Arginine wash in protein purification

Abstract

The present invention relates to a method for isolating a product and / or reducing impurities, such as host cell proteins (HCPs), from a load solution containing the product and one or more impurities by passing the load solution through a medium followed by at least one wash solution comprising arginine or an arginine derivative at a concentration greater than 525 mM and a pH greater than 8, and recovering the product using an eluate.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating a product and / or reducing impurities, such as host cell proteins (HCPs), from a load solution containing the product and one or more impurities by passing the load solution through a chromatography column, followed by at least one wash solution containing arginine or an arginine derivative (e.g., an arginine salt at a concentration greater than 525 mM and a pH greater than 8), and recovering the product using an eluate. [Background technology]

[0002] The present invention relates to protein purification, and in particular to a method for purifying a protein bound to a chromatography resin by passing at least one wash solution containing arginine or an arginine derivative or an arginine salt through a chromatography column containing said resin, followed by recovery of the purified protein in the eluate.

[0003] Using recombinant technology, many proteins, such as therapeutic antibodies, are cultivated in eukaryotic or prokaryotic host cell lines engineered to express the protein. Desirable use of a recombinant protein for pharmaceutical applications is usually contingent on the ability to reliably recover adequate levels of the protein from impurities such as host cell proteins, protein variants, and compounds from the culture medium.

[0004] Traditional protein purification methods are designed to separate a protein of interest from impurities based on differences in size, charge, solubility, and degree of hydrophobicity. Such methods include chromatographic techniques such as affinity chromatography, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, and hydroxyapatite chromatography. These methods often use chromatography resins that can be designed to selectively attach either the protein of interest or the impurities. In the bind-elute mode, the protein of interest selectively binds to the chromatography resin and is differentially eluted from the medium by different solvents. In the flow-through mode, impurities specifically bind to the chromatography resin, but the protein of interest does not, allowing the protein of interest to be recovered 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 may involve an affinity chromatography step that exploits specific interactions between the protein of interest and an immobilized resin. The reduction of impurities is influenced not only by the particular chromatography column used, but also by the wash and elution conditions. The inventors of the present invention have discovered that specific concentrations of arginine (or arginine salts or arginine derivatives) and different pH levels used in the wash steps of the chromatography method, particularly when applied to the antibodies named herein, result in surprisingly high purity and low amounts of host cell protein. Summary of the Invention

[0006] The present invention provides (a) providing a loading solution containing a product that is an Fc-containing monoclonal antibody and one or more impurities; (b) applying the load solution of (a) to a Protein A chromatography column under conditions suitable for product binding; (c) optionally contacting the Protein A chromatography column with a first wash solution; (d) applying one or more wash solutions (e.g., one, two, or three wash solutions) to the Protein A chromatography column, the wash solutions containing arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM and having a pH greater than 8.0; (e) optionally contacting the Protein A chromatography column of d) with a second wash solution; (f) contacting the Protein A chromatography column washed in d) or e) with an eluate under conditions suitable for eluting the product; (g) collecting the product-containing eluate; The present invention relates to a method for reducing impurities in an eluate containing a product, comprising:

[0007] The present invention relates to the purification of antibodies, particularly monoclonal antibodies that bind to calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), and adrenocorticotropic hormone (ACTH) (hereinafter referred to as eptinezumab, PACAP-1, and ACTH-1). [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows the results of an experiment analyzing HCP ("host cell protein") content in samples after a Protein A column step using varying arginine concentrations and pH conditions as outlined in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention provides a method for purifying and recovering a product from a load solution containing one or more impurities using a purification method comprising an arginine wash, or a wash with an arginine salt or arginine derivative. The present invention may be applied to large-scale production of proteins for therapeutic and / or diagnostic purposes.

[0010] In order that the present invention may be more readily understood, certain terms used herein are defined. Additional definitions are set forth throughout the detailed description.

[0011] The term "arginine derivative" or "arginine salt," as used herein, refers to derivatives and salts thereof resulting from reactions at the amino, carboxy, or guanidyl groups of arginine, or by substitution of any hydrogen of arginine with a heteroatom, including inorganic 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, salicylic acid, saccharin, and the like, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, and the like. In some embodiments, the term "arginine derivative" or "arginine salt" herein specifically refers to and includes Arg·HCl, acetylarginine, agmatine, arginic acid, N-α-butyroyl-L-arginine, or N-α-pivaloylarginine.

