Methods for purifying antibodies

Abstract

The present invention relates to the field of manufacturing recombinant antibody molecules. In particular, a method for purifying such recombinant antibody molecules is provided, wherein imidazole or an imidazole analog is added during the elution of the recombinant antibody molecules from an affinity chromatography resin such as a protein A-based resin.

Description

Field of the Invention

[0001] The present invention relates to the field of producing recombinant antibody molecules, and in particular to methods for purifying such recombinant antibody molecules using affinity chromatography and using specific elution conditions. Background of the Invention

[0003] In the therapeutic field, the use of biological entities such as proteins (including especially antibodies and antibody-derived molecules) has been continuously gaining presence and importance, and at the same time, the need for controlled large-scale manufacturing processes has also developed.

[0004] Protein A is a cell wall component produced by several Staphylococcus aureus strains that binds to the Fc region of antibody molecules. Affinity chromatography using immobilized protein A as a ligand has been widely used in antibody purification and remains a core purification step in most antibody purification processes, allowing a high degree of impurity removal from the raw material. In addition, it is also known that protein A binds to the VH3 region present on the antibody molecule, which provides certain strategies for purifying alternative antibody forms lacking the Fc region based on the affinity interaction between the VH3 region and protein A.

[0005] Protein A has a high affinity for Fc domains at neutral pH. Therefore, the starting material containing the antibody to be purified is usually loaded on the protein A resin at neutral pH. The typical process is followed by one or more steps of washing the chromatographic material with a buffer also at neutral pH to ensure that impurities are removed as much as possible. Finally, the elution step is necessary to recover the bound antibody from protein A. The elution step includes using an elution buffer with an acidic pH value (usually about 2.5 to about 4.0), which destroys the interaction between the antibody and protein A. Similarly, WO2016 / 169992 describes a purification method for antibody molecules lacking Fc regions based on the binding of VH3 regions by protein A, which also relies on pH acidification to elute from protein A.

[0006] Typically, lower pH also allows for lower elution volumes, which directly impacts the efficiency of the overall process. Another consideration regarding lower elution volumes is that they enable the use of static binding conditions, as opposed to conventional dynamic binding conditions, where large volumes of elution buffer are washed over the chromatography resin.

[0007] However, a balance must be found between a pH low enough to disrupt the binding of the antibody molecule to Protein A, and a pH not so low as to disrupt the tertiary structure of the protein. Furthermore, aggregation is a common consequence of exposure to acidic conditions, resulting in more impurities that must be removed during purification, ultimately leading to lower process yields. Therefore, for some more unstable antibody molecules, it may be advantageous to find conditions that allow elution from Protein A at a higher pH.

[0008] To overcome these challenges, there have been different attempts to enhance the elution of antibody molecules from Protein A by adding additional components to the elution buffer that can compete with the antibody molecules for binding to Protein A and / or increase the elution pH.

[0009] Arakawa et al. (Elution of antibodies from a protein-A column with an aqueous solution of arginine; 2004; Protein Expression & Purification, 244-248) and US 8,470,328 describe the use of arginine or arginine derivatives in an elution buffer having a pH of 4.0 to 5.0. However, as the pH of the elution buffer increases, the elution volume also increases, because the high pH only leads to a weak dissociation of the antibody from the resin, and then the antibody also easily rebinds to the resin.

[0010] Another consideration for commercial large-scale production processes is the time spent in each chromatography cycle, which in turn is directly affected by the volumes used in loading, washing, and elution of the chromatography support, such as Protein A. Optimizing these parameters over a small range can have a large impact when scaled up to production volumes, leading to improvements in waste that must be disposed of, shorter production times, and therefore an improved cost profile.

[0011] An alternative form of protein purification has also been used which involves expressing the recombinant protein with a histidine tag, then purifying the resulting protein using nickel affinity chromatography (which binds the histidine tag), and eluting the protein with the aid of imidazole. However, a downside to this approach is that for many uses, the tag needs to be removed by proteolysis after purification before the protein can be used.

