5). The effects of these deletions on the native C domain are described in U.S. Pat. Nos. 9,018,305 and 8,329,860, which are hereby incorporated by reference in their entireties.
  • the mutation in SEQ ID Nos: 4-7 is selected from the group consisting of: N11K; N11E; N11Y; N11T; N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; N11E, Q32A; N11E, Q32E, Q40E; N11E, Q32E, K50R; Q9A, N11E, N43A; Q9A, N11E, N28A, N43A; Q9A, N11E, Q40V, A42K, N43E, L44I; Q9A, N11E, Q40V, A42K, N43A, L44I; N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11E, N28A, Q40V, A42K, N43A, N43A,
  • the mutation in SEQ ID Nos: 4-7 can also be selected from the group consisting of N11K; N11Y; N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; Q9A, N11E, N43A; Q9A, N11E, N28A, N43A; Q9A, N11E, Q40V, A42K, N43E, L44I; Q9A, N11E, Q40V, A42K, N43A, L44I; Q9A, N11E, N28A, Q40V, A42K, N43A, L44I; N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11K, H
  • the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
  • SEQ ID NO: 8 SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29.
  • It can also comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.
  • the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 54-70; comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 71-75; or it may comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 76-79. It may further comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 89-95.
  • the polypeptide may e.g. be defined by a sequence selected from the groups above or from subsets of these groups, but it may also comprise additional amino acid residues at the N- and/or C-terminal end, e.g. a leader sequence at the N-terminal end and/or a tail sequence at the C-terminal end.
  • the separation matrix comprises multimers of the immunoglobulin-binding alkali-stabilized Protein A domains.
  • Such multimers comprise, consist essentially of, or consist of a plurality of immunoglobulin-binding alkali-stabilized Protein A domains (polypeptide units) as defined by any embodiment disclosed above.
  • the use of multimers may increase the immunoglobulin binding capacity and multimers may also have a higher alkali stability than monomers.
  • the multimer can e.g. be a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a nonamer.
  • the multimer may be a homomultimer, where all the units in the multimer are identical or it can be a heteromultimer, where at least one unit differs from the others.
  • all the units in the multimer are alkali stable, such as by comprising the mutations/conservations disclosed above.
  • the polypeptides can be linked to each other directly by peptide bonds between the C-terminal and N-terminal ends of the polypeptides.
  • two or more units in the multimer can be linked by linkers comprising oligomeric or polymeric species, such as linkers comprising peptides with up to 25 or 30 amino acids, such as 3-25 or 3-20 amino acids.
  • the linkers may e.g.
  • APKVDAKFDKE SEQ ID NO: 96
  • APKVDNKFNKE SEQ ID NO: 97
  • APKADNKFNKE SEQ ID NO: 98
  • APKVFDKE SEQ ID NO: 99
  • APAKFDKE SEQ ID NO: 100
  • AKFDKE SEQ ID NO: 101
  • APKVDA SEQ ID NO: 102
  • VDAKFDKE SEQ ID NO: 103
  • APKKFDKE SEQ ID NO: 104
  • APKADNKFNKE SEQ ID NO: 98
  • APKVFDKE SEQ ID NO: 99
  • APAKFDKE SEQ ID NO: 100
  • AKFDKE SEQ ID NO: 101
  • APKVDA SEQ ID NO: 102
  • VDAKFDKE SEQ ID NO: 103
  • APKKFDKE SEQ ID NO: 104
  • APK and APKYEDGVDAKFDKE SEQ ID NO: 105.
  • the linkers do not consist of the peptides APKVDAKFDKE (SEQ ID NO: 96) or APKVDNKFNKE (SEQ ID NO: 97), or alternatively do not consist of the peptides APKVDAKFDKE (SEQ ID NO: 96), APKVDNKFNKE (SEQ ID NO: 97), APKFNKE (SEQ ID NO: 107), APKFDKE (SEQ ID NO: 108), APKVDKE (SEQ ID NO: 109) or APKADKE (SEQ ID NO: 110).
  • linker should preferably not destabilize the spatial conformation of the protein units. This can e.g. be achieved by avoiding the presence of proline in the linkers. Furthermore, said linker should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein units. For this purpose, it is advantageous if the linkers do not contain asparagine. It can additionally be advantageous if the linkers do not contain glutamine.
  • the multimer may further at the N-terminal end comprise a plurality of amino acid residues e.g. originating from the cloning process or constituting a residue from a cleaved off signaling sequence. The number of additional amino acid residues may e.g.
  • the multimer may comprise an AQ, AQGT (SEQ ID NO: 111), VDAKFDKE (SEQ ID NO: 103), AQVDAKFDKE (SEQ ID NO: 112) or AQGTVDAKFDKE (SEQ ID NO: 113) sequence at the N-terminal end.
  • the multimer may comprise, or consist essentially, of a sequence selected from the group consisting of: SEQ ID Nos: 80-87. These and additional sequences are listed below and named as Parent(Mutations)n, where n is the number of monomer units in a multimer.
  • the polypeptide and/or multimer further comprises at the C-terminal or N-terminal end one or more coupling elements, selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues.
  • the coupling element(s) may also be located within 1-5 amino acid residues, such as within 1-3 or 1-2 amino acid residues from the C-terminal or N-terminal end.
  • the coupling element may e.g. be a single cysteine at the C-terminal end.
  • the coupling element(s) may be directly linked to the C- or N-terminal end, or it/they may be linked via a stretch comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10 amino acids.
  • This stretch should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein.
  • An advantage of having a C-terminal cysteine is that endpoint coupling of the protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support. This provides excellent mobility of the coupled protein which is important for the binding capacity.
  • the alkali stability of the polypeptide or multimer can be assessed by coupling it to a surface plasmon resonance (SPR) chip, e.g. to Biacore CM5 sensor chips as described in the examples, using e.g. NHS— or maleimide coupling chemistries, and measuring the immunoglobulin-binding capacity of the chip, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/ ⁇ 2° C.
  • the incubation can e.g. be performed in 0.5 M NaOH for a number of 10 min cycles, such as 100, 200 or 300 cycles.
  • the IgG capacity of the matrix after 100 10 min incubation cycles in 0.5 M NaOH at 22+/ ⁇ 2° C. can be at least 55, such as at least 60, at least 80 or at least 90% of the IgG capacity before the incubation.
  • the remaining IgG capacity after 100 cycles for a particular mutant measured as above can be compared with the remaining IgG capacity for the parental polypeptide/multimer.
  • the remaining IgG capacity for the mutant may be at least 105%, such as at least 110%, at least 125%, at least 150% or at least 200% of the parental polypeptide/multimer.
  • the immunoglobulin-binding alkali-stabilized Protein A domains and/or multimers thereof may be encoded by a nucleic acid sequence, such as an RNA sequence or a DNA sequence encoding the polypeptide or multimer.
  • a vector such as a plasmid, which in addition to the coding sequence comprises the required signal sequences, may be used for expression of the polypeptide or multimer.
  • the vector may comprise nucleic acid encoding a multimer as described above, wherein the separate nucleic acids encoding each unit may have homologous or heterologous DNA sequences.
  • An expression system which comprises a nucleic acid or a vector as disclosed above, may be used for expression of the polypertide or multimer.
  • the expression system may e.g. be a gram-positive or gram-negative prokaryotic host cell system, e.g. E. coli or Bacillus sp. which has been modified to express the present polypeptide or multimer.
  • the expression system may be a eukaryotic host cell system, such as a yeast, e.g. Pichia pastoris or Saccharomyces cerevisiae , or mammalian cells, e.g. CHO cells.
  • the separation matrix comprises, consists essentially of, or consists of multimers of immunoglobulin-binding alkali-stabilized Protein A domains as described above, covalently coupled to a porous support.
  • the separation matrix may comprise at least 11, such as 11-21, 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
  • the alkali-stabilized Protein A domain multimers are highly selective for IgG and the separation matrix can suitably have a dissociation constant for human IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5. This can be determined according to the adsorption isotherm method described in N Pakiman et al: J Appl Sci 12, 1136-1141 (2012).
  • the separation matrix comprises at least 15, such as 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein the ligands comprise multimers of alkali-stabilized Protein A domains.
  • Fc-binding ligands covalently coupled to a porous support, wherein the ligands comprise multimers of alkali-stabilized Protein A domains.
  • the separation matrix comprises 5-25, such as 5-20 mg/ml, 5-15 mg/ml, 5-11 mg/ml or 6-11 mg/ml of the polypeptide or multimer coupled to the support.
  • the amount of coupled polypeptide/multimer can be controlled by the concentration of polypeptide/multimer used in the coupling process, by the activation and coupling conditions used and/or by the pore structure of the support used. As a general rule the absolute binding capacity of the matrix increases with the amount of coupled polypeptide/multimer, at least up to a point where the pores become significantly constricted by the coupled polypeptide/multimer.
  • Such a separation matrix is useful for separation of immunoglobulins or other Fc-containing proteins and, due to the improved alkali stability of the polypeptides/multimers, the matrix will withstand highly alkaline conditions during cleaning, which is essential for long-term repeated use in a bioprocess separation setting.
  • the alkali stability of the matrix can be assessed by measuring the immunoglobulin-binding capacity, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/ ⁇ 2° C. The incubation can e.g.
  • the IgG capacity of the matrix after 96-100 15 min incubation cycles or a total incubation time of 24 or 25 h in 0.5 M NaOH at 22+/ ⁇ 2° C. can be at least 80, such as at least 85, at least 90 or at least 95% of the IgG capacity before the incubation.
  • the capacity of the matrix after a total incubation time of 24 h in 1.0 M NaOH at 22+/ ⁇ 2° C. can be at least 70, such as at least 80 or at least 90% of the IgG capacity before the incubation.
  • the 10% breakthrough dynamic binding capacity (Qb10%) for IgG at 2.4 min or 6 min residence time may e.g. be reduced by less than 20% after incubation 31 h in 1.0 M aqueous NaOH at 22+/ ⁇ 2 C.
  • the expressed polypeptide or multimer should be purified to an appropriate extent before being immobilized to a support.
  • purification methods are well known in the field, and the immobilization of protein-based ligands to supports is easily carried out using standard methods. Suitable methods and supports will be discussed below in more detail.
  • the porous support of the separation matrix may be of any suitable well-known kind.
  • a conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy (—COOH), carboxamido (—CONH 2 , possibly in N— substituted forms), amino (—NH 2 , possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces.
  • the porosity of the support can be expressed as a Kav or Kd value (the fraction of the pore volume available to a probe molecule of a particular size) measured by inverse size exclusion chromatography, e.g.
  • Kav is determined as the ratio (V e ⁇ V 0 )/(V t ⁇ V 0 ), where V e is the elution volume of a probe molecule (e.g. Dextran 110 kD), V o is the void volume of the column (e.g. the elution volume of a high Mw void marker, such as raw dextran) and V t is the total volume of the column.
  • Kd can be determined as (V e —V 0 )/V i , where V i is the elution volume of a salt (e.g.
  • both Kd and Kav values always lie within the range 0-1.
  • the Kav value can advantageously be 0.6-0.95, e.g. 0.7-0.90 or 0.6-0.8, as measured with dextran of Mw 110 kDa as a probe molecule.
  • the Kd value as measured with dextran of Mw 110 kDa can suitably be 0.68-0.90, such as 0.68-0.85 or 0.70-0.85.
  • the support has a large fraction of pores able to accommodate both the polypeptides/multimers of the invention and immunoglobulins binding to the polypeptides/multimers and to provide mass transport of the immunoglobulins to and from the binding sites.
  • the polypeptides or multimers may be attached to the porous support via conventional coupling techniques utilising e.g. thiol, amino and/or carboxy groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents. Between the support and the polypeptide/multimer, a molecule known as a spacer can be introduced, which improves the availability of the polypeptide/multimer and facilitates the chemical coupling of the polypeptide/multimer to the support. Depending on the nature of the polypeptide/multimer and the coupling conditions, the coupling may be a multipoint coupling (e.g. via a plurality of lysines) or a single point coupling (e.g. via a single cysteine).
  • thiol amino and/or carboxy groups present in the ligand.
  • the polypeptides or multimers are coupled to the support via thioether bonds.
  • Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment.
  • Thioether bonds are flexible and stable and generally suited for use in affinity chromatography.
  • the thioether bond is via a terminal or near-terminal cysteine residue on the polypeptide or multimer, the mobility of the coupled polypeptide/multimer is enhanced which provides improved binding capacity and binding kinetics.
  • the polypeptide/multimer is coupled via a C-terminal cysteine provided on the protein as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling.
  • the support comprises a polyhydroxy polymer, such as a polysaccharide.
  • polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc.
  • Polysaccharides are inherently hydrophilic with low degrees of nonspecific interactions, they provide a high content of reactive (activatable) hydroxyl groups and they are generally stable towards alkaline cleaning solutions used in bioprocessing.
  • the support comprises agar or agarose.
  • the supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjertén: Biochim Biophys Acta 79(2), 393-398 (1964).
  • the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSETM FF (GE Healthcare).
  • the support has been adapted to increase its rigidity using the methods described in U.S. Pat. No. 6,602,990 or U.S. Pat. No. 7,396,467, which are hereby incorporated by reference in their entireties, and hence renders the matrix more suitable for high flow rates.
  • the support such as a polymer, polysaccharide or agarose support
  • Crosslinker reagents producing such crosslinks can be e.g. epihalohydrins like epichlorohydrin, diepoxides like butanediol diglycidyl ether, allylating reagents like allyl halides or allyl glycidyl ether.
  • Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether crosslinks are alkali stable and do not cause significant nonspecific adsorption.
  • the porous support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc.
  • hydrophobic polymers such as matrices based on divinyl and monovinyl-substituted benzenes
  • the surface of the matrix is often hydrophilised to expose hydrophilic groups as defined above to a surrounding aqueous liquid.
  • Such polymers are easily produced according to standard methods, see e.g. “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)).
  • a commercially available product such as SOURCETM (GE Healthcare) is used.
  • the porous support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide etc.
  • the solid support is in another form such as a surface, a chip, capillaries, or a filter (e.g. a membrane or a depth filter matrix).
  • a filter e.g. a membrane or a depth filter matrix
  • the matrix in one embodiment is in the form of a porous monolith.
  • the matrix in beaded or particle form that can be porous or non-porous.
  • Matrices in beaded or particle form can be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. In case of monoliths, packed bed and expanded beds, the separation procedure commonly follows conventional chromatography with a concentration gradient. In case of pure suspension, batch-wise mode will be used.
  • the separation matrix as disclosed above has excellent alkali stability and may be cleaned and/or sanitized using an alkaline cleaning liquid.
  • the method of cleaning and/or sanitizing the separation matrix comprises the following steps:
  • Cleaning and/or sanitization may be performed on separation matrix not previously used in order to sanitize the matrix prior to use, for example after packing a column comprising the separation matrix for the first time. Cleaning and/or sanitization may also be performed after using the separation matrix to purify an immunoglobulin, i.e. step a) of the method above.
  • the steps of using the separation matrix to purify an immunoglobulin and cleaning and/or sanitizing the separation matrix afterwards may be repeated in order to maximize use of the separation matrix.
  • the steps may be repeated at least 10 times, such as at least 50 times or 50-200 times.
  • the step c) of cleaning/sanitizing may be performed after each step a) of purifying an immunoglobulin, or may be performed less frequently, such as after every second purification, or after every tenth purification.
  • the step c) of cleaning and/or sanitizing the separation matrix need not be performed using the same conditions each time.
  • the separation matrix may be cleaned after each purification step using a first set of conditions, and after every n th purification be sanitized using more stringent conditions.
  • aqueous alkali metal hydroxide solutions are effective cleaning agents and bactericidal in themselves, by cleaning and sanitizing using a cleaning liquid comprising such aqueous alkali metal hydroxide solutions there is a lesser need for using alcohols as cleaning and sanitizing agents.
  • Alkali metal hydroxides are relatively cheap compared to alcohols and are subject to less regulatory burden. Moreover, they are non-flammable and easier to dispose of.
  • the separation matrix is a separation matrix as disclosed above, comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NOs: 51 or 52 are asparagines, and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arg
  • the immunoglobulin-binding alkali-stabilized Protein A domains may comprise further mutations.
  • the glutamine residue at position 1 of SEQ ID Nos: 51 or 52 may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position 35 of SEQ ID Nos: 51 or 52 may be mutated to an alanine.
  • the separation matrix can tolerate extensive cleaning and sanitization procedures, even using concentrated alkali metal hydroxide solutions, without excessive loss of binding capacity.
  • the separation matrix may retain at least 80% of its original dynamic binding capacity after cleaning and/or sanitization.
  • the cleaning liquid comprises at least 50% by volume of an aqueous alkali metal hydroxide solution.
  • the aqueous alkali metal hydroxide solution may comprise a single alkali metal hydroxide or a mixture of alkali metal hydroxides, such as sodium hydroxide, potassium hydroxide, or a mixture of sodium hydroxide and potassium hydroxide.
  • the aqueous alkali metal hydroxide solution may have a molarity of from 500 mM to 5 M, such as from 1 M to 2 M, expressed as the total combined concentration of alkali metal hydroxides if a mixture of alkali metal hydroxides is used.
  • the cleaning liquid may essentially consist of, or consist of, the aqueous alkali metal hydroxide solution.
  • the cleaning liquid may in some embodiments also comprise further components.
  • Such further components may include alcohols, such as a C 2 -C 7 alcohol, such as ethanol, isopropanol or benzoic alcohol.
  • Such further components may include salts, such as sodium chloride. The use of alcohols and/or salts in the cleaning liquid may increase the efficacy of the cleaning liquid in inhibiting or inactivating certain microorganisms, such as spore-forming bacteria.
  • Non-limiting examples of cleaning liquids include:
  • alkali metal hydroxide solution such as 0.5 M-5 M solution
  • a commonly applied measure of sanitization efficacy is a 6-log 10 reduction in the microorganism or contaminant being measured. Sanitizing with concentrated alkali metal hydroxide solution is capable of providing a 6-log 10 reduction in most microorganisms and endotoxins.
  • the cleaning liquid should be passed through the separation matrix with a suitable flow rate, in order to flush contaminants from the matrix.
  • the cleaning liquid is initially passed through the separation matrix with a suitable flow rate until the separation matrix is fully permeated with the cleaning liquid.
  • the cleaning liquid is preferably passed through the separation matrix with a suitable flow rate for the entire duration of the sanitization step.
  • the flow of cleaning liquid through the separation matrix may be stopped once the separation matrix is fully permeated with the cleaning liquid. This reduces the volume of cleaning fluid required to perform a sanitization step, but provides a poorer cleaning of the separation matrix as compared to a procedure where a constant flow of cleaning fluid is applied.
  • the separation matrix may be used as a purification platform for the purification of a variety of immunoglobulins, with a lower risk of host cell protein contamination or carryover.
  • the separation matrix is first cleaned and sanitized thoroughly using a cleaning liquid comprising aqueous alkali metal hydroxide solution prior to use in purification of the second immunoglobulin product.
  • Site-directed mutagenesis was performed by a two-step PCR using oligonucleotides coding for the mutations.
  • a plasmid containing a single domain of either Z, B or C was used as template.
  • the PCR fragments were ligated into an E. coli expression vector.
  • DNA sequencing was used to verify the correct sequence of inserted fragments.
  • Acc I site located in the starting codons (GTA GAC) of the B, C or Z domain was used, corresponding to amino acids VD.
  • the vector for the monomeric domain was digested with Acc I and phosphatase treated.
  • Acc I sticky-ends primers were designed, specific for each variant, and two overlapping PCR products were generated from each template.
  • the PCR products were purified and the concentration was estimated by comparing the PCR products on a 2% agarose gel. Equal amounts of the pair wise PCR products were hybridized (90° C. ⁇ 25° C. in 45 min) in ligation buffer.
  • the resulting product consists approximately to 1 ⁇ 4 of fragments likely to be ligated into an Acc I site (correct PCR fragments and/or the digested vector). After ligation and transformation colonies were PCR screened to identify constructs containing the desired mutant. Positive clones were verified by DNA sequencing.
  • the constructs were expressed in the bacterial periplasm by fermentation of E. coli K12 in standard media. After fermentation the cells were heat-treated to release the periplasm content into the media. The constructs released into the medium were recovered by microfiltration with a membrane having a 0.2 ⁇ m pore size.
  • the permeate was loaded onto a chromatography medium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare).
  • the loaded product was washed with phosphate buffered saline and eluted by lowering the pH.
  • the elution pool was adjusted to a neutral pH (pH 8) and reduced by addition of dithiothreitol.
  • the sample was then loaded onto an anion exchanger. After a wash step the construct was eluted in a NaCl gradient to separate it from any contaminants.
  • the elution pool was concentrated by ultrafiltration to 40-50 mg/ml. It should be noted that the successful affinity purification of a construct on an immobilized IgG medium indicates that the construct in question has a high affinity to IgG.
  • the purified ligands were analyzed with RPC LC-MS to determine the purity and to ascertain that the molecular weight corresponded to the expected (based on the amino acid sequence).
  • the purified monomeric ligands listed in Table 1, further comprising for SEQ ID Nos: 8-16, 23-28 and 36-48 an AQGT leader sequence at the N-terminus and a cysteine at the C terminus, were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore surface plasmon resonance (SPR) instrument (GE Healthcare, Sweden).
  • SPR Biacore surface plasmon resonance
  • the ligands B(Q9A, N11E, Q40V, A42K, N43A, L44I)1 and C(Q9A, N11E, E43A)1 have an improved stability compared to the parental B and C domains, used as references.
  • the Biacore experiment can also be used to determine the binding and dissociation rates between the ligand and IgG. This was used with the set-up as described above and with an IgG1 monoclonal antibody as probe molecule.
  • the on-rate (10 5 M ⁇ 1 s ⁇ 1 ) was 3.1 and the off-rate (10 5 s ⁇ 1 ) was 22.1, giving an affinity (off-rate/on-rate) of 713 pM.
  • Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 SEQ ID NO: 11
  • the on-rate was 4.1 and the off-rate 43.7, with affinity 1070 pM.
  • the IgG affinity was thus somewhat higher for the mutated variant.
  • the purified dimeric, tetrameric and hexameric ligands listed in Table 2 were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore instrument (GE Healthcare, Sweden).
  • GE Healthcare GE Healthcare, Sweden
  • To follow the IgG binding capacity of the immobilized surface 1 mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal strength (proportional to the amount of binding) was noted. The surface was then cleaned-in-place (CIP), i.e.
  • CIP cleaned-in-place
  • the hexameric ligand Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)6 also has improved alkali stability compared to the parental structure Zvar6, used as a reference.
  • Example 2 was repeated with 100 CIP cycles of three ligands using 1 M NaOH instead of 500 mM as in Example 2. The results are shown in Table 3 and show that all three ligands have an improved alkali stability also in 1M NaOH, compared to the parental structure Zvar4 which was used as a reference.
  • the base matrix used was rigid cross-linked agarose beads of 85 micrometers (volume-weighted, d50V) median diameter, prepared according to the methods of U.S. Pat. No. 6,602,990, hereby incorporated by reference in its entirety, and with a pore size corresponding to an inverse gel filtration chromatography Kav value of 0.70 for dextran of Mw 110 kDa, according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.
  • ligand solution 50 mg/mL in a 50 ml Falcon tube
  • 169 mg NaHCO 3 21 mg Na 2 CO 3 , 175 mg NaCl and 7 mg EDTA
  • the Falcon tube was placed on a roller table for 5-10 min, and then 77 mg of DTE was added. Reduction proceeded for >45 min.
  • the ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.
  • the activated gel was washed with 3-5 GV ⁇ 0.1 M phosphate/1 mM EDTA pH 8.6 ⁇ and the ligand was then coupled according to the method described in U.S. Pat. No. 6,399,750, hereby incorporated by reference in its entirety. All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels could be controlled by varying the amount and concentration of the ligand solution.
  • the gels were washed 3 ⁇ GV with distilled water.
  • the gels+1 GV ⁇ 0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6 ⁇ was mixed and the tubes were left in a shaking table at room temperature overnight.
  • the gels were then washed alternately with 3 ⁇ GV ⁇ 0.1 M TRIS/0.15 M NaCl pH 8.6 ⁇ and 0.5 M HAc and then 8-10 ⁇ GV with distilled water.
  • Gel samples were sent to an external laboratory for amino acid analysis and the ligand content (mg/ml gel) was calculated from the total amino acid content.
  • the dynamic binding capacity (DBC) at 10% breakthrough was calculated.
  • the dynamic binding capacity (DBC) was calculated for 10 and 80% breakthrough.
  • the 10% breakthrough DBC (Qb10) was determined both before and after repeated exposures to alkaline cleaning solutions.
  • Each cycle included a CIP step with 0.5 M NaOH pumped through the column at a rate of 0.5/min for 20 min, after which the column was left standing for 4 h. The exposure took place at room temperature (22+/ ⁇ 2° C.). After this incubation, the column was washed with equilibration buffer for 20 min at a flow rate of 0.5 ml/min.
  • Table 4 shows the remaining capacity after six 4 h cycles (i.e. 24 h cumulative exposure time to 0.5 M NaOH), both in absolute numbers and relative to the initial capacity.
  • Example 4 was repeated with the tetrameric ligands shown in Table 5, but with 1.0 M NaOH used in the CIP steps instead of 0.5 M. The results are shown in Table 5 and in FIG. 4 .
  • the base matrices used were a set of rigid cross-linked agarose bead samples of 59-93 micrometers (volume-weighted, d50V) median diameter (determined on a Malvern Mastersizer 2000 laser diffraction instrument), prepared according to the methods of U.S. Pat. No. 6,602,990 and with a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.62-0.82 for dextran of Mw 110 kDa, according to the methods described above, using HR10/30 columns (GE Healthcare) packed with the prototypes in 0.2 M NaCl and with a range of dextran fractions as probe molecules (flow rate 0.2 ml/min).
  • the dry weight of the bead samples ranged from 53 to 86 mg/ml, as determined by drying 1.0 ml drained filter cake samples at 105° C. overnight and weighing.
  • ligand solution 50 mg/mL in a 50 ml Falcon tube
  • 139 mg NaHCO 3 17.4 mg Na 2 CO 3 , 143.8 mg NaCl and 141 mg EDTA
  • the Falcon tube was placed on a roller table for 5-10 min, and then 63 mg of DTE was added. Reduction proceeded for >45 min.
  • the ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.
  • the activated gel was washed with 3-5 GV ⁇ 0.1 M phosphate/1 mM EDTA pH 8.6 ⁇ and the ligand was then coupled according to the method described in U.S. Pat. No. 6,399,750 5.2.2, although with considerably higher ligand amounts (see below). All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min.
  • the ligand content of the gels was controlled by varying the amount and concentration of the ligand solution, adding 5-20 mg ligand per ml gel.
  • the ligand was either a tetramer (SEQ ID NO: 20) or a hexamer (SEQ ID NO: 33) of an alkali-stabilized mutant.
  • the gels were washed 3 ⁇ GV with distilled water.
  • the gels+1 GV ⁇ 0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6 ⁇ was mixed and the tubes were left in a shaking table at room temperature overnight.
  • the gels were then washed alternately with 3 ⁇ GV ⁇ 0.1 M TRIS/0.15 M NaCl pH 8.6 ⁇ and 0.5 M HAc and then 8-10 ⁇ GV with distilled water.
  • Gel samples were sent to an external laboratory for amino acid analysis and the ligand content (mg/ml gel) was calculated from the total amino acid content.
  • the Qb10% dynamic capacity for polyclonal human IgG at 2.4 and 6 min residence time was determined as outlined in Example 4.
  • a series of prototypes, prepared as above, with different ligand content (tetramer, SEQ ID NO: 20) were incubated in 1 M NaOH for 4, 8 and 31 hours at 22+/ ⁇ 2° C. and the dynamic IgG capacity (Qb10%, 6 min residence time) was measured before and after incubation.
  • the prototypes are shown in Table 8 and the results in FIGS. 5 and 6 . It can be seen that the stability towards this harsh alkali treatment increases with increasing ligand content.
  • the ligand content of prototype A was 14.3 mg/ml and of prototype B 18.9 mg/ml.
  • the commercial product MabSelect SuRe® LX (GE Healthcare Life Sciences) was used.
  • the resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of 10 cm, giving bed volumes of 2 ml and the columns were shown to have peak asymmetry within the 0.8-1.5 interval.
  • the mAb peak was collected using a UV watch function and the concentration of the mAb was determined by UV measurement at 280 nm (extinction coefficient 1.5). All absorbance detections were performed using a spectrophotometer, including the measurements for the yield calculations.
  • Samples for HCP (host cell protein) analyses were prepared by adding 10% Preservation buffer (0.2 M NaH 2 PO 4 *H 2 O (5.3%), 0.2 M Na 2 HPO 4 *12 H 2 O (94.7%), 0.5% Tween 20, 1% BSA pH 8) to the samples directly after each run made (e.g. 50 ⁇ l preservation buffer to 450 ⁇ l sample).
  • the HCP content was measured using commercial anti-CHO antibodies (Cygnus Technologies) and a Gyrolab (Gyros AB, Sweden) work station.
  • the resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of 10 cm, giving bed volumes of 2 ml and the columns were shown to have peak asymmetry within the 0.8-1.5 interval.
  • the chromatograms for polyclonal IgG on the reference and the prototype are shown in FIG. 7 and the IgG class analyses are shown in FIG. 8 .
  • the data show that all three classes bind to both resins in a similar way and that the first peak predominantly contains IgG2, while IgG1 and IgG4 elute mainly in the second peak.
  • the anti-IgG3 antibodies cross-reacted with IgG4, so no reliable results for IgG3 were obtained.
  • IgG3 is generally known to show no or only weak binding to Protein A.
  • the dynamic binding capacity for polyclonal IgG was then measured and the process was repeated with another injection of 1 M NaOH.
  • the dynamic capacity was measured after each 240 min incubation cycle with 1 M NaOH.
  • the columns were equilibrated with PBS buffer before the 2 mg/ml sample was loaded (residence time 6 min) until a UV signal of 85% of maximum absorbance was reached. Then the column was washed with PBS buffer, eluted with 500 mM acetic acid pH 3.0 and re-equilibrated.
  • the dynamic binding capacity at 10% and 80% breakthrough was calculated as described above. The results are shown in FIG. 9 and they show that the prototype was significantly more stable than the commercial product.
  • a separation matrix according to the invention was tested for tolerance to repeated CIP cycles using 2M NaOH.
  • the inventive example (Inv. Ex.) was compared to the commercial product MabSelect SuRe (MSS) as a reference.
  • MSS MabSelect SuRe
  • the study was performed for 50 cycles using an ⁇ KTA 4-column periodic counter current (PCC) chromatography setup from GE Healthcare.
  • Each column was packed using a solution of 20% ethanol+0.2 M NaCl, with a flow of 3.5 ml/min for 10 min.
  • the packing tube and top filter were removed and the adaptor was placed on the top of column. Following a further packing flow of 3.5 ml/min for 10 min, the adaptor was adjusted against the bed surface.
  • the packed column volume V c for the inventive example was 1.04 ml, as compared to 1.02 ml for MabSelect SuRe.
  • the ligand stability towards 1 M and 2 M NaOH was studied for a separation matrix according to the invention, using confocal microscopy.
  • the inventive example (Inv. Ex.) was compared to the commercial product MabSelect SuRe LX (MSS LX) as a reference. The study was performed as follows:
  • Separation matrix gels were washed from their storage solution to water through centrifugation, and a 1:1 gel slurry was obtained.
  • 2 ml of the 1:1 slurry in water was added to a 50 mL Falcon tube, centrifuged and decanted.
  • To each tube was added 19 ml of 1 M or 2 M NaOH (2 separation matrices*2 [NaOH] concentrations ⁇ 4 tubes in total).
  • the gels were incubated at room temperature with shaking on Heidolph shaker (1300 rpm).
  • the gels were resuspended with 75 ⁇ l PBS buffer to obtain approximately 1:1 PBS gel slurrys.
  • a Leica SP8 Confocal Microscope was used for the study. Microscope settings were determined using the inventive example and checked using MSS LX in order to make sure saturation was not obtained with either of the gels.
  • FIGS. 11 a MabSelect SuRe LX and 11b The determined relative fluorescence Q rel of the separation matrices as a function of time after incubation in 1M NaOH are shown as FIGS. 11 a MabSelect SuRe LX) and 11b (Inventive example). It can be seen that MSS LX ( FIG. 11 a ) lost approximately 50% of its original binding capacity and reaches saturation faster following incubation for 16 hours in 1 M NaOH, as compared to the reference (no alkali treatment, i.e. 0 hours). Following incubation for 32 hours in 1M NaOH it has lost approximately all Ig-binding capacity. In contrast, the separation matrix of the inventive example ( FIG. 11 b ) is stable to incubation in 1 M NaOH and maintains its original binding capacity after 32 hours incubation in 1 M NaOH, within experimental variation.
  • FIGS. 12 a MabSelect SuRe LX and 12b The determined relative fluorescence Q rel of the separation matrices as a function of time after incubation in 2M NaOH are shown as FIGS. 12 a MabSelect SuRe LX) and 12b (Inventive example). It can be seen that the rate of capacity loss for MSS LX ( FIG. 12 a ) is accelerated with incubation in 2M NaOH. After incubation for 8 hours approximately 50% of Ig-binding capacity is lost, and following incubation for 16 hours in 2M NaOH, more or less all Ig-binding capacity is lost. In contrast, the inventive example ( FIG. 12 b ) is relatively stable to alkali and maintains, at least within experimental variation, its original binding capacity after 16 h or less of incubation in 2 M NaOH.
  • Bacillus subtilis spores were investigated using various cleaning liquids according to the invention.
  • Spore-forming Bacillus subtilis (ATCC No. 6633) is known to be among the more resistant microorganisms to NaOH sanitization.
  • Colonies of B. subtilis were incubated with various cleaning liquids (1M NaOH, 1M NaOH with 2% BnOH, and 1M NaOH with 40% IPA), and the number of colony forming units (CFU/ml) was determined at various predetermined incubation times.
  • FIG. 13 shows the results of the study as CFU/ml as a function of incubation time and cleaning liquid. It can be seen that using 1M NaOH by itself is insufficient to fully inactivate B.
  • subtilis even after 24 hours of sanitization. Adding 2% benzyl alcohol to the cleaning liquid does not provide any significant improvement. However, the use of 1M NaOH with 40% isopropyl alcohol as cleaning liquid allows the reduction of B. subtilis to below detectable levels after 4 hours of treatment.

