Arginine wash in protein purification

Abstract

The present invention relates to a method for isolating a product and / or reducing impurities, such as a host cell protein (HCP), from a loading fluid comprising the product and one or more impurities, said method being carried out by passing the loading fluid through a medium, the product is then passed through at least one wash solution having a pH greater than 8 comprising arginine or an arginine derivative at a concentration greater than about 525 mM, and the product is collected using an eluent solution.

Description

Technical Field

[0001] The present invention relates to a method for separating a product and / or reducing impurities such as host cell proteins (HCP) from a load fluid comprising the product and one or more impurities, the method being carried out by passing the load fluid through a chromatography column and then through at least one washing solution having a pH greater than 8 and comprising arginine or an arginine derivative such as an arginine salt at a concentration greater than 525 mM, and collecting the product using an elution solution. Background Art

[0002] The present invention relates to protein purification and in particular to a method for purifying a protein bound to a chromatography resin, which is carried out by passing at least one washing solution containing arginine or an arginine derivative or an arginine salt through a chromatography column containing the resin and subsequently collecting the purified protein in the eluate.

[0003] By using recombinant techniques, many proteins (such as therapeutic antibodies) are cultured in eukaryotic or prokaryotic host cell lines engineered to express the protein. The use of the desired recombinant protein in pharmaceutical applications often depends on the ability to reliably recover sufficient levels of the protein from impurities such as host cell proteins, protein variants, and compounds in the culture medium.

[0004] Conventional protein purification methods aim to separate the target protein from impurities based on differences in size, charge, solubility, and degree of hydrophobicity. Such methods include chromatography, such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, and hydroxyapatite chromatography. These methods typically employ chromatography resins, which can be designed to selectively adhere to the target protein or impurities. In the bind-and-elute mode, the desired protein selectively binds to the chromatography resin and is differentially eluted from the medium by different solvents. In the flow-through mode, the impurities specifically bind to the chromatography resin while the target protein does not, thereby allowing the recovery of the desired protein in the "flow-through".

[0005] Current methods for purifying proteins (such as antibodies) include two or more chromatography steps. For example, the first step of a protein purification protocol can involve an affinity chromatography step that utilizes a specific interaction between the target protein and an immobilized resin. Not only does the specific chromatography column used affect the reduction of impurities, but the washing and elution conditions also have an impact. The inventors of the present invention have found that certain concentrations of arginine (or arginine salt or arginine derivative) and different pH levels used in the washing step of chromatography produce surprisingly high-purity products and low amounts of host cell proteins, particularly when applied to the antibodies described herein. Summary of the Invention

[0006] The present invention relates to a method for reducing impurities in an eluate containing a product, the method comprising: (a) providing a loading fluid comprising a product and one or more impurities, wherein the product is an Fc-containing monoclonal antibody, (b) applying the loading fluid of (a) to a protein A chromatography column under conditions suitable for binding the product, (c) optionally contacting the protein A chromatography column with a first washing solution, (d) applying one or more washing solutions (e.g., 1, 2, or 3 washing solutions) to the protein A chromatography column, wherein the washing solution comprises arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM, and wherein the pH of the washing solution is greater than 8.0, (e) optionally contacting the protein A chromatography column of (d) with another second washing solution, (f) contacting the washed protein A chromatography column of (d) or (e) with an elution solution under conditions suitable for eluting the product, and (g) collecting the eluate containing the product.

[0007] The present invention relates to the purification of antibodies, in particular monoclonal antibodies that bind to calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), and adrenocorticotropic hormone (ACTH) (e.g., the antibodies named eptinezumab, PACAP-1, and ACTH-1 hereinafter). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The figures shown depict the experimental results of measuring the content of HCP ("host cell protein") in samples after the protein A column step using varying arginine concentrations and pH conditions outlined in Example 1. DETAILED DESCRIPTION

[0008] The present invention provides a method for purifying and recovering a product from a loading fluid containing one or more impurities using a purification method comprising washing with arginine or an arginine salt or an arginine derivative. The present invention can be applied to large-scale preparation of proteins for therapeutic and / or diagnostic purposes.

[0009] To more readily understand the present invention, certain terms as used herein are defined. Additional definitions are set forth throughout the detailed description.

[0010] The term "arginine derivative" or "arginine salt" as used herein refers to derivatives produced by the reaction of arginine at the amino group, carboxyl group, or guanidyl group, or derivatives produced by replacing any hydrogen of arginine with a heteroatom, and salts thereof, which include salts of inorganic acids and / or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulfuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, saccharin, and sulfonic acids (such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid). In some embodiments, the term "arginine derivative" or "arginine salt" as used herein refers to and specifically includes Arg·HCl, acetyl arginine, agmatine, arginine, N-α-butyryl-L-arginine, or N-α-pyvaloyl arginine.

[0011] The term "product" refers to a molecule produced by a natural process (such as by expression in mammalian cells (e.g., CHO cells)). The term "product" includes proteins, such as therapeutic proteins, and in particular monoclonal antibodies capable of binding to protein A resin through their Fc domains, such as the antibodies named epratuzumab, PACAP-1, and ACTH-1 herein. The terms "product" and "protein of interest" are used interchangeably.

