Methods and compositions comprising purified recombinant polypeptides

Abstract

The present invention provides purified recombinant polypeptides isolated from Chinese hamster ovary host cells, including antibodies (e.g., therapeutic antibodies), as well as methods of making and using the polypeptides.

Description

[0001] This application is a divisional application of China Application No. 201480062008.9, filed September 12, 2014, entitled "Methods and Compositions Comprising Purified Recombinant Polypeptides."

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of priority of U.S. Provisional Application No. 61 / 877,517, filed September 13, 2013, which is incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on August 28, 2014, is named 2014.AUG.28P5704R1-WO SequenceListing.txt and is 34,811 bytes in size. TECHNICAL FIELD

[0006] Provided are purified recombinant polypeptides (including antibodies, such as therapeutic antibodies) isolated from Chinese hamster ovary host cells, as well as methods of making and using such polypeptides. BACKGROUND

[0007] A number of drugs for the treatment of asthma and other respiratory disorders are already on the market or in development. One target for asthma therapy is IL-13. IL-13 is a pleiotropic TH2 cytokine produced by activated T cells, NKT cells, basophils, eosinophils, and mast cells, and has been strongly implicated in the pathogenesis of asthma in preclinical models. IL-13 antagonists, including anti-IL-13 antibodies, have been described previously. Certain such antibodies have also been developed for human therapy. Recently, several studies have suggested clinical activity of monoclonal antibodies against IL-13 in the treatment of asthma (see, e.g., Corren et al., 2011, N. Engl. J. Med. 365, 1088-1098; Gauvreau et al., 2011, Am. J. Respir. Crit. Care Med. 183, 1007-1014; Ingram and Kraft, 2012, J. Allergy Clin. Immunol. 130, 829-42; Webb, 2011, Nat Biotechnol 29, 860-863). Among these monoclonal antibodies, lebrikizumab, a humanized IgG4 antibody that neutralizes IL-13 activity, improved lung function in a majority of asthmatic patients who were symptomatic despite treatment with inhaled glucocorticoids and long-acting beta2-adrenergic receptor agonists (Corren et al., 2011, N. Engl. J. Med. 365, 1088-1098).

[0008] In addition, IL-13 has been implicated in a number of other allergic and fibrotic disorders. For example, such diseases and / or conditions mediated by IL13 include, but are not limited to, allergic asthma, non-allergic (intrinsic) asthma, allergic rhinitis, allergic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, and osteoporosis.

[0009] In order for recombinant biopharmaceutical proteins to be accepted for administration to human patients, it is important to remove residual impurities generated in the manufacturing and purification processes from the final biological product. These process components include media proteins, immunoglobulin affinity ligands, viruses, endotoxins, DNA, and host cell proteins. These host cell impurities include process-specific host cell proteins (HCPs), which are process-related impurities / contaminants in biologies from recombinant DNA technology. Although HCPs are typically present in small amounts (in parts per million or nanograms per milligram of intended recombinant protein) in the final drug substance, HCPs are recognized as undesirable and their quantity should be minimized. For example, the U.S. Food and Drug Administration (FDA) requires that biopharmaceuticals for use in humans should be as free as possible of extraneous impurities and requires testing to detect and quantify potential contaminants / impurities such as HCPs.

[0010] The steps for purifying proteins from cell debris first depend on the site of protein expression. Some proteins are secreted directly from the cell into the surrounding growth medium; others are produced intracellularly. For the latter, the first step in the purification process involves cell lysis, which can be performed by a variety of methods, including mechanical shearing, osmotic shock, or enzymatic treatment. This disruption releases the entire contents of the cell into a homogenate and additionally produces subcellular debris that is difficult to remove because of its small size. These are typically removed by centrifugation or filtration. The same problem arises with directly secreted proteins, due to natural cell death and release of intracellular host cell proteins during the operation of the protein production process.

[0011] Once a solution containing the protein of interest is obtained, a combination of different chromatography techniques is typically used to attempt to separate the protein of interest from other proteins produced by the cell. Generally, these techniques separate protein mixtures based on the charge, degree of hydrophobicity, or size of the proteins. Several different chromatography resins can be employed for each of these techniques, which allow for the precise tailoring of purification schemes for the particular protein being included. The essence of each of these separation methods is that either the proteins can be made to move down the column at different rates, allowing for an increase in physical separation as they pass further down the column; or the proteins are selectively adhered to the separation medium, which are then differentially eluted by different solvents. In some cases, the desired protein is separated from the impurities when the impurities specifically adhere to the column, while the protein of interest does not specifically adhere to the column, i.e., the protein of interest is present in the "flow-through."

[0012] Ion exchange chromatography, named for the exchangeable counterions, is a purification method applicable to ionizable molecules. Ionizable molecules are separated based on their charged groups' non-specific electrostatic interactions with oppositely charged molecules attached to a solid phase support matrix, thereby retarding those ionizable molecules that interact more strongly with the solid phase. The net charge of each class of ionizable molecule and its affinity for the matrix varies depending on the number of charged groups, the charge of each group, and the nature of the molecules that compete for interaction with the charged solid phase matrix. These differences result in the separation of different types of molecules by ion exchange chromatography. In a typical protein purification using ion exchange chromatography, a mixture of many proteins from host cells in a mammalian cell culture, for example, is applied to an ion exchange column. After the non-binding molecules are washed away, the non-specifically retained or retarded ionizable proteins of interest are released from the solid phase and separated from proteins with different charge characteristics by adjusting conditions such as pH, counterion concentration, etc. in a stepwise or gradient mode. Anion exchange chromatography involves the competition of anionic molecules of interest with negative counterions for interaction with positively charged molecules attached to a solid phase matrix at the pH and conditions of the particular separation method. In contrast, cation exchange chromatography involves the competition of cationic molecules of interest with positive counterions for interaction with negatively charged molecules attached to a solid phase matrix at the pH and conditions of the particular separation method. Mixed mode ion exchange chromatography (also referred to as multimodal ion exchange chromatography) involves the use of a combination of cation and anion exchange chromatography media in the same step. In particular, "mixed mode" refers to a solid phase support matrix that covalently attaches a mixture of cation exchange, anion exchange, and hydrophobic interaction moieties.

[0013] Hydroxyapatite chromatography of proteins involves the non-specific interaction of the charged amino or carboxyl groups of proteins with oppositely charged groups on hydroxyapatite, where the net charge of the hydroxyapatite and the protein is controlled by the pH of the buffer. Elution is accomplished by displacing the non-specific protein-hydroxyapatite pair with ions such as Ca2+or Mg2+. Negatively charged protein groups are displaced by negatively charged compounds such as phosphates, thereby eluting the net negatively charged proteins.

[0014] Hydrophobic interaction chromatography (HIC) is commonly used for the purification and separation of molecules such as proteins based on differences in their surface hydrophobicity. The hydrophobic groups of proteins non-specifically interact with hydrophobic groups coupled to the chromatography matrix. Differences in the number and nature of the surface hydrophobic groups of proteins result in differential retardation of proteins on HIC columns, thereby separating proteins in a protein mixture.

[0015] Affinity chromatography, which utilizes a specific, structurally dependent (i.e., steric complementarity) interaction between the protein to be purified and an immobilized capture agent, is a standard purification choice for some proteins, such as antibodies. Protein A, for example, is a useful adsorbent for affinity chromatography of proteins, such as antibodies, that comprise an Fc region. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds the Fc region of antibodies with high affinity (about 10 -8 M) for human IgG.

[0016] Purification of recombinant polypeptides is typically performed using either bind-elute chromatography (B / E) or flow-through (F / T) chromatography. These methods are briefly described below.

[0017] Bind-elute chromatography (B / E): In B / E chromatography, the product is typically loaded onto the chromatography material at conditions that maximize dynamic binding capacity (DBC), and then wash and elution conditions are determined to achieve maximum product purity in the eluate.

[0018] Various B / E methods using protein A affinity chromatography have been described, including various intermediate washes. For example, U.S. Patent Nos. 6,127,526 and 6,333,398 describe intermediate wash steps using hydrophobic electrolytes (e.g., tetramethylammonium chloride (TMAC) and tetraethylammonium chloride (TEAC)) during protein A chromatography to remove contaminants that are not bound to the immobilized protein A or the protein of interest of the protein A column. U.S. Patent No. 6,870,034 describes additional methods and washes for using protein A affinity chromatography.

[0019] Flow-through chromatography (F / T): Using F / T chromatography, loading conditions are identified in which impurities strongly bind to the chromatography material while the product flows through. F / T chromatography allows for high loading densities of standard monoclonal antibody preparations (MAbs).

[0020] We identified an enzyme, phospholipase B-like 2, as the single CHOP species present in excess of available antibody in our total CHOP ELISA assay, in the recombinant anti-IL13 MAb preparation produced in CHO cells, and in certain other recombinant polypeptides. As used herein, "PLB2" and "PLBL2" and "PLBD2" are used interchangeably and refer to "phospholipase B-like 2" or its synonym, "phospholipase B domain-like 2." Certain scientific publications regarding PLBL2 include Lakomek, K. et al., BMC Structural Biology 9:56 (2009); Deuschi, et al., FEBS Lett 580:5747-5752 (2006). PLBL2 is synthesized as a proenzyme of about 66,000 MW. It has an initial leader sequence removed and a potential 6 mannose-6-phosphate (M-6-P) group added during post-translational modification. M-6-P is a targeting modification that directs this enzyme to the lysosome via M-6-P receptors. PLBL2 contains 6 cysteines, two of which have free thiol groups and four of which form disulfide bonds. In an acidic environment, PLBL2 is further cleaved into N- and C-terminal fragments with MW of 32,000 and 45,000, respectively. By analogy with other lysosomal enzymes, this cleavage is an activation step that allows access of the substrate to the active site.

[0021] There is about 80% amino acid sequence homology between the hamster and human forms of the enzyme. The activity of the enzyme is thought to be cleaving either fatty acid chain from phospholipids that make up the cell membrane. There are other phospholipases with different substrate cleavage specificities. Similar enzyme activities exist in microorganisms, where they are often virulence factors. Despite the similar enzyme activity in microorganisms, the protein that produces the activity is different, with low sequence homology between the microorganism and mammalian PLBL2 enzymes. Phospholipases produce free fatty acids (FFA) as one product of substrate hydrolysis. Free fatty acids are themselves potential immune signaling factors. Dehydrogenation converts FFA to arachidonic acid, which can participate in inflammatory cascades involving eicosanoids.

[0022] Having identified PLBL2 as a single HCP (CHOP) of recombinant anti-IL13 MAb preparations produced in CHO cells and certain other recombinant polypeptides, we have developed specific, sensitive and quantitative assays, methods and kits for determining PLBL2 levels in anti-IL-13 MAb preparations (and other recombinant polypeptide products) and at various stages of purification. These are briefly described in the Examples below and in U.S. Provisional Patent Application Nos. 61 / 877,503 and 61 / 991,228. In addition, there are formidable challenges in developing methods for large scale, robust and efficient purification of anti-IL-13 MAb (and other recombinant polypeptide products) to obtain MAb (including removal of PLBL2) of sufficient purity for human therapeutic use, including later stage clinical and commercial use. The inventions described herein meet certain of the foregoing needs and provide other benefits.

[0023] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. SUMMARY

[0024] The present invention is based, at least in part, on improvements in methods for purifying recombinant polypeptides produced in Chinese hamster ovary (CHO) cells that provide purified products with substantially reduced levels of hamster PLBL2. Recombinant polypeptides, including therapeutic antibodies such as anti-IL-13 antibodies, purified according to the methods of the present invention can have reduced immunogenicity when administered to human subjects.