[0012] Based on the above, there is a continuing need to provide rapid and robust methods for purifying antibodies in production processes, in particular improved methods for eluting antibodies from affinity chromatography resins. The present invention addresses this need. Summary of the invention

[0013] In a first aspect, the present invention relates to a method for purifying an antibody, comprising:

[0014] a) loading the mixture comprising the antibody to be purified onto an affinity chromatography resin, wherein the affinity chromatography resin is not a nickel-based, zinc-based or cobalt-based resin,

[0015] b) washing the chromatography resin with a wash buffer;

[0016] c) eluting the antibody with an elution buffer comprising 0.01 M to 1.0 M imidazole or an imidazole analog and a pH of 3 to 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Results of static binding studies;

[0018] Figure 2 : Overlay of IgG1 chromatographic elution curves at pH 3.6 + / - imidazole;

[0019] Figure 3 : Overlay of IgG1 chromatographic elution curves at pH 4.0 + / - imidazole;

[0020] Figure 4 : Overlay of IgG4 chromatographic elution curves at pH 3.6 + / - imidazole;

[0021] Figure 5 : Overlay of chromatographic elution curves of TrYbe (VH3) at pH 4.0 + / - imidazole;

[0022] Figure 6 : Dynamic binding research results;

[0023] Figure 7 : Overlay of IgG1 chromatographic elution curves at pH 3.6 + / - 1,3 dimethyl-1H-pyrazole. Specific implementation plan

[0024] The present invention solves the above problems by providing a novel method for purifying antibodies, the method comprising an affinity chromatography step, wherein elution is performed in the presence of imidazole or an imidazole analog. Without being bound by any theory, it is believed that imidazole or its analog binds to the antibody molecules that dissociate from the chromatography resin during the elution step. Therefore, imidazole or its analog can prevent the antibody molecules from rebinding to the resin. This helps to improve the elution kinetics, thereby reducing the elution volume, which is an advantage over methods described in the art.

[0025] The method of the present invention is applicable to antibody purification methods comprising affinity chromatography steps performed under static and dynamic conditions.

[0026] In the context of the present invention, dynamic conditions are considered to be conditions in which the affinity resin is usually in a chromatography column or membrane, a mixture comprising the antibody to be purified, a wash buffer and / or an elution buffer is added to the column, and the eluent is recovered from the column. Static chromatography conditions are conditions that involve mixing the chromatography resin with the mixture comprising the antibody to be purified, followed by incubation for a period of time, followed by separation of the chromatography resin from the liquid phase, and subsequent elution of the bound fraction from the chromatography resin.

[0027] In a preferred embodiment of the process of the invention, one, two or all of steps a), b) and c) are performed under dynamic conditions.

[0028] In a first aspect, the present invention relates to a method for purifying an antibody, comprising:

[0029] a) loading the mixture comprising the antibody to be purified onto an affinity chromatography resin,

[0030] wherein the affinity chromatography resin is not a nickel-based, zinc-based or cobalt-based resin,

[0031] b) washing the chromatography resin with a wash buffer, and

[0032] c) eluting the antibody with an elution buffer comprising 0.01 M to 1.0 M imidazole or an imidazole analog and a pH of 3 to 5.

[0033] The affinity chromatography resin is capable of binding to the antibody, preferably through the Fc region or VH3 domain of the antibody. In one embodiment, the affinity chromatography resin is not a metal ion based resin. In another embodiment, the affinity chromatography resin is selected from: protein A chromatography resin, protein G chromatography resin and protein L chromatography resin.

[0034] A number of affinity chromatography materials containing protein A, protein G or protein L are available to those skilled in the art, for example (GE Healthcare), (Novasep), Captiv (Repligen), Praesto AP (Purolite), or (JSR).

[0035] Buffers suitable for use as washing and elution buffers in Protein A chromatography are readily available in the art and may be selected from the following non-limiting examples: phosphate buffered saline (PBS), Tris, histidine, acetate, formate, citrate buffers or MES (2-(N-morpholino) ethanesulfonic acid imidazole), BES (N,N-(bis-2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)-propanesulfonic acid) or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffers.

[0036] In one embodiment the elution buffer comprises 0.01 M to 0.75 M imidazole or an imidazole analogue, such as 0.01 M to 0.5 M, for example 0.1 M to 0.5 M, such as 0.2 M to 0.5 M, for example 0.2 M to 0.3 M, such as 0.25 M imidazole or an imidazole analogue.

[0037] In one embodiment, the pH of the elution buffer is 3.5-4.5, such as 3.5 to 4.0 or 4.0 to 4.5, or 3.6 to 3.9, such as 3.7 to 3.9.

[0038] In one embodiment, the wash buffer further comprises imidazole or an imidazole analog. In one embodiment, the wash buffer comprises 0.01M to 0.75M imidazole or an imidazole analog, such as 0.01M to 0.5M, for example 0.1M to 0.5M, for example 0.2M to 0.5M, for example 0.2M to 0.3M, such as 0.25M imidazole or an imidazole analog.

[0039] Imidazole is a compound with the chemical formula C 3 N 2 H 4 It is an aromatic heterocyclic ring, classified as an oxadiazole, and has non-adjacent nitrogen atoms.