[0012] The term "product" refers to a molecule produced by a natural process (e.g., expression in mammalian cells such as CHO cells). The term "product" includes proteins such as therapeutic proteins, particularly monoclonal antibodies capable of binding to Protein A resin via their Fc domains, such as the antibodies designated herein as eptinezumab, PACAP-1, and ACTH-1. The terms "product" and "protein of interest" are used interchangeably.

[0013] As used herein, the term "conditioned culture medium" refers to the supernatant produced by removing cells and cell debris by separation methods such as centrifugation and / or microfiltration from cell culture medium exposed to host cells that may secrete a desired product. The supernatant contains selected nutrients (e.g., vitamins, amino acids, cofactors, and minerals); additional growth factors / supplements, including insulin; and additional exogenous or host cell proteins and impurities. The term conditioned culture medium includes clarified conditioned medium, filtered conditioned medium, and conditioned cell culture medium.

[0014] The term "load" refers to a liquid containing the product to be separated and one or more impurities. The load liquid contacts the chromatographic resin (e.g., passes through a chromatographic column) under the operating conditions of the invention described below.

[0015] The term "impurities" refers to foreign or undesired molecules present in a solution, such as a load solution. Impurities can be biopolymers, such as DNA, RNA, or proteins, other than the protein being purified, present in a sample of the protein being purified. Impurities include, for example, aggregated proteins, misfolded proteins, high molecular weight species, low molecular weight species and fragments, and undesired protein variants such as deamidated species; other proteins from the host cells secreting the protein being purified; host cell DNA; components from the cell culture medium; molecules that were part of the absorbent used in affinity chromatography and leached into the sample during a previous purification step (e.g., Protein A); endotoxins; nucleic acids; viruses; or fragments of any of the foregoing.

[0016] The term "resin" refers to an affinity matrix or resin that can undergo a ligand-biopolymer interaction with the product to be separated in a macromolecular separation process. The resin is preferably a Protein A ligand in a Protein A chromatography column.

[0017] The term "host cell proteins (HCPs)" refers to non-product proteins produced by host cells during cell culture or fermentation. Thus, in some embodiments, the product-containing eluate contains HCPs present at less than parts per million (ppm) of HCPs (e.g., less than about 50 ppm, or less than about 20 ppm). The composition of HCPs is highly heterogeneous and depends on the protein product and purification procedure used. Prior to marketing approval of a therapeutic biologic, the levels of contaminating proteins (e.g., HCPs) in the formulation must be quantitatively determined in accordance with ICH and FDA guidelines.

[0018] As used herein, the term "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.

[0019] The term "antibody" refers to any immunoglobulin and encompasses any polypeptide that contains an antigen-binding site. This term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro produced antibodies.

[0020] In a further embodiment of the invention, the product is an antibody having a CH2 / CH3 region and therefore amenable to purification by Protein A chromatography. The term "CH2 / CH3 region" refers to amino acid residues within the Fc region of an immunoglobulin molecule that interact with Protein A. In particular, the antibodies of the invention are monoclonal Fc-containing antibodies capable of binding to a Protein A ligand.

[0021] Examples of Fc-containing monoclonal antibodies of the present invention include the following antibodies that bind to calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), and adrenocorticotropic hormone (ACTH).

[0022] Calcitonin gene-related peptide (CGRP) is produced as a multifunctional neuropeptide 37 amino acids in length. Two forms of CGRP, CGRPα and CGRPβ, exist in humans and have similar activity. 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, each of which has different receptors and biological activities. Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68(2001).

[0023] Migraine is a neurovascular disorder affecting approximately 10% of the adult population in the United States and typically presents with severe headache. Approximately 20–30% of migraine patients experience aura, a localized neurological phenomenon that precedes or coincides with the onset of a migraine headache. CGRP is thought to play an important role in migraine pathogenesis. For example, plasma concentrations of CGRP in the jugular vein, but not other neuropeptides, have been shown to be elevated during the headache phase of a migraine. Furthermore, Arulmozhi et al. (2005) found the following in migraine patients: (1) a strong correlation between plasma CGRP concentrations and migraine; (2) CGRP infusion produced migraine-like headache; (3) baseline CGRP levels were elevated; and (4) changes in plasma CGRP concentrations during a migraine attack were significantly correlated with headache intensity (Arulmozhi, DK, et al., Vas. Pharma., 43:176–187 (2005)).