[0025] Accordingly, in one aspect, there is provided a composition comprising an anti-IL-13 monoclonal antibody purified from CHO cells containing an anti-IL-13 antibody, the composition comprising a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or 0.5 ng / mg and 15 ng / mg, or 0.5 ng / mg and 10 ng / mg, or 0.5 ng / mg and 8 ng / mg, or 0.5 ng / mg and 5 ng / mg, or 0.5 ng / mg and 3 ng / mg, or 0.5 ng / mg and 2 ng / mg, or 0.5 ng / mg and 1 ng / mg, or the limit of quantitation (LOQ) and 1 ng / mg. In certain embodiments, the anti-IL-13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9.In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the amount of hamster PLBL2 in the composition is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0026] In another aspect, there is provided a purified anti-IL-13 monoclonal antibody formulation isolated from CHO cells using a method comprising a hydrophobic interaction chromatography (HIC) step. In certain embodiments, the purified formulation comprises the anti-IL-13 antibody and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) of the assay and 1 ng / mg. In certain embodiments, the HIC step comprises a PHENYL SEPHAROSE TM6Fast Flow (High Sub) resin. In certain embodiments, the HIC step comprises operating the column containing the resin in flow-through mode. In certain embodiments, the HIC step comprises an equilibration buffer and a wash buffer, wherein each of the equilibration buffer and the wash buffer comprises 50 mM sodium acetate pH 5.0. In certain embodiments, the flow-through is monitored at an absorbance of 280 nanometers and between 0.5 OD and 1.5 OD of flow-through is collected. In certain embodiments, up to 8 column volumes of flow-through are collected. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises an ion exchange chromatography step. In certain embodiments, the ion exchange chromatography is anion exchange chromatography. In certain embodiments, the anti-IL-13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0027] In yet another aspect, a purified anti-IL-13 monoclonal antibody preparation isolated from CHO cells is provided. In certain embodiments, the antibody preparation is purified by a method comprising a first protein A affinity chromatography step, a second anion exchange chromatography step, and a third hydrophobic interaction chromatography (HIC) step, thereby producing a purified preparation. In certain embodiments, the purified preparation comprises an anti-IL-13 antibody and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or 0.5 ng / mg and 15 ng / mg, or 0.5 ng / mg and 10 ng / mg, or 0.5 ng / mg and 8 ng / mg, or 0.5 ng / mg and 5 ng / mg, or 0.5 ng / mg and 3 ng / mg, or 0.5 ng / mg and 2 ng / mg, or 0.5 ng / mg and 1 ng / mg, or the limit of quantitation (LOQ) and 1 ng / mg. In certain embodiments, the affinity chromatography step comprises a MABSELECT SURE TM resin, the anion exchange chromatography step comprises a Q SEPHAROSE TM Fast Flow, and the HIC step comprises a PHENYL SEPHAROSE TM 6Fast Flow (High Sub). In certain embodiments, the affinity chromatography step comprises a column comprising a MABSELECT SURE TM resin operated in bind-elute mode, the anion exchange chromatography step comprises a column comprising a Q SEPHAROSE TM Fast Flow resin operated in bind-elute mode, and the HIC step comprises a column comprising a PHENYL SEPHAROSE TM6column of Fast Flow (High Sub) resin. In certain embodiments, the anti-IL-13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0028] In yet another aspect, a method of purifying a recombinant polypeptide produced in CHO cells is provided, wherein the method provides a purified preparation comprising the recombinant polypeptide and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or 0.5 ng / mg and 15 ng / mg, or 0.5 ng / mg and 10 ng / mg, or 0.5 ng / mg and 8 ng / mg, or 0.5 ng / mg and 5 ng / mg, or 0.5 ng / mg and 3 ng / mg, or 0.5 ng / mg and 2 ng / mg, or 0.5 ng / mg and 1 ng / mg, or the limit of quantitation (LOQ) and 1 ng / mg. In certain embodiments, the recombinant polypeptide is selected from the group consisting of a growth factor, a cytokine, an antibody, an antibody fragment, and an immunoadhesin. In certain embodiments, the recombinant polypeptide is an antibody. In certain embodiments, the antibody is a humanized monoclonal antibody. In certain embodiments, the antibody is an IgGl, or IgG2 or IgG3 or IgG4. In certain embodiments, the antibody is an IgGl. In certain embodiments, the antibody is an IgG2. In certain embodiments, the antibody is an IgG3. In certain embodiments, the antibody is an IgG4. In certain embodiments, the method comprises a hydrophobic interaction chromatography (HIC) step. In certain embodiments, the HIC step comprises a PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin.

[0029] In certain embodiments of the above purification method, the purified antibody is an anti-IL-13. In certain embodiments, the antibody is lebrikizumab. In certain embodiments, the HIC step comprises operating the resin-containing column in flow- through mode. In certain embodiments, the HIC step comprises equilibration and wash buffers, wherein each equilibration and wash buffer comprises 50 mM sodium acetate pH 5.0. In certain embodiments, the flow-through is monitored at an absorbance of 280 nanometers and between 0.5 OD and 1.5 OD of flow-through is collected. In certain embodiments, up to 8 column volumes of flow-through is collected. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises an ion exchange chromatography step. In certain embodiments, the ion exchange chromatography is anion exchange chromatography. In certain embodiments, the method comprises a first protein A affinity chromatography step, a second anion exchange chromatography step, and a third hydrophobic interaction chromatography (HIC) step. In certain embodiments, the affinity chromatography step comprises a column containing MABSELECT SURE TM resin, the anion exchange chromatography step comprises a column containing Q SEPHAROSE TM Fast Flow, and the HIC step comprises a column containing PHENYL SEPHAROSE TM 6Fast Flow (high sub). In certain embodiments, the affinity chromatography step comprises operating a column containing MABSELECT SURE TM resin in bind-elute mode, the anion exchange chromatography step comprises operating a column containing Q SEPHAROSE TM Fast Flow resin in bind-elute mode, and the HIC step comprises operating a column containing PHENYL SEPHAROSE TM 6Fast Flow (high sub) resin. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is total Chinese hamster ovary protein ELISA or hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0030] In certain embodiments of the above purification method, the purified antibody is an anti- Aβ. In certain embodiments, the anti-Aβ antibody is crenezumab. In certain embodiments, the anti-Aβ antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 23, CDR-H2 having the amino acid sequence of SEQ ID NO: 24, and CDR-H3 having the amino acid sequence of SEQ ID NO: 25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 26, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the anti-Aβ antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the anti-Aβ antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the anti-Aβ antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the HIC step comprises operating the resin-containing column in flow-through mode. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate pH 5.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate pH 4.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate, 240 mM sodium sulfate pH 4.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate, 240 mM sodium sulfate pH 5.0. In certain embodiments, the loading density is 300 g / L. In certain embodiments, the loading density is 100 g / L. In certain embodiments, the flow-through is monitored at 280 nanometer absorbance and collection of the flow-through is initiated at 0.5 OD, with collection continuing for 10 column volumes. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises a mixed mode chromatography step. In certain embodiments, the method comprises a first protein A affinity chromatography step, a second mixed mode chromatography step, and a third hydrophobic interaction chromatography (HIC) step. In certain embodiments, the affinity chromatography step comprises MABSELECT SURE TM resin, the mixed mode chromatography step comprises CAPTO TMAdhere resin, HIC step includes a column containing PHENYL SEPHAROSE TM 6 Fast Flow (high sub) resin. In certain embodiments, the affinity chromatography step includes a column containing MABSELECT SURE TM resin operated in flow-through mode TM Adhere resin, HIC step includes a column containing PHENYL SEPHAROSE TM 6 Fast Flow (high sub) resin. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is total Chinese hamster ovary protein ELISA or hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0031] In another aspect of the above purification method, the purified antibody is an IgGl. In some embodiments, the antibody is an anti-IL17A / F. In some embodiments, the anti-IL17A / F antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 15, CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and CDR-H3 having the amino acid sequence of SEQ ID NO: 17, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 18, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the anti-IL17A / F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the HIC chromatography step comprises an equilibration buffer and a wash buffer, wherein each of the equilibration buffer and the wash buffer comprises 50 mM sodium acetate pH 5.5. In certain embodiments, the flowthrough is monitored at 280 nanometer absorbance and collection of the flowthrough is initiated at 0.5 OD, with collection continuing for 10 column volumes. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises a cation exchange chromatography step. In certain embodiments, the method comprises a first protein A affinity chromatography step, a second cation exchange chromatography step, and a subsequent hydrophobic interaction chromatography (HIC) step. In certain embodiments, the affinity chromatography step comprises a MABSELECT SURE TM resin, the cation exchange chromatography step comprises a POROS 50HS resin, and the HIC step comprises a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin. In certain embodiments, the affinity chromatography step comprises a column containing a MABSELECT SURE TM resin operated in bind-elute mode, the cation exchange chromatography step comprises a column containing a POROS 50HS resin operated in bind-elute mode, and the HIC step comprises a column containing a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin.

[0032] In yet another aspect, there is provided a preparation of an anti-Abeta monoclonal antibody purified from CHO cells by a method comprising a hydrophobic interaction chromatography (HIC) step. In certain embodiments, the purified preparation comprises the anti-Abeta antibody and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) of the assay and 1 ng / mg. In certain embodiments, the HIC step comprises a PHENYL SEPHAROSE TM6Fast Flow (High Sub) resin. In certain embodiments, the HIC step comprises operating the column containing the resin in flow-through mode. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate pH 5.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate pH 4.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate, 240 mM sodium sulfate pH 4.0. In certain embodiments, the HIC step comprises a equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 150 mM sodium acetate, 240 mM sodium sulfate pH 5.0. In certain embodiments, the loading density is 300 g / L. In certain embodiments, the loading density is 100 g / L. In certain embodiments, the flow-through is monitored at 280 nanometer absorbance and collection of the flow-through is initiated at 0.5 OD, and collection continues for 10 column volumes. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises a mixed mode chromatography step. In certain embodiments, the anti-Abeta antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 23, CDR-H2 having the amino acid sequence of SEQ ID NO: 24, and CDR-H3 having the amino acid sequence of SEQ ID NO: 25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 26, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the anti-Abeta antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0033] In one aspect, there is provided a purified anti-IL17A / F monoclonal antibody formulation isolated and purified from CHO cells by a method comprising a hydrophobic interaction chromatography (HIC) step. In certain embodiments, the purified formulation comprises an anti-IL17A / F antibody and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) and 1 ng / mg of the assay. In certain embodiments, the HIC step comprises a PHENYL SEPHAROSE TM6Fast Flow (High Sub) resin. In certain embodiments, the HIC step comprises operating the column containing the resin in flow-through mode. In certain embodiments, the HIC step comprises an equilibration buffer and a wash buffer, wherein each equilibration buffer and wash buffer comprises 50 mM sodium acetate pH 5.5. In certain embodiments, the flow-through is monitored at an absorbance of 280 nanometers and the flow-through is collected between 0.5 OD, collected for 10 column volumes. In certain embodiments, the method further comprises an affinity chromatography step. In certain embodiments, the affinity chromatography is protein A chromatography. In certain embodiments, the method further comprises a cation exchange chromatography step. In certain embodiments, the anti-IL17A / F antibody comprises three heavy chain CDRs, a CDR-H1 having the amino acid sequence of SEQ ID NO: 15, a CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 17, and three light chain CDRs, a CDR-L1 having the amino acid sequence of SEQ ID NO: 18, a CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the anti-IL17A / F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 32. In certain embodiments, the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay. In certain embodiments, the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA. In certain embodiments, the mass spectrometry assay is LC-MS / MS.

[0034] In yet another aspect, provided is a composition comprising an anti-Abeta monoclonal antibody purified from CHO cells containing an anti-Abeta antibody, the composition comprising a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) and 1 ng / mg of the assay. In certain embodiments, the anti-Abeta antibody is crenezumab. In certain embodiments, the anti-Abeta antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 23, CDR-H2 having the amino acid sequence of SEQ ID NO: 24, and CDR-H3 having the amino acid sequence of SEQ ID NO: 25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 26, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the anti-Abeta antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

[0035] In yet another aspect, provided are compositions comprising an anti-IL17A / F monoclonal antibody purified from CHO cells containing an anti-IL17A / F antibody, the compositions comprising a residual amount of hamster PLBL2. In certain embodiments, the compositions comprise an anti-IL17A / F antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) and 1 ng / mg of the assay. In certain embodiments, the anti-IL17A / F antibody comprises three heavy chain CDRs, a CDR-H1 having the amino acid sequence of SEQ ID NO: 15, a CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 17, and three light chain CDRs, a CDR-L1 having the amino acid sequence of SEQ ID NO: 18, a CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the anti-IL17A / F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 22.In certain embodiments, the anti-IL17A / F antibody comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 21 and a light chain variable region having an amino acid sequence of SEQ ID NO: 22.