[0040] As used herein, the term "imidazole analog" refers to imidazole or other 5-membered ring structures (ie, diazole or triazole) containing at least two nitrogen atoms, such as pyrazole and triazole.

[0041] The ring can be substituted or unsubstituted. If there is a substitution, in one embodiment, they are selected from methyl, ethyl, hydroxyl or hydroxymethyl. In one embodiment, there is only one substituent, such as a methyl. In another embodiment, there are two substitutions, such as two methyl.

[0042] In one embodiment, the ring is unsubstituted at position 1 or 2, such as an imidazole ring (Formula I) that is unsubstituted at position 1 or 2.

[0043]

[0044] (Formula I-imidazole)

[0045] Preferred imidazole analogs include 4(5)-methylimidazole, pyrazole, 1,5-dimethyl-1H-pyrazole, 1,3-dimethyl-1H-pyrazole and 1H-imidazol-1-ylmethanol.

[0046] In another embodiment, the imidazole analog is histidine or a histidine analog, such as n-boc-L-histidine, n-benzyloxycarbonyl-D-histidine, or L-histidine methyl ester dihydrochloride.

[0047] In a preferred embodiment, the ring is unsubstituted, preferably unsubstituted imidazole or pyrazole.

[0048] In one embodiment, the method of the invention comprises the further step of equilibrating the chromatography resin with an equilibration buffer comprising imidazole or an imidazole analogue prior to loading the mixture comprising the antibody onto the chromatography resin.

[0049] In another embodiment, the method of the present invention will include one or more additional chromatographic steps to remove remaining impurities. Typically, such steps will use a non-affinity chromatography step using a solid phase with suitable functionality for gel filtration chromatography, cation chromatography, anion chromatography, mixed mode chromatography, hydrophobic chromatography and hydrophobic charge induction chromatography. These can be operated in a binding and elution mode or a flow-through mode. In the flow-through mode, impurities are combined or have a reduced mobility in the solid phase, and the target protein is recovered in the eluate or flow-through fraction. Those skilled in the art are easy to obtain suitable solid phases for chromatography, such as beaded resins or membranes with suitable functionality. In a specific embodiment according to the method of the present invention, the method additionally includes the step of anion exchange chromatography operated in a flow-through mode.

[0050] In another specific embodiment, the method of the present invention comprises a Protein A chromatography step, followed by a first chromatography step which is an anion exchange chromatography producing a flow-through containing the protein, and a second chromatography step which is a cation exchange chromatography from which an eluate containing the protein is recovered.

[0051] In another embodiment, the method of the present invention comprises Protein A chromatography followed by a first chromatography step of cation exchange chromatography from which an eluate containing the protein is recovered and a second chromatography step of anion exchange chromatography producing a flow-through containing the protein.

[0052] Antibody

[0053] As used herein, the term "antibody" includes monoclonal antibodies and polyclonal antibodies. In addition, as used herein, the term "antibody" includes, but is not limited to, recombinant antibodies produced by recombinant techniques known in the art. "Antibodies" include antibodies of any species, particularly antibodies of mammalian species; such as human antibodies of any isotype, including IgD, IgG 1 IgG 2a IgG 2b IgG 3 IgG 4 IgE and antibodies produced as dimers of this basic structure, including IgGA 1 IgGA 2 or pentamers such as IgM and modified variants thereof, non-human primate antibodies, e.g., antibodies from chimpanzees, baboons, rhesus monkeys, or cynomolgus monkeys; rodent antibodies, e.g., antibodies from mice or rats; rabbit, goat, or horse antibodies; and camelid antibodies (e.g., antibodies from camels or llamas, such as Nanobodies TM) and derivatives thereof; or avian species such as chicken antibodies or fish species such as shark antibodies. The term "antibody" also refers to a "chimeric" antibody, in which the first portion of at least one heavy chain and / or light chain antibody sequence is from a first species, and the second portion of the heavy chain and / or light chain antibody sequence is from a second species. Target chimeric antibodies herein include "primatized" antibodies, which contain variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys, or cynomolgus monkeys) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain sequences derived from non-human antibodies. In most cases, humanized antibodies are human antibodies (acceptor antibodies) in which residues from the hypervariable regions of the recipient are replaced by residues from the hypervariable regions [or complementary determining regions (CDRs)] of non-human species (donor antibodies) such as mice, rats, rabbits, chickens, or non-human primates, with the desired specificity, affinity, and activity. In most cases, residues of human (acceptor) antibodies outside CDR, i.e., in framework regions (FR), are additionally replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the acceptor antibody or the donor antibody. These modifications are performed to further refine antibody properties. Humanization reduces the immunogenicity of non-human antibodies in the human body, thereby promoting the application of antibodies in the treatment of human diseases. Humanized antibodies and several different techniques for producing them are well known in the art. The term "antibody" also refers to human antibodies, which can be produced as a substitute for humanization. For example, it is possible to produce transgenic animals (e.g., mice) that can produce a complete human antibody library without producing endogenous mouse antibodies after immunization. Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technology, such as phage display or ribosome display technology, wherein a recombinant DNA library at least partially artificially produced or from a donor immunoglobulin variable (V) domain gene library is used. Phage and ribosome display technology for producing human antibodies is well known in the art. Human antibodies can also be produced from isolated human B cells that are immunized ex vivo with an antigen of interest and subsequently fused to produce hybridomas that can then be screened for optimal human antibodies. As used herein, the term "antibody" also refers to an aglycosylated antibody.