[0012] As used herein, the term "conditioned medium" refers to the supernatant produced by removing cells and cell debris from a cell culture medium that has been exposed to host cells (which can secrete the desired product) by separation methods (such as centrifugation and / or microfiltration). Conditioned select nutrients (such as vitamins, amino acids, cofactors, and minerals); additional growth factors / supplements including insulin; and additional exogenous or host cell proteins and impurities. The term conditioned medium includes clarified conditioned medium, filtered conditioned medium, and conditioned cell culture medium.

[0013] The term "loading fluid" refers to a liquid containing the product to be separated and one or more impurities. The loading fluid contacts a chromatographic resin (e.g., through a chromatography column) under the operating conditions of the present invention described below.

[0014] The term "impurity" refers to any foreign or unwanted molecule present in a solution (such as a loading fluid). Impurities can be biological macromolecules that are also present in a sample of the protein of interest to be purified, such as DNA, RNA, or proteins other than the protein of interest to be purified. Impurities include, for example, unwanted protein variants such as aggregated proteins, misfolded proteins, high molecular weight substances, low molecular weight substances and fragments, and deamidated substances; other proteins from the host cells secreting the protein to be purified, host cell DNA, components in the cell culture medium, molecules that are part of an absorbent for affinity chromatography leached into the sample during a previous purification step (such as Protein A); endotoxins; nucleic acids; viruses or fragments of any of the foregoing.

[0015] The term "resin" refers to an affinity matrix or resin that can undergo ligand-biological macromolecule interactions with the product to be separated during a macromolecule separation process. The resin is preferably the Protein A ligand in a Protein A chromatography column.

[0016] The term "host cell protein (HCP)" refers to non-product proteins produced by host cells during cell culture or fermentation. Thus, in some embodiments, the eluate containing the product has an HCP of less than 100 ppm (e.g., less than about 50 ppm, or less than about 20 ppm). The HCP composition is highly heterogeneous and depends on the protein product and the purification procedure used. Before any marketing approval of a biological product for therapeutic use, the level of contaminating proteins (such as HCP) in the product must be quantitatively measured according to ICH and FDA guidelines.

[0017] As used herein, the term "protein / protein" refers to one or more polypeptides that can function as a unit. As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. Therapeutic proteins can be, for example, secreted proteins. Therapeutic proteins include antibodies, antigen-binding fragments of antibodies, some of which are described in more detail below.

[0018] The term "antibody" refers to any immunoglobulin and encompasses any polypeptide containing an antigen-binding site. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, and in vitro-generated antibodies.

[0019] In additional embodiments of the invention, the product is an antibody having a CH2 / CH3 region and is thus suitable for purification by protein A chromatography. The term "CH2 / CH3 region" refers to those amino acid residues in the Fc region of the immunoglobulin molecule that interact with protein A. In particular, the antibodies of the invention are Fc-containing monoclonal antibodies capable of binding to a protein A ligand.

[0020] Examples of the Fc-containing monoclonal antibodies of the invention include antibodies that bind to calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), and adrenocorticotropic hormone (ACTH) as described below.

[0021] Calcitonin gene-related peptide (CGRP) is produced as a 37-amino acid multifunctional neuropeptide. Two forms of CGRP, namely CGRP-α and CGRP-β forms, are present in humans and have similar activities. CGRP-α and CGRP-β differ by three amino acids in humans and are derived from different genes. The CGRP peptide family includes amylin, adrenomedullin, and calcitonin, although each peptide has different receptors and biological activities. Doods, H., Curr. Op. Invest. Drugs [Current Opinion in Investigational Drugs], 2(9):1261-68 (2001).

[0022] Migraine is a neurovascular disorder that affects approximately 10% of the adult population in the United States and is typically accompanied by severe headache. Approximately 20%-30% of migraine patients experience aura, including focal neurological phenomena before and / or accompanying the event. CGRP is thought to play an important role in the progression of migraine. For example, it has been found that during the headache phase of migraine, the plasma concentration of CGRP in jugular venous blood is elevated, excluding other neuropeptides. In addition, according to Arulmozhi et al. (2005), the following have been found in migraine patients: (1) a strong correlation between plasma CGRP concentration and migraine; (2) infusion of CGRP produces migraine-like headache; (3) elevated baseline CGRP levels; and (4) changes in plasma CGRP levels during migraine attacks are significantly correlated with headache intensity. (Arulmozhi, D.K., et al., Vas. Pharma. [Vascular Pharmacology], 43:176-187 (2005)).

[0023] In certain embodiments, the invention relates to eptinezumab, a CGRP-binding antibody having the following sequence.