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Abstract

The present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of:
  • a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix;
  • b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and
  • c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time.
The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. application Ser. No. 16/095,753, filed Oct. 23, 2018, which claims the priority benefit of PCT/EP2017/061159, filed on May 10, 2017, which claims priority benefit of Great Britain Application Nos. 1608229.9 and 1608232.3, both of which were filed May 11, 2016, and continuation of U.S. application Ser. No. 15/282,367, filed Sep. 30, 2016, and continuation-in-part of U.S. application Ser. No. 15/348,699, filed Nov. 10, 2016, respectively. The entire contents of which are hereby incorporated by reference herein.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 17, 2023, is named 34428_0769_CON_SL.XML and is 112,000 bytes in size.
    • TECHNICAL FIELD
  • The present invention relates to a method of cleaning and/or sanitizing separation matrices that comprise multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The present invention further relates to a method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, as well as to use of a cleaning liquid comprising at least 70% by volume of an aqueous alkali metal hydroxide solution for the sanitization of a separation matrix.
    • BACKGROUND ART
  • Immunoglobulins represent the most prevalent biopharmaceutical products in either manufacture or development worldwide. The high commercial demand for and hence value of this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective immunoglobulin manufacturing processes whilst controlling the associated costs.
  • Affinity chromatography is used in most cases, as one of the key steps in the purification of these immunoglobulin molecules, such as monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs). A particularly interesting class of affinity reagents is proteins capable of specific binding to invariable parts of an immunoglobulin molecule, such interaction being independent on the antigen-binding specificity of the antibody. Such reagents can be widely used for affinity chromatography recovery of immunoglobulins from different samples such as but not limited to serum or plasma preparations or cell culture derived feed stocks. An example of such a protein is staphylococcal protein A, containing domains capable of binding to the Fc and Fab portions of IgG immunoglobulins from different species. These domains are commonly denoted as the E-, D-, A-, B- and C-domains.
  • Staphylococcal protein A (SpA) based reagents have due to their high affinity and selectivity found a widespread use in the field of biotechnology, e.g. in affinity chromatography for capture and purification of antibodies as well as for detection or quantification. At present, SpA-based affinity medium probably is the most widely used affinity medium for isolation of monoclonal antibodies and their fragments from different samples including industrial cell culture supernatants. Accordingly, various matrices comprising protein A-ligands are commercially available, for example, in the form of native protein A (e.g. Protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden) and also comprised of recombinant protein A (e.g. rProtein A SEPHAROSE™, GE Healthcare). More specifically, the genetic manipulation performed in the commercial recombinant protein A product is aimed at facilitating the attachment thereof to a support and at increasing the productivity of the ligand.
  • These applications, like other affinity chromatography applications, require comprehensive attention to definite removal of contaminants. Such contaminants can for example be non-eluted molecules adsorbed to the stationary phase or matrix in a chromatographic procedure, such as non-desired biomolecules or microorganisms, including for example proteins, carbohydrates, lipids, endotoxins, bacteria and viruses. The removal of such contaminants from the matrix is usually performed after a first elution of the desired product in order to regenerate the matrix before subsequent use. Cleaning the matrix of contaminants usually involves a procedure known as cleaning-in-place (CIP), wherein agents capable of eluting contaminants from the stationary phase are used. Inactivation of microorganisms usually involves a procedure known as sanitization-in-place (SIP) wherein agents capable of inactivating microorganisms are used. Some agents are capable of both cleaning and sanitizing the separation matrix, depending on the concentration of the agent and the contact time with the separation matrix. One such class of agents often used is alkaline solutions that are passed over said stationary phase. At present the most extensively used cleaning and sanitizing agent for most separation matrices is NaOH, and the concentration thereof can range from 0.1 up to e.g. 1 M, depending on the degree and nature of contamination. However, this strategy is associated with exposing the matrix to solutions with pH-values above 13. For many affinity chromatography matrices containing proteinaceous affinity ligands such alkaline environment is a very harsh condition and consequently results in decreased capacities owing to instability of the ligand to the high pH involved.
  • An extensive research has therefore been focused on the development of engineered protein ligands that exhibit an improved capacity to withstand alkaline pH-values. For example, Gülich et al. (Susanne Gülich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober, Journal of Biotechnology 80 (2000), 169-178) suggested protein engineering to improve the stability properties of a Streptococcal albumin-binding domain (ABD) in alkaline environments. Gülich et al. created a mutant of ABD, wherein all the four asparagine residues have been replaced by leucine (one residue), aspartate (two residues) and lysine (one residue). Further, Gülich et al. report that their mutant exhibits a target protein binding behavior similar to that of the native protein, and that affinity columns containing the engineered ligand show higher binding capacities after repeated exposure to alkaline conditions than columns prepared using the parental non-engineered ligand. Thus, it is concluded therein that all four asparagine residues can be replaced without any significant effect on structure and function.
  • Recent work shows that changes can also be made to protein A (SpA) to effect similar properties. US patent application publication US 2005/0143566, which is hereby incorporated by reference in its entirety, discloses that when at least one asparagine residue is mutated to an amino acid other than glutamine or aspartic acid, the mutation confers an increased chemical stability at pH-values of up to about 13-14 compared to the parental SpA, such as the B-domain of SpA, or Protein Z, a synthetic construct derived from the B-domain of SpA (U.S. Pat. No. 5,143,844, incorporated by reference in its entirety). The authors show that when these mutated proteins are used as affinity ligands, the separation media as expected can better withstand cleaning procedures using alkaline agents. Further mutations of protein A domains with the purpose of increasing the alkali stability have also been published in U.S. Pat. No. 8,329,860, JP 2006304633A, U.S. Pat. No. 8,674,073, US 2010/0221844, US 2012/0208234, U.S. Pat. No. 9,051,375, US 2014/0031522, US 2013/0274451 and WO 2014/146350, all of which are hereby incorporated by reference in their entireties. However, the currently available mutants are still sensitive to alkaline pH and the NaOH concentration during cleaning is usually limited to 0.1 M, which means that complete cleaning is difficult to achieve. Moreover, such a concentration of NaOH is insufficient in itself for effective sanitization of the affinity separation matrix, and therefore sanitization is often performed using solutions comprising alcohols such as ethanol or isopropanol. Higher NaOH concentrations, which would improve the cleaning and sanitization, lead to unacceptable capacity losses of the affinity separation matrix.
  • There is thus still a need in this field to obtain a separation matrix containing protein ligands having a further improved stability towards alkaline cleaning and sanitization procedures. There is also a need for such separation matrices with an improved binding capacity to allow for economically efficient purification of therapeutic antibodies.
    • SUMMARY OF THE INVENTION
  • The inventors of the present invention have recognised that alcohol solutions are suboptimal for the sanitization of affinity separation matrices. Alcohols are flammable, subject to regulation, and difficult to dispose of. Moreover, the inventors of the present invention have recognised that low concentration NaOH, such as 0.1 M NaOH, provides insufficient cleaning in order to allow the separation matrix to be used for the purification of a variety of different immunoglobulin products. Instead, the separation matrix is typically only used to purify separate batches of a single product in order to prevent carryover of impurities from one product to another. The inventors have recognised that concentrated aqueous alkali metal hydroxide solutions have a number of advantages as cleaning and storage solutions. Concentrated alkali metal hydroxide solutions are bacteriostatic and can inactive most viruses, bacteria, yeasts, fungi and endotoxins. They are capable of effectively stripping the separation matrix of impurities to such a degree that the same separation matrix may be used to purify a variety of immunoglobulins. This facilitates the use of the separation matrix as a separation platform. Moreover, concentrated alkali metal hydroxide solutions are relatively cheap, easily disposed of, and removal from the separation matrix is simple to detect using pH and/or conductivity measurements.
  • It is therefore an object of the present invention to provide a method for cleaning and sanitizing an affinity separation matrix for the purification of immunoglobulins which permits the use of concentrated alkali metal hydroxide solution.
  • This object is achieved by the method according to the appended claims of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine. The method comprises the steps of:
      • a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix;
      • b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and
      • c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time.
  • By using a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains as defined above, a highly alkali-stable separation matrix having a high dynamic binding capacity is obtained. The inventors of the present invention have observed that such separation matrices are relatively stable upon impregnation with concentrated aqueous alkali metal hydroxide solution, and substantially retain dynamic binding capacity after contact with such concentrated alkali metal hydroxide solution. This means that such a separation matrices are suitable for cleaning and/or sanitization with concentrated alkali metal hydroxide solutions.
  • Further mutations to the immunoglobulin-binding alkali-stabilized Protein A domains may provide further enhancement of properties such as enhanced alkali stability. For example, the glutamine residue at position 1 of SEQ ID Nos: 51 or 52 may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position 35 of SEQ ID Nos: 51 or 52 may be mutated to an alanine.
  • The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or nonamers. By using an appropriate multimer, the immunoglobulin binding capacity and alkali stability of the separation matrix may be increased.
  • The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may each comprise a C-terminal cysteine residue for covalent coupling to the porous support. The multimers of immunoglobulin-binding alkali-stabilized Protein A domains may be coupled to the porous support via thioether links. This provides a robust, alkali-stable and well-proven method of attaching the ligands to the solid support.
  • The separation matrix may comprise at least 11 mg/ml, such as at least 15 mg/ml, of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support. This ensures a separation matrix with a good binding capacity.
  • The porous support may comprise cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml. The porous support may for example be highly cross-linked agarose beads.
  • The aqueous alkali metal hydroxide solution used in the cleaning liquid may be sodium hydroxide solution, potassium hydroxide solution or a mixture thereof, preferably sodium hydroxide solution. Sodium hydroxide solution is relatively cheap, readily available and widely accepted for use as a cleaning and sanitization solution. The aqueous alkali metal hydroxide solution may have a molarity of from 500 mM to 5 M, such as from 1 M to 2 M. This ensures a solution with good bactericidal properties.
  • The cleaning liquid may in some instances further comprise a C2-C7 alcohol, such as ethanol, isopropanol or benzyl alcohol. A cleaning liquid combining an alcohol and an alkali metal hydroxide may be more effective in inactivating certain microorganisms, such as some spore-forming bacteria.
  • The cleaning liquid may comprise at least 70% by volume aqueous alkali metal hydroxide solution, such as at least 90% by volume aqueous alkali metal hydroxide solution, preferably at least 99% by volume aqueous alkali metal hydroxide solution.
  • In some instances the cleaning liquid may consist of, or consist essentially of, aqueous alkali metal hydroxide solution.
  • The predetermined contact time may be a time sufficient to provide a 6-log10 reduction in endotoxin concentration and/or microorganism concentration in the separation matrix. The predetermined contact time may be from 10 minutes to 50 hours, such as from 30 minutes to 24 hours, or such as from 1 hour to 12 hours. Such contact times are typically sufficient to inactivate a wide range of microorganisms.
  • Steps a)-c) of the method above may be repeated at least 10 times, such as at least 50 times or 50-200 times. Thus, the separation matrix may be reused many times while still retaining sufficient capacity to achieve an acceptable purification.
  • The object of the invention is further achieved by a method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, as defined by the appended claims. The method comprises the steps of purifying a first immunoglobulin and cleaning and sanitizing the separation matrix by performing the steps a)-c) of the method of cleaning and/or sanitizing a separation matrix as described above. The method further comprises the step of d) purifying a mixture comprising a second immunoglobulin using the separation matrix, wherein the second immunoglobulin is different from the first immunoglobulin.
  • The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.
  • Further objects, advantages and novel features of the present invention will become apparent to one skilled in the art from the following detailed description.
    • Definitions
  • The terms “antibody” and “immunoglobulin” are used interchangeably herein, and are understood to include also fragments of antibodies, fusion proteins comprising antibodies or antibody fragments and conjugates comprising antibodies or antibody fragments.
  • The terms an “Fc-binding polypeptide”, “Fc-binding domain” and “Fc-binding protein” mean a polypeptide, domain or protein respectively, capable of binding to the crystallisable part (Fc) of an antibody and includes e.g. Protein A and Protein G, or any fragment or fusion protein thereof that has maintained said binding property.
  • The term “linker” herein means an element linking two polypeptide units, monomers or domains to each other in a multimer.
  • The term “spacer” herein means an element connecting a polypeptide or a polypeptide multimer to a support.
  • The term “% identity” with respect to comparisons of amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST™) described in Altshul et al. (1990) J. Mol. Biol., 215: 403-410. A web-based software for this is freely available from the US National Library of Medicine. Here, the algorithm “blastp (protein-protein BLAST)” is used for alignment of a query sequence with a subject sequence and determining i.a. the % identity.
  • As used herein, the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • For a fuller understanding of the present invention and further objects and advantages of it, the detailed description set out below should be read together with the accompanying figures, and in which:
  • FIG. 1 shows an alignment of the Fc-binding domains as defined by SEQ ID NOs: 1-7 and 51-52.
  • FIG. 2 shows results from Example 2 for the alkali stability of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to an SPR biosensor chip.
  • FIG. 3 shows results from Example 4 for the alkali stability (0.5 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to agarose beads.
  • FIG. 4 shows results from Example 4 for the alkali stability (1.0 M NaOH) of parental and mutated tetrameric Zvar (SEQ ID NO: 7) polypeptide variants coupled to agarose beads.
  • FIG. 5 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO: 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qb10%, 6 min residence time) vs. incubation time in 1 M NaOH.
  • FIG. 6 shows results from Example 7 for the alkali stability (1.0 M NaOH) of agarose beads with different amounts of mutated multimer variants (SEQ ID NO: 20) coupled. The results are plotted as the relative remaining dynamic capacity (Qb10%, 6 min residence time) after 31 h incubation in 1 M NaOH vs. the ligand content of the prototypes.
  • FIG. 7 shows results from a pH gradient elution of polyclonal human IgG a) from the reference matrix MabSelect SuRe LX and b) a matrix according to the invention.
  • FIG. 8 shows analyses of the IgG1, IgG2 and IgG4 components in fractions from the chromatograms of FIG. 7 . a) reference matrix and b) matrix according to the invention. For each fraction, the first bar (blue) represents IgG1, the second (red) IgG 4 and the third (green) IgG 2.
  • FIG. 9 shows results from accelerated alkali stability measurements with 1 M NaOH incubation for the reference matrix MabSelect SuRe LX (MSS LX) and a matrix according to the invention. The stability is expressed as the percentage of the 10% breakthrough capacity remaining after incubation.
  • FIG. 10 shows results from repeated CIP cycles using 2 M NaOH as the cleaning liquid for the reference matrix MabSelect SuRe (MSS) and a separation matrix according to the invention (Inv. Ex). The capacity of each separation matrix relative to initial capacity (determined as load volume to 35% breakthrough DBC) is shown as a function of cycle number.
  • FIG. 11 shows results from incubation with 1 M NaOH the reference matrix MabSelect SuRe LX (MSS LX) and a separation matrix according to the invention (Inv. Ex). The relative fluorescence as a function of time due to binding of labelled hIgG is shown for each of the separation matrices incubated for set periods of 0, 16 and 32 hours.
  • FIG. 12 shows results from incubation with 1 M NaOH the reference matrix MabSelect SuRe LX (MSS LX) and a separation matrix according to the invention (Inv. Ex). The relative fluorescence as a function of time due to binding of labelled hIgG is shown for each of the separation matrices incubated for set periods of up to 32 hours.
  • FIG. 13 shows results from the inactivation of B. subtilis spores using cleaning fluids comprising 1 M NaOH.
    •  DETAILED DESCRIPTION
  • One aspect of the present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support.
  • Throughout this detailed description, two separate numbering conventions may be used. Unless otherwise stated, the amino acid residue position numbering convention of FIG. 1 is used, and the position numbers are designated as corresponding to those in SEQ ID Nos: 4-7. This applies also to multimers, where the position numbers designate the positions in the polypeptide units or monomers according to the convention of FIG. 1 , unless otherwise stated. However, throughout the claims, summary of invention and on occasion in the detailed description, the position numbers corresponding to those of SEQ ID Nos: 51 and 52 are used. Note that position 1 of SEQ ID NO: 51 or SEQ ID NO: 52 corresponds to position 9 of SEQ ID Nos: 4-7, and in this manner the different numbering conventions may be interconverted.
  • The immunoglobulin-binding alkali-stabilized Protein A domains of the invention, also termed herein as “the polypeptide”, comprise, consist essentially of, or consist of mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID Nos: 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.
  • SEQ ID NO: 51 (truncated Zvar)
    QQ NAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQ
    SEQ ID NO 52: (truncated C domain)
    QQ NAFYEILHLP NLTEEQRNGF IQSLKDDPSV 
    SKEILAEAKK LNDAQ
  • Such immunoglobulin-binding alkali-stabilized Protein A domains may comprise, consist essentially of, or consist of mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 90%, at least 95% or at least 98% identity to, SEQ ID NO: 1 (E-domain), SEQ ID NO: 2 (D-domain), SEQ ID NO: 3 (A-domain), SEQ ID NO: 22 (variant A-domain), SEQ ID NO: 4 (B-domain), SEQ ID NO: 5 (C-domain), SEQ ID NO: 6 (Protein Z), SEQ ID NO: 7 (Zvar), SEQ ID NO: 51 (Zvar without the linker region amino acids 1-8 and 56-58) or SEQ ID NO: 52 (C-domain without the linker region amino acids 1-8 and 56-58) as illustrated in FIG. 1 , wherein at least the asparagine (or serine, in the case of SEQ ID NO: 2) residue at the position corresponding to position 11 in SEQ ID NOs: 4-7 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine, and wherein the asparagine residues corresponding to positions 21 and 52 in SEQ ID NOs: 4-7 (positions 13 and 44 of SEQ ID Nos: 51 or 52) are conserved.