[0054] As used herein, the term "antibody" refers not only to full-length antibodies of any species, including full-length antibodies from humans (e.g., IgG) and other mammalian species, but also to antibody fragments. Antibody fragments contain at least one heavy chain or light chain immunoglobulin domain known in the art and bind to one or more antigens. Examples of antibody fragments according to the present invention include Fab, Fab', F(ab')2, and Fv and scFv fragments; as well as di-antibodies, tri-antibodies, tetra-antibodies, mini-antibodies, domain antibodies (dAbs), such as sdAbs, V H H and VNAR Fragments, single-chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-fv constructs. Antibody fragments as defined above are known in the art.

[0055] In one embodiment, the antibody purified using the method of the invention does not contain any of the following motifs: a polyhistidine motif, an HQ motif, an HN motif, or a hat motif, wherein a polyhistidine motif is a sequence of five or more consecutive histidine residues, an HQ motif is a sequence comprising at least three alternations of histidine and glutamine (HQHQHQ (SEQ ID NO:7)), an HN motif is a sequence comprising at least three alternations of histidine and asparagine (HNHNHN (SEQ ID NO:8)), and a HAT motif is the sequence KDHLIHNVHKEEHAHAHNK (SEQ ID NO:9).

[0056] In one embodiment of the method of the present invention, the antibody to be purified is an antibody comprising an Fc region.

[0057] In one embodiment, the antibody to be purified is an antibody comprising a CH2 and a CH3 domain.

[0058] In one embodiment, the antibody to be purified is an antibody containing a VH3 region and bound to an affinity chromatography resin via the VH3 region.

[0059] In another embodiment, the antibody is selected from the group consisting of: IgG, Fab', F(ab')2, scFv, Fab-Fv, Fab-scFv, Fab-(scFv)2, Fab-(Fv)2, diabody, triabody and tetrabody.

[0060] In one embodiment of the methods of the invention, the antibody is a FabFv or a disulfide-stabilized form thereof as disclosed in PCT / EP2014 / 074409 (which is incorporated herein by reference).

[0061] In one embodiment, the antibody comprises a binding domain specific for human serum albumin, in particular a binding domain of a CDR or variable region as disclosed in WO2013 / 068563 (which is incorporated herein by reference).

[0062] In one embodiment, the antibody, e.g., Fab-dsFv format, is an antibody disclosed in PCT / EP2014 / 074409 or WO2014 / 019727 (which are incorporated herein by reference).

[0063] In another embodiment, the antibody is in the form of a Fab-scFv fusion protein disclosed in WO2013 / 068571 (which is incorporated herein by reference).

[0064] In another embodiment, the antibody is a multispecific antibody molecule comprising or consisting of:

[0065] a) a polypeptide chain of formula (I):

[0066] VH-CH1-X-V1; and

[0067] b) a polypeptide chain of formula (II):

[0068] VL-CL-Y-V2;

[0069] in:

[0070] VH stands for heavy chain variable domain;

[0071] CH1 represents a domain of the heavy chain constant region, such as domain 1 thereof;

[0072] X represents a bond or joint;

[0073] Y represents a bond or joint;

[0074] V1 represents dsFv, sdAb, scFv or dsscFv;

[0075] VL stands for light chain variable domain;

[0076] CL represents a domain from the light chain constant region, such as Ckappa;

[0077] V2 represents dsFv, sdAb, scFv or dsscFv;

[0078] wherein at least one of V1 or V2 is a dsFv or dsscFv as described in WO2015 / 197772 (which is incorporated herein by reference).

[0079] As used herein, "single-chain variable fragment" or "scFv" refers to a single-chain variable fragment comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL), which is stabilized by a peptide linker between the VH and VL variable domains. The VH and VL variable domains can be in any suitable orientation, for example, the C-terminus of VH can be connected to the N-terminus of VL, or the C-terminus of VL can be connected to the N-terminus of VH.