[0024] The heavy chain CDRs of eptinezumab CDR-H1: GYYMN SEQ ID No.: 1 CDR-H2: VIGINGATYYASWAKG SEQ ID No.:2 CDR-H3: GDI SEQ ID No.:3

[0025] The heavy chain variable region of epratuzumab contains EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYA SWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSSEQ ID No.:4

[0026] The heavy chain of epratuzumab contains EVQLVESGGG LVQPGGSLRL SCAVSGIDLS GYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTIS RDNSKTTVYL QMNSLRAEDT AVYFCARGDI WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDARVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY ASTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG SEQ ID No.:5

[0027] In the case of processing in a system where the heavy chain is not cleaved, such as in yeast like Pichia, a C-terminal lysine (K) may be present, but the C-terminal lysine is typically cleaved in the CHO expression system: EVQLVESGGGLVQPGGSLRL SCAVSGIDLS GYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTIS RDNSKTTVYLQMNSLRAEDT AVYFCARGDI WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDARVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY ASTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK SEQ ID No.:6

[0028] CDRs of the light chain of epratuzumab CDR-L1: QASQSVYHNTYLA SEQ ID No.:7 CDR-L2: DASTLAS SEQ ID No.:8 CDR-L3: LGSYDCTNGDCFV SEQ ID No.:9

[0029] The variable region of the light chain of epratuzumab contains QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLA SGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKRSEQ ID No.:10

[0030] The light chain of eptinezumab comprises QVLTQSPSSL SASVGDRVTI NCQASQSVYH NTYLAWYQQK PGKVPKQLIY DASTLASGVP SRFSGSGSGT DFTLTISSLQ PEDVATYYCL GSYDCTNGDC FVFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.:11

[0031] Pituitary adenylate cyclase-activating polypeptide (“PACAP”) is a member of the secretin / vasoactive intestinal peptide (“VIP”) / growth hormone-releasing hormone (“GHRH”) family. PACAP is a multifunctional vasodilatory peptide that exists in two α-amidated active forms, one with 38 amino acids and the other with 27 amino acids. Both peptides have the same 27 N-terminal amino acids and are synthesized from the same precursor protein, preproPACAP (see, Moody et al., Curr. Opin. Endocrinol. Diabetes Obes. [Current Opinion in Endocrinology, Diabetes and Obesity], 18(1):61-67, 2011). PACAP38 is the more prevalent active form, accounting for up to 90% of the PACAP forms in mammalian tissues (see, Kaiser and Russo, Neuropeptides [Neuropeptides], 47:451-461, 2013). The sequence of PACAP38 is identical in all mammals and differs from its orthologs in birds and amphibians by only one amino acid (see, Vaudry et al., Pharmacol. Rev. [Pharmacological Reviews], 52:269-324, 2000). The secretin / VIP / GHRH family includes the mammalian peptide histidine methionine (“PHM”), secretin, glucagon, glucagon-like peptide-1 (“GLP1”), glucagon-like peptide-2 (“GLP2”), glucose-dependent insulinotropic polypeptide (“GIP”), and growth hormone-releasing factor (“GRF”). PACAP27 has 68% sequence identity with VIP at the amino acid level (see, Vaudry et al., 2000).

[0032] It is hypothesized that PACAP plays a role in a variety of diseases and conditions, including but not limited to migraine, headache, and pain, but such a role of PACAP has not been clinically proven. Migraine is thought to have a neurovascular component. Migraine affects approximately 10% of the adult population in the United States and is typically accompanied by severe headache. Approximately 20%-30% of migraine patients experience aura, including focal neurological phenomena before and / or accompanying the event. Several observations suggest a role of PACAP in migraine: (1) during human migraine attacks (episodes), plasma levels of PACAP are elevated compared to interictal levels (see, Tuka et al., Cephalalgia [Headache], 33(13):1085-1095, 2013); (2) infusion of PACAP38 induces headache in healthy subjects and migraine-like attacks after headache in migraine patients (see, Schytz et al., Brain [Brain], 132:16-25, 2009; and Amin et al., Brain [Brain], 137:779-794, 2014), respectively; (3) PACAP-induced vasodilation may play a role in neurogenic inflammation (see, Kaiser and Russo, Neuropeptides [Neuropeptides], 47:451-461, 2013); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and is responsive to triptans (see, Amin et al., Brain [Brain], 32:140-149, 2012). PACAP has also been shown to induce vasodilation, photophobia, mast cell degranulation, and neuronal activation (see, Markovics et al., Neurobiology of Disease [Neurobiology of Disease], 45:633-644, 2012; Baun et al., Cephalalgia [Headache], 32(4):337-345, 2012; Chan et al., Pharmacology & Therapeutics [Pharmacology & Therapeutics], 129:332-351, 2011).

[0033] In certain embodiments, the invention relates to an antibody (designated PACAP-1) that binds to PACAP and has the following sequences.