[0036] In one aspect, methods of treating IL-13 mediated disorders are provided, the methods comprising administering a therapeutic composition comprising an anti-IL-13 monoclonal antibody purified from CHO cells and a residual amount of hamster PLBL2. In certain embodiments, the amount of hamster PLBL2 is less than 20 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 15 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 10 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 8 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 3 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 2 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 1 ng / mg. In certain embodiments, the amount of hamster PLBL2 is less than 0.5 ng / mg. In certain embodiments, the amount of hamster PLBL2 is between 0.5 ng / mg and 20 ng / mg, or between 0.5 ng / mg and 15 ng / mg, or between 0.5 ng / mg and 10 ng / mg, or between 0.5 ng / mg and 8 ng / mg, or between 0.5 ng / mg and 5 ng / mg, or between 0.5 ng / mg and 3 ng / mg, or between 0.5 ng / mg and 2 ng / mg, or between 0.5 ng / mg and 1 ng / mg, or between the limit of quantitation (LOQ) and 1 ng / mg of the assay. In certain embodiments, the anti-IL-13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9.In certain embodiments, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the therapeutic composition is administered subcutaneously once every four weeks. In certain embodiments, the therapeutic composition is administered subcutaneously once every eight weeks. In certain embodiments, the therapeutic composition is administered subcutaneously once every 12 weeks. In certain embodiments, the patient is treated once every four weeks for at least one month. In certain embodiments, the patient is treated once every four weeks for at least three months. In certain embodiments, the patient is treated once every four weeks for at least six months. In certain embodiments, the patient is treated once every four weeks for at least nine months. In certain embodiments, the patient is treated once every four weeks for at least 12 months. In certain embodiments, the patient is treated once every four weeks for at least 18 months. In certain embodiments, the patient is treated once every four weeks for at least two years. In certain embodiments, the patient is treated once every four weeks for more than two years. In certain embodiments, the IL-13-mediated disorder is asthma. In certain embodiments, the IL-13-mediated disorder is idiopathic pulmonary fibrosis. In certain embodiments, the IL-13-mediated disorder is atopic dermatitis. In certain embodiments, the IL-13-mediated disorder is selected from the group consisting of allergic asthma, non-allergic asthma, allergic rhinitis, allergic conjunctivitis, eczema, urticaria, food allergy, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, and osteoporosis.

[0037] In another aspect, the therapeutic composition administered to the patient according to any of the above methods has less immunogenicity to hamster PLBL2 than a reference composition, wherein the reference composition comprises an anti-IL-13 monoclonal antibody purified from Chinese hamster ovary host cells and a residual amount of hamster PLBL2 greater than 30 ng / mg. In certain embodiments, the amount of hamster PLBL2 in the reference composition is greater than 50 ng / mg. In certain embodiments, the amount of hamster PLBL2 in the reference composition is greater than 100 ng / mg. In certain embodiments, the amount of hamster PLBL2 in the reference composition is greater than 200 ng / mg. In certain embodiments, the amount of hamster PLBL2 in the reference composition is greater than 300 ng / mg. In certain embodiments, the amount of hamster PLBL2 in the reference composition is between 30 ng / mg and 300 ng / mg, or between 30 ng / mg and 200 ng / mg, or between 30 ng / mg and 100 ng / mg, or between 30 ng / mg and 50 ng / mg. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 shows the level of total CHOP in octanoic acid treated Protein A pools of anti-IL-13 MAbs as described in Example 2. (A) Protein A pools were octanoic acid precipitated at pH 4.5. (B) Protein A pools were octanoic acid precipitated at pH 5.0. The level of CHOP is indicated along the vertical axis in ng / mg; the percentage of octanoic acid is shown along the horizontal axis, each bar represents the value shown from a 2-fold serial dilution.

[0039] Figure 2 Figure 2 shows the level of total CHOP in Protein A pools of anti-IL-13 MAbs as described in Example 2, following Protein A chromatography and subsequent cation exchange chromatography on SP Sepharose® 50HS. The level of total CHOP in additive treated HCCF anti-IL-13 MAbs following cation exchange chromatography on SP Sepharose® 50HS is shown. The vertical axis shows the corrected CHOP level in ng / ml; the horizontal axis indicates the additive (control, 0.6 M guanidine, or 0.6 M arginine), each bar represents the value shown from a 2-fold serial dilution.

[0040] Figure 3 shows the level of total CHOP in UFDF pools of anti-IL-13 MAbs following different HIC resins under different salt and pH conditions as described in Example 2. (A) OCTYL- Sepharose® Fast Flow resin; (B) PHENYL SEPHAROSE® Fast Flow (low sub) resin; (C) BUTYL- SEPHAROSE® Fast Flow (low sub) resin; (D) PHENYL SEPHAROSE® Fast Flow (high sub) resin; (E) SEPHACRYL® S-200 HR gel filtration resin. Fast Flow resin; (B) PHENYL SEPHAROSE TM 6Fast Flow(low sub) resin; (C)BUTYL- 4Fast Flow resin; (D) PHENYL SEPHAROSE TM 6Fast Flow(high sub) resin; the level of CHOP in ppm for the highest dilution is shown along the vertical axis, the concentration of sodium sulfate is shown along the horizontal axis; the pH value (5.5, 6.0, 7.0 or 8.0 is indicated by the legend.

[0041] DETAILED DESCRIPTION

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th Ed., John Wiley & Sons (New York, N.Y. 1992), provide one of skill with a general guide to many of the terms used in this application.

[0043] Certain Definitions

[0044] For the purposes of interpreting this specification, the following definitions will apply, whenever appropriate. The terms used in singular form also include the plural, and vice versa, unless the context clearly indicates otherwise. To the extent that any definition provided herein conflicts with any document incorporated herein by reference, the definition provided herein controls.

[0045] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a protein" or "an antibody" includes a plurality of proteins or antibodies, respectively; reference to "a cell" includes a mixture of cells, and the like.

[0046] The term "detecting" is used herein in the broadest sense, including qualitative and quantitative measurement of a target molecule. Detecting includes merely recognizing the presence of a target molecule in a sample, as well as determining whether the target molecule is present in the sample at a detectable level.

[0047] "Sample" refers to a small portion of a larger quantity of material. Typically, a sample is tested according to the methods described herein. A sample is typically obtained from a preparation of recombinant polypeptides, e.g., from a culture of host cells from which the preparation of recombinant polypeptides is obtained. A sample can be obtained, for example, but not limited to, from a harvested cell culture fluid, from a particular step in a purification process, an in-process pool, or from a final purified product.

[0048] The term "product" as described herein is the substance to be purified by various chromatographic methods; e.g., a polypeptide.

[0049] The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be straight or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified, naturally or by intervention; for example, disulfide- formed, glycosylated, lipoylated, acetylated, phosphorylated, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), and other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

[0050] A "purified" polypeptide (e.g., an antibody or immunoadhesin) refers to a polypeptide that has been increased in purity, such that it exists in a form that is more pure than it existed when in its natural environment and / or when initially synthesized and / or when amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0051] The term "tag epitope" as used herein refers to a chimeric polypeptide comprising a polypeptide fused to a "tag polypeptide." The tag polypeptide has enough residues to provide epitopes against which antibodies can be made, but it is also short enough that it does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is preferably also sufficiently unique so that antibodies do not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues, often between about 8 and 50 amino acid residues (in some cases, between about 10 and 20 amino acid residues).

[0052] "Active" or "activity" herein refers to a form of a polypeptide that retains a biological and / or immunological activity of interest, where "biological" activity refers to a biological function (inhibited or stimulated) elicited by the polypeptide, and "immunological" activity refers to the ability to induce the production of antibodies against an antigenic epitope possessed by the polypeptide.

[0053] The term "antagonist" is used in the broadest sense, and includes any molecule which partially or fully blocks, suppresses, or neutralizes a biological activity of a native polypeptide (e.g., a cytokine). In a similar manner, the term "agonist" is used in the broadest sense, and includes any molecule which mimics a biological activity of a native polypeptide. Suitable agonist or antagonist molecules specifically include agonistic or antagonistic antibodies or antibody fragments, fragments or amino acid sequence variants of the native polypeptide, and the like. Methods for identifying agonists or antagonists of a polypeptide can include contacting the polypeptide with a candidate agonist or antagonist molecule, and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

[0054] A polypeptide that "binds" a target antigen (e.g., a tumor-associated polypeptide antigen target) is a polypeptide that binds the antigen with sufficient affinity so that the polypeptide is useful as a detection reagent, diagnostic, and / or therapeutic agent for targeting samples containing the antigen, cells or tissues expressing the antigen, and does not significantly cross-react with other polypeptides.

[0055] With respect to the binding of a polypeptide to a target molecule, the terms "specific binding" or "specifically binds" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding to a detectable greater extent than a non-specific interaction. Specific binding can be determined, for example, by comparing the binding of a molecule to a control molecule, which is typically a structurally similar molecule that does not have binding activity. Specific binding can also be determined, for example, by competition with an excess of unlabeled target, by competition with a control molecule that is similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of labeled target to the probe is competitively inhibited by the excess of unlabeled target.

[0056] The term "antibody" herein is used in the broadest sense and includes various antibody structures including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0057] Antibodies are naturally occurring immunoglobulin molecules having different structures, all based on the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains that are disulfide bond linked, forming a functional antibody. Each heavy and light chain itself comprises a "constant region" (C) and a "variable region" (V). The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen specific interactions with immune effectors. The antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of the antibody to specifically bind to a particular antigen.

[0058] The antigen-binding specificity of an antibody is determined by the structural properties of the V region. The variability is not evenly distributed throughout the 110 amino acid span of the variable domain. Instead, the V region consists of a relatively invariant stretch called the framework region (FR) of 15-30 amino acids interrupted by regions of extreme variability called "hypervariable regions" of 9-12 amino acids each that are interposed between the framework regions. Each of the four FRs is largely beta sheet that forms a framework for the antigen binding loop. Each of the four FRs is largely beta sheet that forms a framework for the antigen binding loop. The hypervariable regions in each chain are held together and held in proximity to the hypervariable regions of the other chain by the FRs to form an antigen binding site. (See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit the effector functions, such as antibody-dependent cellular cytotoxicity (ADCC).

[0059] Each V region typically comprises three complementarity determining regions ("CDRs", each of which comprises a "hypervariable loop") and four framework regions. The antibody binding site (the smallest structural unit necessary to bind a particular desired antigen with a high affinity) thus typically includes three CDRs, and at least three, and preferably four, framework regions interspersed therebetween, to hold the CDRs in and present them in the proper conformation. A classical four-chain antibody has V H and V LA domain-defined antigen-binding site. Some antibodies, such as camel and shark antibodies, lack a light chain and rely on binding sites formed solely by heavy chains. Single-domain engineered immunoglobulins can be prepared where the binding site is formed solely by either the heavy or light chain, without V... H and V L Collaboration between them.

[0060] When used herein, the term "hypervariate region" refers to certain amino acid residues of the antibody responsible for antigen binding. The hypervariate region may contain amino acid residues from the "complementarity-determining region" or "CDR" discussed above (e.g., in V...). L The middle part is approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) and in V H The residues are approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the “hypervariant ring” (e.g., in V). L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H 26-32(H1), 52A-55(H2) and 96-101(H3) (Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0061] "Frame region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined in the text.

[0062] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; tandem biantibodies (taDb); linear antibodies (e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-arm antibodies, single variable domain antibodies, microantibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv, or tandem (di,tri)-scFv); and bispecific T-cell binders (BiTE).

[0063] Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments, each with one antigen-combining site) and a residual "Fc" fragment, which contains the constant regions of both H chains and is responsible for antibody effector functions. C Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0064] "Fv" is the minimum antibody fragment that contains a complete antigen- recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen- binding site on the surface of the H -V L dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0065] Fab fragments also contain the constant domains of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains carry(s) at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0066] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0067] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0068] "Single-chain Fv" or "scFv" antibody fragments comprise the V H and V Ldomains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, enabling the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0069] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V H -V L ) connected to a light chain variable domain (V L ) in the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0070] The term "multispecific antibody" is used in the broadest sense, and specifically covers antibodies with polyepitopic specificity. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V H ) and a light chain variable domain (V L ), wherein the V H V L unit has polyepitopic specificity; antibodies with two or more V L and V H domains, wherein each V H V LA unit binds a distinct epitope; an antibody having two or more single variable domains, wherein each single variable domain binds a distinct epitope; a full length antibody; an antibody fragment such as a Fab, Fv, dsFv, scFv, diabody, bispecific diabody, triabody, trifunctional antibody, antibody fragments that have been covalently or noncovalently linked. "Polyepitopic specificity" refers to the ability to specifically bind two or more different epitopes on the same or different targets. "Monospecific" refers to the ability to bind only one epitope. According to one specific embodiment, the multispecific antibody is an IgG antibody that binds each epitope with an affinity of 5 pM to 0.001 pM, 3 pM to 0.001 pM, 1 pM to 0.001 pM, 0.5 pM to 0.001 pM, or 0.1 pM to 0.001 pM.