  • A number of the Fc-binding domains listed above are shown aligned in FIG. 1 . The parental, i.e. non-engineered, Staphylococcus Protein A (SpA) comprises five Fc-dining domains termed domain E (SEQ ID NO: 1), D (SEQ ID NO: 2), A (SEQ ID NO: 3), B (SEQ ID NO: 4) and C (SEQ ID NO: 5). Protein Z (SEQ ID NO: 6) is a mutated B-domain as disclosed in U.S. Pat. No. 5,143,844, hereby incorporated by reference in its entirety. SEQ ID NO: 7 denotes a further mutated variant of Protein Z, here called Zvar, with the mutations N3A, N6D, N23T. SEQ ID NO: 22 (not shown in FIG. 1 ) is a natural variant of the A-domain in Protein A from Staphylococcus aureus strain N315, having an A46S mutation, using the position terminology of FIG. 1 . SEQ ID NO: 51 is Zvar (SEQ ID NO: 7) without the linker region amino acids at positions 1-8 and 56-58. SEQ ID NO: 52 is the C-domain of protein A without the linker region amino acids 1-8 and 56-58) as illustrated in FIG. 1 .
  • The mutation of N11 (N3 of SEQ ID Nos: 5-52) in these domains, together with the conservation of the asparagine residues N21 and N52 (N13 and N44 of SEQ ID Nos: 51-52) confers an improved alkali stability in comparison with the parental domain/polypeptide, without impairing the immunoglobulin-binding properties. Hence, the polypeptide can also be described as an Fc- or immunoglobulin-binding polypeptide, or alternatively as an Fc- or immunoglobulin-binding polypeptide unit.
  • Described in alternative language, the immunoglobulin-binding alkali-stabilized Protein A domains may comprise, consist essentially of, or consist of a sequence as defined by, or having at least 90%, at least 95% or at least 98% identity to SEQ ID NO 53.
  • SEQ ID NO: 53
    X1Q X2AFYEILX3LP NLTEEQRX4X5F IX6X7LKDX8PSX9
    SX10X11X12LAEAKX13 X14NX15AQ
  • wherein individually of each other:
      • X1=A, Q or is deleted
      • X2=E,K,Y,T,F,L,W,I,M,V,A,H or R
      • X3=H or K
      • X4=A or N
      • X5=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such as S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R
      • or K
      • X6=Q or E
      • X7=S or K
      • X8=E or D
      • X9=Q, V or is deleted
      • X10=K, R, A or is deleted
      • X11=A, E, N or is deleted
      • X12=l or L
      • X13=K or R
      • X14=L or Y
      • X15=D,F,Y,W,K or R
  • Specifically, the amino acid residues in SEQ ID NO: 53 may individually of each other be:
      • X1=A or is deleted
      • X2=E
      • X3=H
      • X4=N
      • X6=Q
      • X7=S
      • X8=D
      • X9=V or is deleted
      • X10=K or is deleted
      • X11=A or is deleted
      • X12=I
      • X13=K
      • X14=L
  • In certain embodiments, the amino acid residues in SEQ ID NO: 53 may be: X1=A, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L. In some embodiments X2=E, X3=H, X4=N, X8=A, X6=Q, X7=S, X8=D, X12=I, X13=K, X14=L and X15=D and one or more of X1, X9, X10 and X11 is deleted. In further embodiments, X1=A, X2=E, X3=H, X4=N, X8=S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D, or alternatively X1=A, X2=E, X3=H, X4=N, X8=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=F,Y,W,K or R.
  • In some embodiments, the amino acid residues may individually of each other be:
      • a) X1=A or is deleted, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V or is deleted, X10=K or is deleted, X11=A or is deleted, X12=1, X13=K, X14=L;
      • b) X1=A, X2=E, X3=H, X4=N, X8=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D;
      • c) X1 is A, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D; or
      • d) X1 is A, X3=H, X4=N, X5=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=Land X15=D.
  • The N11 (X2) mutation (e.g. a N11E or N11K mutation) may be the only mutation or the polypeptide may also comprise further mutations, such as substitutions in at least one of the positions corresponding to positions 3, 6, 9, 10, 15, 18, 23, 28, 29, 32, 33, 36, 37, 40, 42, 43, 44, 47, 50, 51, 55 and 57 in SEQ ID NOs: 4-7. In one or more of these positions, the original amino acid residue may e.g. be substituted with an amino acid which is not asparagine, proline or cysteine. The original amino acid residue may e.g. be substituted with an alanine, a valine, a threonine, a serine, a lysine, a glutamic acid or an aspartic acid. Further, one or more amino acid residues may be deleted, e.g. from positions 1-6 and/or from positions 56-58.
  • In some embodiments, the amino acid residue at the position corresponding to position 9 in SEQ ID NOs: 4-7 (X1) is an amino acid other than glutamine, asparagine, proline or cysteine, such as an alanine or it can be deleted. The combination of the mutations at positions 9 and 11 provides particularly good alkali stability, as shown by the examples. In specific embodiments, in SEQ ID NO: 7 the amino acid residue at position 9 is an alanine and the amino acid residue at position 11 is a lysine or glutamic acid, such as a lysine. Mutations at position 9 are also discussed in copending application PCT/SE2014/050872, which is hereby incorporated by reference in its entirety.
  • In some embodiments, the amino acid residue at the position corresponding to position 50 in SEQ ID NOs: 4-7 (X13) is an arginine or a glutamic acid.
  • In certain embodiments, the amino acid residue at the position corresponding to position 3 in SEQ ID NOs: 4-7 is an alanine and/or the amino acid residue at the position corresponding to position 6 in SEQ ID NOs: 4-7 is an aspartic acid. One of the amino acid residues at positions 3 and 6 may be an asparagine and in an alternative embodiment both amino acid residues at positions 3 and 6 may be asparagines.
  • In some embodiments the amino acid residue at the position corresponding to position 43 in SEQ ID NOs: 4-7 (X11) is an alanine or a glutamic acid, such as an alanine or it can be deleted. In specific embodiments, the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acid respectively, while the amino acid residue at position 43 is alanine or glutamic acid.
  • In certain embodiments the amino acid residue at the position corresponding to position 28 in SEQ ID Nos: 4-7 (X5) is an alanine or an asparagine, such as an alanine.
  • In some embodiments the amino acid residue at the position corresponding to position 40 in SEQ ID NOs: 4-7 (X9) is selected from the group consisting of asparagine, alanine, glutamic acid and valine, or from the group consisting of glutamic acid and valine, or valine, or it can be deleted. In specific embodiments, the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and glutamic acid respectively, while the amino acid residue at position 40 is valine. Optionally, the amino acid residue at position 43 may then be alanine or glutamic acid.
  • In certain embodiments, the amino acid residue at the position corresponding to position 42 in SEQ ID NO: 4-7 (X10) is an alanine, lysine or arginine or it can be deleted.
  • In some embodiments the amino acid residue at the position corresponding to position 18 in SEQ ID NO: 4-7 (X3) is a lysine or a histidine, such as a lysine.
  • In certain embodiments the amino acid residue at the position corresponding to position 33 in SEQ ID NO: 4-7 (X7) is a lysine or a serine, such as a lysine.
  • In some embodiments the amino acid residue at the position corresponding to position 37 in SEQ ID NO: 4-7 (X8) is a glutamic acid or an aspartic acid, such as a glutamic acid.
  • In certain embodiments the amino acid residue at the position corresponding to position 51 in SEQ ID NO: 4-7 (X14) is a tyrosine or a leucine, such as a tyrosine.
  • In some embodiments, the amino acid residue at the position corresponding to position 44 in SEQ ID NO: 4-7 (X12) is a leucine or an isoleucine. In specific embodiments, the amino acid residues at positions 9 and 11 in SEQ ID NO: 7 are alanine and lysine/glutamic acid respectively, while the amino acid residue at position 44 is isoleucine. Optionally, the amino acid residue at position 43 may then be alanine or glutamic acid.
  • In some embodiments, the amino acid residues at the positions corresponding to positions 1, 2, 3 and 4 or to positions 3, 4, 5 and 6 in SEQ ID NOs: 4-7 have been deleted. In specific variants of these embodiments, the parental polypeptide is the C domain of Protein A (SEQ ID NO: 5). The effects of these deletions on the native C domain are described in U.S. Pat. Nos. 9,018,305 and 8,329,860, which are hereby incorporated by reference in their entireties.
  • In certain embodiments, the mutation in SEQ ID Nos: 4-7, such as in SEQ ID NO: 7, is selected from the group consisting of: N11K; N11E; N11Y; N11T; N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; N11E, Q32A; N11E, Q32E, Q40E; N11E, Q32E, K50R; Q9A, N11E, N43A; Q9A, N11E, N28A, N43A; Q9A, N11E, Q40V, A42K, N43E, L44I; Q9A, N11E, Q40V, A42K, N43A, L44I; N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11E, N28A, Q40V, A42K, N43A, L44I; Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R; Q9A, N11K, H18K, D37E, A42R; Q9A, N11E, D37E, Q40V, A42K, N43A, L44I and Q9A, N11E, D37E, Q40V, A42R, N43A, L44I. These mutations provide particularly high alkaline stabilities. The mutation in SEQ ID Nos: 4-7, such as in SEQ ID NO: 7, can also be selected from the group consisting of N11K; N11Y; N11F; N11L; N11W; N11I; N11M; N11V; N11A; N11H; N11R; Q9A, N11E, N43A; Q9A, N11E, N28A, N43A; Q9A, N11E, Q40V, A42K, N43E, L44I; Q9A, N11E, Q40V, A42K, N43A, L44I; Q9A, N11E, N28A, Q40V, A42K, N43A, L44I; N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y; N11K, H18K, D37E, A42R, N43A, L44I; Q9A, N11K, H18K, D37E, A42R, N43A, L44I and Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R.
  • In some embodiments, the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. It may e.g. comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29. It can also comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48.
  • In certain embodiments, the polypeptide comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 54-70; comprises or consists essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 71-75; or it may comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 76-79. It may further comprise or consist essentially of a sequence defined by or having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 89-95.
  • The polypeptide may e.g. be defined by a sequence selected from the groups above or from subsets of these groups, but it may also comprise additional amino acid residues at the N- and/or C-terminal end, e.g. a leader sequence at the N-terminal end and/or a tail sequence at the C-terminal end.
  • SEQ ID NO: 8 Zvar(Q9A,N11E,N43A)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SAALLAEAKK LNDAQAPK
    SEQ ID NO: 9 Zvar(Q9A, N11E, N28A, N43A)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ 
    SAALLAEAKK LNDAQAPK
    SEQ ID NO: 10 Zvar(Q9A, N11E, Q40V, A42K, 
    N43E, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKEILAEAKK LNDAQAPK
    SEQ ID NO: 11 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 12 Zvar(N11E, Q32A)
    VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IASLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 13 Zvar(N11E)
    VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 14 Zvar(N11E, Q32E, Q40E)
    VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSE 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 15 Zvar(N11E, Q32E, K50R)
    VDAKFDKEQQ EAFYEILHLP NLTEEQRNAF IESLKDDPSQ 
    SANLLAEAKR LNDAQAPK
    SEQ ID NO: 16 Zvar(N11K)
    VDAKFDKEQQ KAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 23 Zvar(N11K, H18K, S33K, D37E, 
    A42R, N43A, L44I, K50R, L51Y)
    VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ 
    SRAILAEAKR YNDAQAPK
    SEQ ID NO: 24 Zvar(Q9A, N11E, N28A, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 25 Zvar(Q9A, N11K, H18K, S33K, 
    D37E, A42R, N43A, L44I, K50R, L51Y)
    VDAKFDKEAQ KAFYEILKLP NLTEEQRAAF IQKLKDEPSQ 
    SRAILAEAKR YNDAQAPK
    SEQ ID NO: 26 Zvar(N11K, H18K, D37E, A42R, 
    N43A, L44I)
    VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ 
    SRAILAEAKK LNDAQAPK
    SEQ ID NO: 27 Zvar(Q9A, N11K, H18K, D37E, 
    A42R, N43A, L44I)
    VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ 
    SRAILAEAKK LNDAQAPK
    SEQ ID NO: 28 Zvar(Q9A, N11K, H18K, D37E, 
    A42R, N43A, L44I, K50R)
    VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ 
    SRAILAEAKR LNDAQAPK
    SEQ ID NO: 29 Zvar(Q9A, N11K, H18K, D37E, 
    A42R)
    VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ 
    SRNLLAEAKK LNDAQAPK
    SEQ ID NO: 36 B(Q9A, N11E, Q40V, A42K, N43A, 
    L44I)
    ADNKFNKEAQ EAFYEILHLP NLNEEQRNGF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 37 C(Q9A, N11E, E43A)
    ADNKFNKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 38 Zvar(N11Y)
    VDAKFDKEQQ YAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 39 Zvar(N11T)
    VDAKFDKEQQ TAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 40 Zvar(N11F)
    VDAKFDKEQQ FAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 41 Zvar(N11L)
    VDAKFDKEQQ LAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 42 Zvar(N11W)
    VDAKFDKEQQ WAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 43 Zvar(N11I)
    VDAKFDKEQQ IAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 44 Zvar(N11M)
    VDAKFDKEQQ MAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 45 Zvar(N11V)
    VDAKFDKEQQ VAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 46 Zvar(N11A)
    VDAKFDKEQQ AAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 47 Zvar(N11H)
    VDAKFDKEQQ HAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 48 Zvar(N11R)
    VDAKFDKEQQ RAFYEILHLP NLTEEQRNAF IQSLKDDPSQ 
    SANLLAEAKK LNDAQAPK
    SEQ ID NO: 49 Zvar(Q9A, N11E, D37E, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 50 Zvar(Q9A, N11E, D37E, Q40V, 
    A42R, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDEPSV 
    SRAILAEAKK LNDAQAPK
    SEQ ID NO: 54 Zvar(Q9A, N11E, A29G, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNGF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 55 Zvar(Q9A, N11E, A29S, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNSF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 56 Zvar(Q9A, N11E, A29Y, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNYF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 57 Zvar(Q9A, N11E, A29Q, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNQF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 58 Zvar(Q9A, N11E, A29T, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNTF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 59 Zvar(Q9A, N11E, A29N, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNNF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 60 Zvar(Q9A, N11E, A29F, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNFF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 61 Zvar(Q9A, N11E, A29L, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNLF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 62 Zvar(Q9A, N11E, A29W, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNWF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 63 Zvar(Q9A, N11E, A29I, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNIF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 64 Zvar(Q9A, N11E, A29M, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNMF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 65 Zvar(Q9A, N11E, A29V, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNVF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 66 Zvar(Q9A, N11E, A29D, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNDF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 67 Zvar(Q9A, N11E, A29E, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNEF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 68 Zvar(Q9A, N11E, A29H, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNHF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 69 Zvar(Q9A, N11E, A29R, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNRF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 70 Zvar(Q9A, N11E, A29K, Q40V, 
    A42K, N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNKF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 71 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, D53F)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNFAQAPK
    SEQ ID NO: 72 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, D53Y)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNYAQAPK
    SEQ ID NO: 73 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, D53W)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNWAQAPK
    SEQ ID NO: 74 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, D53K)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNKAQAPK
    SEQ ID NO: 75 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, D53R)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNRAQAPK
    SEQ ID NO: 76 Zvar(Q9del, N11E, Q40V, A42K, 
    N43A, L44I)
    VDAKFDKE_Q EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 77 Zvar(Q9A, N11E, Q40del, A42K, 
    N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPS_ 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 78 Zvar(Q9A, N11E, Q40V, A42del, 
    N43A, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    S_AILAEAKK LNDAQAPK
    SEQ ID NO: 79 Zvar(Q9A, N11E, Q40V, A42K, 
    N43del, L44I)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SK_ILAEAKK LNDAQAPK
    SEQ ID NO: 89 Zvar(D2del, A3del, K4del, Q9A, 
    N11E, Q40V, A42K, N43A, L44I)
    V___FDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 90 Zvar(V1del, D2del, Q9A, N11E, 
    Q40V, A42K, N43A, L44I, K58del)
    __AKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAP_
    SEQ ID NO: 91 Zvar(K4del, F5del, D6del, K7del, 
    E8del, Q9A, N11E, Q40V, A42K, N43A, L44I)
    VDA_____AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO: 92 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, A56del, P57del, K58del)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQ
    SEQ ID NO: 93 Zvar(V1del, D2del, A3del, Q9A, 
    N11E, Q40V, A42K, N43A, L44I)
    ___KFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO 94: Zvar(V1del, D2del, A3del, K4del, 
    F5del, D6del, K7del, E8del, Q9A, N11E, Q40V, 
    A42K, N43A, L44)
    ________AQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK
    SEQ ID NO 95: Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I, K58_insYEDG)
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPKYE DG
  • The separation matrix comprises multimers of the immunoglobulin-binding alkali-stabilized Protein A domains. Such multimers comprise, consist essentially of, or consist of a plurality of immunoglobulin-binding alkali-stabilized Protein A domains (polypeptide units) as defined by any embodiment disclosed above. The use of multimers may increase the immunoglobulin binding capacity and multimers may also have a higher alkali stability than monomers. The multimer can e.g. be a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a nonamer. The multimer may be a homomultimer, where all the units in the multimer are identical or it can be a heteromultimer, where at least one unit differs from the others. Advantageously, all the units in the multimer are alkali stable, such as by comprising the mutations/conservations disclosed above. The polypeptides can be linked to each other directly by peptide bonds between the C-terminal and N-terminal ends of the polypeptides. Alternatively, two or more units in the multimer can be linked by linkers comprising oligomeric or polymeric species, such as linkers comprising peptides with up to 25 or 30 amino acids, such as 3-25 or 3-20 amino acids. The linkers may e.g. comprise or consist essentially of a peptide sequence defined by, or having at least 90% identity or at least 95% identity, with an amino acid sequence selected from the group consisting of APKVDAKFDKE (SEQ ID NO: 96), APKVDNKFNKE (SEQ ID NO: 97), APKADNKFNKE (SEQ ID NO: 98), APKVFDKE (SEQ ID NO: 99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO: 101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ ID NO: 103), APKKFDKE (SEQ ID NO: 104), APK, APKYEDGVDAKFDKE (SEQ ID NO: 105) and YEDG (SEQ ID NO: 106) or alternatively selected from the group consisting of APKADNKFNKE (SEQ ID NO: 98), APKVFDKE (SEQ ID NO: 99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO: 101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ ID NO: 103), APKKFDKE (SEQ ID NO: 104), APKYEDGVDAKFDKE (SEQ ID NO: 105) and YEDG (SEQ ID NO: 106). They can also consist essentially of a peptide sequence defined by or having at least 90% identity or at least 95% identity with an amino acid sequence selected from the group consisting of APKADNKFNKE (SEQ ID NO: 98), APKVFDKE (SEQ ID NO: 99), APAKFDKE (SEQ ID NO: 100), AKFDKE (SEQ ID NO: 101), APKVDA (SEQ ID NO: 102), VDAKFDKE (SEQ ID NO: 103), APKKFDKE (SEQ ID NO: 104), APK and APKYEDGVDAKFDKE (SEQ ID NO: 105). In some embodiments the linkers do not consist of the peptides APKVDAKFDKE (SEQ ID NO: 96) or APKVDNKFNKE (SEQ ID NO: 97), or alternatively do not consist of the peptides APKVDAKFDKE (SEQ ID NO: 96), APKVDNKFNKE (SEQ ID NO: 97), APKFNKE (SEQ ID NO: 107), APKFDKE (SEQ ID NO: 108), APKVDKE (SEQ ID NO: 109) or APKADKE (SEQ ID NO: 110).
  • The nature of such a linker should preferably not destabilize the spatial conformation of the protein units. This can e.g. be achieved by avoiding the presence of proline in the linkers. Furthermore, said linker should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein units. For this purpose, it is advantageous if the linkers do not contain asparagine. It can additionally be advantageous if the linkers do not contain glutamine. The multimer may further at the N-terminal end comprise a plurality of amino acid residues e.g. originating from the cloning process or constituting a residue from a cleaved off signaling sequence. The number of additional amino acid residues may e.g. be 20 or less, such as 15 or less, such as 10 or less or 5 or less. As a specific example, the multimer may comprise an AQ, AQGT (SEQ ID NO: 111), VDAKFDKE (SEQ ID NO: 103), AQVDAKFDKE (SEQ ID NO: 112) or AQGTVDAKFDKE (SEQ ID NO: 113) sequence at the N-terminal end.