[0024] In a specific embodiment, the invention relates to the CGRP-binding antibody eptinezumab, having the sequence: eptinezumab heavy chain CDR CDR-H1: GYYMN SEQ ID NO: 1 CDR-H2: VIGINGATYYASWAKG SEQ ID NO: 2 CDR-H3: GDI SEQ ID NO: 3

[0025] The variable heavy chain of eptinezumab comprises: EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS SEQ ID NO: 4

[0026] The heavy chain of eptinezumab comprises: [ka]

[0027] The C-terminal lysine (K) may be present if the heavy chain is not processed in a system that cleaves it (e.g., yeast such as Pichia), but the C-terminal lysine is typically cleaved in CHO expression systems: [ka]

[0028] eptinezumab light chain CDR CDR-L1: QASQSVYHNTYLA SEQ ID NO: 7 CDR-L2: DASTLAS SEQ ID NO: 8 CDR-L3: LGSYDCTNGDCFV SEQ ID NO: 9

[0029] The variable light chain of eptinezumab comprises: QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR SEQ ID NO: 10

[0030] The light chain of eptinezumab comprises: [ka]

[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 vasodilator peptide that exists in two α-amidated active forms: one containing 38 amino acids and the other containing 27 amino acids. Both peptides have the same N-terminal 27 amino acids and are synthesized from the same precursor protein, preproPACAP (see Moody et al., Curr. Opin. Endocrinol. Diabetes Obes., 18(1):61-67, 2011). PACAP38 is the more common active form, accounting for up to 90% of PACAP forms in mammalian tissues (see Kaiser and Russo, Neuropeptides, 47:451-461, 2013). The sequence of PACAP38 is identical in all mammals and differs from its avian and amphibian orthologues by only one amino acid (see Vaudry et al., Pharmacol. Rev., 52:269-324, 2000). The secretin / VIP / GHRH family includes 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 shares 68% sequence identity with VIP at the amino acid level (see Vaudry et al., 2000).

[0032] PACAP has been hypothesized to be involved in numerous diseases and disorders, including but not limited to migraine, headache, and pain, but such a role for 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 usually accompanied by severe headache. Approximately 20-30% of migraine patients experience aura, a focal neurological phenomenon that precedes or coincides with the onset of a migraine. A role for PACAP in migraine has been suggested by several observations: (1) in humans, plasma concentrations of PACAP are elevated during migraine attacks (ictal period) compared with interictal concentrations (see Tuka et al., Cephalalgia, 33(13):1085-1095, 2013); (2) infusion of PACAP38 induced headache in healthy subjects and headache followed by migraine-like attacks in migraine patients (see Schytz et al., Brain, 132:16-25, 2009; and Amin et al., Brain, 137:779-794, 2014, respectively); (3) PACAP-induced vasodilation may play a role in neurogenic inflammation (Kaiser and (See Russo, Neuropeptides, 47:451-461, 2013); and (4) PACAP-induced migraine, accompanied by photophobia, phonophobia, and nausea, is responsive to triptans (See Amin et al., Brain, 32:140-149, 2012). PACAP has also been shown to cause vasodilation, photophobia, mast cell degranulation, and neuronal activation (See Markovics et al., Neurobiology of Disease, 45:633-644, 2012; Baun et al., Cephalalgia, 32(4):337-345, 2012; Chan et al., Pharmacology & Therapeutics, 129:332-351, 2011).

[0033] In a particular embodiment, the present invention relates to an antibody that binds to PACAP (designated PACAP-1) having the following sequence: PACAP-1 heavy chain CDR CDR-H1: SYYMT SEQ ID NO: 12 CDR-H2: FIDAGGDAYYASWAKG SEQ ID NO: 13 CDR-H3: DLDL SEQ ID NO: 14

[0034] The variable heavy chain of PACAP-1 comprises: EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSS SEQ ID NO: 15

[0035] The heavy chain of PACAP-1 comprises: [ka]

[0036] PACAP-1 light chain CDR CDR-L1: QSSESVYGNYLA SEQ ID NO: 17 CDR-L2: EASKLES SEQ ID NO: 18 CDR-L3: AGGDISEGVA SEQ ID NO: 19