[0071] The expression "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain in order to recognize a target antigen. Examples of single domain antibodies include those from Camelidae (lamas and camels) and cartilaginous fishes (e.g., nurse sharks) and those derived from human and mouse antibodies by recombinant methods (Nature (1989) 341 :544-546; Dev Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235; Trends Biotechnol (2003): 21 :484-490; WO 2005 / 035572; WO 03 / 035694; Febs Lett (1994) 339:285-290; WO 00 / 29004; WO 02 / 051870).

[0072] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein each antibody in the population is identical and / or binds the same epitope, except for possible variants that can arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the methods provided herein can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0073] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, or Monkey) and human constant region sequences (U.S. Patent No. 5,693,780).

[0074] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0075] For purposes herein, "intact antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. The constant domains can be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant of the same. Preferably, the intact antibody has one or more effector functions.

[0076] A "native antibody" is generally an heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among the different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by various constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0077] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0078] When ALIGN-2 is used in the context of a comparison of amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or stated alternatively, a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0079] 100 times the fraction X / Y

[0080] where X is the number of amino acid residues scored by the sequence comparison program ALIGN-2 as identical matches in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that % amino acid sequence identity of A to B will not equal % amino acid sequence identity of B to A, when the length of amino acid sequence A is not equal to the length of amino acid sequence B. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0081] The terms "anti-IL-13 antibody" and "antibody that binds IL-13" refer to an antibody that is capable of binding IL-13 with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent for targeting IL-13. In some embodiments, an anti-IL-13 antibody binds to IL-13 with an affinity that is less than about 10% of the binding of the antibody to an unrelated, non-IL-13 protein as measured, e.g., by radioimmunoassay (RIA). In certain embodiments, an antibody that binds IL-13 has a dissociation constant (Kd) of < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M). In particular embodiments, an anti-IL-13 antibody binds an epitope of IL-13 that is conserved among IL-13 from different species.

[0082] An "IL-13-mediated disorder" refers to a disorder associated with an excess level or activity of IL-13, wherein atypical symptoms can manifest due to IL-13 levels or activity in the body locally and / or systemically. Examples of IL-13-mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease, Crohn's disease, inflammatory disorders of the lung (including pulmonary fibrosis, such as IPF), COPD, and liver fibrosis.

[0083] The term "respiratory disorder" includes, but is not limited to, asthma (e.g., allergic and non-allergic asthma (e.g., due to, e.g., respiratory syncytial virus (RSV) infection, e.g., in young children)); bronchitis (e.g., chronic bronchitis); chronic obstructive pulmonary disease (COPD) (e.g., emphysema (e.g., cigarette-induced emphysema); conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis, pulmonary fibrosis, and allergic rhinitis. Examples of diseases that can be characterized by airway inflammation, excess airway secretion, and airway obstruction include asthma, chronic bronchitis, bronchiectasis, and cystic fibrosis.

[0084] The term "therapeutic agent" refers to any agent used in the treatment of a disease. A therapeutic agent can be, e.g., a polypeptide (e.g., an antibody, immunoadhesin, or peptibody), an aptamer or small molecule that can bind a protein or nucleic acid molecule (which can bind a nucleic acid molecule encoding a target (i.e., an siRNA)), etc.

[0085] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule such as a cytotoxic moiety or radiolabel.

[0086] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells in which an exogenous nucleic acid has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom without regard for the number of passages. The progeny can not be identical to the parent cell in nucleic acid content, but can contain mutations that are not present in the parent cell.

[0087] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0088] An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0089] The term "sequential" as used herein with respect to chromatography refers to having a first chromatography and a subsequent second chromatography. There can be additional steps between the first chromatography and the second chromatography.

[0090] The term "sequential" as used herein with respect to chromatography refers to having a first chromatography and a subsequent second chromatography. There can be additional steps between the first chromatography and the second chromatography.

[0091] "Contaminants" and "impurities" refer to substances other than the desired polypeptide product. Contaminants and impurities include, but are not limited to, host cell material, such as CHOP, including single CHOP species; filtered protein A; nucleic acids; variants, fragments, aggregates, or derivatives of the desired polypeptide; other polypeptides; endotoxins; viral contaminants; cell culture media components, etc. In some examples, the contaminants can be host cell proteins (HCPs) from, for example, but not limited to, bacterial cells, such as E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells.

[0092] The terms "Chinese hamster ovary cell protein" and "CHOP" are used interchangeably to refer to a mixture of host cell proteins ("HCPs") from Chinese hamster ovary ("CHO") cell cultures. HCPs or CHOPs are typically present as contaminants in a cell culture medium or lysate (e.g., harvested cell culture fluid ("HCCF")) comprising a protein of interest (e.g., an antibody or immunoadhesin expressed in CHO cells). The amount of CHOP present in a mixture comprising a protein of interest provides a measure of the degree of purity of the protein of interest. HCPs or CHOPs include, but are not limited to, proteins of interest expressed by host cells, such as CHO host cells. Typically, the amount of CHOP in a mixture of proteins is expressed as parts per million relative to the amount of protein of interest in the mixture. It is understood that where the host cell is another mammalian cell type, E. coli, yeast, insect cell, or plant cell, HCP refers to proteins present in the host cell lysate that are not the target protein.

[0093] The terms "parts per million" or "ppm" are used interchangeably herein to refer to a measure of the purity of a protein of interest purified by the methods of the application. The unit ppm refers to the amount of HCP or CHOP in nanograms per milliliter per milligram per milliliter of protein of interest (i.e., CHOP ppm = (CHOP ng / ml) / (protein of interest mg / ml), where the proteins are in solution). Where the proteins are dried (e.g., by lyophilization), ppm refers to (CHOP ng) / (protein of interest mg). Impurities can also be expressed as "ng / mg," which is used interchangeably with ppm.

[0094] To "purify" a polypeptide from a composition comprising the polypeptide and one or more impurities means to increase the degree of purity of the polypeptide in the composition by removing (completely or partially) at least one impurity from the composition.

[0095] A "purification step" can be part of an overall purification process that results in a "homogeneous" composition, which is used herein to refer to a composition that contains less than 100 ppm HCP (100 ng / mg), or less than 90 ppm (90 ng / mg), or less than 80 ppm (80 ng / mg), or less than 70 ppm (70 ng / mg), or less than 60 ppm (60 ng / mg), or less than 50 ppm 50 ng / mg), or less than 40 ppm (40 ng / mg), or less than 30 ppm (30 ng / mg), or less than 20 ppm (20 ng / mg), or less than 10 ppm (10 ng / mg), or less than 5 ppm (5 ng / mg), or less than 3 ppm (3 ng / mg) or less than 1 ppm (1 ng / mg) of impurities in the composition containing the protein of interest. In certain embodiments, the HCP is a single HCP species. In one embodiment, the single HCP species is hamster PLBL2.

[0096] A "composition" to be purified herein contains a polypeptide of interest and one or more impurities or contaminants. The composition can be "partially purified" (i.e., already subjected to one or more purification steps), or can be obtained directly from a host cell or organism that produces the polypeptide (e.g., the composition can comprise a harvested cell culture fluid).

[0097] The terms "protein A" and "ProA" are used interchangeably herein to include protein A recovered from its natural source, protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and variants thereof that retain the ability to bind proteins having CH2 / CH3 regions, such as Fc regions. Protein A is commercially available from various sources. Protein A is typically immobilized on a solid phase support material. The term "ProA" also refers to an affinity chromatography resin or column that contains a chromatographic solid support matrix having covalently attached protein A.

[0098] The term "chromatography" refers to a method by which a solute of interest in a mixture is separated from other solutes in the mixture by the differential rates of migration of the solutes in the mixture through a stationary medium under the influence of a mobile phase, or in a binding-elution process.

[0099] The terms "affinity chromatography" and "protein affinity chromatography" are used interchangeably herein to refer to a protein separation technique in which a protein of interest or an antibody of interest is reversibly and specifically bound to a biological specific ligand. Typically, the biological specific ligand is covalently attached to a chromatography solid phase material, and the protein of interest in solution has access to the biological specific ligand when the solution is contacted with the chromatography solid phase material. The protein of interest (e.g., an antibody, enzyme or receptor protein) retains its specific binding affinity for the biological specific ligand (e.g., an antigen, substrate, co-factor, or hormone) during the chromatography step, while other solutes and / or proteins in the mixture do not appreciably or specifically bind to the ligand. The binding of the protein of interest to the immobilized ligand allows the contaminating proteins or protein impurities to flow through the chromatography media while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest in active form is then removed from the immobilized ligand using low pH, high pH, high salt, competitive ligand, etc., and the protein of interest is flowed through the chromatography column using an elution buffer that does not contain the contaminating proteins or protein impurities that were earlier allowed to flow through the column. Any component can serve as a ligand for purification of its respective specifically binding protein, e.g., an antibody.

[0100] The terms "non-affinity chromatography" and "non-affinity purification" refer to purification methods in which affinity chromatography is not utilized. Non-affinity chromatography includes chromatography techniques that rely on non-specific interactions between a molecule of interest (e.g., a protein, such as an antibody) and a solid phase matrix.

[0101] The term "specifically binds," as used herein in the context of chromatography, e.g., to describe the interaction between a molecule of interest and a ligand bound to a solid phase matrix, means that the protein of interest typically reversibly binds the ligand by a combination of the effects of spatial complementarity of the protein and ligand structures at the binding site plus electrostatic forces, hydrogen bonding, hydrophobic forces, and / or van der Waals forces at the binding site. The greater the spatial complementarity, the stronger the other forces at the binding site, the greater the specificity of binding of the protein to its corresponding ligand. Non-limiting examples of specific binding include antibody-antigen binding, enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation, DNA binding protein-DNA binding, regulatory protein-protein interactions, etc. Typically, specific binding in affinity chromatography occurs in free solution with affinities of about 10 -4 to 10 -8 M.

[0102] The term "non-specific binding," as used in the context of chromatography herein, as describing the interaction between a protein of interest and a ligand or other compound bound to a solid phase matrix, refers to the binding of the protein of interest to the ligand or compound on the solid phase matrix through electrostatic forces, hydrogen bonds, hydrophobic forces, and / or van der Waals forces at the site of interaction, but lacking structural complementarity that enhances the effect of non-structural forces. Examples of non-specific interactions include, but are not limited to, electrostatic forces, hydrophobic forces, and van der Waals forces, as well as hydrogen bonding.

[0103] A "salt" is a compound formed from the interaction of an acid and a base. Exemplary salts include, but are not limited to, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), chloride (e.g., sodium chloride), sulfate (e.g., sodium sulfate), or potassium salt.

[0104] As used herein, "solvent" refers to a liquid substance that is capable of dissolving or dispersings one or more other substances to provide a solution. Solvents include water and organic solvents, where certain organic solvents include non-polar solvents, ethanol, methanol, isopropanol, acetonitrile, hexylene glycol, propylene glycol, and 2,2-thiodiethanol.

[0105] The term "detergent" refers to ionic and non-ionic surfactants, such as polysorbate (e.g., polysorbate 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfonate; octoxynol; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauryl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauryl amidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl laurate; and the MONAQUAT(tm) series (Mona Industries, Inc., Paterson, New Jersey), polysorbates, such as polysorbate 20 (TWEEN 20(r)) or polysorbate 80 (TWEEN 80(r)).

[0106] A "polymer" herein is a molecule formed from the covalent joining of two or more monomers, where the monomers are not amino acid residues. Examples of polymers include, but are not limited to, polyethylene glycol, polypropylene glycol, and copolymers (e.g., PLURONICS TM , PF68, etc.), polyethylene glycol (PEG), such as PEG 400 and PEG 8000.