  • In certain embodiments, the multimer may comprise, or consist essentially, of a sequence selected from the group consisting of: SEQ ID Nos: 80-87. These and additional sequences are listed below and named as Parent(Mutations)n, where n is the number of monomer units in a multimer.
  • SEQ ID NO: 17 Zvar(Q9A, N11E, N43A)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSQ SAALLAEAKK 
    LNDAQAPKC
    SEQ ID NO: 18 Zvar(Q9A, N11E, N28A, N43A)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF 
    IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF 
    IQSLKDDPSQ SAALLAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRAAF IQSLKDDPSQ SAALLAEAKK 
    LNDAQAPKC
    SEQ ID NO: 19 Zvar(Q9A, N11E, Q40V, A42K, 
    N43E, L44I)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKEILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKEILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 20 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 30 Zvar(N11K, H18K, S33K, D37E, 
    A42R, N43A, L44I, K50R, L51Y)4
    AQGT VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF 
    IQKLKDEPSQ SRAILAEAKR YNDAQAPK VDAKFDKEQQ 
    KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR 
    YNDAQAPK VDAKFDKEQQ KAFYEILKLP NLTEEQRNAF 
    IQKLKDEPSQ SRAILAEAKR YNDAQAPK VDAKFDKEQQ 
    KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR 
    YNDAQAPKC
    SEQ ID NO: 31 Zvar(Q9A, N11K, H18K, D37E, 
    A42R)4
    AQGT VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF 
    IQSLKDEPSQ SRNLLAEAKK LNDAQAPK VDAKFDKEAQ 
    KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK 
    LNDAQAPK VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF 
    IQSLKDEPSQ SRNLLAEAKK LNDAQAPK VDAKFDKEAQ 
    KAFYEILKLP NLTEEQRNAF IQSLKDEPSQ SRNLLAEAKK 
    LNDAQAPKC
    SEQ ID NO: 32 Zvar(Q9A, N11E, N28A, Q40V, 
    A42K, N43A, L44I)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRAAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRAAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 33 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)6
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 34 Zvar(Q9A, N11E, D37E, Q40V, 
    A42K, N43A, L44I)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDEPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDEPSV SKAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SKAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 35 Zvar(Q9A, N11E, D37E, Q40V, 
    A42R, N43A, L44I)4
    AQGT VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDEPSV SRAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK 
    LNDAQAPK VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDEPSV SRAILAEAKK LNDAQAPK VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDEPSV SRAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 80 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with D2, A3 and K4 in linker
    deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK VFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 81 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with K58, V1 and D2 in linker
    deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAP AKFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 82 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with P57, K58, V1, D2 and A3
    in linker deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAP AKFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 83 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with K4, F5, D6, K7 and E8
    in linker deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK VDAAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 84 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with A56, P57 and K58 in
    linker deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQ VDAKFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 85 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with V1, D2 and A3 in linker 
    deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK KFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 86 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with V1, D2, A3, K4, F5, D6, 
    K7 and E8 in linker deleted 
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV
    SKAILAEAKK LNDAQAPK AQ EAFYEILHLP NLTEEQRNAF 
    IQSLKDDPSV SKAILAEAKK LNDAQAPKC
    SEQ ID NO: 87 Zvar(Q9A, N11E, Q40V, A42K, 
    N43A, L44I)2 with YEDG inserted in linker 
    between K58 and V1
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK YEDG VDAKFDKEAQ 
    EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK 
    LNDAQAPKC
    SEQ ID NO: 88 Zvar2
    VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV 
    SKAILAEAKK LNDAQAPK VDAKFDKEAQ EAFYEILHLP 
    NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPKC
  • In some embodiments, the polypeptide and/or multimer, as disclosed above, further comprises at the C-terminal or N-terminal end one or more coupling elements, selected from the group consisting of one or more cysteine residues, a plurality of lysine residues and a plurality of histidine residues. The coupling element(s) may also be located within 1-5 amino acid residues, such as within 1-3 or 1-2 amino acid residues from the C-terminal or N-terminal end. The coupling element may e.g. be a single cysteine at the C-terminal end. The coupling element(s) may be directly linked to the C- or N-terminal end, or it/they may be linked via a stretch comprising up to 15 amino acids, such as 1-5, 1-10 or 5-10 amino acids. This stretch should preferably also be sufficiently stable in alkaline environments not to impair the properties of the mutated protein. For this purpose, it is advantageous if the stretch does not contain asparagine. It can additionally be advantageous if the stretch does not contain glutamine. An advantage of having a C-terminal cysteine is that endpoint coupling of the protein can be achieved through reaction of the cysteine thiol with an electrophilic group on a support. This provides excellent mobility of the coupled protein which is important for the binding capacity.
  • The alkali stability of the polypeptide or multimer can be assessed by coupling it to a surface plasmon resonance (SPR) chip, e.g. to Biacore CM5 sensor chips as described in the examples, using e.g. NHS— or maleimide coupling chemistries, and measuring the immunoglobulin-binding capacity of the chip, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/−2° C. The incubation can e.g. be performed in 0.5 M NaOH for a number of 10 min cycles, such as 100, 200 or 300 cycles. The IgG capacity of the matrix after 100 10 min incubation cycles in 0.5 M NaOH at 22+/−2° C. can be at least 55, such as at least 60, at least 80 or at least 90% of the IgG capacity before the incubation. Alternatively, the remaining IgG capacity after 100 cycles for a particular mutant measured as above can be compared with the remaining IgG capacity for the parental polypeptide/multimer. In this case, the remaining IgG capacity for the mutant may be at least 105%, such as at least 110%, at least 125%, at least 150% or at least 200% of the parental polypeptide/multimer.
  • The immunoglobulin-binding alkali-stabilized Protein A domains and/or multimers thereof may be encoded by a nucleic acid sequence, such as an RNA sequence or a DNA sequence encoding the polypeptide or multimer. A vector, such as a plasmid, which in addition to the coding sequence comprises the required signal sequences, may be used for expression of the polypeptide or multimer. The vector may comprise nucleic acid encoding a multimer as described above, wherein the separate nucleic acids encoding each unit may have homologous or heterologous DNA sequences.
  • An expression system, which comprises a nucleic acid or a vector as disclosed above, may be used for expression of the polypertide or multimer. The expression system may e.g. be a gram-positive or gram-negative prokaryotic host cell system, e.g. E. coli or Bacillus sp. which has been modified to express the present polypeptide or multimer. Alternatively, the expression system may be a eukaryotic host cell system, such as a yeast, e.g. Pichia pastoris or Saccharomyces cerevisiae, or mammalian cells, e.g. CHO cells.
  • The separation matrix comprises, consists essentially of, or consists of multimers of immunoglobulin-binding alkali-stabilized Protein A domains as described above, covalently coupled to a porous support.
  • The separation matrix may comprise at least 11, such as 11-21, 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein:
      • a) the ligands comprise multimers of alkali-stabilized Protein A domains,
      • b) the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70, such as 56-66, micrometers and a dry solids weight of 55-80, such as 60-78 or 65-78, mg/ml. The cross-linked polymer particles may further have a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.69-0.85, such as 0.70-0.85 or 0.69-0.80, for dextran of Mw 110 kDa. The multimers may e.g. comprise tetramers, pentamers, hexamers or heptamers of alkali-stabilized Protein A domains, such as hexamers of alkali-stabilized Protein A domains. The combination of the high ligand contents with the particle size range, the dry solids weight range and the optional Kd range provides for a high binding capacity, e.g. such that the 10% breakthrough dynamic binding capacity for IgG is at least 45 mg/ml, such as at least 50 or at least 55 mg/ml at 2.4 min residence time. Alternatively, or additionally, the 10% breakthrough dynamic binding capacity for IgG may be at least 60 mg/ml, such as at least 65, at least 70 or at least 75 mg/ml at 6 min residence time.
  • The alkali-stabilized Protein A domain multimers are highly selective for IgG and the separation matrix can suitably have a dissociation constant for human IgG2 of below 0.2 mg/ml, such as below 0.1 mg/ml, in 20 mM phosphate buffer, 180 mM NaCl, pH 7.5. This can be determined according to the adsorption isotherm method described in N Pakiman et al: J Appl Sci 12, 1136-1141 (2012).
  • In certain embodiments the separation matrix comprises at least 15, such as 15-21 or 15-18 mg/ml Fc-binding ligands covalently coupled to a porous support, wherein the ligands comprise multimers of alkali-stabilized Protein A domains. These multimers can suitably be as disclosed in any of the embodiments described above or as specified below.
  • In some embodiments the separation matrix comprises 5-25, such as 5-20 mg/ml, 5-15 mg/ml, 5-11 mg/ml or 6-11 mg/ml of the polypeptide or multimer coupled to the support. The amount of coupled polypeptide/multimer can be controlled by the concentration of polypeptide/multimer used in the coupling process, by the activation and coupling conditions used and/or by the pore structure of the support used. As a general rule the absolute binding capacity of the matrix increases with the amount of coupled polypeptide/multimer, at least up to a point where the pores become significantly constricted by the coupled polypeptide/multimer. Without being bound by theory, it appears though that for the Kd values recited for the support, the constriction of the pores by coupled ligand is of lower significance. The relative binding capacity per mg coupled polypeptide/multimer will decrease at high coupling levels, resulting in a cost-benefit optimum within the ranges specified above.
  • Such a separation matrix is useful for separation of immunoglobulins or other Fc-containing proteins and, due to the improved alkali stability of the polypeptides/multimers, the matrix will withstand highly alkaline conditions during cleaning, which is essential for long-term repeated use in a bioprocess separation setting. The alkali stability of the matrix can be assessed by measuring the immunoglobulin-binding capacity, typically using polyclonal human IgG, before and after incubation in alkaline solutions at a specified temperature, e.g. 22+/−2° C. The incubation can e.g. be performed in 0.5 M or 1.0 M NaOH for a number of 15 min cycles, such as 100, 200 or 300 cycles, corresponding to a total incubation time of 25, 50 or 75 h. The IgG capacity of the matrix after 96-100 15 min incubation cycles or a total incubation time of 24 or 25 h in 0.5 M NaOH at 22+/−2° C. can be at least 80, such as at least 85, at least 90 or at least 95% of the IgG capacity before the incubation. The capacity of the matrix after a total incubation time of 24 h in 1.0 M NaOH at 22+/−2° C. can be at least 70, such as at least 80 or at least 90% of the IgG capacity before the incubation. The the 10% breakthrough dynamic binding capacity (Qb10%) for IgG at 2.4 min or 6 min residence time may e.g. be reduced by less than 20% after incubation 31 h in 1.0 M aqueous NaOH at 22+/−2 C.
  • As the skilled person will understand, the expressed polypeptide or multimer should be purified to an appropriate extent before being immobilized to a support. Such purification methods are well known in the field, and the immobilization of protein-based ligands to supports is easily carried out using standard methods. Suitable methods and supports will be discussed below in more detail.
  • The porous support of the separation matrix may be of any suitable well-known kind. A conventional affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy (—COOH), carboxamido (—CONH2, possibly in N— substituted forms), amino (—NH2, possibly in substituted form), oligo- or polyethylenoxy groups on their external and, if present, also on internal surfaces. The porosity of the support can be expressed as a Kav or Kd value (the fraction of the pore volume available to a probe molecule of a particular size) measured by inverse size exclusion chromatography, e.g. according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13. Kav is determined as the ratio (Ve−V0)/(Vt−V0), where Ve is the elution volume of a probe molecule (e.g. Dextran 110 kD), Vo is the void volume of the column (e.g. the elution volume of a high Mw void marker, such as raw dextran) and Vt is the total volume of the column. Kd can be determined as (Ve—V0)/Vi, where Vi is the elution volume of a salt (e.g. NaCl) able to access all the volume except the matrix volume (the volume occupied by the matrix polymer molecules). By definition, both Kd and Kav values always lie within the range 0-1. The Kav value can advantageously be 0.6-0.95, e.g. 0.7-0.90 or 0.6-0.8, as measured with dextran of Mw 110 kDa as a probe molecule. The Kd value as measured with dextran of Mw 110 kDa can suitably be 0.68-0.90, such as 0.68-0.85 or 0.70-0.85. An advantage of this is that the support has a large fraction of pores able to accommodate both the polypeptides/multimers of the invention and immunoglobulins binding to the polypeptides/multimers and to provide mass transport of the immunoglobulins to and from the binding sites.
  • The polypeptides or multimers may be attached to the porous support via conventional coupling techniques utilising e.g. thiol, amino and/or carboxy groups present in the ligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS) etc are well-known coupling reagents. Between the support and the polypeptide/multimer, a molecule known as a spacer can be introduced, which improves the availability of the polypeptide/multimer and facilitates the chemical coupling of the polypeptide/multimer to the support. Depending on the nature of the polypeptide/multimer and the coupling conditions, the coupling may be a multipoint coupling (e.g. via a plurality of lysines) or a single point coupling (e.g. via a single cysteine).
  • In certain embodiments the polypeptides or multimers are coupled to the support via thioether bonds. Methods for performing such coupling are well-known in this field and easily performed by the skilled person in this field using standard techniques and equipment. Thioether bonds are flexible and stable and generally suited for use in affinity chromatography. In particular when the thioether bond is via a terminal or near-terminal cysteine residue on the polypeptide or multimer, the mobility of the coupled polypeptide/multimer is enhanced which provides improved binding capacity and binding kinetics. In some embodiments the polypeptide/multimer is coupled via a C-terminal cysteine provided on the protein as described above. This allows for efficient coupling of the cysteine thiol to electrophilic groups, e.g. epoxide groups, halohydrin groups etc. on a support, resulting in a thioether bridge coupling.
  • In certain embodiments the support comprises a polyhydroxy polymer, such as a polysaccharide. Examples of polysaccharides include e.g. dextran, starch, cellulose, pullulan, agar, agarose etc. Polysaccharides are inherently hydrophilic with low degrees of nonspecific interactions, they provide a high content of reactive (activatable) hydroxyl groups and they are generally stable towards alkaline cleaning solutions used in bioprocessing.
  • In some embodiments the support comprises agar or agarose. The supports used in the present invention can easily be prepared according to standard methods, such as inverse suspension gelation (S Hjertén: Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the base matrices are commercially available products, such as crosslinked agarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). In an embodiment, which is especially advantageous for large-scale separations, the support has been adapted to increase its rigidity using the methods described in U.S. Pat. No. 6,602,990 or U.S. Pat. No. 7,396,467, which are hereby incorporated by reference in their entireties, and hence renders the matrix more suitable for high flow rates.
  • In certain embodiments the support, such as a polymer, polysaccharide or agarose support, is crosslinked, such as with hydroxyalkyl ether crosslinks. Crosslinker reagents producing such crosslinks can be e.g. epihalohydrins like epichlorohydrin, diepoxides like butanediol diglycidyl ether, allylating reagents like allyl halides or allyl glycidyl ether. Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether crosslinks are alkali stable and do not cause significant nonspecific adsorption.
  • Alternatively, the porous support is based on synthetic polymers, such as polyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides etc. In case of hydrophobic polymers, such as matrices based on divinyl and monovinyl-substituted benzenes, the surface of the matrix is often hydrophilised to expose hydrophilic groups as defined above to a surrounding aqueous liquid. Such polymers are easily produced according to standard methods, see e.g. “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988)). Alternatively, a commercially available product, such as SOURCE™ (GE Healthcare) is used. In another alternative, the porous support according to the invention comprises a support of inorganic nature, e.g. silica, zirconium oxide etc.
  • In yet another embodiment, the solid support is in another form such as a surface, a chip, capillaries, or a filter (e.g. a membrane or a depth filter matrix).
  • As regards the shape of the matrix according to the invention, in one embodiment the matrix is in the form of a porous monolith. In an alternative embodiment, the matrix is in beaded or particle form that can be porous or non-porous. Matrices in beaded or particle form can be used as a packed bed or in a suspended form. Suspended forms include those known as expanded beds and pure suspensions, in which the particles or beads are free to move. In case of monoliths, packed bed and expanded beds, the separation procedure commonly follows conventional chromatography with a concentration gradient. In case of pure suspension, batch-wise mode will be used.
  • The separation matrix as disclosed above has excellent alkali stability and may be cleaned and/or sanitized using an alkaline cleaning liquid. The method of cleaning and/or sanitizing the separation matrix comprises the following steps:
      • a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix;
      • b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and
      • c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time.
  • Cleaning and/or sanitization may be performed on separation matrix not previously used in order to sanitize the matrix prior to use, for example after packing a column comprising the separation matrix for the first time. Cleaning and/or sanitization may also be performed after using the separation matrix to purify an immunoglobulin, i.e. step a) of the method above. The steps of using the separation matrix to purify an immunoglobulin and cleaning and/or sanitizing the separation matrix afterwards may be repeated in order to maximize use of the separation matrix. The steps may be repeated at least 10 times, such as at least 50 times or 50-200 times. The step c) of cleaning/sanitizing may be performed after each step a) of purifying an immunoglobulin, or may be performed less frequently, such as after every second purification, or after every tenth purification. The step c) of cleaning and/or sanitizing the separation matrix need not be performed using the same conditions each time. For example, the separation matrix may be cleaned after each purification step using a first set of conditions, and after every nth purification be sanitized using more stringent conditions.
  • Because aqueous alkali metal hydroxide solutions are effective cleaning agents and bactericidal in themselves, by cleaning and sanitizing using a cleaning liquid comprising such aqueous alkali metal hydroxide solutions there is a lesser need for using alcohols as cleaning and sanitizing agents. Alkali metal hydroxides are relatively cheap compared to alcohols and are subject to less regulatory burden. Moreover, they are non-flammable and easier to dispose of.