[0034] The heavy chain CDRs of PACAP-1 CDR-H1: SYYMT SEQ ID No.: 12 CDR-H2: FIDAGGDAYYASWAKG SEQ ID No.: 13 CDR-H3: DLDL SEQ ID No.: 14

[0035] The variable region of the heavy chain of PACAP-1 contains EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSS SEQ ID No.:15

[0036] The heavy chain of PACAP-1 contains EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ IDNo.:16

[0037] The CDRs of the light chain of PACAP-1 CDR-L1: QSSESVYGNYLA SEQ ID No.:17 CDR-L2: EASKLES SEQ ID No.:18 CDR-L3: AGGDISEGVA SEQ ID No.:19

[0038] The variable region of the light chain of PACAP-1 contains DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAWFQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKRSEQ ID No.:20

[0039] The light chain of PACAP-1 contains DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAWFQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQID No.:21

[0040] POMC peptides (including ACTH (adrenocorticotropic hormone)) are thought to act mainly through the melanocortin receptors (MCRs), which are a family of five G-protein coupled receptors (i.e., MC1R, MC2R, MC3R, MC4R, and MC5R). MCRs are expressed in different tissues and have different physiological functions. MC1R (expressed on melanocytes, macrophages, and adipocytes) is involved in pigmentation and inflammation. MC2R (expressed in the adrenal cortex) is involved in adrenal steroidogenesis. MC3R (expressed in the central nervous system (CNS), gastrointestinal (GI) tract, and kidney) is involved in energy homeostasis and inflammation. MC4R (expressed in the CNS and spinal cord) is involved in energy homeostasis, appetite regulation, and erectile function. MC5R (expressed on lymphocytes and exocrine cells) is involved in the exocrine function and regulation of sebaceous glands. See Ramachandrappa et al., Frontiers in Endocrinology 4:19 (2013).

[0041] ACTH (one of the major end products of POMC processing) is a hormone crucial for normal steroidogenesis and maintaining normal adrenal weight. ACTH is secreted by the pituitary gland under physiological or psychological stress, and its main role is to increase the production and release of corticosteroids. In particular, ACTH is secreted by corticotroph cells of the anterior pituitary (or adenohypophysis) in response to the hypothalamic releasing hormone corticotropin-releasing hormone (CRH). Once secreted, ACTH travels to the adrenal cortex, where it binds to and activates MC2R. Activation of MC2R leads to the production of cAMP in adrenal cells. cAMP binds to and activates protein kinase (PKA), thus activating the conversion of lipid cholesterol to the steroid hormone cortisol.

[0042] Cortisol is a hormone that affects many biological processes to restore homeostasis after stress. Exemplary processes regulated by cortisol include regulating glucose homeostasis, increasing blood pressure, gluconeogenesis, promoting the metabolism of glycogen, lipids, and proteins, and suppressing the immune system. Under normal physiological conditions, cortisol levels are tightly regulated. However, in some medical conditions (including diseases and disorders further described herein), cortisol levels are elevated. Excessive production of cortisol has been shown to have many negative effects, such as damaging the hippocampus (a region of the brain crucial for cognitive function and regulation of the hypothalamic-pituitary-adrenal axis); increasing fat accumulation, elevated blood pressure levels, and blood glucose levels; osteoporosis; muscle weakness; and suppressing the immune system. Thus, elevated cortisol levels may play a role in: ACTH-driven hypercortisolism (such as Cushing's Disease or Cushing's Syndrome), obesity, diabetes, sleep apnea, depression, anxiety disorders, cancer (such as Cushing's Syndrome caused by ectopic ACTH expression, such as in small cell lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, neuro-oncology, or thymoma), muscle atrophy, hypertension, cognitive dysfunction, galactorrhea, and metabolic syndrome.

[0043] Aldosterone is a hormone released by the adrenal glands that helps regulate blood pressure. In particular, aldosterone increases the reabsorption of sodium and water and the release of potassium by the kidneys. In some disease conditions, aldosterone levels are elevated. For example, primary and secondary hyperaldosteronism occur when the adrenal glands release too much of the hormone aldosterone. Primary hyperaldosteronism (such as Conn's syndrome) is caused by a problem in the adrenal glands themselves, resulting in the release of too much aldosterone, while in secondary hyperaldosteronism, the excess aldosterone is caused by a condition outside the adrenal glands similar to a primary condition, such as, resulting in the adrenal glands releasing too much aldosterone. Primary hyperaldosteronism was once considered a rare disease, but some experts believe it may be the cause of hypertension in some patients. Most cases of primary hyperaldosteronism

[0044] In certain embodiments, the present invention relates to an antibody (named ACTH-1) that binds to ACTH and has the following sequences.

[0045] Heavy chain CDRs of ACTH-1 CDR-H1: SGYDIC SEQ ID No.:22 CDR-H2: CIDTGSGNTYYASSAKG SEQ ID No.:23 CDR-H3: GISSI SEQ ID No.:24

[0046] The heavy chain variable region of ACTH-1 comprises EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAKGISSIWGQG TLVTVSS SEQ ID No.:25

[0047] The heavy chain of ACTH-1 comprises EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAKGISSIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQID No.:26

[0048] If expressed in yeast (such as Pichia pastoris cells), the heavy chain of ACTH-1 may comprise a terminal lysine (K) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAKGISSIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQID No.:32

[0049] The light chain CDR of ACTH-1 CDR-L1: QASQTISSDLA SEQ ID No.:27 CDR-L2: AASKLTS SEQ ID No.:28 CDR-L3: QTYYDIIDDGAT SEQ ID No.:29

[0050] The light chain variable region of ACTH-1 contains DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKL TSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDIIDDGATFGGGTKVEIKR SEQ ID No.:30

[0051] The light chain of ACTH-1 contains DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDIIDDGATFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No.:31

[0052] Antibody preparations for use with the methods described herein can be from a variety of sources, including but not limited to serum from immunized animals, ascites, hybridoma or myeloma supernatants, conditioned media derived from cultured recombinant cell lines expressing antibody molecules or cell extracts from antibody-producing cells. In one embodiment of the invention, the product is an antibody from conditioned media of a recombinant cell line producing the antibody. Although there can be some variation from cell line to cell line and among various antibody products, based on the disclosure herein, adapting the invention herein to a particular combination of antibody protein and production cell line is well within the scope of one of ordinary skill in the art.