[0107] The terms "ion exchange" and "ion exchange chromatography" refer to chromatographic methods in which a target solute (such as a protein) in a mixture interacts with charged compounds linked (e.g., covalently) to a solid-phase ion exchange material, such that the target solute interacts with the charged compounds more or less nonspecifically than solute impurities or contaminants in the mixture. Contaminating solutes in the mixture elute from the ion exchange material column faster or slower than the target solute, or bind to the resin or efflux from the resin relative to the target solute. "Ion exchange chromatography" specifically includes cation exchange, anion exchange, and mixed-mode chromatography.

[0108] The phrase "ion exchange material" refers to a solid phase that is negatively charged (i.e., a cation exchange resin) or positively charged (i.e., anion exchange resin). The charge can be provided by attaching (e.g., covalently linking) one or more charged ligands to the solid phase. Alternatively, or additionally, the charge can be an inherent property of the solid phase (e.g., in the case of silica, which has an overall negative charge).

[0109] A "solid phase" refers to a non-aqueous matrix that may have one or more charged ligands attached to it. A solid phase can be a purification column, a discontinuous phase of discrete particles, a membrane, or a filter, etc. Examples of materials used to form a solid phase include polysaccharides (such as agarose and cellulose); and other mechanically stable matrices such as silica (e.g., controlled-porosity glass), poly(divinyl)styrene, polyacrylamide, ceramic particles, and any derivatives thereof.

[0110] "Cation exchange resin" refers to a negatively charged solid phase that allows free cations to exchange with cations in an aqueous solution flowing through or across the solid phase. The negatively charged ligands attached to the solid phase to form the cation exchange resin can be, for example, carboxylates or sulfonates. Commercially available cation exchange resins include, but are not limited to: carboxymethyl cellulose immobilized on agarose, sulfopropyl (SP) (e.g., SP-SEPHAROSE FAST FLOW (or SP-SEPHAROSE HIGHPERFORMANCE)), and resins immobilized on agarose and... The sulfonyl group on HS (e.g., S-SEPHAROSE FASTFLOW).

[0111] "Mixed-mode ion exchange resins" refer to solid phases covalently modified with cation, anion, and hydrophobic moieties. Mixed-mode ion exchange is also known as "multi-mode ion exchange." Commercially available mixed-mode ion exchange resins are available, such as BAKERBOND ABX, which contains weak cation exchange groups, a low concentration of anion exchange groups, and aqueous ligands attached to a silica solid phase support matrix. Other exemplary mixed-mode ion exchange resins include, but are not limited to, CAPTO. TMAdhere resin, QMA resin, CAPTO TM MMC resin, MEP HyperCel resin, HEA HyperCel resin, PPA HyperCel resin, or ChromaSorb membrane or Sartobind STIC. In some embodiments, the mixed mode material is CAPTO TM Adhere resin.

[0112] The term "anion exchange resin" is used herein to refer to a solid phase that is positively charged, e.g., that has one or more positively charged ligands, such as quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX, and FAST Q SEPHAROSE TM and Q SEPHAROSE TM FAST FLOW.

[0113] A "buffer" is a solution that resists changes in pH through the action of its acid-base conjugate components. Various buffers that can be used, e.g., depending on the desired buffer pH, are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). In certain instances, the buffer has a pH value ranging from about 2 to about 9, or from about 3 to about 8, or from about 4 to about 7, or from about 5 to about 7. Non-limiting examples of buffers that control the pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof.

[0114] The term "hydrophobic interaction chromatography" or "HIC" is used herein to refer to a chromatographic method that separates molecules based on their hydrophobicity. Exemplary resins that can be used in HIC include, but are not limited to, phenyl-, butyl-, octyl- SEPHAROSE, BUTYL 4Fast Flow, PHENYL SEPHAROSE TM High Performance, PHENYL SEPHAROSE TM 6Fast Flow (low sub), and PHENYL SEPHAROSE TM6Fast Flow (high sub). Typically, sample molecules in a high salt buffer are loaded onto a HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing the hydrophobic regions of the sample molecules, which are then adsorbed by the HIC column. The stronger the hydrophobicity of the molecule, the less salt is required to promote binding. Typically, a decreasing salt gradient is used to elute the sample from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecule increases, and the molecule elutes from the column to increase the hydrophobicity. Elution of the sample can also be achieved by adding a mild organic modifier or detergent to the elution buffer.

[0115] A "loading buffer" is a buffer used to load a composition comprising a polypeptide molecule of interest and one or more impurities onto an ion exchange resin. The loading buffer has a conductivity and / or pH such that the polypeptide molecule of interest (and typically one or more impurities) binds to the ion exchange resin, or the protein of interest flows through the column while the impurities bind to the resin.

[0116] An "intermediate buffer" is used to elute one or more impurities from an ion exchange resin prior to eluting the polypeptide molecule of interest. The conductivity and / or pH of the intermediate buffer is such that one or more impurities elute from the ion exchange resin, but the amount of the polypeptide of interest does not significantly.

[0117] As used herein, the term "wash buffer" refers to a buffer used to wash or re-equilibrate an ion exchange resin prior to elution of the polypeptide molecule of interest. In some cases, the wash buffer and the loading buffer can be the same for convenience, although this is not required.

[0118] An "elution buffer" is used to elute the polypeptide of interest from a solid phase. The conductivity and / or pH of the elution buffer is such that the polypeptide of interest elutes from the ion exchange resin.

[0119] A "regeneration buffer" can be used to regenerate an ion exchange resin so that it can be reused. The regeneration buffer has a conductivity and / or pH required to substantially remove all impurities and the polypeptide of interest from the ion exchange resin.

[0120] The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, the electric current flows through the transport of ions. Thus, as the ion content present in an aqueous solution increases, the solution will have a higher conductivity. The measurement unit for conductivity is milliSeimens (mS / cm) per centimeter, and can be measured using, for example, a conductivity meter sold by Orion. The conductivity of a solution can be changed by changing the ion concentration therein. For example, to achieve a desired conductivity, the buffer concentration and / or salt (e.g., NaCl or KCl) concentration in the solution can be changed.

[0121] The "pi" or "isoelectric point" of a polypeptide refers to the pH at which the net charge of the polypeptide is zero. The pi can be calculated from the net charge of the amino acid residues, or from the sialic acid residues of carbohydrate attached to the polypeptide, or can be determined by isoelectric focusing.

[0122] "Binding" of a molecule to an ion exchange material refers to exposing the molecule to the ion exchange material under suitable conditions (pH / conductivity) such that the molecule is reversibly immobilized within or on the ion exchange material through ionic interactions between the molecule and the charged group(s) of the ion exchange material.

[0123] "Washing" an ion exchange material refers to flowing or passing an appropriate buffer through or over the ion exchange material.

[0124] "Eluting" a molecule (e.g., a polypeptide or an impurity) from an ion exchange material refers to removing the molecule from the ion exchange material by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

[0125] "Ultrafiltration" is a form of membrane filtration in which hydrostatic pressure forces liquid through a semipermeable membrane. Suspended solids and high molecular weight solutes are retained, while water and low molecular weight solutes flow through the membrane. In some examples, ultrafiltration membranes have a pore size in the range of 1 to 100 nm. The terms "ultrafiltration membrane" and "ultrafiltration filter" can be used interchangeably.

[0126] "Diafiltration" is a method of removing salts or other sparingly soluble agents from a solution in conjunction with an ultrafiltration membrane. Small molecules are separated from the solution while larger molecules are retained in the retentate. This method selectively utilizes a permeable (porous) membrane filter to separate components of solutions and suspensions based on their molecular size.

[0127] As used herein, "filtrate" refers to the portion of a sample that passes through a filtration membrane.

[0128] As used herein, "retentate" refers to the portion of a sample that is substantially retained by a filtration membrane.

[0129] The term "pharmaceutical formulation" refers to a preparation which is effective for the biological activity of the active ingredient contained therein, and which is amenable to pharmaceutical manufacture and administration to a subject in need thereof.

[0130] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient which is not toxic to the subject to which the formulation is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0131] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention aimed at altering the natural course of a treated individual and can be used in preventative or clinicopathological processes. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some implementations, antibodies are used to delay disease development or slow disease progression.

[0132] The use of "about" in the text to refer to a value or parameter includes (and describes) the implementation scheme involving that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0133] Anti-IL-13 antibody

[0134] In some embodiments, isolated and purified antibodies that bind IL-13 are provided. Exemplary anti-IL-13 antibodies are known, including but not limited to, for example, lebrikizumab, IMA-026, IMA-638 (also known as anrukinzumab, INN Number 910649-32-0; QAX-576), tralokinumab (also known as CAT-354, CAS Number 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody. Examples of such anti-IL-13 antibodies and other IL13 inhibitors are disclosed, for example, in WO 2005 / 062967, WO 2008 / 086395, WO 2006 / 085938, US 7,615,213, US 7,501,121, WO 2007 / 036745, WO 2010 / 073119, WO 2007 / 045477. In one embodiment, the anti-IL-13 antibody is a humanized IgG4 antibody. In one embodiment, the anti-IL-13 antibody is lebrikizumab. In one embodiment, the anti-IL-13 antibody comprises three heavy chain CDRs, CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2), and CDR-H3 (SEQ ID NO: 3). In one embodiment, the anti-IL-13 antibody comprises three light chain CDRs, CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), and CDR-L3 (SEQ ID NO: 6). In one embodiment, the anti-IL-13 antibody comprises three heavy chain CDRs and three light chain CDRs, CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2), CDR-H3 (SEQ ID NO: 3), CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), and CDR-L3 (SEQ ID NO: 6). In one embodiment, the anti-IL-13 antibody comprises a variable heavy chain region, VH, having an amino acid sequence selected from SEQ ID NOs: 7 and 8. In one embodiment, the anti-IL-13 antibody comprises a variable light chain region, VL, having an amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-IL-13 antibody comprises a variable heavy chain region, VH, having an amino acid sequence selected from SEQ ID NOs: 7 and 8, and a variable light chain region, VL, having an amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-IL-13 antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO.: 13.In one embodiment, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and a light chain having the amino acid sequence of SEQ ID NO: 14.

[0135] In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-IL-13 antibody comprising that sequence retains the ability to bind IL-13. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 9. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-IL-13 antibody comprises the VL sequence in SEQ ID NO: 9, including post-translational modifications of that sequence.

[0136] In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-IL-13 antibody comprising that sequence retains the ability to bind IL-13. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 9. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-IL-13 antibody comprises the VL sequence in SEQ ID NO: 9, including post-translational modifications of that sequence.

[0137] In yet another embodiment, the anti-IL-13 antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0138] The following table shows the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of lebrikizumab, as well as the VH, VL, heavy chain sequence, and light chain sequence. As shown in Table 1 below, the VH and heavy chain can include an N-terminal glutamine, and the heavy chain can also include a C-terminal lysine. As is well known in the art, the N-terminal glutamine residue can form pyroglutamic acid and the C-terminal lysine residue can be cleaved off during the manufacturing process.

[0139] Table 1. Amino acid sequences of anti-IL-13 antibody (lebrikizumab).

[0140]

[0141] Other recombinant polypeptides

[0142] Recombinant polypeptides produced in CHO cells can be purified according to the methods described herein, eliminating or reducing hamster PLBL2 levels such that only a residual or undetectable amount remains. Such polypeptides include, but are not limited to, growth factors, cytokines, immunoglobulins, antibodies, peptibodies, and the like.

[0143] Certain exemplary antibodies include Aβ antibodies, IL17A / F antibodies, and CMV antibodies. Exemplary anti-Aβ antibodies and methods of producing such antibodies have been previously described, for example, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408. Exemplary anti-IL17A / F antibodies and methods of producing such antibodies have been previously described, for example, in WO2009136286 and U.S. Patent No. 8,715,669. Exemplary anti-CMV antibodies, including anti-CMV-MSL, and methods of producing such antibodies have been previously described in WO 2012047732.