  • The separation matrix is a separation matrix as disclosed above, comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by, or having at least 80% such as at least 90%, 95% or 98% identity to, SEQ ID NO: 51 or SEQ ID NO: 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NOs: 51 or 52 are asparagines, and wherein at least the asparagine residue at position 3 of SEQ ID Nos: 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine. The immunoglobulin-binding alkali-stabilized Protein A domains may comprise further mutations. For example, the glutamine residue at position 1 of SEQ ID Nos: 51 or 52 may be mutated to an alanine; and/or the asparagine or glutamic acid residue at position 35 of SEQ ID Nos: 51 or 52 may be mutated to an alanine.
  • Due to the high alkaline stability of the alkali-stabilized Protein A domains, the separation matrix can tolerate extensive cleaning and sanitization procedures, even using concentrated alkali metal hydroxide solutions, without excessive loss of binding capacity. For example, the separation matrix may retain at least 80% of its original dynamic binding capacity after cleaning and/or sanitization.
  • The cleaning liquid comprises at least 50% by volume of an aqueous alkali metal hydroxide solution. The aqueous alkali metal hydroxide solution may comprise a single alkali metal hydroxide or a mixture of alkali metal hydroxides, such as sodium hydroxide, potassium hydroxide, or a mixture of sodium hydroxide and potassium hydroxide. The aqueous alkali metal hydroxide solution may have a molarity of from 500 mM to 5 M, such as from 1 M to 2 M, expressed as the total combined concentration of alkali metal hydroxides if a mixture of alkali metal hydroxides is used. The cleaning liquid may essentially consist of, or consist of, the aqueous alkali metal hydroxide solution. However, the cleaning liquid may in some embodiments also comprise further components. Such further components may include alcohols, such as a C2-C7 alcohol, such as ethanol, isopropanol or benzoic alcohol. Such further components may include salts, such as sodium chloride. The use of alcohols and/or salts in the cleaning liquid may increase the efficacy of the cleaning liquid in inhibiting or inactivating certain microorganisms, such as spore-forming bacteria.
  • Non-limiting examples of cleaning liquids include:
      • Sodium hydroxide solution (0.5 M, 1 M, 2 M or 5 M);
      • Potassium hydroxide solution (0.5 M, 1 M, 2 M or 5 M);
      • Sodium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 10-20% by volume ethanol;
      • Sodium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 10-50% by volume isopropanol;
      • Sodium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 1-5% by volume benzyl alcohol;
      • Potassium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 10-20% by volume ethanol;
      • Potassium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 10-50% by volume isopropanol; or
      • Potassium hydroxide solution (0.5 M, 1 M, 2 M or 5 M) with 1-5% by volume benzyl alcohol.
  • The use of relatively concentrated alkali metal hydroxide solution, such as 0.5 M-5 M solution, provides exceptional removal of matrix contaminants such as proteins and nucleic acids, as well as quickly and effectively inactivating viruses, bacteria, yeast and fungi. A commonly applied measure of sanitization efficacy is a 6-log10 reduction in the microorganism or contaminant being measured. Sanitizing with concentrated alkali metal hydroxide solution is capable of providing a 6-log10 reduction in most microorganisms and endotoxins.
  • During cleaning of the separation matrix, the cleaning liquid should be passed through the separation matrix with a suitable flow rate, in order to flush contaminants from the matrix. During sanitization, the cleaning liquid is initially passed through the separation matrix with a suitable flow rate until the separation matrix is fully permeated with the cleaning liquid. The cleaning liquid is preferably passed through the separation matrix with a suitable flow rate for the entire duration of the sanitization step. However, if desired the flow of cleaning liquid through the separation matrix may be stopped once the separation matrix is fully permeated with the cleaning liquid. This reduces the volume of cleaning fluid required to perform a sanitization step, but provides a poorer cleaning of the separation matrix as compared to a procedure where a constant flow of cleaning fluid is applied.
  • Because aqueous alkali metal hydroxide solutions are so effective in removing contaminants from the separation matrix, the separation matrix may be used as a purification platform for the purification of a variety of immunoglobulins, with a lower risk of host cell protein contamination or carryover. After purification of a first immunoglobulin product, the separation matrix is first cleaned and sanitized thoroughly using a cleaning liquid comprising aqueous alkali metal hydroxide solution prior to use in purification of the second immunoglobulin product.
    • EXAMPLES
  • Mutagenesis of Protein
  • Site-directed mutagenesis was performed by a two-step PCR using oligonucleotides coding for the mutations. As template a plasmid containing a single domain of either Z, B or C was used. The PCR fragments were ligated into an E. coli expression vector. DNA sequencing was used to verify the correct sequence of inserted fragments.
  • To form multimers of mutants an Acc I site located in the starting codons (GTA GAC) of the B, C or Z domain was used, corresponding to amino acids VD. The vector for the monomeric domain was digested with Acc I and phosphatase treated. Acc I sticky-ends primers were designed, specific for each variant, and two overlapping PCR products were generated from each template. The PCR products were purified and the concentration was estimated by comparing the PCR products on a 2% agarose gel. Equal amounts of the pair wise PCR products were hybridized (90° C.→25° C. in 45 min) in ligation buffer. The resulting product consists approximately to ¼ of fragments likely to be ligated into an Acc I site (correct PCR fragments and/or the digested vector). After ligation and transformation colonies were PCR screened to identify constructs containing the desired mutant. Positive clones were verified by DNA sequencing.
  • Construct Expression and Purification
  • The constructs were expressed in the bacterial periplasm by fermentation of E. coli K12 in standard media. After fermentation the cells were heat-treated to release the periplasm content into the media. The constructs released into the medium were recovered by microfiltration with a membrane having a 0.2 μm pore size.
  • Each construct, now in the permeate from the filtration step, was purified by affinity. The permeate was loaded onto a chromatography medium containing immobilized IgG (IgG Sepharose 6FF, GE Healthcare). The loaded product was washed with phosphate buffered saline and eluted by lowering the pH.
  • The elution pool was adjusted to a neutral pH (pH 8) and reduced by addition of dithiothreitol. The sample was then loaded onto an anion exchanger. After a wash step the construct was eluted in a NaCl gradient to separate it from any contaminants. The elution pool was concentrated by ultrafiltration to 40-50 mg/ml. It should be noted that the successful affinity purification of a construct on an immobilized IgG medium indicates that the construct in question has a high affinity to IgG.
  • The purified ligands were analyzed with RPC LC-MS to determine the purity and to ascertain that the molecular weight corresponded to the expected (based on the amino acid sequence).
    • Example 1
  • The purified monomeric ligands listed in Table 1, further comprising for SEQ ID Nos: 8-16, 23-28 and 36-48 an AQGT leader sequence at the N-terminus and a cysteine at the C terminus, were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore surface plasmon resonance (SPR) instrument (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface 1 mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal strength (proportional to the amount of binding) was noted. The surface was then cleaned-in-place (CIP), i.e. flushed with 500 mM NaOH for 10 minutes at room temperature (22+/−2° C.). This was repeated for 96-100 cycles and the immobilized ligand alkaline stability was followed as the remaining IgG binding capacity (signal strength) after each cycle. The results are shown in Table 1 and indicate that at least the ligands Zvar(N11K)1, Zvar(N11E)1, Zvar(N11Y)1, Zvar(N11T)1, Zvar(N11F)1, Zvar(N11L)1, Zvar(N11W)1, ZN11I)1, Zvar(N11M)1, Zvar(N11V)1, Zvar(N11A)1, Zvar(N11H1), Zvar(N11R)1, Zvar(N11E, Q32A)1, Zvar(N11E, Q32E, Q40E)1 and Zvar(N11E, Q32E, K50R)1, Zvar(Q9A, N11E, N43A)1, Zvar(Q9A, N11E, N28A, N43A)1, Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)1, Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1, Zvar(Q9A, N11E, N28A, Q40V, A42K, N43A, L44I)1, Zvar(N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y)1, Zvar(Q9A, N11K, H18K, S33K, D37E, A42R, N43A, L44I, K50R, L51Y)1, Zvar(N11K, H18K, D37E, A42R, N43A, L44I)1, Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I)1 and Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, L44I, K50R)1, as well as the varieties of Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 having G,S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K in position 29, the varieties of Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 having F,Y,W,K or R in position 53 and the varieties of Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 where Q9, Q40, A42 or N43 has been deleted, have an improved alkali stability compared to the parental structure Zvar1, used as the reference. Further, the ligands B(Q9A, N11E, Q40V, A42K, N43A, L44I)1 and C(Q9A, N11E, E43A)1 have an improved stability compared to the parental B and C domains, used as references.
  • TABLE 1
    Monomeric ligands, evaluated by Biacore (0.5 M NaOH).
    Reference
    Capacity capacity Capacity
    after after relative
    Ligand 96-100 96-100 to
    Sequence cycles cycles reference
    Zvar(N11E,Q32A)1 SEQ ID 57% 55% 1.036
    NO: 12
    Zvar(N11E)1 SEQ ID 59% 55% 1.073
    NO: 13
    Zvar(N11E,Q32E,Q40E)1 SEQ ID 52% 51% 1.020
    NO: 14
    Zvar(N11E,Q32E,K50R)1 SEQ ID 53% 51% 1.039
    NO: 15
    Zvar(N11K)1 SEQ ID 62% 49% 1.270
    NO: 16
    Zvar(N11Y)1 SEQ ID 55% 46% 1.20
    NO: 38
    Zvar(N11T)1 SEQ ID 50% 46% 1.09
    NO: 39
    Zvar(N11F)1 SEQ ID 55% 46% 1.20
    NO: 40
    Zvar(N11L)1 SEQ ID 57% 47% 1.21
    NO: 41
    Zvar(N11W)1 SEQ ID 57% 47% 1.21
    NO: 42
    Zvar(N11I)1 SEQ ID 57% 47% 1.21
    NO: 43
    Zvar(N11M)1 SEQ ID 58% 46% 1.26
    NO: 44
    Zvar(N11V)1 SEQ ID 56% 46% 1.22
    NO: 45
    Zvar(N11A)1 SEQ ID 58% 46% 1.26
    NO: 46
    Zvar(N11H)1 SEQ ID 57% 46% 1.24
    NO: 47
    Zvar(N11R)1 SEQ ID 59% 46% 1.28
    NO: 48
    Zvar(Q9A,N11E,N43A)1 SEQ ID 70% 47% 1.49
    NO: 8
    Zvar(Q9A,N11E,N28A,N43A)1 SEQ ID 68% 47% 1.45
    NO: 9
    Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)1 SEQ ID 67% 47% 1.43
    NO: 10
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID 66% 47% 1.40
    NO: 11
    Zvar(Q9A,N11E,N28A,Q40V,A42K,N43A,LA4I)1 SEQ ID 65% 48% 1.35
    NO: 24
    Zvar(N11K,H18K,S33K,D37E,A42R,N43A,LA4I,K50R,L51Y)1 SEQ ID 67% 46% 1.46
    NO: 23
    Zvar(Q9A,N11K,H18K,S33K,D37E,A42R,N43A,L44I,K50R,L51Y)1 SEQ ID 59% 46% 1.28
    NO: 25
    Zvar(N11K, H18K, D37E, A42R, N43A, L44I)1 SEQ ID 59% 45% 1.31
    NO: 26
    Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, LA4I)1 SEQ ID 63% 45% 1.40
    NO: 27
    Zvar(Q9A, N11K, H18K, D37E, A42R, N43A, LA4I, K50R)1 SEQ ID 67% 45% 1.49
    NO: 28
    B(Q9A,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID 39% 35% 1.11
    NO: 36
    C(Q9A,N11E,E43A)1 SEQ ID 60% 49% 1.22
    NO: 37
    Zvar(Q9A,N11E,A29G,Q40V,A42K,N43A,L44I)1 SEQ ID 69% 48% 1.44
    NO: 54
    Zvar(Q9A,N11E,A29S,Q40V,A42K,N43A,L44I)1 SEQ ID 66% 48% 1.38
    NO: 55
    Zvar(Q9A,N11E,A29Y,Q40V,A42K,N43A,L44I)1 SEQ ID 61% 48% 1.27
    NO: 56
    Zvar(Q9A,N11E,A29Q,Q40V,A42K,N43A,L44I)1 SEQ ID 60% 47% 1.28
    NO: 57
    Zvar(Q9A,N11E,A29T,Q40V,A42K,N43A,L44I)1 SEQ ID 60% 47% 1.28
    NO: 58
    Zvar(Q9A,N11E,A29N,Q40V,A42K,N43A,L44I)1 SEQ ID 61% 47% 1.30
    NO: 59
    Zvar(Q9A,N11E,A29F,Q40V,A42K,N43A,L44I)1 SEQ ID 62% 46% 1.35
    NO: 60
    Zvar(Q9A,N11E,A29L,Q40V,A42K,N43A,L44I)1 SEQ ID 61% 46% 1.33
    NO: 61
    Zvar(Q9A,N11E,A29W,Q40V,A42K,N43A,L44I)1 SEQ ID 60% 46% 1.30
    NO: 62
    Zvar(Q9A,N11E,A29I,Q40V,A42K,N43A,L44I)1 SEQ ID 58% 47% 1.23
    NO: 63
    Zvar(Q9A,N11E,A29M,Q40V,A42K,N43A,L44I)1 SEQ ID 62% 47% 1.32
    NO: 64
    Zvar(Q9A,N11E,A29V,Q40V,A42K,N43A,L44I)1 SEQ ID 62% 47% 1.32
    NO: 65
    Zvar(Q9A,N11E,A29D,Q40V,A42K,N43A,LA4I)1 SEQ ID 56% 47% 1.19
    NO: 66
    Zvar(Q9A,N11E,A29E,Q40V,A42K,N43A,L44I)1 SEQ ID 57% 47% 1.21
    NO: 67
    Zvar(Q9A,N11E,A29H,Q40V,A42K,N43A,L44I)1 SEQ ID 57% 47% 1.21
    NO: 68
    Zvar(Q9A,N11E,A29R,Q40V,A42K,N43A,L44I)1 SEQ ID 58% 46% 1.26
    NO: 69
    Zvar(Q9A,N11E,A29K,Q40V,A42K,N43A,L44I)1 SEQ ID 59% 46% 1.28
    NO: 70
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53F)1 SEQ ID 58% 46% 1.26
    NO: 71
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53Y)1 SEQ ID 59% 46% 1.28
    NO: 72
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53W)1 SEQ ID 62% 46% 1.35
    NO: 73
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53K)1 SEQ ID 65% 46% 1.41
    NO: 74
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I,D53R)1 SEQ ID 60% 46% 1.30
    NO: 75
    Zvar(Q9del,N11E,Q40V,A42K,N43A,L44I)1 SEQ ID 60% 46% 1.30
    NO: 76
    Zvar(Q9A,N11E,Q40del,A42K,N43A,L44I)1 SEQ ID 59% 46% 1.28
    NO: 77
    Zvar(Q9A,N11E,Q40V,A42del,N43A,L44I)1 SEQ ID 57% 46% 1.24
    NO: 78
    Zvar(Q9A,N11E,Q40V,A42K,N43del,L44I)1 SEQ ID 55% 46% 1.20
    NO: 79
  • The Biacore experiment can also be used to determine the binding and dissociation rates between the ligand and IgG. This was used with the set-up as described above and with an IgG1 monoclonal antibody as probe molecule. For the reference Zvar1, the on-rate (105 M−1s−1) was 3.1 and the off-rate (105 s−1) was 22.1, giving an affinity (off-rate/on-rate) of 713 pM. For Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)1 (SEQ ID NO: 11), the on-rate was 4.1 and the off-rate 43.7, with affinity 1070 pM. The IgG affinity was thus somewhat higher for the mutated variant.
    • Example 2
  • The purified dimeric, tetrameric and hexameric ligands listed in Table 2 were immobilized on Biacore CM5 sensor chips (GE Healthcare, Sweden), using the amine coupling kit of GE Healthcare (for carbodiimide coupling of amines on the carboxymethyl groups on the chip) in an amount sufficient to give a signal strength of about 200-1500 RU in a Biacore instrument (GE Healthcare, Sweden). To follow the IgG binding capacity of the immobilized surface 1 mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and the signal strength (proportional to the amount of binding) was noted. The surface was then cleaned-in-place (CIP), i.e. flushed with 500 mM NaOH for 10 minutes at room temperature (22+/−2° C.). This was repeated for 300 cycles and the immobilized ligand alkaline stability was followed as the remaining IgG binding capacity (signal strength) after each cycle. The results are shown in Table 2 and in FIG. 2 and indicate that at least the ligands Zvar(Q9A, N11E, N43A)4, Zvar(Q9A, N11E, N28A, N43A)4, Zvar(Q9A, N11E, Q40V, A42K, N43E, L44I)4 and Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4, Zvar(Q9A, N11E, D37E, Q40V, A42K, N43A, L44I)4 and Zvar(Q9A, N11E, D37E, Q40V, A42R, N43A, L44I)4 have an improved alkali stability compared to the parental structure Zvar4, which was used as a reference. The hexameric ligand Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)6 also has improved alkali stability compared to the parental structure Zvar6, used as a reference. Further, Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I) dimers with deletions of a) D2, A3, K4; b) K58, V1, D2; c) P57, K58, V1, D2, A3; d) K4, F5, D6, K7, E8; e) A56, P57, K58; V1, D2, A3 or f) V1, D2, A3, K4, F5, D6, K7, E8 from the linker region between the two monomer units have improved alkali stability compared to the parental structure Zvar2, used as a reference. Also Zvar(Q9A, NE, Q4V, A42K, N43A, L44I) dimers with an insertion of YEDG between K58 and V1 in the linker region have improved alkali stability compared to Zvar2.
  • TABLE 2
    Dimeric, tetrameric and hexameric ligands, evaluated by Biacore (0.5M NaOH).
    Remaining Capacity Remaining Capacity Remaining Capacity
    capacity relative capacity relative capacity relative
    SEQ
    100 to ref. 200 to ref. 300 to ref.
    ID cycles 100 cycles 200 cycles 300
    Ligand NO: (%) cycles (%) cycles (%) cycles
    Zvar4 21 67 1 36 1 16 1
    Zvar(Q9A, N11E, N43A)4 17 81 1.21 62 1.72 41 2.56
    Zvar(Q9A, N11E, N28A, 18 80 1.19 62 1.72 42 2.62
    N43A)4
    Zvar(Q9A, N11E, Q40V, 19 84 1.25 65 1.81 48 3.00
    A42K, N43E, L44I)4
    Zvar(Q9A, N11E, Q40V, 20 90 1.34 74 2.06 57 3.56
    A42K, N43A, LA41)4
    Zvar(Q9A, N11E, N28A, 32 84 1.24 Not tested Not Not tested Not
    Q40V, A42K, N43A, L44I)4 tested tested
    Zvar(Q9A, N11E, Q40V, 33 87 1.30 Not tested Not Not tested Not
    A42K, N43A, L44I)6 tested tested
    Zvar(Q9A, N11E, D37E, 34 81 1.13 Not tested Not Not tested Not
    Q40V, A42K, N43A, L44I)4 tested tested
    Zvar(Q9A, N11E, D37E, 35 84 1.17 Not tested Not Not tested Not
    Q40V, A42R, N43A, LA4I)4 tested tested
    Zvar(Q9A, N11E, Q40V, 80 70 1.27 Not tested Not Not tested Not
    A42K, N43A, LA4I)2 with tested tested
    D2, A3 and K4 in linker
    deleted
    Zvar(Q9A, N11E, Q40V, 81 76 1.38 Not tested Not Not tested Not
    A42K, N43A, L44I)2 with tested tested
    K58, V1 and D2 in linker
    deleted
    Zvar(Q9A, N11E, Q40V, 82 74 1.35 Not tested Not Not tested Not
    A42K, N43A, L44I)2 with tested tested
    P57, K58, V1, D2 and A3
    in linker deleted
    Zvar(Q9A, N11E, Q40V, 83 70 1.30 Not tested Not Not tested Not
    A42K, N43A, L44I)2 with tested tested
    K4, F5, D6, K7 and E8 in
    linker deleted
    Zvar(Q9A, N11E, Q40V, 84 68 1.26 Not tested Not Not tested Not
    A42K, N43A, LA4I)2 with tested tested
    A56, P57 and K58 in
    linker deleted
    Zvar(Q9A, N11E, Q40V, 85 75 1.39 Not tested Not Not tested Not
    A42K, N43A, L44I)2 with tested tested
    V1, D2 and A3 in linker
    deleted
    Zvar(Q9A, N11E, Q40V, 86 62 1.13 Not tested Not Not tested Not
    A42K, N43A, LA4I)2 with tested tested
    V1, D2, A3, K4, F5, D6,
    K7 and E8 in linker
    deleted
    Zvar(Q9A, N11E, Q40V, 87 72 1.31 Not tested Not Not tested Not
    A42K, N43A, L44I)2 with tested tested
    YEDG inserted in linker
    between K58 and V1
    Zvar2 88 55 1 Not tested Not Not tested Not
    tested tested
    • Example 3
  • Example 2 was repeated with 100 CIP cycles of three ligands using 1 M NaOH instead of 500 mM as in Example 2. The results are shown in Table 3 and show that all three ligands have an improved alkali stability also in 1M NaOH, compared to the parental structure Zvar4 which was used as a reference.
  • TABLE 3
    Tetrameric ligands, evaluated by Biacore (1M NaOH).
    Remaining Capacity
    capacity relative to
    Ligand Sequence 100 cycles (%) ref. 100 cycles
    Zvar4 SEQ ID NO: 21 27 1
    Zvar(Q9A,N11E,N28A,N43A)4 SEQ ID NO: 18 55 2.04
    Zvar(Q9A,N11E,Q40V,A42K,N43E,L44I)4 SEQ ID NO: 19 54 2.00
    Zvar(Q9A,N11E,Q40V,A42K,N43A,L44I)4 SEQ ID NO: 20 56 2.07
    • Example 4
  • The purified tetrameric ligands of Table 2 (all with an additional N-terminal cysteine) were immobilized on agarose beads using the methods described below and assessed for capacity and stability. The results are shown in Table 4 and FIG. 3 .
  • TABLE 4
    Matrices with tetrametric ligands, evaluated in columns (0.5M NaOH).
    Remaining
    IgG Remaining Capacity
    capacity IgG retention
    Initial Qb10 capacity relative
    IgG after after to ref.
    SEQ Ligand capacity six 4 h six 4 h after
    ID content Qb10 cycles cycles six 4 h
    Ligand NO. (mg/ml) (mg/ml) (mg/ml) (%) cycles
    Zvar4 21 7 52.5 36.5 60 1
    Zvar4 21 12 61.1 43.4 71 1
    Zvar(Q9A, N11E, N28A, N43A)4 18 7.0 49.1 44.1 90 1.50
    Zvar(Q9A, N11E, N28A, N43A)4 18 12.1 50.0 46.2 93 1.31
    Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4 20 7.2 49.0 44.2 90 1.50
    Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4 20 12.8 56.3 53.6 95 1.34
    Zvar(N11K, H18K, S33K, D37E, A42R, N43A, 30 9.7 56.3 52.0 92 1.53
    L44I, K50R, L51Y)4
    Zvar(Q9A, N11K, H18K, D37E, A42R)4 31 10.8 56.9 52.5 92 1.30
  • Activation
  • The base matrix used was rigid cross-linked agarose beads of 85 micrometers (volume-weighted, d50V) median diameter, prepared according to the methods of U.S. Pat. No. 6,602,990, hereby incorporated by reference in its entirety, and with a pore size corresponding to an inverse gel filtration chromatography Kav value of 0.70 for dextran of Mw 110 kDa, according to the methods described in Gel Filtration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.
  • 25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 g NaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 min at 25° C. 4.0 mL of epichlorohydrin was added and the reaction progressed for 2 hours. The activated gel was washed with 10 gel sediment volumes (GV) of water.
  • Coupling
  • To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mg NaHCO3, 21 mg Na2CO3, 175 mg NaCl and 7 mg EDTA, was added. The Falcon tube was placed on a roller table for 5-10 min, and then 77 mg of DTE was added. Reduction proceeded for >45 min. The ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.
  • The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH 8.6} and the ligand was then coupled according to the method described in U.S. Pat. No. 6,399,750, hereby incorporated by reference in its entirety. All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels could be controlled by varying the amount and concentration of the ligand solution.
  • After immobilization the gels were washed 3×GV with distilled water. The gels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3×GV {0.1 M TRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilled water. Gel samples were sent to an external laboratory for amino acid analysis and the ligand content (mg/ml gel) was calculated from the total amino acid content.
  • Protein