[0080] "Disulfide-stabilized single-chain variable fragment" or "dsscFv" refers to a single-chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains, and further comprises an interdomain disulfide bond between VH and VL.

[0081] "Disulfide-stabilized variable fragment" or "dsFv" refers to a single chain variable fragment that does not comprise a peptide linker between the VH and VL variable domains, but is instead stabilized by an interdomain disulfide bond between VH and VL.

[0082] In a specific embodiment, the antibody is a multispecific antibody in the Fab-2 x dsscFv format described in WO2015 / 197772 (which is incorporated herein by reference).

[0083] In yet another specific embodiment, the multispecific antibody in the format of Fab-2 x dsscFv is a trivalent antibody, ie each Fv binds a different epitope.

[0084] In yet another specific embodiment, the multispecific antibody has the format of Fab-dsscFv-dsFv as described in WO2015 / 197772, which is incorporated herein by reference.

[0085] In one embodiment, the antibody to be purified comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0086] SEQ ID NO: 1: (a) Heavy chain variable domain of anti-albumin antibody (without ds)

[0087] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS

[0088] SEQ ID NO:2:(b) Heavy chain variable domain of anti-albumin antibody (ds)

[0089] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS

[0090] SEQ ID NO:3:(c) light chain variable domain of anti-albumin antibody (without ds)

[0091] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKRT

[0092] SEQ ID NO:4:(d) light chain variable domain of anti-albumin antibody (ds)

[0093] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKRT

[0094] SEQ ID NO:5: 645gH5gL4 specific for albumin

[0095] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKRT

[0096] SEQ ID NO:6: 645gH5gL4ds specific for albumin

[0097] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKRT

[0098] In many embodiments, the mixture comprising antibodies loaded onto the affinity chromatography resin in step a) of the method of the invention is derived directly or indirectly from a cell culture (eg, mammalian or bacterial culture), wherein the antibodies are recombinantly produced.

[0099] Recombinant antibodies or antibody derivatives, such as antibody fragments, can be produced for large-scale commercial purposes by culturing eukaryotic host cells transfected with one or more expression vectors encoding the recombinant antibodies. The eukaryotic host cells are preferably mammalian cells, more preferably Chinese hamster ovary (CHO) cells.

[0100] Mammalian cells can be cultured in any medium that supports their growth and antibody expression, preferably a medium that is a chemically defined medium that does not contain animal-derived products such as animal serum and peptone. Different cell culture media are available to those skilled in the art, which contain different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or equivalent energy sources, present in appropriate concentrations to allow cell growth and protein production. It is known to those skilled in the art that at different times in the cell culture cycle, additional cell culture media components can be included in the cell culture media at appropriate concentrations.

[0101] Mammalian cell culture can be carried out in any suitable container, such as a shake flask or a bioreactor, which may or may not be operated in a fed-batch mode, depending on the desired production scale. These bioreactors may be, for example, stirred tanks or airlift reactors. Various large-scale bioreactors are available, with capacities ranging from greater than 1,000 L to 50,000 L, preferably from 5,000 L to 20,000 L, or to 10,000 L. Alternatively, smaller-scale bioreactors, such as 2 L to 100 L, may also be used to produce antibodies to be purified according to the methods of the invention.

[0102] The antibody or antigen binding fragment thereof that can be manufactured according to the method of the present invention is generally present in the supernatant of a mammalian host cell culture (generally a CHO cell culture). For a CHO culture process in which a target protein such as an antibody or an antigen binding fragment thereof is secreted in the supernatant, the supernatant is collected by methods known in the art, generally by centrifugation. For the avoidance of doubt, supernatant refers to the liquid above the precipitated cells after the cell culture is centrifuged.

[0103] In one embodiment of the present invention, the method comprises culturing CHO cells expressing an antibody of interest, recovering the supernatant, and purifying the antibody from the mixture, wherein the purification comprises at least one affinity chromatography step performed according to the method of the present invention.

[0104] Alternatively, the host cell is preferably a prokaryotic cell, preferably a gram-negative bacterium. More preferably, the host cell is an Escherichia coli (E. coli) cell. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72, 211-222.). Host cells are recombinant cells that have been genetically engineered to produce target proteins, such as antibody fragments. Recombinant E. coli host cells can be derived from any suitable E. coli strain, including from MC4100, TG1, TG2, DHB4, DH5α, DH1, BL21, K12, XL1Blue and JM109. An example is E. coli strain W3110 (ATCC 27,325), a common host strain for recombinant protein fermentation. Antibody fragments can also be produced by culturing modified E. coli strains, such as metabolic mutants or E. coli strains lacking proteases.