[0144] Exemplary polypeptides include mammalian proteins, such as, for example, CD4; integrins and their subunits, such as β7; growth hormones, including human growth hormone and bovine growth hormone; growth hormone-releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; ct-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulation factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or tissue-type plasminogen activator (t-PA, e.g., ) ; bombazine; thrombin; tumor necrosis factor alpha and -beta; enkephalin; RANTES (regulated on activation, normal T cell expressed and secreted); human macrophage inflammatory protein (MIP-I-A); serum albumin such as human serum albumin; Mullerian-inhibiting substance; mouse gonadotropin-associated peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); IgE, receptors for hormones or growth factors; integrins; protein A or D; rheumatoid factors; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4 or TGF-beta 5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I); insulin-like growth factor binding proteins; other CD proteins such as CD3, CD8, CD19, and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon-alpha, -beta, or -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, etc.; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor (DAF); viral antigens, such as, for example, a portion of HIV envelope; transporters; homing receptors; addressins; regulatory proteins; integrins such as CDlla, CDllb, CDllc, CD18, integrin subunits such as alpha 4, alpha E, beta 7; cell adhesion molecules such as ICAM, VLA-4 and VCAM; tumor-associated antigens such as HER1, (EGFR), HER2, HER3 or HER4 receptors; Apo2L / TRAIL, and fragments of any of the polypeptides listed above; and immunoadhesins and antibodies that bind to any of the proteins listed above; and biologically active fragments or variants of any of the proteins listed above.

[0145] Other exemplary polypeptides include brain polypeptides including, but not limited to, beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), P-selectin, and caspase 6, and fragments of any of the polypeptides listed above; and immunoadhesins and antibodies that bind any of the proteins listed above; and biologically active fragments or variants of any of the proteins listed above.

[0146] Other exemplary polypeptides include therapeutic antibodies and immunoadhesins, including but not limited to, antibodies, including antibody fragments, to one or more of the following antigens: HER1 (EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF (e.g., bevacizumab, ranibizumab), MET (e.g., onartuzumab), CD20 (e.g., rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, VCAM, IL-17A and / or F, IgE (e.g., omalizumab), DR5, CD40, Apo2L / TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7 (e.g., etrolizumab), IL-13 (e.g., lebrikizumab) Abeta (e.g., crenezumab, gantenerumab), P-selectin (e.g., inclacumab), IL-6R (e.g., tociluzumab), IFNa (e.g., rontalizumab), M1 prime (e.g., quilizumab), mitogen-activated protein kinase (MAPK), OX40L, TSLP, Factor D (e.g., lampalizumab) and receptors, e.g.: IL-9 receptor, IL-5 receptor, IL-4 receptor alpha, IL-13 receptor alpha 1 and IL-13 receptor alpha 2, OX40, TSLP-R, IL-7R alpha (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcepsilonRI and FcepsilonRII / CD23 (receptors for IgE).Other exemplary antibodies include, but are not limited to, those selected from the group consisting of anti-estrogen receptor antibodies, anti-progesterone receptor antibodies, anti-p53 antibodies, anti-cathepsin D antibodies, anti-Bcl-2 antibodies, anti-E-cadherin antibodies, anti-CA 125 antibodies, anti-CA 15-3 antibodies, anti-CA 19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras oncoprotein antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9 / p24 antibodies, anti-CD10 antibodies, anti-CDllc antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies, anti-CD19 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, anti-CD31 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD35 antibodies, anti-CD38 antibodies, anti-CD41 antibodies, anti-LCA / CD45 antibodies, anti-CD45RO antibodies, anti-CD45RA antibodies, anti-CD39 antibodies, anti-CD100 antibodies, anti-CD95 / Fas antibodies, anti-CD99 antibodies, anti-CD106 antibodies, anti-ubiquitin antibodies, anti-CD71 antibodies, anti-c-myc antibodies, anti-cytokeratin antibodies, anti- vimentin antibodies, anti-HPV protein antibodies, anti-kappa light chain antibodies, anti-lambda light chain antibodies, anti-melanin antibodies, anti-prostate specific antigen antibodies, anti-S-100 antibodies, anti-tau antigen antibodies, anti-fibrin antibodies, anti-keratin antibodies, and anti-Tn antigen antibodies.

[0147] Certain purification methods

[0148] The proteins purified using the methods described herein are generally produced using recombinant techniques. Methods for producing recombinant proteins are described, for example, in U.S. Patent Nos. 5,534,615 and 4,816,567, which are specifically incorporated by reference herein. In certain embodiments, the protein of interest is produced in CHO cells (see, e.g., WO 94 / 11026). An example of a protein includes an anti-IL-13 monoclonal antibody (anti-IL-13 MAb), which can be purified using the methods already described herein above.

[0149] When using recombinant techniques, the protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. In the case of secretion into the medium, the recombinant host cells can be separated from the medium, for example, by centrifugation or ultrafiltration.

[0150] Protein A immobilized on a solid phase is used to purify the anti-IL-13 Mab preparation. In certain embodiments, the solid phase is a column comprising a glass, silica, agarose or polystyrene surface for immobilizing the protein A. In certain embodiments, the solid phase is a controlled pore glass column or a silica column. Sometimes, the column has been coated with a reagent, such as glycerol, in an attempt to prevent non-specific sticking to the column. PROSEP A TM column is an example of a protein A controlled pore glass column coated with glycerol. Other examples of columns contemplated herein include 50ATM (polystyrene) column or rProtein A SEPHAROSE FAST FLOW TM (Methylated dextran) column or MABSELECT SURE TM (Methylated dextran) column, which is available from GE Healthcare Life Sciences (Methylated dextran).

[0151] The solid phase for protein A chromatography is equilibrated with a suitable buffer. For example, the equilibration buffer can be 25 mM Tris, 25 mM NaCl, pH 7.7 + 0.20.

[0152] The preparation from the recombinant host cell and containing impurities and / or contaminants is loaded onto the solid phase equilibrated with a loading buffer, which can be the same as the equilibration buffer. As the impurity / contaminant-containing preparation flows through the solid phase, the protein is adsorbed to the immobilized protein A and other impurities / contaminants (such as Chinese hamster ovary protein, CHOP, in the case of a protein produced in CHO cells) can non-specifically bind to the solid phase.

[0153] The next steps, which are performed sequentially, require removal of the impurities / contaminants bound to the solid phase, the antibody and / or protein A, by washing the solid phase in intermediate wash steps. After loading, the solid phase can be equilibrated with the equilibration buffer before starting the intermediate wash steps.

[0154] The intermediate wash buffer can include a salt and optionally other compounds, for example (a) a detergent (e.g., a polysorbate, such as polysorbate 20 or polysorbate 80); (b) a solvent (such as hexylene glycol); and (c) a polymer (such as polyethylene glycol {PEG}).

[0155] The salt used can be selected based on the protein of interest. Exemplary salts include, but are not limited to, sodium acetate, sodium citrate, and potassium phosphate.

[0156] The amount of salt and other compounds, if any, in the composition is a combined amount that elutes the impurities / pollutants, but does not substantially remove the protein of interest. Exemplary salt concentrations in such wash buffers are about 0.1 to about 2 M, or about 0.2 M to about 0.6 M. Useful detergent concentrations are about 0.01 to about 5%, or about 0.1% to 1%, or about 0.5%, e.g., where the detergent is a polysorbate. Exemplary solvent concentrations are about 1% to 40%, or about 5 to about 25%. Where the other compound is a polymer (e.g., PEG 400 or PEG 8000), its concentration can be, e.g., about 1% to about 20%, or about 5% to about 15%.

[0157] The pH of the intermediate wash buffer is typically about 4 to about 8, or about 4.5 to about 5.5, or about 5.0. In one embodiment, the pH is 7.00 ± 0.10.

[0158] Following the intermediate wash step described above, the protein of interest is recovered from the column. This is typically achieved using a suitable elution buffer. The protein can be eluted from the column, e.g., using an elution buffer having a low pH (also referred to as acidic conditions), e.g., a pH in the range of about 2 to about 5, or in the range of about 2.5 to about 3.5. Examples of elution buffers for this purpose include citrate or acetate buffers.

[0159] The eluted protein preparation can be subjected to additional purification steps, either before or after the protein A chromatography step. Exemplary further purification steps include hydroxyapatite chromatography; dialysis; affinity chromatography using antibody capture proteins; hydrophobic interaction chromatography (HIC); ammonium sulfate precipitation; anion or cation exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica; focusing; ultrafiltration diafiltration (UFDF), and gel filtration. In the examples herein, the protein A chromatography step is followed by downstream anion exchange (e.g., Q-Sepharose-Fast Flow) or multimodal (e.g., mixed mode) ion exchange (e.g., CAPTO TM Adhere) and HIC (e.g., PHENYL SEPHAROSE TM 6fast flow-highsub) purification steps.

[0160] The proteins recovered thereby can be formulated in pharmaceutically acceptable carriers for various diagnostic, therapeutic, or other uses known for these molecules.

[0161] In some embodiments of any of the methods described herein, the chromatography material is an ion exchange chromatography material; for example, an anion exchange chromatography material. In some embodiments, the anion exchange chromatography material is a positively charged solid phase with free anions exchanged with anions in an aqueous solution flowing through or over the solid phase. In some embodiments of any of the methods described herein, the anion exchange material can be a membrane, monolith, or resin. In one embodiment, the anion exchange material can be a resin. In some embodiments, the anion exchange material can include primary amine, secondary amine, tertiary amine, or quaternary ammonium ion functional groups, polyamine functional groups, or diethylaminoaethyl functional groups. In some embodiments described above, the anion exchange chromatography material is an anion exchange chromatography column. In some embodiments described above, the anion exchange chromatography material is an anion exchange chromatography membrane.

[0162] In some embodiments of any of the methods described herein, the ion exchange material can utilize a conventional chromatography material or a convective chromatography material. Conventional chromatography materials include, for example, perfusive materials (e.g., poly(styrene-divinylbenzene) resins) and diffusive materials (e.g., cross-linked agarose resins). In some embodiments, the poly(styrene-divinylbenzene) resins can be

[0163] Examples of anion exchange materials include, but are not limited to, HQ 50, PI 50, D, Mustang Q, Q SEPHAROSE TM FF, and DEAE Sepharose.

[0164] ​In some aspects, the chromatography material is a hydrophobic interaction chromatography material. Hydrophobic interaction chromatography (HIC) is a liquid chromatography technique that separates biomolecules based on hydrophobicity. Examples of HIC chromatography materials include, but are not limited to, Toyopearl hexyl 650, Toyopearl butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source 30T, Resource 15, Sepharose Hi-Trap, Octyl sepharose, PHENYL SEPHAROSE TM high performance, PHENYL SEPHAROSE TM 6 fast flow (low sub) and PHENYL SEPHAROSE TM 6 fast flow (high sub). In some embodiments described above, the HIC chromatography material is a HIC chromatography column. In some embodiments described above, the HIC chromatography material is a HIC chromatography membrane.

[0165] In some aspects, the chromatography material is an affinity chromatography material. Examples of affinity chromatography materials include, but are not limited to, chromatography materials derivatized with protein A or protein G. Examples of affinity chromatography materials include, but are not limited to, Prosep-VA, Prosep-VA Ultra Plus, Protein A sepharose fast flow, Tyopearl Protein A, MAbSelect, MABSELECT SURE TM LX, and MABSELECT SURE TM In some embodiments described above, the affinity chromatography material is an affinity chromatography column. In some embodiments described above, the affinity chromatography material is an affinity chromatography membrane.

[0166] Various buffers can be used depending on, for example, the desired buffer pH, the desired buffer conductivity, the characteristics of the protein of interest, and the purification method. In some embodiments of any of the methods described herein, the method comprises using a buffer. The buffer can be a loading buffer, an equilibration buffer, or a wash buffer. In some embodiments, one or more of the loading buffer, the equilibration buffer, and / or the wash buffer are the same. In some embodiments, the loading buffer, the equilibration buffer, and / or the wash buffer are different. In some embodiments of any of the methods described herein, the buffer comprises a salt. The loading buffer can comprise sodium chloride, sodium acetate, or a mixture thereof. In some embodiments, the loading buffer is a sodium chloride buffer. In some embodiments, the loading buffer is a sodium acetate buffer.

[0167] As used herein, load is a composition that is loaded onto a chromatography material. A loading buffer is a buffer used to load a composition comprising a product of interest onto a chromatography material. A chromatography material can be equilibrated with an equilibration buffer prior to loading a composition to be purified. In some embodiments, a wash buffer is used after a composition is loaded onto a chromatography material and before a polypeptide of interest is eluted from the solid phase. However, some products of interest, such as polypeptides, can be removed from a chromatography material by a wash buffer (e.g., flow-through mode).