[0037] The variable light chain of PACAP-1 comprises: DIQLTQSPSTLSASVGDRVTITCQSSESVYGNYLAWFQQKPGKAPKFLIYEASKLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGDISEGVAFGGGTKVEIKR SEQ ID NO: 20

[0038] The light chain of PACAP-1 comprises: [ka]

[0039] POMC peptides, including ACTH (adrenocorticotropic hormone), are thought to act primarily through melanocortin receptors (MCRs), a family of five G protein-coupled receptors (MCIR, MC2R, MC3R, MC4R, and MC5R). MCRs are expressed in diverse tissues and perform distinct physiological functions. MCIRs, expressed in melanocytes, macrophages, and adipocytes, are involved in pigmentation and inflammation. MC2Rs, expressed in the adrenal cortex, are involved in adrenal steroidogenesis. MC3Rs, expressed in the central nervous system (CNS), gastrointestinal tract (GI), and kidneys, are involved in energy homeostasis and inflammation. MC4Rs, expressed in the CNS and spinal cord, are involved in energy homeostasis, appetite regulation, and erectile function. MC5Rs, expressed in lymphocytes and exocrine cells, are involved in exocrine function and sebaceous gland regulation. See Ramachandrappa et al., Frontiers in Endocrinology 4:19 (2013).

[0040] ACTH, one of the major end products of POMC processing, is a hormone essential for normal steroidogenesis and the maintenance of normal adrenal mass. ACTH is secreted from the pituitary gland in response to physiological or psychological stress, and its primary effect is to increase the production and release of corticosteroids. Specifically, ACTH is secreted from corticotrophic cells in the anterior lobe of the pituitary gland (or adrenal pituitary gland) in response to the release of corticotropin-releasing hormone (CH) from the hypothalamus. Upon secretion, ACTH travels to the adrenal cortex, where it binds to and activates MC2R. Activation of MC2R results in the production of cAMP in adrenal cells. cAMP binds to and activates protein kinase A (PICA), which activates the conversion of lipid cholesterol to the steroid hormone cortisol.

[0041] Cortisol is a hormone that affects numerous biological processes to restore homeostasis after stress. Typical processes regulated by cortisol include regulating glucose homeostasis, increasing blood pressure, promoting gluconeogenesis, glycogen, lipid, and protein metabolism, and suppressing the immune system. Under normal physiological conditions, cortisol levels are tightly controlled. However, in certain situations (including diseases and disorders described further herein), cortisol levels are elevated. Excessive secretion of cortisol has been shown to have many adverse effects, including damaging the hippocampus, a brain region important for regulating cognitive function and the hypothalamic-pituitary-adrenal axis; increasing fat deposition, blood pressure, and blood glucose levels; reducing bone mass; reducing muscle strength; and suppressing the immune system. Thus, elevated cortisol levels may play a role in ACTH-driven cortisone excess (such as Cushing's disease or syndrome), obesity, diabetes, sleep apnea, depression, anxiety disorders, cancer (such as Cushing's syndrome due to ectopic ACTH expression in small cell lung cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, neuronal tumors, or thymoma), muscle atrophy, hypertension, cognitive dysfunction, galactorrhea, and metabolic syndrome.

[0042] Aldosterone is a hormone secreted by the adrenal glands that helps regulate blood pressure. In particular, aldosterone increases the reabsorption of sodium and water and the release of potassium in the kidneys. Several conditions result in elevated aldosterone levels. For example, primary and secondary hyperaldosteronism are caused by excessive secretion of the hormone aldosterone by the adrenal glands. While primary hyperaldosteronism, such as Conn's syndrome, results from a problem with the adrenal glands themselves that causes excessive aldosterone release, the excess aldosterone in secondary hyperaldosteronism results from something outside the adrenal glands mimicking the primary condition, such as the adrenal glands releasing too much aldosterone. Primary hyperaldosteronism was previously considered a rare condition, but some experts believe it may be the cause of high blood pressure in some patients. Most cases of primary hyperaldosteronism

[0043] In a particular embodiment, the present invention relates to an antibody that binds to ACTH (designated ACTH-1) having the following sequence: ACTH-1 heavy chain CDR CDR-H1: SGYDIC SEQ ID NO: 22 CDR-H2: CIDTGSGNTYYASSAKG SEQ ID NO: 23 CDR-H3: GISSI SEQ ID NO: 24

[0044] The variable heavy chain of ACTH-1 comprises: EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAKGISSIWGQGTLVTVSS SEQ ID NO: 25