[0105] Antibody fragments that can be purified according to the methods of the present invention are usually present in the periplasm of E. coli host cells or in the host cell culture supernatant, depending on the nature of the protein, the scale of production and the E. coli strain used. Methods for targeting proteins to these compartments are well known in the art (Makrides, SC (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60, 512-538.). Examples of suitable signal sequences for directing proteins to the periplasm of E. coli include E. coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences. Proteins can be targeted to the supernatant by relying on the natural secretory pathway or by inducing limited leakage of the outer membrane to cause protein secretion, examples of which are the use of pelB leader sequences, protein A leader sequences, co-expression of bacteriocin release proteins, mitomycin-induced bacteriocin release proteins, and the addition of glycine to the culture medium and the co-expression of the kil gene for membrane permeabilization. Most preferably, in the methods of the present invention, the antibodies are expressed in the periplasm of the host E. coli.

[0106] The expression of antibodies in Escherichia coli host cells can also be controlled by inducible systems, and thus the expression of recombinant antibodies in Escherichia coli is controlled by inducible promoters. Many inducible promoters suitable for Escherichia coli are well known in the art, and depending on the promoter, the expression of recombinant protein can be induced by different factors such as the concentration of specific substances in temperature or growth medium. The example of inducible promoter includes available lactose or non-hydrolyzable lactose analogs, isopropyl-bD-1-thiogalactoside (IPTG)-induced Escherichia coli lac, tac and trc promoters, and phoA, trp and araBAD promoters induced by phosphate, tryptophan and L-arabinose, respectively. Expression can be induced by, for example, adding an inducing agent or temperature variation (wherein induction is temperature-dependent). When the induction of recombinant protein expression is realized by adding an inducing agent in the culture, an inducing agent can be added by any suitable method according to the fermentation system and the inducing agent.

[0107] The E. coli host cell culture (fermentation) can be cultured in any medium that supports the growth of E. coli and the expression of recombinant proteins. The medium can be any chemically defined medium, such as the medium described in Durany O, CGdMCL-SJ (2004) Studies on the expression of recombinant fuculose-1-phosphate aldolase in Escherichia coli. Process Biochem 39, 1677-1684.

[0108] The cultivation of E. coli host cells can be carried out in any suitable container such as a shake flask or a fermentor, depending on the desired production scale. Various large fermentors are available, with capacities ranging from more than 1,000 liters to up to 100,000 liters. Preferably, a fermentor of 1,000 liters to 50,000 liters is used, more preferably a fermentor of 1,000 liters to 25,000 liters, 20,000 liters, 15,000 liters, 12,000 liters or 10,000 liters. Small fermentors of 0.5 liters to 1000 liters in capacity can also be used.

[0109] Colibacillary fermentation can be carried out in any suitable system (for example continuously, in batches or fed-batch mode), and this depends on required protein and productive rate.If desired, batch mode can be used together with the rapid interpolation of nutrient agent or inducing agent.Or, can use fed-batch culture, and grow with maximum specific growth rate with batch mode pre-induction culture, and described maximum specific growth rate can use the nutrient that is initially present in fermentation tank and one or more nutrient feed modes that are used to control growth rate to maintain, until fermentation is finished.Fed-batch mode can also be used for pre-induction, to control the metabolism of Escherichia coli host cells, and allows to reach higher cell density.

[0110] If desired, the host cells may be collected from the fermentation medium, for example, by centrifugation, filtration or concentration of the sample.

[0111] In one embodiment, the method according to the invention comprises the steps of centrifugation and cell recovery prior to extraction of antibodies.

[0112] For E. coli fermentation processes in which a target protein, such as an antibody fragment, is present in the periplasmic space of a host cell, it is desirable to release the protein from the host cell. Release can be achieved by any suitable method, such as cell lysis by mechanical or pressure treatment, freeze-thaw treatment, osmotic shock, extractants, or heat treatment. Such extraction methods for protein release are well known in the art.

[0113] In yet another embodiment, the method according to the present invention further comprises recovering the host cells from the cell culture medium, harvesting the protein using a protein extraction step performed in the presence of a reducing agent, recovering the antibody-containing mixture produced by the protein extraction step, and purifying the antibody from the mixture, wherein the purification comprises at least one affinity chromatography step performed according to the method of the present invention.

[0114] Example

[0115] Example 1: Static binding MODDE study

[0116] method

[0117] PrA MabSelect SuRe resin, GE, was used in static binding mode (CV = 1 mL). The MODDE DoE study was designed to test four imidazole elution buffers (N1-N4), with a fifth buffer used for center points (N5-N7) in triplicate (Table 1). The resin was equilibrated with 100 mM sodium phosphate at pH 7.0. Monoclonal IgG1 antibody was loaded onto the resin at 40 g / L resin. The resin was then washed with 100 mM sodium phosphate at pH 7.0. Elution was performed with one of the five buffers, with four consecutive elution cycles to maximize recovery. The total amount eluted per elution cycle was then calculated.