[0053] In certain embodiments, at least one impurity is present in the loading fluid applied to the chromatography column, and at least one wash solution containing arginine or an arginine salt or an arginine derivative is used to remove the impurity bound to the resin of the chromatography column. In one embodiment of the invention, after washing and eluting the chromatography column, the purified product contains less than 60% impurities (e.g., host cell proteins), in one embodiment about 40% impurities, in one embodiment about 20% impurities, in one embodiment about 10% impurities, in one embodiment about 5% impurities, in one embodiment less than 3% impurities, and in another embodiment less than 1% impurities. Impurities include but are not limited to unwanted protein variants such as aggregated proteins, high molecular weight substances, low molecular weight substances and fragments, and deamidated substances; other proteins from the host cells secreting the protein being purified; host cell DNA; components of the cell culture medium, molecules that are part of the absorbent for affinity chromatography leached into the sample during a prior purification step (e.g., protein A and protein G); endotoxins; nucleic acids; viruses or fragments of any of the foregoing.

[0054] The chromatography columns used in the methods described herein are, for example, affinity chromatography columns, hydrophobic interaction chromatography columns, immobilized metal affinity chromatography columns, size exclusion chromatography columns, diafiltration, ultrafiltration, virus removal filtration, and / or ion exchange chromatography columns, protein A chromatography columns or protein G chromatography columns. Protein A chromatography columns can be, for example, PROSEP-A TM (Millipore, UK), Protein A Sepharose FAST FLOW TM (GE Healthcare, Piscataway, N.J.), TOYOPEARL TM 650M Protein A (TosoHass Co., Philadelphia, Pa.), or MabSelect TM Column (GE Healthcare, Piscataway, N.J.) or MabSelect SuRe (AKTA Avant).

[0055] Before contacting the resin in the chromatography column with the loading fluid, it may be necessary to adjust parameters such as pH value, ionic strength, and temperature, and in some cases, different types of substances may also need to be added. Therefore, equilibration of the chromatography column by washing it with a solution (e.g., a buffer for adjusting pH value, ionic strength, etc., or a detergent for introduction) is an optional step to bring about the properties required for the binding and purification of the product.

[0056] In one embodiment of the present invention, the protein A chromatography column is equilibrated and washed with a washing solution to bring about the properties required for the purified product. In one embodiment of the present invention, the protein A chromatography column can be equilibrated with a solution containing salts (e.g., about 10 mM to about 30 mM NaPO4, and about 100 mM to about 150 mM NaCl). The pH of the equilibration buffer can range from about 6.0 to about 7.0. In one embodiment, the pH of the equilibration buffer is about 6.8. After contacting the resin in the chromatography column (e.g., protein A chromatography column) with the loading fluid, the resin is first washed. According to one embodiment of the present invention, the first wash can be performed with the above equilibration solution.

[0057] According to the present invention, after equilibration of the protein A chromatography column and optionally performing a first wash without using arginine or arginine salts or arginine derivatives, the washing solution used subsequently in the methods described herein can contain arginine or arginine derivatives. Arginine derivatives can be, but are not limited to, acetylarginine, agmatine, arginine, N-α-butyryl-L-arginine, or N-α-pivaloylarginine.

[0058] The concentration of arginine or an arginine derivative in the washing solution is from about 500 mM to about 600 mM, for example, 500 mM, 525 mM, 550 mM, 575 mM or 600 mM. In certain embodiments, the concentration of arginine or an arginine derivative in the washing solution is from about 500 mM to about 600 mM or from about 525 mM to about 575 mM, or from about 550 mM to about 575 mM. In certain embodiments, the concentration of arginine or an arginine derivative in the washing solution is greater than about 500 mM and less than about 600 mM.

[0059] The pH of the washing solution is typically from about 8.0 to about 8.7, for example, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6 and 8.7. In some cases, the pH of the washing solution is greater than 8.0 and less than about 8.7. The washing solution may contain 20 mM to 50 mM sodium phosphate (Na2HPO4) (for example, 20 mM, 30 mM, 40 mM or 50 mM). In one embodiment, the bound medium is washed with 5 column volumes of the washing solution and then subjected to an elution step.

[0060] After the washing step using arginine or an arginine derivative, there may be one or more washing steps that do not use arginine or an arginine derivative. According to one embodiment, this may use a sodium acetate-containing washing medium having a pH of about 5 to about 6 (for example, pH about 5.4) (for example, a sodium acetate concentration of from about 20 mM to about 50 mM, for example, 20 mM, 30 mM, 40 mM or 50 mM).