[0045] The heavy chain of ACTH-1 comprises: [ka]

[0046] The heavy chain of ACTH-1 may include a terminal lysine (K) when expressed in yeast (eg, Pichia pastoris cells). [ka]

[0047] ACTH-1 light chain CDR CDR-L1: QASQTISSDLA SEQ ID NO: 27 CDR-L2: AASKLTS SEQ ID NO: 28 CDR-L3: QTYYDIIDDGAT SEQ ID NO: 29

[0048] The variable light chain of ACTH-1 comprises: DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDIIDDGATFGGGTKVEIKR SEQ ID NO: 30

[0049] The light chain of ACTH-1 comprises: [ka]

[0050] Antibody preparations used in the methods described herein can be obtained from several sources, including, but not limited to, serum from immunized animals, ascites fluid, hybridoma or myeloma supernatant, conditioned culture medium from culturing a recombinant cell line expressing the antibody molecule, or cell extracts of antibody-producing cells. In one embodiment of the present invention, the product is antibody from the conditioned culture medium of an antibody-producing recombinant cell line. While there may be some variation from cell line to cell line and for various antibody products, it is within the knowledge of one of ordinary skill in the art, based on the disclosure herein, to adapt the invention to a particular combination of antibody protein and producing cell line.

[0051] In certain embodiments, the fluid load applied to the chromatography column includes at least one impurity, and at least one wash solution containing arginine or an arginine salt or arginine derivative is used to remove impurities bound to the resin of the chromatography column. In one embodiment of the present invention, 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 after washing and elution from the chromatography column. Impurities include, but are not limited to, undesired protein variants such as aggregated proteins, high molecular weight species, low molecular weight species and fragments, and deamidated species; other proteins from the host cells secreting the protein being purified; host cell DNA; components from the cell culture medium; molecules that were part of the absorbent used in affinity chromatography and leached into the sample during previous purification steps (e.g., Protein A and Protein G); endotoxins; nucleic acids; viruses; or fragments of any of the foregoing.

[0052] The chromatography column used in the methods described herein may be, for example, an affinity chromatography column, a hydrophobic interaction chromatography column, an immobilized metal affinity chromatography column, a size-exclusion chromatography column, a diafiltration, an ultrafiltration, a virus removal filtration, and / or an ion-exchange chromatography column, a Protein A chromatography column, or a Protein G chromatography column. Protein A chromatography columns may include, for example, PROSEP-A™ (Millipore, UK), Protein A Sepharose FAST FLOW™ (GE Healthcare, Piscataway, NJ), TOYOPEARL™ 650M Protein A (TosoHass Co., Philadelphia, Pa.), a MabSelect™ column (GE Healthcare, Piscataway, NJ), or MabSelect SuRe (AKTA Avant).

[0053] Before contacting the resin of the chromatography column with the load solution, it may be necessary to adjust parameters such as pH, ionic strength, and temperature, and in some cases, to add different types of substances. Therefore, equilibration of the chromatography column by washing with a solution that provides the necessary properties for product binding and purification (e.g., a buffer to adjust pH, ionic strength, etc., or a solution to introduce a detergent) is an optional step.

[0054] In one embodiment of the present invention, a Protein A chromatography column is equilibrated and washed with a wash solution to provide the properties required for product purification. In one embodiment of the present invention, the Protein A chromatography column may be equilibrated using a solution containing salts, such as, for example, about 10 mM to about 30 mM NaPO4 and about 100 mM to about 150 mM NaCl. The pH of the equilibration buffer may 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 of a chromatography column (e.g., a Protein A chromatography column) with a load solution, the resin is first washed. According to one embodiment of the present invention, the first wash may be performed using the equilibration solution described above.

[0055] According to the present invention, after equilibration of the Protein A chromatography column and, optionally, an initial wash that does not contain arginine or an arginine salt or an arginine derivative, subsequent wash solutions used in the methods described herein may contain arginine or an arginine derivative, including, but not limited to, acetylarginine, agmatine, arginic acid, N-α-butyroyl-L-arginine, or N-α-pivaloylarginine.

[0056] The concentration of arginine or an arginine derivative in the washing solution is 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 about 500 mM to about 600 mM, or about 525 mM to about 575 mM, or 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.