[0118] Table 1 – Static Binding MODDE Study Data

[0119]

[0120] Results and Conclusions

[0121] Figure 1 is a coefficient plot generated in the MODDE analysis software from the data in Table 1. Each bar represents one of the parameters being studied and what happens to the measured factor when the parameter value increases. A positive bar indicates that a higher value of the parameter increases the value of the measured factor, while a negative bar indicates that a higher value of the parameter decreases the measured factor.

[0122] Figure 1It is shown that imidazole has a statically significant effect on the amount recovered at cycles 1 and 2. However, there is no effect on cycles 3 and 4, because most of the product has eluted in these steps, and the amount produced is comparable to that in the absence of imidazole. High pH has a negative impact on elution amount and recovery, but the addition of imidazole can offset this effect. As more and more IgG1 is recovered in the earlier cycles, imidazole allows the number of cycles, and thus the elution volume, to decrease due to its faster elution kinetics. For example, at pH 4.0, only 3 cycles with imidazole are required to achieve the same amount, compared to 4 cycles without imidazole.

[0123] Example 2: IgG chromatogram elution curve

[0124] method

[0125] Two MabSelect SuRe HiScreen columns were used in dynamic binding mode. The resin was balanced with 100mM sodium phosphate at pH 7.0. Monoclonal antibody IgG1 or IgG4 was loaded onto the resin with 50g / L resin. The resin was then washed with 100mM sodium phosphate at pH 7.0, followed by salt washing with 100mM sodium phosphate and 500mM sodium chloride at pH 6.9. Elution was performed with 100mM sodium citrate at pH 3.6 or pH 4.0 (adjusted with NaOH) or 100mM sodium citrate and 300mM imidazole at pH 3.6 or pH 4.0 (adjusted with HCl).

[0126] Results and Conclusions

[0127] Figure 2 and Figure 3 Results obtained for an IgG1 monoclonal antibody are shown: the citrate eluent recovered 83% of the product at pH 3.6 and 39% at pH 4.0. However, these yields were increased to 94% and 65%, respectively, by the addition of imidazole. In addition, at pH 3.6, the elution volume decreased with the addition of imidazole. At pH 4.0, the elution volume with imidazole was greater than that with the buffer without imidazole because more product was eluted, and for the run without imidazole, a nearly infinite elution volume would be required to achieve the same recovery because of its slower elution kinetics. Figure 4 Equivalent results obtained with IgG4 are shown.

[0128] Example 3: VH3 chromatographic elution curve

[0129] method

[0130] Two MabSelect HiScreen columns, GE, (9.4 mL CV) were used in dynamic binding mode. The resin was equilibrated with 100 mM sodium phosphate, pH 7.0. As described in WO2015 / 197772 The multispecific trivalent antibody molecules of the format Fab-2 x dsscFv described in were loaded onto the resin at 30 g / L resin. The resin was then washed with 100 mM sodium phosphate at pH 7.0. Elution was performed with 100 mM sodium citrate at pH 4.0 (adjusted with NaOH) or with 100 mM sodium citrate at pH 4.0 plus 300 mM imidazole (adjusted with HCl).

[0131] Results and Conclusions

[0132] Figure 5 It is shown that citrate elution recovered 36% of the product at pH 4.0. However, by adding imidazole, the yield increased to 49% in the same volume of elution buffer. Therefore, the addition of imidazole increased the elution kinetics, allowing more product to elute faster, thus requiring less buffer to achieve the same recovery. Additionally, this experiment shows that imidazole can prevent the product from rebinding to the resin ligand through the Fc and VH3 binding sites.

[0133] Example 4: IgGMODDE Study

[0134] method

[0135] Two MabSelect SuRe HiScreen posts, GE, (9.4mL CV) were used in dynamic binding mode. The resin was balanced with 100mM sodium phosphate at pH 7.0. Monoclonal antibody IgG1 was loaded onto the resin with 50g / L resin. The resin was then washed with 100mM sodium phosphate at pH 7.0, followed by salt washing with 100mM sodium phosphate and 500mM sodium chloride at pH 6.9. A series of different 0.1M citrate elution buffers (table 2) were tested in DoE with different imidazole concentrations and pH values. Extra imidazole wash buffer was tested, replacing final pre-elution washing, to study whether pre-loading the post with imidazoles has a greater impact on elution kinetics.