[0061] In certain embodiments of the present invention, the product can be eluted from the washed resin from, for example, a Protein A chromatography column. To elute the product from the Protein A chromatography column, the washed resin from the column is contacted with an elution buffer. In some embodiments, the elution buffer contains from about 15 mM to about 50 mM acetic acid. In additional embodiments, the elution buffer may also contain 20 mM to 50 mM glycine. The pH of the elution buffer can range from about 2.0 to about 5.0. In one embodiment, the pH of the elution buffer is about 4.0. In another embodiment, the pH of the buffer is about 3.65 or from 3.55 to 3.57.

[0062] After antibody elution, the resin of the column can optionally be cleaned, i.e., stripped and regenerated. This procedure is typically carried out periodically to minimize the accumulation of impurities on the solid phase surface and / or to sterilize the matrix to avoid microbial contamination of the product.

[0063] The buffer components can be adjusted according to the knowledge of those of ordinary skill in the art. The range of the sample buffer composition is provided in the examples below. Not all buffers or steps are necessary, but these are provided only for illustration. The high-throughput screening as described in the examples can be used to effectively optimize the buffer conditions for protein A column chromatography.

[0064] The eluate can contain the product, and the ratio of the product to host cell proteins is increased compared to the corresponding method where no detectable amount of arginine or arginine derivative is used in the wash solution.

[0065] The present invention also relates to products prepared by the methods described herein. In general, it is typically desirable to further isolate and / or purify the products isolated according to the present invention and formulate them for pharmaceutical use according to standard methods. For proteins, see, e.g., Protein Purification Principles and Practice, 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames, B.D. (Eds.), and Deutscher, M.P., Simon, M.I., Abelson, J.N. (Eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, which are incorporated herein by reference. Those of ordinary skill in the art will understand that the exact techniques used will vary depending on the characteristics of the product. The products of the present invention having pharmacological activity will be useful for preparing pharmaceuticals. These pharmaceuticals can be administered to a subject or can first be formulated for delivery by any available route including but not limited to: parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic, transdermal (topical), transmucosal, rectal, and vaginal.

[0066] The pharmaceutical composition of the product is formulated according to methods known in the art to be compatible with its intended route of administration, see, for example, "Remington: The Science & Practice of Pharmacy", 19th Edition, Williams & Williams, (1995) and "Physician's Desk Reference", 52nd Edition, Medical Economics, Montvale, N.J. (1998). In some embodiments, the product is formulated using sterile water (e.g., SWFI), buffered saline (e.g., phosphate buffered saline), polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), or a suitable mixture thereof.