[0057] The pH of the wash solution is generally about 8.0 to about 8.7, e.g., 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, and 8.7. In some cases, the pH of the wash solution is greater than 8.0 and less than about 8.7. The wash solution may contain 20 mM to 50 mM sodium phosphate (NaHPO) (e.g., 20 mM, 30 mM, 40 mM, or 50 mM). In one embodiment, the bound medium is washed with 5 column volumes of wash solution, followed by an elution step.

[0058] The arginine or arginine derivative wash step may be followed by one or more wash steps that do not contain arginine or an arginine derivative, which, according to one embodiment, may use a sodium acetate-containing wash medium (e.g., sodium acetate at a concentration of about 20 mM to about 50 mM, e.g., 20 mM, 30 mM, 40 mM, or 50 mM) at a pH of about 5 to about 6 (e.g., about pH 5.4).

[0059] In certain embodiments of the present invention, the product may be eluted from, for example, a washed resin from a Protein A chromatography column. To elute the product from a Protein A chromatography column, the washed resin from the chromatography column is contacted with an elution buffer. In some embodiments, the elution buffer contains about 15 mM to about 50 mM acetic acid. In further embodiments, the elution buffer may contain 20 mM to 50 mM glycine. The pH of the elution buffer may 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 3.55 to 3.57.

[0060] The resin from the chromatography column may optionally be cleaned, i.e., stripped and regenerated, after elution of the antibody. This procedure is usually performed periodically to minimize the accumulation of impurities on the solid surface and / or to sterilize the matrix to prevent microbial contamination of the product.

[0061] Buffer components may be adjusted according to the knowledge of those skilled in the art. Ranges of sample buffer compositions are shown in the Examples below. Not all buffers or steps are required and are provided for illustrative purposes only. Buffer conditions for Protein A column chromatography may be efficiently optimized using high-throughput screening as described in the Examples.

[0062] The eluate can contain the product, and the ratio of product to host cell protein is increased compared to a corresponding method that does not use detectable amounts of arginine or an arginine derivative in the wash solution.

[0063] The present invention also relates to products prepared according to the methods described herein. Generally, it will be desirable to further isolate and / or purify products isolated according to the present invention and formulate them for pharmaceutical use according to standard methods. For proteins, see, for example, Protein Purification Principles and Practice, 2nd Edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, which are incorporated herein by reference. Those skilled in the art will understand that the exact techniques used will depend on the properties of the product. Products of the present invention that have pharmacological activity are useful in the preparation of pharmaceuticals. They may be administered to a subject and initially 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.

[0064] The product pharmaceutical composition is formulated to be compatible with the intended route of administration according to methods known in the art, see, e.g., "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995), and "Physician's Desk Reference," 52nd ed., Medical Economics, Montvale, NJ (1998). In some embodiments, the product is formulated using sterile water (e.g., SW1), buffered saline (e.g., phosphate buffered saline), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), or suitable mixtures thereof.

[0065]

[0087] Non-limiting examples of products that may 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 aforementioned products.

[0066] Embodiment The present invention further relates to the following embodiment (E). E1. (a) providing a loading solution containing a product that is an Fc-containing monoclonal antibody and one or more impurities; (b) applying the load solution of (a) to a Protein A chromatography column under conditions suitable for product binding; (c) optionally contacting the Protein A chromatography column with a first wash solution; (d) applying one or more wash solutions (e.g., one, two, or three wash solutions) to the Protein A chromatography column, the wash solutions containing arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM and having a pH greater than 8.0; (e) optionally contacting the Protein A chromatography column of d) with a second wash solution; (f) contacting the Protein A chromatography column washed in d) or e) with an eluate under conditions suitable for eluting the product; (g) collecting the product-containing eluate; 1. A method for reducing impurities in a product-containing eluate, comprising:

[0067] E2. The method of embodiment 1, wherein the loading solution in step (a) is obtained from conditioned culture medium from CHO cells expressing eptinezumab, PACAP-1, or ACTH-1, e.g., CHO cells expressing the antibody.

[0068] E3. The method of embodiment 1, wherein the impurities in step (a) are selected from the group consisting of DNA, RNA, endotoxin, proteins (excluding eptinezumab, PACAP-1, or ACTH-1), and host cell proteins (such as undesired proteins from CHO cells).

[0069] E4. The concentration of arginine or arginine salt or arginine derivative is (I) About 500mM to about 600mM; (II) about 525mM to about 575mM, (III) Approximately 550mM to approximately 575mM or (IV) about 575 mM; 2. The method of embodiment 1.