  • Gammanorm 165 mg/ml (Octapharma), diluted to 2 mg/ml in Equilibration buffer.

  • Equilibration Buffer

  • PBS Phosphate buffer 10 mM+0.14 M NaCl+0.0027 M KCl, pH 7,4 (Medicago)

  • Adsorption Buffer

  • PBS Phosphate buffer 10 mM+0.14 M NaCl+0.0027 M KCl, pH 7,4 (Medicago)

  • Elution Buffers

  • 100 mM acetate pH 2.9
  • Dynamic Binding Capacity
  • 2 ml of resin was packed in TRICORN™ 5 100 columns. The breakthrough capacity was determined with an ÄKTAExplorer 10 system at a residence time of 6 minutes (0.33 ml/min flow rate). Equilibration buffer was run through the bypass column until a stable baseline was obtained. This was done prior to auto zeroing. Sample was applied to the column until a 100% UV signal was obtained. Then, equilibration buffer was applied again until a stable baseline was obtained.
  • Sample was loaded onto the column until a UV signal of 85% of maximum absorbance was reached. The column was then washed with 5 column volumes (CV) equilibration buffer at flow rate 0.5 ml/min. The protein was eluted with 5 CV elution buffer at a flow rate of 0.5 ml/min. Then the column was cleaned with 0.5M NaOH at flow rate 0.2 ml/min and re-equilibrated with equilibration buffer.
  • For calculation of breakthrough capacity at 10%, the equation below was used. That is the amount of IgG that is loaded onto the column until the concentration of IgG in the column effluent is 10% of the IgG concentration in the feed.
  • q 10 ⁢ % = C 0 V C [ V app - V sys - ∫ V sys V app A ⁡ ( V ) - A sub A 100 ⁢ % - A sub * dv ]
      • A100%=100% UV signal;
      • sub=absorbance contribution from non-binding IgG subclass;
      • A(V)=absorbance at a given applied volume;
      • Vc=column volume;
      • Vapp=volume applied until 10% breakthrough;
      • Vsys=system dead volume;
      • 0=feed concentration.
  • The dynamic binding capacity (DBC) at 10% breakthrough was calculated. The dynamic binding capacity (DBC) was calculated for 10 and 80% breakthrough.
  • CIP—0.5 M Naoh
  • The 10% breakthrough DBC (Qb10) was determined both before and after repeated exposures to alkaline cleaning solutions. Each cycle included a CIP step with 0.5 M NaOH pumped through the column at a rate of 0.5/min for 20 min, after which the column was left standing for 4 h. The exposure took place at room temperature (22+/−2° C.). After this incubation, the column was washed with equilibration buffer for 20 min at a flow rate of 0.5 ml/min. Table 4 shows the remaining capacity after six 4 h cycles (i.e. 24 h cumulative exposure time to 0.5 M NaOH), both in absolute numbers and relative to the initial capacity.
    • Example 5
  • Example 4 was repeated with the tetrameric ligands shown in Table 5, but with 1.0 M NaOH used in the CIP steps instead of 0.5 M. The results are shown in Table 5 and in FIG. 4 .
  • TABLE 5
    Matrices with tetrametric ligands, evaluated in columns-1.0M NaOH.
    Remaining
    IgG Remaining Capacity
    capacity IgG retention
    Initial Qb10 capacity relative
    IgG after after to ref.
    SEQ Ligand capacity six 4 h six 4 h after
    ID content Qb10 cycles cycles six 4 h
    Ligand NO. (mg/ml) (mg/ml) (mg/ml) (%) cycles
    Zvar4 21 12 60.1 33.5 56 1
    Zvar(Q9A, N11E, Q40V, A42K, N43A, L44I)4 20 12.8 60.3 56.0 93 1.67
    Zvar(N11K, H18K, S33K, D37E, A42R, N43A, 30 9.7 62.1 48.1 77 1.44
    L44I, K50R, L51Y)4
    • Example 6
  • Base Matrices
  • The base matrices used were a set of rigid cross-linked agarose bead samples of 59-93 micrometers (volume-weighted, d50V) median diameter (determined on a Malvern Mastersizer 2000 laser diffraction instrument), prepared according to the methods of U.S. Pat. No. 6,602,990 and with a pore size corresponding to an inverse gel filtration chromatography Kd value of 0.62-0.82 for dextran of Mw 110 kDa, according to the methods described above, using HR10/30 columns (GE Healthcare) packed with the prototypes in 0.2 M NaCl and with a range of dextran fractions as probe molecules (flow rate 0.2 ml/min). The dry weight of the bead samples ranged from 53 to 86 mg/ml, as determined by drying 1.0 ml drained filter cake samples at 105° C. overnight and weighing.
  • TABLE 6
    Base matrix samples
    Base d50v Dry weight
    matrix Kd (μm) (mg/ml)
    A18 0.704 59.0 56.0
    A20 0.70 69.2 55.8
    A27 0.633 87.2 74.2
    A28 0.638 67.4 70.2
    A29 0.655 92.6 57.5
    A32 0.654 73.0 70.5
    A33 0.760 73.1 55.5
    A38 0.657 70.9 56.2
    A39 0.654 66.0 79.1
    A40 0.687 64.9 74.9
    A41 0.708 81.7 67.0
    A42 0.638 88.0 59.4
    A43 0.689 87.5 77.0
    A45 0.670 56.6 66.0
    A52 0.620 53.10 63.70
    A53 0.630 52.6 86.0
    A54 0.670 61.3 75.3
    A55 0.640 62.0 69.6
    A56 0.740 61.0 56.0
    A56-2 0.740 51.0 56.0
    A62a 0.788 48.8 70.1
    A62b 0.823 50.0 46.9
    A63a 0.790 66.8 59.6
    A63b 0.765 54.0 79.0
    A65a 0.796 58.0 60.0
    A65b 0.805 57.3 46.0
    B5 0.793 69.0 84.4
    C1 0.699 71.0 73.4
    C2 0.642 66.5 81.1
    C3 0.711 62.0 82.0
    C4 0.760 62.0 82.0
    H31 0.717 82.0 59.0
    H35 0.710 81.1 61.0
    H40 0.650 52.8 65.0
    I1 0.640 50.0 67.0
    41 0.702 81.6 60.6
  • Coupling
  • 100 ml base matrix was washed with 10 gel volumes distilled water on a glass filter. The gel was weighed (1 g=1 ml) and mixed with 30 ml distilled water and 8.08 g NaOH (0.202 mol) in a 250 ml flask with an agitator. The temperature was adjusted to 27+/−2° C. in a water bath. 16 ml epichlorohydrin (0.202 mol) was added under vigorous agitation (about 250 rpm) during 90+/−10 minutes. The reaction was allowed to continue for another 80+/−10 minutes and the gel was then washed with >10 gel volumes distilled water on a glass filter until neutral pH was reached. This activated gel was used directly for coupling as below.
  • To 16.4 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 139 mg NaHCO3, 17.4 mg Na2CO3, 143.8 mg NaCl and 141 mg EDTA, was added. The Falcon tube was placed on a roller table for 5-10 min, and then 63 mg of DTE was added. Reduction proceeded for >45 min. The ligand solution was then desalted on a PD10 column packed with Sephadex G-25. The ligand content in the desalted solution was determined by measuring the 276 nm UV absorption.
  • The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH 8.6} and the ligand was then coupled according to the method described in U.S. Pat. No. 6,399,750 5.2.2, although with considerably higher ligand amounts (see below). All buffers used in the experiments had been degassed by nitrogen gas for at least 5-10 min. The ligand content of the gels was controlled by varying the amount and concentration of the ligand solution, adding 5-20 mg ligand per ml gel. The ligand was either a tetramer (SEQ ID NO: 20) or a hexamer (SEQ ID NO: 33) of an alkali-stabilized mutant.
  • After immobilization the gels were washed 3×GV with distilled water. The gels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixed and the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3×GV {0.1 M TRIS/0.15 M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilled water. Gel samples were sent to an external laboratory for amino acid analysis and the ligand content (mg/ml gel) was calculated from the total amino acid content.
  • Evaluation
  • The Qb10% dynamic capacity for polyclonal human IgG at 2.4 and 6 min residence time was determined as outlined in Example 4.
  • TABLE 7
    Prototype results
    Ligand Qb10% Qb10%
    Base content 2.4 min 6 min
    Prototype matrix (mg/ml) Multimer (mg/ml) (mg/ml)
    N1 A38 7.45 tetramer 44.4 58.25
    N2 A20 7.3 tetramer 45.12 57.21
    N3 A42 6.72 tetramer 33.56 50.02
    N4 A29 7.3 tetramer 36.34 51.8
    N5 A28 7.9 tetramer 42.38 58.25
    N6 A39 6.96 tetramer 41.88 54.67
    N7 A27 7.5 tetramer 29.19 48.73
    N8 A43 6.99 tetramer 33.43 49.79
    N9 A38 11.34 tetramer 48.1 72.78
    N10 A20 10.6 tetramer 50.66 70.07
    N11 A42 11.1 tetramer 32.25 57.78
    N12 A29 11 tetramer 34.85 64.68
    N13 A28 11.9 tetramer 39.92 63.75
    N14 A39 10.48 tetramer 44.37 64.79
    N15 A27 12.1 tetramer 24.8 55.56
    N16 A43 10.51 tetramer 31.82 58.04
    N17 A41 8.83 tetramer 38.5 56.8
    N18 A41 8.83 tetramer 37.84 58.6
    N19 A41 8.83 tetramer 35.06 57.23
    N20 A41 5.0 tetramer 35.64 46.04
    N21 A41 13.0 tetramer 34.95 62.23
    N22 A40 13.15 tetramer 56.85 71.09
    N23 A33 7.33 tetramer 48.69 55.76
    N24 A40 11.03 tetramer 54.96 73.8
    033A A38 7.5 tetramer 44 58
    033B A38 11.3 tetramer 48 73
    097A A20 7.3 tetramer 45 57
    097B A20 10.6 tetramer 51 70
    003A A28 7.9 tetramer 42 58
    003B A28 11.9 tetramer 40 64
    003C A28 15.8 tetramer 37 67
    038A A39 7.0 tetramer 42 55
    038B A39 10.5 tetramer 44 65
    074 A40 13.2 tetramer 57 71
    093 A33 7.3 tetramer 49 56
    058A A40 11.0 tetramer 55 74
    077 A18 8.2 tetramer 52 59
    010 A32 10.7 tetramer 40 57
    099 A32 13.3 tetramer 37 66
    030A B5 6.3 tetramer 32 38
    030B B5 9.6 tetramer 45 47
    293A C1 5.4 tetramer 38 47
    293B C1 10.8 tetramer 43 60
    294A C2 5.1 tetramer 39 46
    294B C2 10.5 tetramer 42 57
    336A H40 5.6 tetramer 47 52
    336B H40 9.1 tetramer 52 67
    091 A18 13.4 tetramer N/A 63
    092 A20 12.8 tetramer 49 67
    080 A33 9.4 tetramer 51 58
    089 A40 6.1 tetramer 49 59
    688A A62a 6.6 tetramer 41 46
    688B A62a 14.8 tetramer 55 62
    871 A62a 9.7 tetramer 48 60
    934A A63a 6.6 tetramer 40 44
    934B A63a 14.0 tetramer 48 56
    017B A65a 13.1 tetramer 56 64
    041A A62b 5.2 tetramer 40 N/A
    041B A62b 11.1 tetramer 52 N/A
    116A A65b 5.8 tetramer 42 46
    116B A65b 8.8 tetramer 49 56
    017A A65a 6.1 tetramer 40 44
    387A A62a 6.4 tetramer 43 45
    387B A62a 7.5 tetramer 47 56
    432 A63a 6.1 tetramer 39 44
    433A A65a 6.6 tetramer 42 47
    433B A65a 13.6 tetramer 52 61
    579A I1 6.1 tetramer 45 51
    579B I1 11.2 tetramer 57 68
    064A C3 5.9 tetramer 44 52
    064B C3 9.0 tetramer 49 62
    064C C3 14.3 tetramer 51 70
    352A C4 10.1 tetramer 55 63
    352B C4 14.4 tetramer 59 67
    066A C3 6.8 hexamer 48 59
    066B C3 11.9 hexamer 51 73
    066C C3 15.1 hexamer 43 61
    353A C4 11.2 hexamer 62 74
    353B C4 15.2 hexamer 57 82
    872A A62a 9.6 hexamer 56 72
    872B A62a 14.5 hexamer 62 84
    869A H40 6.9 hexamer 50 56
    869B H40 14.3 hexamer 56 75
    869C H40 23.0 hexamer 41 65
    962A H35 6.8 hexamer 36 49
    962B H35 12.3 hexamer 31 54
    962C H35 20.3 hexamer 20 43
    112A A56 7.9 hexamer 47 55
    112B A56 12.4 hexamer 57 73
    112C A56 19.2 hexamer 55 80
    113A A56 7.1 hexamer 48 57
    113B A56 12.4 hexamer 53 73
    113C A56 15.2 hexamer 48 76
    212A H31 6.5 hexamer 37 38
    212B H31 10.4 hexamer 50 61
    212C H31 20.0 hexamer 31 52
    213A A33 6.5 hexamer 44 53
    213B A33 10.9 hexamer 50 65
    213C A33 11.1 hexamer 50 68
    432A A20 6.4 hexamer 41 56
    432B A20 12.4 hexamer 38 64
    432C A20 21.1 hexamer 44 43
    433A A38 5.9 hexamer 47 57
    433B A38 11.6 hexamer 48 72
    433C A38 15.8 hexamer 36 62
    742A A54 6.7 hexamer 38 46
    742B A54 12.6 hexamer 45 52
    742C A54 21.1 hexamer 38 65
    726A A63b 6.4 hexamer 42 46
    726B A63b 10.6 hexamer 49 60
    726C A63b 16.7 hexamer 53 69
    793A A56-2 6.8 hexamer 50 58
    793B A56-2 12.5 hexamer 59 72
    793C A56-2 19.2 hexamer 61 82
    • Example 7
  • A series of prototypes, prepared as above, with different ligand content (tetramer, SEQ ID NO: 20) were incubated in 1 M NaOH for 4, 8 and 31 hours at 22+/−2° C. and the dynamic IgG capacity (Qb10%, 6 min residence time) was measured before and after incubation. The prototypes are shown in Table 8 and the results in FIGS. 5 and 6 . It can be seen that the stability towards this harsh alkali treatment increases with increasing ligand content.
  • TABLE 8
    Samples for incubation in 1 M NaOH
    Qb10%,
    6 min,
    Ligand before
    content incubation
    Prototype (mg/ml) (mg/ml)
    N1 12 78
    LE28 13 79
    N17 16 73
    N16 20 73
    • Example 8
  • Two crosslinked agarose bead prototypes, prepared as above, with different ligand content (hexamer, SEQ ID NO: 33), median bead diameter (d50,v) 62 μm and Kd 0.70 for dextran of Mw 110 kD, were evaluated with a real mAb feed. The ligand content of prototype A was 14.3 mg/ml and of prototype B 18.9 mg/ml. For comparison, the commercial product MabSelect SuRe® LX (GE Healthcare Life Sciences) was used. The resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of 10 cm, giving bed volumes of 2 ml and the columns were shown to have peak asymmetry within the 0.8-1.5 interval. The sample loaded was a clarified CHO cell supernatant with 4.9 mg/ml monoclonal IgG1 antibody at physiological pH and the experimental conditions were as listed below in Table 9 (CV=column volumes, RT=residence time).
  • TABLE 9
    Conditions for evaluation with real feed.
    Equilibration: 3 CV 20 mM phosphate, 150 mM NaCl pH 7.4, RT = 3.4 min
    Sample loading: 43 mg mAb/ml resin, RT = 6 min
    Wash 1: 5 CV 20 mM phosphate, 500 mM NaCl pH 7.4, 1.5 CV at
    RT = 6 min and 3.5 CV at RT = 3.4 min
    Wash 2: 1 CV 50 mM acetate pH 6.0, RT = 3.4 min
    Elution: 3 CV 50 mM acetate pH 3.5, RT = 6 min, peak collected
    between 150 mAU-150 mAU
    Strip: 2 CV 100 mM acetate, RT = 3.4 min
    CIP: 3 CV 0.1 M NaOH, RT = 6 min
    Re-equilibration: 5 CV 20 mM phosphate, 150 mM NaCl pH 7.4, RT = 3.4 min
  • The mAb peak was collected using a UV watch function and the concentration of the mAb was determined by UV measurement at 280 nm (extinction coefficient 1.5). All absorbance detections were performed using a spectrophotometer, including the measurements for the yield calculations.
  • Samples for HCP (host cell protein) analyses were prepared by adding 10% Preservation buffer (0.2 M NaH2PO4*H2O (5.3%), 0.2 M Na2HPO4*12 H2O (94.7%), 0.5 % Tween 20, 1% BSA pH 8) to the samples directly after each run made (e.g. 50 μl preservation buffer to 450 μl sample). The HCP content was measured using commercial anti-CHO antibodies (Cygnus Technologies) and a Gyrolab (Gyros AB, Sweden) work station.
  • The results are presented in Table 10 below and show that the performance of the prototypes is in the same range as for the commercial product. The HCP content in the feed was 331 000 ppm.
  • TABLE 10
    Results from real feed evaluation
    Yield Elution pool HCP in pool
    Resin (%) (CV) (ppm)
    MabSelect SuRe LX 90 1.5 914
    MabSelect SuRe LX 95 1.6 1021
    Prototype A 96 1.3 1076
    Prototype A 95 1.3 1105
    Prototype B 96 1.3 1040
    Prototype B 93 1.3 1104
    • Example 9
  • A crosslinked agarose bead matrix prototype, prepared as above, with 14.5 mg/ml ligand (hexamer, SEQ ID NO: 33), median bead diameter (d50,v) 57.4 μm, Kd 0.72 for dextran of Mw 110 kD and dry weight 70.3 mg/ml, was evaluated for elution pH with two real mAb feeds (mAb1 2.4 g/l and mAb2 4.9 g/l) IgG1, physiological pH, and a sample of polyclonal human IgG (Gammanorm, Octapharma). For comparison, the commercial product MabSelect SuRe® LX (GE Healthcare Life Sciences) was used. The resins were packed in Tricorn columns (GE Healthcare Life Sciences) to bed heights of 10 cm, giving bed volumes of 2 ml and the columns were shown to have peak asymmetry within the 0.8-1.5 interval. The samples loaded were clarified CHO cell supernatants with IgG1 mAbs at physiological pH and the experimental conditions were as listed below in Table 11 (CV=column volumes, RT=residence time).
  • TABLE 11
    Conditions for elution pH evaluation.
    Equilibration: 5 CV 20 mM phosphate, 150 mM NaCl pH 7.4,
    RT = 3.4 min
    Sample loading: 10 mg mAb/ml resin, RT = 6 min
    Wash: 6 CV 20 mM phosphate, 150 mM NaCl pH 7.4,
    RT = 3.4 min
    Elution: 30 CV 100 mM citrate pH 6-3 gradient,
    RT = 6 min
    CIP: 3 CV 0.1 M NaOH, RT = 6 min
    Re-equilibration: 8 CV 20 mM phosphate,
    150 mM NaCl pH 7.4, RT = 3.4 min
  • The results are shown below in Table 12 and indicate that the antibodies elute at similar pH levels as on the reference, although with some individual variation depending on the particular antibody-resin combination.
  • TABLE 12
    Results from elution pH evaluation
    Elution pH
    MabSelect Elution pH
    Sample SuRe LX prototype
    mAb
    1 3.67 3.53
    mAb 2 3.68 3.80
    Polyclonal 4.01 4.24
    IgG (peak 1) (peak 1)
    3.70 3.81
    (peak 2) (peak 2)
  • Fractions from the pH-gradient elution of polyclonal IgG were also analysed with respect to content of IgG1, IgG2 and IgG4, using a Biacore SPR instrument (GE Healthcare Life Sciences) with antibodies against the four different IgG classes immobilized on a CM5 Biacore chip.
  • The chromatograms for polyclonal IgG on the reference and the prototype are shown in FIG. 7 and the IgG class analyses are shown in FIG. 8 . The data show that all three classes bind to both resins in a similar way and that the first peak predominantly contains IgG2, while IgG1 and IgG4 elute mainly in the second peak. The anti-IgG3 antibodies cross-reacted with IgG4, so no reliable results for IgG3 were obtained. IgG3 is generally known to show no or only weak binding to Protein A.
    • Example 10
  • A crosslinked agarose bead matrix prototype, prepared as above, with 12.6 mg/ml ligand (tetramer, SEQ ID NO: 20), 84.9 μm median bead diameter (d50,v), Kd 0.71 for dextran Mw 110 kD and 62.2 mg/ml dry weight, was evaluated with respect to alkali stability, using the commercial product MabSelect SuRe LX as a reference. Tricorn 5 columns packed with the resins to 10 cm bed height were flushed with 3 column volumes of 1 M NaOH. The flow was then stopped for 240 minutes (corresponding to 16 normal CIP cycles of 15 min/cycle) before washing out the NaOH solution by 3 column volumes of PBS buffer. The dynamic binding capacity for polyclonal IgG (Gammanorm, Octapharma) was then measured and the process was repeated with another injection of 1 M NaOH. The dynamic capacity was measured after each 240 min incubation cycle with 1 M NaOH. In the capacity measurements, the columns were equilibrated with PBS buffer before the 2 mg/ml sample was loaded (residence time 6 min) until a UV signal of 85% of maximum absorbance was reached. Then the column was washed with PBS buffer, eluted with 500 mM acetic acid pH 3.0 and re-equilibrated. The dynamic binding capacity at 10% and 80% breakthrough was calculated as described above. The results are shown in FIG. 9 and they show that the prototype was significantly more stable than the commercial product.
    • Example 11
  • A separation matrix according to the invention was tested for tolerance to repeated CIP cycles using 2M NaOH. The inventive example (Inv. Ex.) was compared to the commercial product MabSelect SuRe (MSS) as a reference. The study was performed for 50 cycles using an ÄKTA 4-column periodic counter current (PCC) chromatography setup from GE Healthcare.
  • Each column was packed using a solution of 20% ethanol+0.2 M NaCl, with a flow of 3.5 ml/min for 10 min. The packing tube and top filter were removed and the adaptor was placed on the top of column. Following a further packing flow of 3.5 ml/min for 10 min, the adaptor was adjusted against the bed surface. The packed column volume Vc for the inventive example was 1.04 ml, as compared to 1.02 ml for MabSelect SuRe.
  • The PCC setup was then run using the following conditions:
  •
    Equilibration: 3 column volumes (CV) 20 mM phosphate,
    150 mM NaCl pH 7.4
    Sample loading: mAb 4.28 mg/ml; load until 35% breakthrough DBC;
    RT = 2.4 min
    Wash 1: 1.5 CV 20 mM phosphate,
    500 mM NaCl pH 7.0 200 cm/h + 3.5 CV
    Wash 2: 1 CV 50 mM acetate pH 6.0
    Elution: 3 CV 50 mM acetate pH 3.5
    Strip: 2 CV 100 mM acetic acid pH 2.9
    CIP: 3 CV 2 M NaOH (contact time 15 min)
    Re-equilibration: 10 CV 20 mM phosphate, 150 mM NaCl pH 7.4
  • The results are shown in FIG. 10 , where the load volume to 35% breakthrough dynamic binding capacity of each matrix is normalized relative to its load volume to 35% DBC. It can be seen that the commercial reference MabSelect SuRe (MSS) loses a proportion of its immunoglobulin binding capacity each cycle and after 16 cycles has less than 80% of its original capacity remaining. The inventive example (Inv. Ex.) on the other hand shows little loss in binding capacity and has over 95% of its original binding capacity remaining after 50 CIP cycles in 2 M NaOH, equivalent to a contact time of 750 minutes.
    • Example 12
  • The ligand stability towards 1 M and 2 M NaOH was studied for a separation matrix according to the invention, using confocal microscopy. The inventive example (Inv. Ex.) was compared to the commercial product MabSelect SuRe LX (MSS LX) as a reference. The study was performed as follows:
  • Separation Matrix Preparation
  • Separation matrix gels were washed from their storage solution to water through centrifugation, and a 1:1 gel slurry was obtained. For each gel, 2 ml of the 1:1 slurry in water was added to a 50 mL Falcon tube, centrifuged and decanted. To each tube was added 19 ml of 1 M or 2 M NaOH (2 separation matrices*2 [NaOH] concentrations→4 tubes in total). The gels were incubated at room temperature with shaking on Heidolph shaker (1300 rpm). A sample of 1500 μl of gel slurry was taken after 2, 4, 6, 8, 16, 24 and 32 h of incubation and washed in 2 ml Eppendorf tubes through centrifugation (13000 rpm, Eppendorf centrifuge). The washing steps were as follows:

  • 2×1.8 mL MQ water

  • 1×1.8 mL HAc buffer

  • 2×1.8 mL Tris buffer

  • 2×1.8 mL PBS buffer
  • After final decantation, the gels were resuspended with 75 μl PBS buffer to obtain approximately 1:1 PBS gel slurrys.
  • Preincubation
  • A sample of each gel that had not been incubated in NaOH, i.e. the original 1:1 gel water slurry, was exchanged to give a 1:1 PBS slurry. A 16 μl sample of each gel slurry was added to 500 μl of Cy5-hIgG solution (Cy5-labelled human immunoglobulin G) and mixed end-over-end over the weekend, at room temperature, covered in tin foil.
  • Microsope Settings
  • A Leica SP8 Confocal Microscope was used for the study. Microscope settings were determined using the inventive example and checked using MSS LX in order to make sure saturation was not obtained with either of the gels.
      • Objective: 63×/130 Glyc 21C (Leica)
      • Laser: 638 nm
      • Detector PTM gain: 629.6 V
      • Detector offset: −0.8%
      • Scanning speed: 400 Hz
      • Frame size: 512×512 pixels
      • Frame average: 2
      • Kinetics experiment (Adsorption of hIgG in gels as a function of time)
  • A sample of 15 μl of each NaOH-incubated and washed 1:1 gel slurry was added to 500 μl of Cy5-hIgG, giving a mixture of >300 mg Ab/ml gel. These mixtures were Incubated at room temperature on Heidolph shaker at 1300 rpm. At predetermined times (5, 10, 15, 30, 60, 90, 120, 180 and 240 minutes), 15 μl samples were taken out and imaged using the microscope. The confocal images were integrated and adsorption curves were obtained by calculating the relative fluoresence, Qrel, of each bead, see eq 1.
  • Q rel = F ring / volume · ( r out 3 - r in 3 ) r out 3 ( eq . 1 )
      • where rout and rin denote the outer and inner boundary radius of the fluorescent region of the bead (determined using the line tool in the microscope software, LAS-X) and
  • F ring / volume = F ring 4 3 ⁢ π ⁢ r out 3 ( eq . 2 )
      • where Fring is the total fluoresence of the fluorescent region of the bead, determined using the circle tool in LAS-X.
      • Combining eq 1 and 2 yields eq 3:
  • Q rel = F ring · ( r out 3 - r in 3 ) 4 3 ⁢ π ⁢ r out 6 . ( eq . 3 )
      • This procedure follows from the work presented in A. Ljunglöf, J. Thömmes J. Chromatogr. A 813 (1998) 387-395.
  • Results, Analysis and Conclusions
  • The determined relative fluorescence Qrel of the separation matrices as a function of time after incubation in 1M NaOH are shown as FIGS. 11 a MabSelect SuRe LX) and 11b (Inventive example). It can be seen that MSS LX (FIG. 11 a ) lost approximately 50% of its original binding capacity and reaches saturation faster following incubation for 16 hours in 1 M NaOH, as compared to the reference (no alkali treatment, i.e. 0 hours). Following incubation for 32 hours in 1M NaOH it has lost approximately all Ig-binding capacity. In contrast, the separation matrix of the inventive example (FIG. 11 b ) is stable to incubation in 1 M NaOH and maintains its original binding capacity after 32 hours incubation in 1 M NaOH, within experimental variation.
  • The determined relative fluorescence Qrel of the separation matrices as a function of time after incubation in 2M NaOH are shown as FIGS. 12 a MabSelect SuRe LX) and 12b (Inventive example). It can be seen that the rate of capacity loss for MSS LX (FIG. 12 a ) is accelerated with incubation in 2M NaOH. After incubation for 8 hours approximately 50% of Ig-binding capacity is lost, and following incubation for 16 hours in 2M NaOH, more or less all Ig-binding capacity is lost. In contrast, the inventive example (FIG. 12 b ) is relatively stable to alkali and maintains, at least within experimental variation, its original binding capacity after 16 h or less of incubation in 2 M NaOH. However, it can be seen that after 24 h or more of incubation in 2 M NaOH the inventive example has lost binding capacity, and that approximately 50% of the binding capacity has been lost after 32 h of incubation. Also, saturation is reached faster as the binding capacity is lost, most clearly seen with the 32 h incubated samples.
  • These results demonstrate that the separation matrix of the inventive example is significantly more alkali stable than MabSelect SuRe LX and appears to withstand up to 16 h of 2 M NaOH incubation without significantly affecting mass transport and binding capacity.
    • Example 13
  • The inactivation of Bacillus subtilis spores was investigated using various cleaning liquids according to the invention. Spore-forming Bacillus subtilis (ATCC No. 6633) is known to be among the more resistant microorganisms to NaOH sanitization. Colonies of B. subtilis were incubated with various cleaning liquids (1M NaOH, 1M NaOH with 2% BnOH, and 1M NaOH with 40% IPA), and the number of colony forming units (CFU/ml) was determined at various predetermined incubation times. FIG. 13 shows the results of the study as CFU/ml as a function of incubation time and cleaning liquid. It can be seen that using 1M NaOH by itself is insufficient to fully inactivate B. subtilis even after 24 hours of sanitization. Adding 2% benzyl alcohol to the cleaning liquid does not provide any significant improvement. However, the use of 1M NaOH with 40% isopropyl alcohol as cleaning liquid allows the reduction of B. subtilis to below detectable levels after 4 hours of treatment.

Claims (24)

1. A method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the method comprises the steps of:
a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix;
b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and
c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time;
wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), wherein the parental Fc-binding domain of SpA is defined by SEQ ID NO: 53
X1Q X2AFYEILX3LP NLTEEQRX4X5F IX6X7LKDX8PSX9  SX10X11X12LAEAKX13 X14NX15AQ
wherein individually of each other:
X1=A, Q or is deleted
X2=E,K,Y,T,F,L,W,I,M,V,A,H or R
X3=H or K
X4=A or N
X5=A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such as S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K
X6=Q or E
X7=S or K
X8=E or D
X9=Q, V or is deleted
X10=K, R, A or is deleted
X11=A, E, N or is deleted
X12=I or L
X13=K or R
X14=L or Y
X15=D, F,Y,W,K or R.
2. The method according to claim 1, wherein individually of each other:
X1=A or is deleted
X2=E
X3=H
X4=N
X6=Q
X7=S
X8=D
X9=V or is deleted
X10=K or is deleted
X11=A or is deleted
X12=I
X13=K
X14=L.
3. The method according to claim 1, wherein individually of each other:
X1=A, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L.
4. The method according to claim 1, wherein individually of each other:
X2=E, X3=H, X4=N, X5=A, X6=Q, X7=S, X8=D, X12=I, X13=K, X14=L and X15=D and one or more of X1, X9, X10 and X11 is deleted.
5. The method according to claim 1, wherein individually of each other:
X1=A, X2=E, X3=H, X4=N, X8=S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D, or alternatively X1=A, X2=E, X3=H, X4=N, X8=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=F,Y,W,K or R.
6. The method according to claim 1, wherein individually of each other:
a) X1=A or is deleted, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V or is deleted, X10=K or is deleted, X11=A or is deleted, X12=1, X13=K, X14=L;
b) X1=A, X2=E, X3=H, X4=N, X8=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D;
c) X1 is A, X2=E, X3=H, X4=N, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=Land X15=D; or
d) X1 is A, X3=H, X4=N, X5=A, X6=Q, X7=S, X8=D, X9=V, X10=K, X11=A, X12=I, X13=K, X14=L and X15=D.
7. The method according to claim 1, wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50.
8. The method according to claim 1, wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29.
9. The method according to claim 1, wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.
10. The method according to claim 1, wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers and nonamers.
11. The method according to claim 1, wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains each comprise a C-terminal cysteine residue for covalent coupling to the porous support.
12. The method according to claim 1, wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are coupled to the porous support via thioether links.
13. The method according to claim 1, wherein the separation matrix comprises at least 11 mg/ml of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support.
14. The method according to claim 1, wherein the porous support is highly cross-linked agarose beads.
15. The method according to claim 1, wherein the aqueous alkali metal hydroxide solution is sodium hydroxide solution, potassium hydroxide solution or a mixture thereof.
16. The method according to claim 1, wherein the aqueous alkali metal hydroxide solution has a molarity of from 500 mM to 5 M.
17. The method according to claim 1, wherein the cleaning liquid further comprises a C2-C7 alcohol.
18. The method according to claim 1, wherein the cleaning liquid comprises at least 70% by volume aqueous alkali metal hydroxide solution.
19. The method according to claim 1, wherein the predetermined contact time is a time sufficient to provide a 6-log 10 reduction in an endotoxin concentration and/or a microorganism concentration in the separation matrix.
20. The method according to claim 1, wherein the predetermined contact time is from 10 minutes to 50 hours.
21. The method according to claim 1, wherein the steps a)-c) are repeated at least 10 times.
22. A method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), wherein the parental Fc-binding domain of SpA is defined by claim 1; and wherein the method comprises:
purifying a first immunoglobulin and cleaning and sanitizing the separation matrix by performing the steps a)-c) of the method of cleaning and/or sanitizing a separation matrix according to claim 1; and further comprises the step of
d) purifying a mixture comprising a second immunoglobulin using the separation matrix, wherein the second immunoglobulin is different from the first immunoglobulin.
23. The method according to claim 22, wherein the separation matrix retains at least 80% of its original dynamic binding capacity after step c).
24. A method of using a cleaning liquid comprising at least 70% by volume of an aqueous alkali metal hydroxide solution for the sanitization of a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the method comprises cleaning and/or sanitizing the separation matrix by containing the cleaning liquid with the separation matrix for a predetermined contact time; and
wherein the Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA) as defined by claim 1.
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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201904125D0 (en) 2019-03-26 2019-05-08 Ge Healthcare Bio Sciences Ab Method for sanitization of a chromatography column
GB202018588D0 (en) * 2020-11-26 2021-01-13 Cytiva Bioprocess R & D Ab Separation matrix
WO2024137698A1 (en) * 2022-12-19 2024-06-27 Eli Lilly And Company Methods of cleaning chromatography matrices