[0067] Non-limiting examples of products that can be recovered using the methods described herein include proteins or peptides, such as antibodies, antibody fragments, recombinant proteins, naturally secreted proteins, proteins or peptides engineered to be secreted, non-protein products produced by cells, or combinations of the foregoing. Embodiments The present invention also relates to the following embodiment (E): E1. A method for reducing impurities in an eluate containing a product, the method comprising: (a) providing a loading fluid comprising the product and one or more impurities, wherein the product is an Fc-containing monoclonal antibody, (b) applying the loading fluid of (a) to a protein A chromatography column under conditions suitable for binding the product, (c) optionally contacting the protein A chromatography column with a first wash solution, (d) applying one or more wash solutions (e.g., 1, 2, or 3 wash solutions) to the protein A chromatography column, wherein the wash solution comprises arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM, and wherein the pH of the wash solution is greater than 8.0, (e) optionally contacting the protein A chromatography column of (d) with another second wash solution, (f) contacting the washed protein A chromatography column of (d) or (e) with an elution solution under conditions suitable for eluting the product, and (g) collecting the eluate containing the product. E2. The method according to embodiment 1, wherein the loading fluid in step (a) is obtained from CHO cells expressing eptinezumab, PACAP-1, or ACTH-1, such as the conditioned medium from CHO cells expressing said antibody. E3. The method according to embodiment 1, wherein the impurities in step (a) are selected from the group consisting of: DNA, RNA, endotoxin, proteins (proteins other than eptinezumab, PACAP-1 or ACTH-1), and host cell proteins (e.g., unwanted proteins from CHO cells). E4. The method according to embodiment 1, wherein the concentration of the arginine or the arginine salt or arginine derivative ranges from: (I) about 500 mM to about 600 mM; (II) about 525 mM to about 575 mM, (III) about 550 mM to about 575 mM or (IV) is about 575 mM. E5. The method according to embodiment 1 or 2, wherein the pH of the one or more washing solutions in (d): (i) ranges from about 8.0 to about 8.7; (ii) ranges from about 8.2 to about 8.7; (iii) ranges from about 8.5 to about 8.7; (iv) is about 8.5; (v) is about 8.6; or (vi) is about 8.7. E6. The method according to embodiments 1-5, wherein the arginine salt or arginine derivative comprises Arg·HCl, acetyl arginine, agmatine, arginine, N-α-butyryl-L-arginine, or N-α-pivaloyl arginine. E7. The method according to any one of embodiments 1-6, wherein the monoclonal antibody is specific for CGRP, such as eptinezumab as defined by: Variable heavy chain Seq ID NO.: 4 and variable light chain Seq ID No.: 10 and / or Heavy chain Seq ID No.: 5 or 6 and light chain Seq ID No.: 11. E8. The method according to any one of embodiments 1-6, wherein the monoclonal antibody is specific for PACAP, such as PACAP-1 as defined by: Variable heavy chain Seq ID NO.: 15 and variable light chain Seq ID No.: 20 and / or Heavy chain Seq ID No.: 16 and light chain Seq ID No.: 21. E9. The method according to any one of embodiments 1-6, wherein the monoclonal antibody is specific for ACTH, such as ACTH-1 as defined by: Variable heavy chain Seq ID NO.: 25 and variable light chain Seq ID No.: 30 and / or Heavy chain Seq ID No.: 26 or heavy chain Seq ID No.: 32 and light chain Seq ID No.: 31. E10. The method according to any one of embodiments 1 - 9, wherein in d), the concentration of arginine in the washing solution is about 575 mM and the pH is 8.5 or 8.7. E11. The method according to any one of embodiments 1 - 9, wherein in d), the concentration of arginine in the one or more washing solutions is about 525 mM and the pH is 8.5 or 8.7. E12. The method according to any one of embodiments 1 - 11, wherein one or more of the impurities are host cell proteins, nucleic acids, product variants, and / or endotoxins. E13. The method according to any one of embodiments 1 - 12, wherein the elution solution contains about 15 mM to about 50 mM acetic acid and optionally contains about 20 mM to 50 mM glycine. E14. The method according to any one of embodiments 1 - 13, wherein the elution solution has a pH of about 2 to about 5. E15. The method according to any one of embodiments 1 - 14, wherein the elution solution has a pH of about 4. E16. The method according to any one of embodiments 1 - 15, wherein in c) and e), the one or more washing solutions do not contain arginine or arginine salts or arginine derivatives. E17. A pharmaceutical composition comprising eptinezumab, PACAP - 1, or ACTH - 1 obtained by the method according to embodiment 1. E18. Use of the pharmaceutical composition comprising eptinezumab, PACAP - 1, or ACTH - 1 according to embodiment 17 as a medicine. E19. The pharmaceutical composition comprising eptinezumab according to embodiment E18 for use in the treatment of headache, migraine (such as chronic migraine or episodic migraine), and cluster headache. E20. The pharmaceutical composition comprising ACTH - 1 according to embodiment 17 for use in the treatment of Cushing's disease or congenital adrenal hyperplasia. E21. The pharmaceutical composition comprising PACAP - 1 according to embodiment 17 for use in the treatment of headache, migraine (such as chronic migraine or episodic migraine), and cluster headache. E22. A method for treating headache, migraine (such as chronic migraine or episodic migraine) and cluster headache, the method comprising administering a therapeutically effective amount of eptinezumab or PACAP-1 obtained by the method as described in embodiment 1. E23. A method for treating Cushing's disease or congenital adrenal hyperplasia, the method comprising administering a therapeutically effective amount of ACTH-1 obtained by the method as described in embodiment 1. Examples Example 1 To explore the effects of different arginine concentrations and pH levels in the wash solution on the Protein A column, the inventors of the present invention tested different pH and arginine conditions and measured different impurities, including HCP concentration. The ranges tested were pH 7.6–8.7, arginine 375 - 575 mM, and the wash length varied between 5 - 10 column volumes (CV). The Protein A column was loaded with filtered clarified harvest medium from CHO cells expressing eptinezumab. Eptinezumab was harvested from 6 x 3 L bioreactors and harvested by depth filtration at 4.34 g / L. The Protein A column was run under the following conditions: The Protein A column used was MabSelect SuRE resin and instrument AKTAAvant (Column name: 20.0 cm / 10 ml / 0.8 cm--MabSelect SuRe Lx 10253604--Column number 817) The measurement of HCP was carried out according to the following protocol: · Immunoenzymatic assay for measuring CHO host cell proteins (Cygnus Technologies catalog number 550-1) Reagents and materials Anti-CHO:HRP (F551-1) affinity purified goat antibody conjugated to HRP in a protein matrix containing preservatives. 1 x 12 mL Anti-CHO coated microtiter strips F552-1 * 12 x 8 well strips packed in bags containing desiccant CHO HCP standard F553-1 CHO HCP in bovine serum albumin containing preservatives. Standards of 0, 1, 3, 12, 40, and 100 ng / mL. 1 mL / vial Stop Solution 0.5M sulfuric acid. 1x 12 mL TMB Substrate F005 3,3’,5,5’-Tetramethylbenzidine. 1x 12 mL Wash Concentrate (20X) with Preservative F004 Tris Buffered Saline. 1x 50 mL · Measurement – Host Cell Protein (HCP) · Assay Protocol · 1. Pipette 100 μL of anti-CHO:HRP (#F551-1) into each well. · 2. Pipette 50 μL of standards, controls, and samples into the wells as indicated on the working list. · 3. Cover the lid and incubate for 2 hours at 400–600 rpm on an orbital shaker at room temperature 24 °C + 4 °C. · 4. Pour the contents of the wells into the waste. Blot and gently but firmly tap the absorbent paper to remove most of the residual liquid. There is no need to over-tap the plate or use a vacuum aspiration device to try to remove all of the residual liquid, which may cause variable dissociation of the antibody-binding material, resulting in lower OD and poorer precision. Fill the wells to overflow with approximately 350 μL of diluted wash solution using a squirt bottle or by pipetting. Pour and tap again. Repeat the washing a total of 4 times. Wipe off any liquid at the bottom outside the microtiter wells, as any residue may interfere with the reading step. Do not let the wash solution remain in the wells for more than a few seconds. · 5. Pipette 100 μL of TMB Substrate (#F005). · 6. Incubate for 30 minutes at room temperature. · 7. Pipette 100 μL of Stop Solution (#F006). · 8. Read the absorbance at 450 / 650 nm. Table 1 shows the wash conditions, yield, and HCP content in the eluate for the wash experiments. Table 1 As described in the present invention, similar results were obtained by purifying PACAP and ACTH antibodies (referred to as PACAP-1 and ACTH-1 in this application). After purification under the same conditions as above, the HCP content of the PACAP samples ranged from 156.9 to 294.7, while for ACTH, an HCP content of approximately 245 was obtained when running at a pH of approximately 8.5 and an arginine concentration of approximately 575 mM. In further studies using pH 8.5 and arginine (575 mM), in two separate runs of PACAP-1, the HCP content decreased from approximately 307684 ng HCP / mg protein and 700688 ng HCP / mg protein to approximately 224 ng HCP / mg protein and 220 ng HCP / mg protein, respectively. Additionally, ACTH-1 purification showed a decrease in HCP ng / mg protein from 891432 to 326 ng / mg (or from 2104671 ng / mL HCP to 10002 ng / mL HCP), and a decrease from 10042454 ng / mg to 232 ng / mg (or from 2435063 ng / mL HCP to 7062 ng / mL).