[0070] E5. The pH of one or more of the cleaning solutions in (d) is (i) in the range of about 8.0 to about 8.7; (ii) in the range of about 8.2 to about 8.7; (iii) in the range of about 8.5 to about 8.7; (iv) about 8.5; (v) about 8.6; or (vi) is approximately 8.7; 3. The method of embodiment 1 or 2.

[0071] E6. The method of any one of embodiments 1 to 5, wherein the arginine salt or arginine derivative comprises Arg·HCl, acetylarginine, agmatine, arginine acid, N-α-butyroyl-L-arginine, or N-α-pivaloylarginine.

[0072] E7. The method of any one of embodiments 1-6, wherein the monoclonal antibody is specific for CGRP, such as eptinezumab, 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.

[0073] E8. The method of any one of embodiments 1 to 6, wherein the monoclonal antibody is specific for PACAP, such as PACAP-1, 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.

[0074] E9. The method of any one of embodiments 1-6, wherein the monoclonal antibody is specific for ACTH, such as ACTH-1, 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.

[0075] The method according to any one of embodiments 1 to 9, wherein the concentration of arginine in the washing solution in E10.d) is about 575 mM and the pH is 8.5 or 8.7.

[0076] 10. The method according to any one of the preceding claims, wherein the concentration of arginine in the one or more washing solutions in E11.d) is about 525 mM and the pH is 8.5 or 8.7.

[0077] E12. The method of any one of embodiments 1-11, wherein one or more of the impurities is a host cell protein, nucleic acid, product variant, and / or endotoxin.

[0078] E13. The method of any one of embodiments 1-12, wherein the elution solution comprises about 15 mM to about 50 mM acetic acid, and optionally about 20 mM to 50 mM glycine.

[0079] E14. The method of any one of embodiments 1-13, wherein the elution solution has a pH of about 2 to about 5.

[0080] E15. The method of any one of embodiments 1-14, wherein the elution solution has a pH of about 4.

[0081] E16. The method according to any one of embodiments 1 to 15, wherein one or more of the washing solutions in c) and e) does not contain arginine or an arginine salt or an arginine derivative.

[0082] E17. A pharmaceutical composition comprising eptinezumab, PACAP-1 or ACTH-1 obtained using the method of embodiment 1.

[0083] E18. Use of the pharmaceutical composition of embodiment 17, comprising eptinezumab, PACAP-1, or ACTH-1, as a medicament.

[0084] E19. A pharmaceutical composition comprising eptinezumab according to embodiment E18 for use in the treatment of headache, migraine (such as chronic or episodic migraine), and cluster headache.

[0085] E20. A pharmaceutical composition comprising ACTH-1 according to embodiment 17 for use in the treatment of Cushing's disease or congenital adrenal hyperplasia.

[0086] E21. A pharmaceutical composition comprising PACAP-1 according to embodiment E17 for use in the treatment of headache, migraine (such as chronic or episodic migraine), and cluster headache.

[0087] E22. A method for treating headache, migraine (such as chronic or episodic migraine), and cluster headache, comprising administering a therapeutically effective amount of eptinezumab or PACAP-1 obtained by the method of embodiment 1.

[0088] E23. A method for treating Cushing's disease or congenital adrenal hyperplasia, comprising administering a therapeutically effective amount of ACTH-1 obtained by the method of embodiment 1. [Example]

[0089] Example 1 To investigate the effect of different arginine concentrations and pH levels in the wash solution on a Protein A column, the inventors of the present invention tested different pH and arginine conditions and measured different impurities, including HCP concentration.

[0090] The ranges tested were pH 7.6 to -8.7, arginine 375 to 575 mM, and wash times varied between 5 and 10 column volumes (CV).

[0091] The Protein A column was loaded with clarified media harvested from CHO cells expressing eptinezumab. eptinezumab was harvested from 6 x 3 L bioreactors and depth filtered at 4.34 g / L.

[0092] The Protein A column was run under the following conditions:

[0093] [Table 1]

[0094] The Protein A column used was MabSelect SuRE resin, and the device was AKTA Avant (column name: 20.0 cm / 10 ml / 0.8 cm--MabSelect SuRe Lx10253604--Col#817).