[0168] As used herein, elution is the removal of a product, such as a polypeptide, from a chromatography material. An elution buffer is a buffer used to elute a polypeptide or other product of interest from a chromatography material. In many cases, an elution buffer has different physical characteristics than a loading buffer. For example, an elution buffer can have a different conductivity than a loading buffer or a different pH than a loading buffer. In some embodiments, an elution buffer has a lower conductivity than a loading buffer. In some embodiments, an elution buffer has a higher conductivity than a loading buffer. In some embodiments, an elution buffer has a lower pH than a loading buffer. In some embodiments, an elution buffer has a higher pH than a loading buffer. In some embodiments, an elution buffer has a different conductivity and a different pH than a loading buffer. An elution buffer can have any combination of a higher or lower conductivity and a higher or lower pH.

[0169] Conductivity refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, the electric current is transmitted through ions. Thus, as the amount of ions present in an aqueous solution increases, the aqueous solution will have a higher conductivity. The basic unit of measurement for conductivity is the Siemen (or mho), mho (mS / cm), and can be measured using a conductivity meter, such as the various models of the Orion conductivity meter. Since the conductivity of an electrolyte is the ability of the ions in the solution to carry an electric current, the conductivity of a solution can be changed by changing the concentration of ions in it. For example, the concentration of a buffer and / or the concentration of a salt (such as sodium chloride, sodium acetate, or potassium chloride) in a solution can be changed to obtain a desired conductivity. Preferably, the salt concentration of various buffers is changed to obtain a desired conductivity.

[0170] In some embodiments of any of the methods described herein, the flow rate is less than about any of 50 CV / hr, 40 CV / hr, or 30 CV / hr. The flow rate can be any between about 5 CV / hr and 50 CV / hr, 10 CV / hr and 40 CV / hr, or 18 CV / hr and 36 CV / hr. In some embodiments, the flow rate is about any of 9 CV / hr, 18 CV / hr, 25 CV / hr, 30 CV / hr, 36 CV / hr, or 40 CV / hr. In some embodiments of any of the methods described herein, the flow rate is less than about any of 100 cm / hr, 75 cm / hr, or 50 cm / hr. The flow rate can be any between about 25 cm / hr and 150 cm / hr, 25 cm / hr and 100 cm / hr, 50 cm / hr and 100 cm / hr, or 65 cm / hr and 85 cm / hr, or 50 cm / hr and 250 cm / hr, or 100 cm / hr and 250 cm / hr, or 150 cm / hr and 250 cm / hr.

[0171] Bed height is the height of the chromatography material used. In some embodiments of any of the methods described herein, the bed height is higher than about any of 3 cm, 10 cm, or 15 cm. The bed height can be any between about 3 cm and 35 cm, 5 cm and 15 cm, 3 cm and 10 cm, or 5 cm and 8 cm. In some embodiments, the bed height is about any of 3 cm, 5 cm, 10 cm, or 15 cm. In some embodiments, the bed height is determined according to the amount of polypeptide or contaminant in the load.

[0172] In some embodiments, the chromatography is in a column of a vessel having a volume greater than about 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 75 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 25 L, 50 L, 100 L, 200 L, 400 L, or 450 L.

[0173] In some embodiments, fractions are collected from the chromatography. In some embodiments, the collected fractions are greater than about 0.01 CV, 0.02 CV, 0.03 CV, 0.04 CV, 0.05 CV, 0.06 CV, 0.07 CV, 0.08 CV, 0.09 CV, 0.1 CV, 0.2 CV, 0.3 CV, 0.4 CV, 0.5 CV, 0.6 CV, 0.7 CV, 0.8 CV, 0.9 CV, 1.0 CV, 2.0 CV, 3.0 CV, 4.0 CV, 5.0 CV. In some embodiments, fractions containing the product (e.g., polypeptide) are pooled. In some embodiments, fractions containing polypeptide from the load fraction and elution fraction are pooled. The amount of polypeptide in a fraction can be determined by one of skill in the art; for example, the amount of polypeptide in a fraction can be determined by UV spectroscopy. In some embodiments, fractions containing detectable fragments of the polypeptide are pooled.

[0174] In some embodiments of any of the methods described herein, the at least one impurity or contaminant is host cell material, such as CHOP; filtered protein A; nucleic acid; a variant, fragment, aggregate, or derivative of the desired polypeptide; another polypeptide; endotoxin; viral contaminant; cell culture media component, gentamicin, or any one or more of the foregoing. In some embodiments, the impurity or contaminant can be host cell protein (HCP) from, for example, but not limited to, bacterial cells such as E. coli cells, insect cells, prokaryotic cells, eukaryotic cells, yeast cells, mammalian cells, avian cells, fungal cells.

[0175] Host cell proteins (HCPs) are proteins from the cells in which the polypeptide is produced. For example, CHOP is a protein from the host cell, i.e., Chinese hamster ovary protein. The amount of CHOP can be measured by enzyme-linked immunosorbent assay ("ELISA") or mass spectrometry. In some embodiments of any of the methods described herein, the amount of HCPs (e.g., CHOP) is reduced by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The amount of HCPs can be reduced by any of between about 10% and 99%, between 30% and 95%, between 30% and 99%, between 50% and 95%, between 50% and 99%, between 75% and 99%, or between 85% and 99%. In some embodiments, the amount of HCPs is reduced by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the reduction is determined by comparing the amount of HCPs in the composition recovered from a purification step to the amount of HCPs in the composition prior to the purification step.

[0176] In some embodiments of any of the methods described herein, the method further comprises recovering the purified polypeptide. In some embodiments, the purified polypeptide is recovered from any of the purification steps described herein. The chromatography step can be anion exchange chromatography, HIC, or protein A chromatography. In some embodiments, the first chromatography step is protein A, followed by anion exchange or multimodal ion exchange, followed by HIC.

[0177] In some embodiments, the polypeptide is further purified by viral filtration following chromatography. Viral filtration is the removal of viral impurities in a polypeptide purification feed stream. Examples of viral filtration include ultrafiltration and microfiltration. In some embodiments, the polypeptide is purified using a parvovirus filter.

[0178] In some embodiments, the polypeptide is concentrated following chromatography. Examples of concentration methods are known in the art, including but not limited to ultrafiltration and diafiltration.

[0179] In some embodiments of any of the methods described herein, the method further comprises combining the purified polypeptide in the purification method with a pharmaceutically acceptable carrier.

[0180] Monoclonal antibodies

[0181] In some embodiments, the antibody purified according to the methods of the application is a monoclonal antibody. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody, such variants being present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.

[0182] For example, the monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0183] In the hybridoma method, a mouse or other appropriate host animal, e.g., a hamster, is immunized as herein described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to a polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, e.g., polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0184] Hybridoma cells thus produced are seeded and grown in a suitable culture medium, preferably in the presence of one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminoimidazole carboxylic acid, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0185] In some embodiments, myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to medium such as HAT medium. Among these, in some embodiments, the myeloma cell line is murine myeloma line, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0186] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

[0187] The binding affinity of the monoclonal antibody, e.g., can be determined by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0188] After the identification of hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, e.g., D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal.

[0189] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, polypeptide A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0190] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, the hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin polypeptides, to obtain the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188 (1992).

[0191] In further embodiments, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nm range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as the construction of very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are applicable to the production of numerous "designer antibodies".

[0192] The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or a portion of the coding sequence for a non-immunoglobulin polypeptide.

[0193] Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

[0194] In some embodiments of any of the methods described herein, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgG monoclonal antibody.

[0195] Humanized antibodies

[0196] In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues derived from a non-human source introduced therein. These non-human amino acid residues are often referred to as "import" residues, which generally are derived from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0197] The selection of human variable domains (including both light and heavy chains) to be used in making humanized antibodies is important to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151 :2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151 :2623 (1993)).

[0198] More importantly, the humanized antibody must retain its high affinity for the antigen and other favorable biological properties. To achieve this goal, in certain embodiments of the method, the humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products, using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the binding loy and therefore which residues may need to be preserved. This analysis can be made with reference to the modeling of a candidate immunoglobulin sequence. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristics, such as increased affinity for the target antigen, are achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0199] Human antibodies

[0200] In some embodiments, the antibody is a human antibody. Rather than humanizing, it is now possible to generate human antibodies and antibody fragments using transgenic mice (e.g., mice that are incapable of expressing endogenous immunoglobulins, but whose genomes have been reprogrammed to express human antibody H ) genes in response to antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

[0201] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and expressed as functional antibody fragments when the phage displays the gene product(s) on the phage surface. Because the filamentous phage comprises a single-stranded DNA copy of the phage genome, selections based on antibody function are directly reflected by selection of the appropriate phage genomes. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazone antibodies from a large human combinatorial library generated in phage by annexing the V-genes from a selection of sodium mice. Combinatorial human V-gene libraries can be constructed and antibodies to a diverse array of antigens (including self-antigens) isolated substantially following the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0202] Human antibodies can also be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

[0203] Antibody fragments

[0204] In some embodiments, the antibody is an antibody fragment. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived from whole antibodies via proteolytic digestion (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments are known to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. The antibody fragment can also be a "linear antibody", e.g., as described in U.S. Patent 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0205] In some embodiments, an antibody fragment described herein is provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, an Fv, and a diabody.

[0206] Chimeric polypeptides

[0207] The polypeptides described herein can be altered in a manner to form chimeric molecules comprising the polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises the polypeptide fused to a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is typically located at the amino- or carboxy-terminus of the polypeptide. The presence of such an epitope tag form of the polypeptide can be detected using an antibody to the tag polypeptide. In addition, the provision of an epitope tag enables the polypeptide to be readily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds the epitope tag.

[0208] Other

[0209] Another type of covalent modification of the polypeptide comprises linking the polypeptide to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The polypeptides can also be encapsulated in liposomes, microparticles, microcapsules, or macrocapsules, for example, prepared from biodegradable polymers such as polylactide-polyglycolide. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, A.R., Ed., (1990).

[0210] Obtaining polypeptides

[0211] The polypeptides used in the purification methods described herein can be obtained using methods well known in the art, including recombinant methods. The following section provides guidance regarding these methods.

[0212] Polynucleotides

[0213] "Polynucleotide" or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0214] Polynucleotides encoding polypeptides can be obtained from any source, including, but not limited to, cDNA libraries prepared from tissues believed to have mRNA for the polypeptide and to express it at detectable levels. Thus, polynucleotides encoding polypeptides can be readily obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries, or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0215] For example, a polynucleotide can encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only the heavy chain variable region (V H ), but also the heavy chain constant region (C H ), which generally comprises three constant domains, C H 1, C H 2 and C H 3; and a "hinge" region. In some cases, the presence of the constant region is desirable.

[0216] Other polypeptides that can be encoded by a polynucleotide include antigen binding fragments of antibodies, such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and F(ab')2, and "minibodies." Minibodies (in the general case) are C H 1 and C K or C LA minibody fragment from which a domain has been excised. Since minibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic applications, but as bivalent they should retain higher binding affinity than monovalent antibody fragments (e.g., dAbs). Accordingly, the term "antibody" as used herein includes not only whole antibody molecules, but also antigen-binding fragments of antibodies of the types discussed above, unless the context indicates otherwise. Preferably, each framework region present in the encoded polypeptide comprises at least one amino acid substitution relative to the corresponding human acceptor framework region. Thus, for example, the framework regions can comprise, in total, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions relative to the acceptor framework regions.

[0217] Suitably, the polynucleotides described herein can be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0218] The term "isolated polynucleotide" is intended to mean a molecule that is removed or separated from its normal or natural environment, or produced in a manner that it does not exist in nature. In some embodiments, the polynucleotide is a purified polynucleotide. The term purified is intended to mean that at least some contaminant molecules or substances are removed.

[0219] Suitably, the polynucleotides are substantially purified, such that the relevant polynucleotide constitutes the predominant (i.e., most abundant) polynucleotide present in the composition.

[0220] Expression of polynucleotides

[0221] The following description primarily relates to the production of polypeptides by culturing cells transformed or transfected with vectors containing polynucleotides encoding the polypeptides. Of course, alternative methods well known in the art are contemplated to be used to make the polypeptides. For example, the appropriate amino acid sequence, or portions thereof, can be produced by direct peptide synthesis using solid- phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using the manufacturer's instructions. Individual portions of the polypeptide can be chemically synthesized independently and the portions combined using chemical or enzymatic methods to produce the desired polypeptide.

[0222] Polynucleotides as described herein are inserted into expression vectors for production of polypeptides. The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences include, but are not limited to, a promoter, such as a naturally associated or heterologous promoter, a signal sequence, an enhancer element, and a transcription termination sequence.