Claims

1. A method for reducing impurities in an eluate containing a product, the method comprising: (a) providing a loading fluid containing a product and one or more impurities, wherein the product is an Fc-containing monoclonal antibody, (b) applying the loading fluid in (a) to a protein A chromatography column under conditions suitable for binding the product, (c) optionally contacting the protein A chromatography column with a first washing solution, (d) applying one or more washing solutions (e.g., 1, 2, or 3 washing solutions) to the protein A chromatography column, wherein the washing solution contains arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM, and wherein the pH of the washing solution is greater than 8.0, (e) optionally contacting the protein A chromatography column in (d) with another second washing solution, (f) contacting the washed protein A chromatography column in (d) or (e) with an elution solution under conditions suitable for eluting the product, and (g) collecting the eluate containing the product.

2. The method according to claim 1, wherein the concentration of the arginine or the arginine salt or arginine derivative comprises: (I) from about 500 mM to about 600 mM; (II) from about 525 mM to about 575 mM, (III) from about 550 mM to about 575 mM or (IV) is about 575 mM.

3. The method according to claim 1 or 2, wherein the pH of the one or more washing solutions in (d): (i) ranges from about 8.0 to about 8.7; (ii) ranges from about 8.2 to about 8.7; (iii) ranges from about 8.5 to about 8.7; (iv) is about 8.5; (v) is about 8.6; or (vi) is about 8.

7.

4. The method according to claim 1, 2, or 3, wherein the arginine salt or arginine derivative comprises Arg·HCl, acetyl arginine, agmatine, arginine, N-α-butyryl-L-arginine, or N-α-pivaloyl arginine.

5. The method according to any one of claims 1-4, wherein the monoclonal antibody is specific for CGRP, such as eptinezumab.

6. The method according to any one of claims 1-4, wherein the monoclonal antibody is specific for PACAP, such as PACAP-1.

7. The method according to any one of claims 1-4, wherein the monoclonal antibody is specific for ACTH, such as ACTH-1.

8. The method according to any one of claims 1-7, wherein the concentration of arginine in the washing solution in (d) is about 575 mM, and the pH is 8.5 or 8.

7.

9. The method according to any one of claims 1-7, wherein the concentration of arginine in the one or more washing solutions in (d) is about 525 mM, and the pH is 8.5 or 8.

7.

10. The method according to any one of claims 1-9, wherein one or more of the impurities are host cell proteins, nucleic acids, product variants, and / or endotoxins.

11. The method according to any one of claims 1-9, wherein the elution solution comprises from about 15 mM to about 50 mM acetic acid and optionally from about 20 mM to 50 mM glycine.

12. The method according to any one of claims 1-10, wherein the elution solution has a pH of from about 2 to about 5.

13. The method according to any one of claims 1-10, wherein the elution solution has a pH of about 4.

14. The method according to any one of claims 1-13, wherein the one or more washing solutions in c) and e) do not contain arginine or arginine salts or arginine derivatives.

Images