[0095] Measurements of HCP were performed according to the protocol: Immunoenzymatic assay for measurement of CHO host cell proteins (Cygnus Technologies catalog number 550-1)

[0096] Reagents and materials Anti-CHO:HRP (F551-1) Affinity-purified goat antibody conjugated to HRP in a preservative-added protein matrix. 1 x 12 mL > Anti-CHO coated microtiter strips F552-1*12 x 8 well strips, desiccant bag included CHO HCP Standard F553-1 CHO HCP in preservative-added bovine serum albumin. Standards at 0, 1, 3, 12, 40, and 100 ng / mL. 1 mL / vial >Stop solution 0.5M sulfuric acid. 1 x 12mL >TMB Substrate F005 3,3',5,5'Tetramethylbenzidine, 1 x 12 mL > Cleaning Concentrate (20x) F004 Tris-buffered saline with preservative. 1 x 50mL

[0097] Host cell protein (HCP) measurement 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 listed on the worklist. 3. Cover and incubate on an orbital shaker at 400-600 rpm (24°C + 4°C) for 2 hours. 4. Discard the contents of the wells as waste. Tap gently but firmly on absorbent paper to soak up the liquid and remove most of the remaining liquid. There is no need to tap the plate too hard or use a vacuum to remove all of the remaining liquid, as this can cause various dissociations of antibody-bound substances, resulting in a lower OD and reduced accuracy. Using a spray bottle or pipette, add approximately 350 μL of diluted wash solution to each well until it overflows. Discard and tap gently again. Repeat this process four times in total. Wipe off any liquid remaining on the outside bottom of the microtiter wells, as it will interfere with the reading process. Do not allow the wash solution to remain in the wells for more than a few seconds. 5. Pipette 100 μL of TMB substrate (#F005). 6. Incubate at room temperature for 30 minutes. 7. Pipette 100 μL of Stop Solution (#F006). 8. Read the absorbance at 450 / 650nm.

[0098] [Table 2]

[0099] Similar results were obtained by purifying PACAP and ACTH antibodies (designated PACAP-1 and ACTH-1 in this application) as described in this invention.

[0100] The HCP content of PACAP samples ranged from 156.9 to 294.7 after purification under the same conditions as above, whereas for ACTH, an HCP content of approximately 245 was obtained when performed at a pH of approximately 8.5 and an arginine concentration of approximately 575 mM.

[0101] Further testing using pH 8.5 and 575 mM arginine reduced the HCP content in two separate runs of PACAP-1 from approximately 307,684 ng HCP / mg protein and 700,688 ng HCP / mg protein to approximately 224 ng HCP / mg protein and 220 ng HCP / mg protein, respectively. Furthermore, purification of ACTH-1 reduced the HCP content from 891,432 to 326 ng / mg protein (2,104,671 ng / mL HCP to 10,002 ng / mL HCP) and from 10,042,454 ng / mg to 232 ng / mg (2,435,063 ng / mL HCP to 7,062 ng / mL).

Claims

1. (a) providing a load fluid containing a product that is an Fc-containing monoclonal antibody and one or more impurities; (b) applying the load solution of a) to a Protein A chromatography column under conditions suitable for product binding; (c) optionally contacting the Protein A chromatography column with a first wash solution; (d) applying one or more wash solutions (e.g., one, two, or three wash solutions) to the Protein A chromatography column, the wash solutions comprising arginine or an arginine salt or other arginine derivative at a concentration greater than 500 mM and having a pH greater than 8.0; (e) optionally contacting the Protein A chromatography column of d) with a second wash solution; (f) contacting the Protein A chromatography column washed in d) or e) with an eluate under conditions suitable to elute the product; (g) collecting an eluate containing the product; 1. A method for reducing impurities in a product-containing eluate, comprising:

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

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

4. 4. The method of any one of claims 1, 2 or 3, wherein the arginine salt or arginine derivative comprises Arg.HCl, acetylarginine, agmatine, arginine acid, N-α-butyroyl-L-arginine, or N-α-pivaloylarginine.

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

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

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

8. The method according to any one of claims 1 to 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. 8. The method of any one of claims 1 to 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. 10. The method of any one of claims 1 to 9, wherein one or more of the impurities is a host cell protein, a nucleic acid, a product variant, and / or an endotoxin.

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

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

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

14. The method according to any one of claims 1 to 13, wherein one or more of the washing solutions in c) and e) does not contain arginine or an arginine salt or an arginine derivative.

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