Family Cites Families (184)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE451946B (en) 1982-12-10 1987-11-09 Gambro Lundia Ab DEVICE FOR THE DISPOSAL OF ONE OR MORE FRACTIONS OF WHOLE BLOOD, PLASMA OR SIMILAR BODY WASHES
US4704366A (en) 1984-06-22 1987-11-03 Bio-Rad Laboratories, Inc. Process for binding IgG to protein A
SE8505922D0 (en) 1985-12-13 1985-12-13 Kabigen Ab CONSTRUCTION OF AN IGG BINDING PROTEIN TO FACILITATE DOWNSTREAM PROCESSING USING PROTEIN ENGINEERING
US4801687A (en) 1986-10-27 1989-01-31 Bioprobe International, Inc. Monoclonal antibody purification process using protein A
AU612370B2 (en) 1987-05-21 1991-07-11 Micromet Ag Targeted multifunctional proteins
US5011686A (en) 1987-09-21 1991-04-30 Creative Biomolecules, Inc. Thrombus specific conjugates
US5084398A (en) 1987-11-20 1992-01-28 Creative Biomolecules Selective removal of immune complexes
US4933435A (en) 1989-04-05 1990-06-12 Bioprobe International Antibody purification process
JP2500329B2 (en) 1991-05-09 1996-05-29 工業技術院長 ALPHA.-Amino acid polymer having crown compound residue and method for producing the same
SE9400088D0 (en) 1994-01-14 1994-01-14 Kabi Pharmacia Ab Bacterial receptor structures
SE9503925D0 (en) 1995-11-07 1995-11-07 Pharmacia Biotech Ab Separation medium for IgG
SE9601368D0 (en) 1996-04-11 1996-04-11 Pharmacia Biotech Ab Process for the production of a porous cross-linked polysaccharide gel
SI0950067T1 (en) 1996-11-27 2007-12-31 Genentech Inc Affinity purification of polypeptide on protein a matrix
AU2003210802B2 (en) 2002-02-05 2009-09-10 Genentech Inc. Protein purification
SE0200943D0 (en) 2002-03-25 2002-03-25 Amersham Biosciences Ab Mutant protein
US7709209B2 (en) 2002-03-25 2010-05-04 Ge Healthcare Bio-Sciences Ab Protein ligands
CN1777435B (en) 2002-09-13 2011-01-12 拜奥根Idec公司 Method of purifying polypeptides by simulated moving bed chromatography
WO2004050847A2 (en) 2002-11-27 2004-06-17 Gtc Biotherapeutics, Inc. Modified antibodies stably produced in milk and methods of producing same
CN1524957A (en) * 2003-02-26 2004-09-01 本元正阳基因技术股份有限公司 Recombinant protein a gene ,its expression products and use
WO2005033144A2 (en) 2003-10-03 2005-04-14 Brigham And Women's Hospital Tim-3 polypeptides
US8084032B2 (en) 2004-01-21 2011-12-27 Ajinomoto Co., Inc. Purification method which prevents denaturation of an antibody
SE0400274D0 (en) 2004-02-09 2004-02-09 Affibody Ab New polypeptide
SE0402322D0 (en) 2004-09-22 2004-09-22 Amersham Biosciences Ab Method of preparing a chromatography matrix
JP2006304633A (en) 2005-04-26 2006-11-09 Apro Life Science Institute Inc Immunoglobulin-binding protein
JP5148484B2 (en) 2005-05-24 2013-02-20 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ Chromatographic matrix regeneration
AR053633A1 (en) 2005-06-17 2007-05-09 Wyeth Corp METHODS TO PURIFY PROTEINS CONTAINING AN FC REGION
WO2007021807A1 (en) 2005-08-12 2007-02-22 Schering Corporation Mcp1 fusions
EP1974215B1 (en) 2006-01-06 2017-12-27 EMD Millipore Corporation Affinity chromatography matrices and methods of making and using the same
CA2636109A1 (en) 2006-01-06 2007-07-19 Amgen Inc. Methods and systems for isolating target molecules from complex solutions by column-chromatography using eluants containing organic solvents
CA2637252A1 (en) 2006-01-17 2007-07-26 Biolex Therapeutics, Inc. Plants and plant cells having inhibited expression of .alpha.1,3-fucosyltransferase and .beta.1,2-xylosyltransferase
WO2007097361A1 (en) 2006-02-21 2007-08-30 Protenova Co., Ltd. Immunoglobulin affinity ligand
WO2007109163A2 (en) 2006-03-16 2007-09-27 Amgen Inc Wash buffer and method of using
PL2650305T3 (en) 2006-03-24 2024-09-16 Bioverativ Therapeutics Inc. PC5 as a factor IX propeptide processing enzyme
US8377448B2 (en) 2006-05-15 2013-02-19 The Board Of Trustees Of The Leland Standford Junior University CD47 related compositions and methods for treating immunological diseases and disorders
WO2007144476A1 (en) 2006-06-16 2007-12-21 Groupe Novasep Multi-column sequence separation method
DK1873166T3 (en) 2006-06-30 2010-12-20 Conaris Res Inst Ag Improved sgp 130Fc dimers
ZA200900836B (en) 2006-08-28 2010-05-26 Ares Trading Sa Process for the purification of FC-fusion proteins
ES2764222T3 (en) 2006-09-08 2020-06-02 Wyeth Llc Arginine wash in protein purification using affinity chromatography
CN106422418B (en) 2006-09-29 2022-03-01 思拓凡生物工艺研发有限公司 Chromatography ligand comprising domain C from staphylococcus aureus protein a for antibody isolation
DK2089429T3 (en) 2006-10-19 2015-01-12 Janssen Biotech Inc PROCEDURE FOR MANUFACTURING NON-AGGREGATED ANTIBODY-FC DOMAINS
US20080167450A1 (en) 2007-01-05 2008-07-10 Hai Pan Methods of purifying proteins
WO2008137471A2 (en) 2007-05-02 2008-11-13 Ambrx, Inc. Modified interferon beta polypeptides and their uses
JP5571552B2 (en) 2007-06-15 2014-08-13 ジーイー・ヘルスケア・バイオ−サイエンシズ・アーベー Chromatographic method
SG189793A1 (en) 2008-04-23 2013-05-31 Symphogen As Methods for manufacturing a polyclonal protein
US8592555B2 (en) 2008-08-11 2013-11-26 Emd Millipore Corporation Immunoglobulin-binding proteins with improved specificity
WO2010019493A1 (en) 2008-08-14 2010-02-18 Merck Sharp & Dohme Corp. Methods for purifying antibodies using protein a affinity chromatography
JP5229888B2 (en) 2008-09-30 2013-07-03 独立行政法人産業技術総合研究所 Protein A mutant protein and antibody capture agent with improved easy dissociation in weakly acidic region
WO2010044885A2 (en) 2008-10-17 2010-04-22 Whitehead Institute For Biomedical Research Soluble mtor complexes and modulators thereof
US8183207B2 (en) 2008-11-26 2012-05-22 Five Prime Therapeutics, Inc. Treatment of osteolytic disorders and cancer using CSF1R extracellular domain fusion molecules
US8080246B2 (en) 2008-11-26 2011-12-20 Five Prime Therapeutics, Inc. Colony stimulating factor 1 receptor (CSF1R) extracellular domain fusion molecules
SG195555A1 (en) 2008-12-24 2013-12-30 Emd Millipore Corp Caustic stable chromatography ligands
WO2010080065A1 (en) 2009-01-12 2010-07-15 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
EP2208681B1 (en) 2009-01-16 2013-07-03 Tetra Laval Holdings & Finance S.A. A unit for sterilizing a web of packaging material for a machine for packaging food products
EP2412809B1 (en) 2009-03-24 2017-08-09 Kaneka Corporation Protein having affinity for immunoglobulin, and immunoglobulin-binding affinity ligand
US8728479B2 (en) 2009-03-31 2014-05-20 The Trustees Of The University Of Pennsylvania Antigen-binding proteins comprising recombinant protein scaffolds
SG175004A1 (en) 2009-04-02 2011-11-28 Roche Glycart Ag Multispecific antibodies comprising full length antibodies and single chain fab fragments
AU2010240569A1 (en) 2009-04-20 2011-10-20 Pfizer Inc. Control of protein glycosylation and compositions and methods relating thereto
KR101747103B1 (en) 2009-06-26 2017-06-14 리제너론 파마슈티칼스 인코포레이티드 Readily isolated bispecific antibodies with native immunoglobulin format
EP3702775A1 (en) 2009-06-26 2020-09-02 Cytiva Sweden AB Method for determining binding capacities in a chromatography system
EP3219723B1 (en) 2009-08-27 2021-10-06 Novo Nordisk A/S Targeting tissue factor to activated platelets
DK2483310T3 (en) 2009-09-29 2014-09-01 Roche Glycart Ag BISPECIFIC DEATH RECEPTOR AGONISTIC ANTIBODIES
SG10201408384PA (en) 2009-12-18 2015-01-29 Novartis Ag Wash solution and method for affinity chromatography
ES2492681T3 (en) 2010-01-22 2014-09-10 Boehringer Ingelheim International Gmbh Chromatographic method to purify proteins containing FC
CA2791918C (en) 2010-03-05 2019-11-19 Boehringer Ingelheim International Gmbh Selective enrichment of antibodies
CN102844432A (en) 2010-03-24 2012-12-26 株式会社钟化 Protein capable of binding specifically to immunoglobulin, and immunoglobulin-binding affinity ligand
FR2958936A1 (en) 2010-04-14 2011-10-21 Sanofi Aventis ROBO1-FC FUSION PROTEIN AND ITS USE IN THE TREATMENT OF TUMORS
EP2558497A2 (en) 2010-04-15 2013-02-20 Amgen Inc. Human fgf receptor and beta-klotho binding proteins
ES2617777T5 (en) 2010-04-23 2022-10-13 Hoffmann La Roche Production of heteromultimeric proteins
KR101152036B1 (en) 2010-04-27 2012-06-08 (주)셀트리온 A method to improve the sorbent efficiency of protein A chromatography using switching column with continuous feeding
US20130231464A1 (en) 2010-04-28 2013-09-05 Oncolmmune, Inc. Methods of use of soluble cd24 for therapy of rheumatoid arthritis
US9290573B2 (en) 2010-05-06 2016-03-22 Novartis Ag Therapeutic low density lipoprotein-related protein 6 (LRP6) multivalent antibodies
EP2580238A1 (en) 2010-06-09 2013-04-17 Zymogenetics, Inc. Dimeric vstm3 fusion proteins and related compositions and methods
WO2011162210A1 (en) 2010-06-21 2011-12-29 協和発酵キリン株式会社 Method for purifying protein using amino acid
KR101333958B1 (en) 2010-10-20 2013-11-27 주식회사 한독 Human Interleukin-1 receptor antagonist-hybrid Fc fusion protein
AR083705A1 (en) 2010-11-04 2013-03-13 Abbott Lab IMMUNOGLOBULINS WITH DUAL VARIABLE DOMAIN AND USES OF THE SAME
JP6105477B2 (en) 2010-11-05 2017-03-29 ライナット ニューロサイエンス コーポレイション Engineered polypeptide conjugates and methods of making them using transglutaminase
WO2012068317A2 (en) 2010-11-16 2012-05-24 Excelimmune, Inc. Methods for producing recombinant proteins
WO2012074463A1 (en) 2010-11-29 2012-06-07 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
TWI424121B (en) 2010-12-10 2014-01-21 Prosol Corp Turbo molecular pump with improved blade structures
WO2012087231A1 (en) 2010-12-20 2012-06-28 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
JP5974342B2 (en) 2010-12-20 2016-08-23 ジーイー・ヘルスケア・バイオプロセス・アールアンドディ・アクチボラグ Affinity chromatography matrix
US9051375B2 (en) 2010-12-21 2015-06-09 The University Of Western Ontario Alkali-resistant variants of protein A and their use in affinity chromatography
KR101461772B1 (en) 2010-12-21 2014-12-03 제이에스알 가부시끼가이샤 Support for affinity chromatography and method for isolating immunoglobulin
EP2658979B1 (en) 2010-12-27 2018-02-14 Alexion Pharmaceuticals, Inc. Compositions comprising natriuretic peptides and methods of use thereof
US20120238730A1 (en) 2011-03-15 2012-09-20 Abbott Laboratories Integrated approach to the isolation and purification of antibodies
JP5933526B2 (en) 2011-03-25 2016-06-08 株式会社カネカ Novel immunoglobulin-binding polypeptide
WO2012133349A1 (en) 2011-03-25 2012-10-04 株式会社カネカ Protein for affinity-separation matrix
DK2691411T3 (en) 2011-03-29 2020-05-11 Glaxosmithkline Llc PROTEIN PURIFICATION BUFFER SYSTEM
EP3428176A1 (en) 2011-06-01 2019-01-16 Novartis AG Wash solution and method for affinity chromatography
EP2728000B1 (en) 2011-06-03 2018-12-05 National Institute of Advanced Industrial Science And Technology Protein a mutant protein having reduced affinity in acidic region, and antibody capture agent
SG10201604559TA (en) 2011-06-08 2016-07-28 Emd Millipore Corp Chromatography matrices including novel staphylococcus aureus protein a based ligands
WO2013006244A1 (en) 2011-06-08 2013-01-10 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Bispecific monoclonal antibody therapeutics against west nile virus with improved cns penetration
US20130011394A1 (en) 2011-06-22 2013-01-10 Hoffmann-La Roche Inc. Complexes comprising mhc class i fusion polypeptides and antigen-specific antibodies and methods of use
MX2014000031A (en) 2011-07-01 2014-07-09 Bayer Ip Gmbh Relaxin fusion polypeptides and uses thereof.
KR20140043934A (en) 2011-07-08 2014-04-11 머크 샤프 앤드 돔 코포레이션 Method for purifying fc-fusion protein
AU2012283235A1 (en) 2011-07-08 2014-01-09 Bayer Intellectual Property Gmbh Fusion proteins releasing Relaxin and uses thereof
SG11201400236YA (en) 2011-09-01 2014-03-28 Human Genome Sciences Inc Viral clearance methods
BR112014006537A2 (en) 2011-09-23 2017-11-28 Roche Glycart Ag bispecific antibodies, pharmaceutical formulation, uses of a bispecific antibody, treatment method, nucleic acid, expression vectors, host cell and method for producing a bispecific antibody
AU2012315956B2 (en) 2011-09-29 2016-01-21 Seagen Inc. Intact mass determination of protein conjugated agent compounds
EP2578286A1 (en) 2011-10-04 2013-04-10 Merck Patent GmbH Method and apparatus for chromatographic purification
AU2012328322A1 (en) 2011-10-27 2014-06-12 Genmab A/S Production of heterodimeric proteins
WO2013075740A1 (en) 2011-11-23 2013-05-30 Sanofi Antibody purification method
WO2013081540A1 (en) 2011-11-30 2013-06-06 Ge Healthcare Bio-Sciences Ab Affinity chromatography matrix
BR112014014418A2 (en) 2011-12-15 2019-09-24 Nat Res Council Canada soluble igf receptor fc fusion proteins and uses thereof
CN104220461A (en) 2011-12-29 2014-12-17 Atyr医药公司 Aspartyl-trna synthetase-fc conjugates
JP6420756B2 (en) 2012-03-28 2018-11-07 ジーイー・ヘルスケア・バイオプロセス・アールアンドディ・アクチボラグ Affinity chromatography matrix
WO2013150043A1 (en) 2012-04-05 2013-10-10 F. Hoffmann-La Roche Ag Bispecific antibodies against human tweak and human il17 and uses thereof
EP2656892A1 (en) 2012-04-23 2013-10-30 Merck Patent GmbH Chromatography method
US20140154270A1 (en) 2012-05-21 2014-06-05 Chen Wang Purification of non-human antibodies using kosmotropic salt enhanced protein a affinity chromatography
EP2854845B1 (en) 2012-06-01 2018-03-28 IBC Pharmaceuticals, Inc. Multimeric complexes with improved in vivo stability, pharmacokinetics and efficacy
EP2682168A1 (en) 2012-07-02 2014-01-08 Millipore Corporation Purification of biological molecules
RS62509B1 (en) 2012-07-13 2021-11-30 Roche Glycart Ag Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases
PL2880170T3 (en) 2012-08-02 2017-02-28 F.Hoffmann-La Roche Ag METHOD FOR PRODUCING SOLUBLE FcR AS Fc-FUSION WITH INERT IMMUNOGLOBULIN Fc-REGION AND USES THEREOF
JP2015524821A (en) 2012-08-02 2015-08-27 ジェイエヌ バイオサイエンシーズ エルエルシー Antibody or fusion protein multimerized via cysteine mutation and μ tail
KR20150037959A (en) 2012-08-02 2015-04-08 에프. 호프만-라 로슈 아게 Method for producing monomeric and multimeric molecules and uses thereof
CN104379169A (en) 2012-08-14 2015-02-25 Ibc药品公司 T-cell redirecting bispecific antibodies for treatment of disease
PL2895503T3 (en) 2012-09-13 2019-09-30 Bristol-Myers Squibb Company Fibronectin based scaffold domain proteins that bind to myostatin
SG11201502238PA (en) 2012-09-21 2015-05-28 Kaneka Corp Protein ligand for affinity isolation matrix
WO2015012904A2 (en) 2012-12-13 2015-01-29 Immunomedics, Inc. Antibody-sn-38 immunoconjugates with a cl2a linker
WO2014124326A1 (en) 2013-02-08 2014-08-14 Stem Centrx, Inc. Novel multispecific constructs
DK2772289T3 (en) 2013-03-01 2015-08-10 Chromacon Ag Chromatographic method for the enrichment and isolation
JP6285532B2 (en) 2013-03-14 2018-02-28 イー・エム・デイー・ミリポア・コーポレイシヨン Method for increasing protein purity using chromatography based on protein A
LT2970422T (en) 2013-03-15 2018-06-25 F. Hoffmann-La Roche Ag Il-22 polypeptides and il-22 fc fusion proteins and methods of use
US9587235B2 (en) 2013-03-15 2017-03-07 Atyr Pharma, Inc. Histidyl-tRNA synthetase-Fc conjugates
AU2013203043B2 (en) 2013-03-15 2016-10-06 Takeda Pharmaceutical Company Limited Methods to produce a human plasma-derived igg preparation enriched in brain disease-related natural iggs
US20140302037A1 (en) 2013-03-15 2014-10-09 Amgen Inc. BISPECIFIC-Fc MOLECULES
CN104059133B (en) 2013-03-18 2019-03-15 南京金斯瑞生物科技有限公司 The albumin A with high alkaline-resisting characteristic of one kind mutation and its application
JP6192390B2 (en) 2013-07-05 2017-09-06 キヤノン株式会社 Photoelectric conversion device, photoelectric conversion system
US10239951B2 (en) 2013-05-08 2019-03-26 Zymeworks Inc. Bispecific HER2 and HER3 antigen binding constructs
CA2910065C (en) 2013-05-15 2023-09-19 Medimmune Limited Purification of recombinantly produced polypeptides
JP6147340B2 (en) 2013-05-29 2017-06-14 Jsr株式会社 Cleaning composition, protein purification method, and protein
US20160304617A1 (en) 2013-06-17 2016-10-20 Asana Biosciences, Llc 5t4-targeted immunofusion molecule and methods
EP3020731B1 (en) 2013-07-09 2019-06-12 ABLBio Novel dual-targeted protein specifically binding to dll4 and vegf, and use thereof
EP3019520B1 (en) 2013-07-10 2019-12-11 GE Healthcare BioProcess R&D AB Mutated immunoglobulin-binding polypeptides
US9340582B2 (en) 2013-07-25 2016-05-17 Novartis Ag Bioconjugates of synthetic apelin polypeptides
CN113683695A (en) 2013-08-02 2021-11-23 辉瑞公司 anti-CXCR 4 antibodies and antibody-drug conjugates
WO2015035180A1 (en) 2013-09-05 2015-03-12 Genentech, Inc. Method for chromatography reuse
HUE057005T2 (en) 2013-09-25 2022-04-28 Bioverativ Therapeutics Inc On-column viral inactivation methods
JP6491600B2 (en) 2013-09-27 2019-03-27 株式会社カネカ Method for producing porous cellulose beads using aqueous alkali solution
CN105764503A (en) 2013-10-15 2016-07-13 西雅图基因公司 PEGylated drug-linkers for improved ligand-drug conjugate pharmacokinetics
CA2931356A1 (en) 2013-11-27 2015-06-04 Zymeworks Inc. Bispecific antigen-binding constructs targeting her2
KR102178323B1 (en) 2013-11-29 2020-11-13 삼성전자주식회사 Anti-c-Met/anti-Ang2 bispecific antibody
PT3082877T (en) 2013-12-17 2019-12-03 Novartis Ag Cytotoxic peptides and conjugates thereof
PL3099692T3 (en) 2014-01-27 2019-08-30 Pfizer Inc. Bifunctional cytotoxic agents
US20160152725A1 (en) 2014-02-25 2016-06-02 Memorial Sloan-Kettering Cancer Center Antigen-binding proteins specific for hla-a2-restricted wilms tumor 1 peptide
HUE055424T2 (en) 2014-03-17 2021-11-29 Mitsubishi Tanabe Pharma Corp Antibody-fynomer conjugates
CN106456714A (en) 2014-03-28 2017-02-22 纽约大学 FGF23 fusion proteins
KR102430829B1 (en) 2014-04-25 2022-08-09 리나트 뉴로사이언스 코프. Antibody-drug conjugates with high drug loading
WO2015166072A1 (en) 2014-04-30 2015-11-05 Novo Nordisk A/S Methods for the purification of proteins using caprylic acid
EP3143049B1 (en) 2014-05-14 2019-08-21 F. Hoffmann-La Roche AG Her3/her2 bispecific antibodies binding to the beta-hairpin of her3 and domain ii of her2
WO2015181805A1 (en) 2014-05-28 2015-12-03 Zymeworks Inc. Modified antigen binding polypeptide constructs and uses thereof
AU2015273098B2 (en) 2014-06-13 2018-05-10 Novartis Ag Auristatin derivatives and conjugates thereof
BR112016029157A8 (en) 2014-06-13 2021-07-06 Lupin Ltd process for purifying the tnfr:fc fusion protein
US9777073B2 (en) 2014-07-21 2017-10-03 Wuhan Yzy Biopharma Co., Ltd. Construction and application of bispecific antibody EpCAM×CD3
AR101262A1 (en) 2014-07-26 2016-12-07 Regeneron Pharma PURIFICATION PLATFORM FOR Bispecific Antibodies
KR102259232B1 (en) 2014-08-25 2021-05-31 삼성전자주식회사 Anti-c-Met/anti-Ang2 bispecific antibody
WO2016030791A1 (en) 2014-08-28 2016-03-03 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
CN105481954B (en) * 2014-09-16 2021-03-26 亿一生物制药(北京)有限公司 Recombinant protein A and application thereof
CN106687482B (en) 2014-09-17 2022-01-25 诺和诺德股份有限公司 Antibodies capable of binding two epitopes on tissue factor pathway inhibitor (1-161)
JP6369807B2 (en) 2014-10-21 2018-08-08 株式会社プロテイン・エクスプレス Protein L mutant
JP2016079153A (en) 2014-10-21 2016-05-16 株式会社プロテイン・エクスプレス Protein g mutant
SG11201607162QA (en) 2014-10-24 2016-10-28 Gemtier Medical Shanghai Inc Disposable Safe Vein Transfusion Puncture Needle
US11566082B2 (en) 2014-11-17 2023-01-31 Cytiva Bioprocess R&D Ab Mutated immunoglobulin-binding polypeptides
MX2017007637A (en) 2014-12-09 2017-09-05 Abbvie Inc Bcl-xl inhibitory compounds and antibody drug conjugates including the same.
MX2017007641A (en) 2014-12-09 2017-10-02 Abbvie Inc Antibody drug conjugates with cell permeable bcl-xl inhibitors.
DK3233907T3 (en) 2014-12-19 2021-06-07 Genmab As Bispecific heterodimeric proteins in rodents
EP3280453A1 (en) 2015-04-07 2018-02-14 Immunomedics, Inc. Y-90-labeled anti-cd22 antibody (epratuzumab tetraxetan) in refractory/relapsed adult cd22+ b-cell acute lymphoblastic leukemia
US11820807B2 (en) 2015-06-12 2023-11-21 Ubi Pharma Inc Immunoglobulin fusion proteins and uses thereof
WO2017011342A1 (en) 2015-07-10 2017-01-19 Abbvie Inc. Igm-or-ige-modified binding proteins and uses thereof
BR112018000477A2 (en) 2015-07-16 2018-09-18 Navigo Proteins Gmbh unnatural immunoglobulin binding protein, composition, use of unnatural ig binding protein, immunoglobulin affinity purification method, method of generating an unnatural immunoglobulin binding protein, nucleic acid molecule, vector, host cell or a non-human host, and method for producing an unnatural immunoglobulin binding protein
JP2017037070A (en) 2015-08-10 2017-02-16 三菱化学株式会社 Separating agent and liquid chromatography column
GB201515339D0 (en) 2015-08-28 2015-10-14 Ge Healthcare Bio Sciences Ab A seperation matrix and a method of seperating antibodies
WO2017050889A1 (en) 2015-09-25 2017-03-30 F. Hoffmann-La Roche Ag Recombinant immunoglobulin heavy chains comprising a sortase conjugation loop and conjugates thereof
AR106365A1 (en) 2015-10-02 2018-01-10 Hoffmann La Roche BISPECIFIC MOLECULES OF ANTIGEN BINDING TO T-CELL ACTIVATORS
MA43017A (en) 2015-10-02 2018-08-08 Hoffmann La Roche BISPECIFIC ANTIBODIES SPECIFIC TO A TNF CO-STIMULATION RECEPTOR
CN108271377B (en) 2015-10-07 2021-11-19 豪夫迈·罗氏有限公司 Bispecific antibodies having a tetravalent targeting co-stimulatory TNF receptor
TWI812873B (en) 2015-11-30 2023-08-21 美商輝瑞股份有限公司 Antibodies and antibody fragments for site-specific conjugation
US10889615B2 (en) 2016-05-11 2021-01-12 Cytiva Bioprocess R&D Ab Mutated immunoglobulin-binding polypeptides
US10654887B2 (en) 2016-05-11 2020-05-19 Ge Healthcare Bio-Process R&D Ab Separation matrix
US10703774B2 (en) 2016-09-30 2020-07-07 Ge Healthcare Bioprocess R&D Ab Separation method
JP7106187B2 (en) 2016-05-11 2022-07-26 サイティバ・バイオプロセス・アールアンドディ・アクチボラグ How to save the separation matrix
US10730908B2 (en) 2016-05-11 2020-08-04 Ge Healthcare Bioprocess R&D Ab Separation method
EP3455242B1 (en) 2016-05-11 2021-06-23 Cytiva BioProcess R&D AB Separation method
EP3455243B1 (en) 2016-05-11 2021-03-24 Cytiva BioProcess R&D AB Separation matrix

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