[0136] Table 2 – Dynamic binding MODDE study data

[0137]

[0138] Results and Conclusions

[0139] Figure 6 In MODDE analysis software, Figure 1The coefficients plot was generated in the same manner but using the data in Table 2. "ImE" is the concentration of imidazole in the elution buffer, and "ImW" is the concentration of imidazole in the wash buffer. "*" indicates an interaction between the terms.

[0140] Figure 6 Shown the result of MODDE research, this result further supported previous experiment, because it determined that imidazoles had a static significant effect on reducing elution volume, therefore increased the product concentration in the eluent.This research has also determined the square term (ImE*ImE) of imidazole concentration, and it shows that there is optimal working concentration, departs from this scope and will cause elution volume to increase.In addition, before elution, can further reduce elution volume with imidazole buffer washing column.Before the elution step, imidazoles are pre-loaded on the post to prevent the elution front moving downward along the post from being in the environment that does not contain imidazoles.Therefore, can prevent all aspects of the elution step from being re-bonded on the resin with imidazole pre-loading, and this therefore increases elution kinetics.

[0141] Example 5: Elution curve of imidazole analogs

[0142] method

[0143] Two MabSelect HiScreen columns, GE (9.4 mL CV) were used in dynamic binding mode. The resin was equilibrated with 100 mM sodium phosphate, pH 7.0. Monoclonal antibody IgG1 was loaded onto the resin at 35 g / L resin. The resin was then washed with 100 mM sodium phosphate, pH 7.0. Elution was performed with either 100 mM sodium citrate, pH 3.6 or 100 mM sodium citrate, pH 3.6 plus 250 mM 1,3-dimethyl-1H-pyrazole. In preliminary analog studies, this analog was found to be effective in reducing elution volume.

[0144] Results and Conclusions

[0145] Figure 7 The control citrate elution is shown to recover 95% of the product in 2.85 column volumes at pH 3.6. However, by adding 1,3-dimethyl-1H-pyrazole, the elution volume was reduced to 1.76 columns with a comparable recovery of 94%. Therefore, the addition of 1,3-dimethyl-1H-pyrazole improves the elution kinetics, allowing the product to elute faster, thereby requiring less buffer to achieve similar recovery. Sequence Listing <110> UCB Biopharma Srl <120> Methods for purifying antibodies <130> PF0185 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable domain of anti-albumin antibody (without DS) <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable domain of anti-albumin antibody (ds) <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable domain of anti-albumin antibody (without DS) <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 <210> 4 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Light chain variable domain of anti-albumin antibody (DS) <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn 20 25 30 Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile 85 90 95 Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 110 <210> 5 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 645 gH5gL4 specific to albumin <400> 5 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu 180 185 190 Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr 225 230 235 240 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 245 250 <210> 6 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> 645 gH5gL4ds specific to albumin <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile 35 40 45 Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln 130 135 140 Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val 145 150 155 160 Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser 165 170 175 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu 180 185 190 Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr 225 230 235 240 Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr 245 250

Claims

1. A method for purifying an antibody, comprising: a) loading the mixture comprising the antibody to be purified onto an affinity chromatography resin, wherein the affinity chromatography resin is selected from the group consisting of: protein A chromatography resin, protein G chromatography resin and protein L chromatography resin, b) washing the chromatography resin with a wash buffer; c) eluting the antibody with an elution buffer comprising 0.01 M to 1.0 M imidazole or an imidazole analog and a pH of 3 to 5, wherein the imidazole analog is selected from 4(5)-methylimidazole, pyrazole, 1,5-dimethyl-1H-pyrazole, 1,3-dimethyl-1H-pyrazole and 1H-imidazol-1-ylmethanol.

2. The method of claim 1, wherein the elution buffer comprises 0.01 M to 0.5 M imidazole or an imidazole analog.

3. The method according to claim 1 or 2, wherein the wash buffer comprises imidazole or an imidazole analog. The method according to claim 3 , wherein the wash buffer comprises 0.01 M to 0.5 M imidazole or an imidazole analog.

5. The method according to claim 1 or 2, wherein the method comprises the further step of equilibrating the chromatography resin with an equilibration buffer comprising imidazole or an imidazole analogue before loading the mixture comprising the antibody onto the chromatography resin.

6. The method according to claim 1 or 2, wherein one, two or all of steps a), b) and c) are performed under dynamic conditions.

7. The method according to claim 1 or 2, wherein the antibody is selected from the group consisting of: IgG, Fab', F(ab')2, scFv, Fab-Fv, Fab-scFv, Fab-(scFv)2, Fab-(Fv)2, diabody, triabody and tetrabody.

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