[0223] A polynucleotide is "operably linked" when it is in a functional relationship with another nucleotide sequence. For instance, a presequence or secretion leader nucleic acid is operably linked to a polypeptide nucleic acid if it influences the transcription of the sequence; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the nucleic acid sequences being linked are contiguous and, in the case of a secretion leader, are contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0224] For antibodies, the light and heavy chains can be cloned into the same or different expression vectors. The nucleic acid segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector to ensure expression of the immunoglobulin polypeptides.

[0225] Vectors containing polynucleotide sequences (e.g., variable heavy and / or variable light encoding sequences and optional expression control sequences) can be transferred into host cells by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, the transgene can be microinjected into a fertilized oocyte, or can be incorporated into the genome of an embryonic stem cell, and the nucleus of such cell transferred into an enucleated oocyte.

[0226] Vectors

[0227] The term "vector" includes expression vectors and transformation vectors and shuttle vectors.

[0228] The term "expression vector" refers to a construct capable of expressing in vivo or in vitro.

[0229] The term "transformation vector" refers to a construct that is capable of being transferred from one entity to another - the entities can be entities of the same species or can be entities of different species. If the construct is capable of being transferred from one species to another - for example, from an E. coli plasmid to a bacterium such as a Bacillus sp., the transformation vector is sometimes referred to as a "shuttle vector". It can even be a construct that is capable of being transferred from an E. coli plasmid to an Agrobacterium into a plant.

[0230] As described below, the vectors can be transformed into suitable host cells to provide for expression of the polypeptides. Various vectors are publicly available. The vector, for example, can be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Standard ligation techniques are employed to construct suitable vectors containing one or more of these components.

[0231] The vector can be, for example, a plasmid, virus, or phage vector, which has an origin of replication, optionally a promoter for expression of the polynucleotide, and optionally a regulator of the promoter. The vector can contain one or more selectable marker genes well known in the art.

[0232] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome DNA.

[0233] Host cells

[0234] The host cells can be, for example, bacterial, yeast or other fungal cells, insect cells, plant cells, or mammalian cells.

[0235] Transgenic multicellular host organisms that have been genetically engineered can be used to produce the polypeptides. The organisms can be, for example, transgenic mammalian organisms (e.g., transgenic goat or mouse lines).

[0236] Suitable prokaryotes include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P), Pseudomonas, such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is a particularly preferred host or parent host because it is a commonly used host strain for fermentation of recombinant polynucleotide products. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 can be modified to effect a genetic mutation in a gene encoding a host endogenous polypeptide, examples of such hosts include E. coli W3110 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan'; E. coli strain W3110 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kana resistant degP deletion mutation; and E. coli strains with mutated periplasmic proteases. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0237] Expression vectors can be prepared in these prokaryotic hosts, which generally contain expression control sequences (e.g., replication origins) compatible with the host cell. In addition, there will be any number of various well-known promoters, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters generally control expression, optionally with operator sequences, and ribosome binding site sequences, etc., for initiation and termination of transcription and translation.

[0238] Eukaryotic microbes can be used for expression. Eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding polypeptides. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe, Yarrowia lipolytica, and others as described in Bennett, J.W. and La Ta, A. 1993, "Fungi for Industrial Production," Biotechnology and zosaccharomyces pombe); Kluyveromyces hosts such as, for example, K. lactis (MW98-8C, CBS 683, CBS 4574), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402226); Pichia pastoris; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces such as S. occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. Methylotrophic yeasts are suitable here, including but not limited to, yeasts capable of growth on methanol selected from the group consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces is the preferred yeast host, with suitable vectors having, as needed, expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like. Typical promoters include those for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, those from alcohol dehydrogenase, isocitrate

[0239] In addition to microorganisms, mammalian tissue cell cultures can be used to express and produce polypeptides as described herein, and in some cases are preferred (see Winnacker, From Genes to Clones VCH Publishers, N.Y., N.Y. (1987). For some embodiments, eukaryotic cells can be preferred because of the development of a number of suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins), including CHO cell lines, various Cos cell lines, HeLa cells, preferably, myeloma cell lines, or transformed B cells or hybridomas. In some embodiments, the mammalian host cell is a CHO cell.

[0240] In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO or CHO-DP-12 line); mouse Sertoli cells; monkey kidney cells (CV1 ATCC CRL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2 cells).

[0241] Formulations and methods of making formulations

[0242] Provided herein are formulations and methods of making formulations comprising a polypeptide (e.g., an antibody) purified using the methods described herein. For example, the purified polypeptide can be combined with a pharmaceutically acceptable carrier.

[0243] For storage, the polypeptide formulations in some embodiments can be prepared by mixing a polypeptide having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0244] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer, which is nontoxic to the cells or mammal being exposed thereto at the dosages and concentrations employed. Often physiologically acceptable carriers are aqueous pH buffered solutions.

[0245] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants TM , PLURONICS TM or polyethylene glycol (PEG).

[0246] In some embodiments, the polypeptides in the polypeptide formulation remain functionally active.

[0247] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through a sterile filtration membrane.

[0248] The formulations herein can also contain more than one active agent necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to a polypeptide, it can be desirable to include another polypeptide (e.g., an antibody) in one formulation. Alternatively, or additionally, the composition can further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an anti-hormonal agent, and / or a cardioprotective agent. Such molecules are suitably present in the combination in amounts that are effective for the purpose intended.

[0249] Exemplary formulations of the anti-IL-13 antibodies described herein are provided in International Patent Publication No. WO 2013 / 066866.

[0250] Product

[0251] The purified polypeptides described herein and / or formulations comprising the purified polypeptides described herein can be included in a product. The product can include a container that holds the polypeptide and / or polypeptide formulation. In certain embodiments, the product comprises: (a) a container that holds a composition comprising a polypeptide described herein and / or a polypeptide formulation; and (b) a label or package insert on or associated with the container. The label or package insert can indicate that the composition is used for the treatment of a subject.

[0252] The product comprises a container and a label or package insert that accompanies the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds or contains the formulation and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a polypeptide. The label or package insert indicates that the composition is used for the treatment of a subject, with specific guidance regarding dosage amounts and intervals for providing the polypeptide and other drugs. The product can further include other materials such as other buffers, diluents, filters, needles, and syringes as can be necessary or desirable for commercial and user purposes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further contained within an injection device. In some embodiments, the injection device is an auto-injector.

[0253] "Package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, adverse reactions associated with the product in the package, and / or warnings concerning the use of such products.

[0254] Exemplary products containing formulations of anti-IL-13 antibodies described herein are provided in International Patent Publication WO 2013 / 066866.

[0255] Further details of the application are illustrated by the following non-limiting examples. All cited references are expressly incorporated herein by reference. Specific Embodiments

[0256] 1. A composition comprising an anti-IL-13 monoclonal antibody purified from Chinese hamster ovary host cells, wherein the composition comprises the anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0257] 2. The composition of embodiment 1, wherein the anti-IL-13 antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0258] 3. The composition of embodiment 2, wherein the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7.

[0259] 4. The composition of embodiment 2, wherein the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9.

[0260] 5. The composition of embodiment 3, wherein the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10.

[0261] 6. The composition of embodiment 4, wherein the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14.

[0262] 7. The composition of embodiment 2, wherein the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9.

[0263] 8. The composition of embodiment 7, wherein the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14.

[0264] 9. The composition of embodiment 1, wherein the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay.

[0265] 10. The composition of embodiment 9, wherein the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA.

[0266] 11. The composition of embodiment 9, wherein the mass spectrometry assay is LC-MS / MS.

[0267] 12. A purified anti-IL-13 monoclonal antibody preparation isolated from Chinese hamster ovary host cells, wherein the preparation is purified by a method comprising a hydrophobic interaction chromatography (HIC) step, thereby producing a purified preparation, wherein the purified preparation comprises anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0268] 13. The anti-IL-13 antibody preparation of embodiment 12, wherein the HIC step comprises a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin.

[0269] 14. The anti-IL-13 antibody preparation of embodiment 13, wherein the HIC step comprises operating a column containing a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin in flow-through mode.

[0270] 15. The anti-IL-13 antibody preparation of embodiment 14, wherein the HIC step comprises an equilibration buffer and a wash buffer, wherein the equilibration buffer and the wash buffer each comprise 50 mM sodium acetate pH 5.0.

[0271] 16. The anti-IL-13 antibody preparation of embodiment 15, wherein the flow-through is monitored at an absorbance of 280 nanometers and the flow-through is collected between 0.5 OD and 1.5 OD.

[0272] 17. The anti-IL-13 antibody preparation of embodiment 15, wherein up to 8 column volumes of flow-through are collected.

[0273] 18. The anti-IL-13 antibody preparation of embodiment 12, wherein the method further comprises an affinity chromatography step.

[0274] 19. The anti-IL-13 antibody preparation of embodiment 18, wherein the affinity chromatography is protein A chromatography.

[0275] 20. The anti-IL-13 antibody preparation of embodiment 12, wherein the method further comprises an ion exchange chromatography step.

[0276] 21. The anti-IL-13 antibody preparation of embodiment 20, wherein the ion exchange chromatography is anion exchange chromatography.

[0277] 22. A purified anti-IL-13 monoclonal antibody preparation isolated from Chinese hamster ovary cells, wherein the antibody preparation is purified by a method comprising a first Protein A affinity chromatography step, a second anion exchange chromatography step, and a third hydrophobic interaction chromatography (HIC) step, thereby producing a purified preparation, wherein the purified preparation comprises anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0278] 23. The anti-IL-13 antibody preparation according to embodiment 22, wherein the affinity chromatography step comprises a MABSELECT SURE TM resin, the anion exchange chromatography step comprises a Q SEPHAROSE TM Fast Flow resin, and the HIC step comprises a PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin.

[0279] 24. The anti-IL-13 antibody preparation according to embodiment 23, wherein

[0280] the affinity chromatography step comprises operating a column containing MABSELECT SURE TM resin in a bind-elute mode;

[0281] the anion exchange chromatography step comprises operating a column containing Q SEPHAROSE TM Fast Flow resin in a bind-elute mode;

[0282] the HIC step comprises operating a column containing PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin in a flow-through mode.

[0283] 25. The anti-IL-13 antibody formulation of either embodiment 12 or embodiment 22, wherein the anti-IL-13 antibody comprises three heavy chain CDRs and three light chain CDRs, wherein the three heavy chain CDRs are CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and the three light chain CDRs are CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0284] 26. The anti-IL-13 antibody formulation of embodiment 25, wherein the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7.

[0285] 27. The anti-IL-13 antibody formulation of embodiment 25, wherein the anti-IL-13 antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 9.

[0286] 28. The anti-IL-13 antibody formulation of embodiment 26, wherein the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10.

[0287] 29. The anti-IL-13 antibody formulation of embodiment 27, wherein the anti-IL-13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 14.

[0288] 30. The anti-IL-13 antibody formulation of embodiment 25, wherein the anti-IL-13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO: 9.

[0289] 31. The anti-IL-13 antibody formulation of embodiment 30, wherein the anti-IL-13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10 and a light chain having the amino acid sequence of SEQ ID NO: 14.

[0290] 32. The anti-IL-13 antibody formulation of either embodiment 12 or embodiment 22, wherein the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay.

[0291] 33. The anti-IL-13 antibody formulation of embodiment 32, wherein the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA.

[0292] 34. The anti-IL-13 antibody preparation of embodiment 32, wherein the mass spectrometry assay is LC-MS / MS.

[0293] 35. A method of purifying a recombinant polypeptide produced in a Chinese hamster ovary host cell, wherein the method provides a purified preparation comprising the recombinant polypeptide and a residual amount of hamster PLBL2.

[0294] 36. The method of embodiment 35, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0295] 37. The method of embodiment 36, comprising a hydrophobic interaction chromatography (HIC) step.

[0296] 38. The method of embodiment 37, wherein the HIC step comprises a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin.

[0297] 39. The method of embodiment 38, wherein the HIC step comprises operating a column containing a PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin in flow-through mode.

[0298] 40. The method of embodiment 37, wherein the recombinant polypeptide is selected from the group consisting of a growth factor, a cytokine, an antibody, an antibody fragment, and an immunoadhesin.

[0299] 41. The method of embodiment 40, wherein the recombinant polypeptide is an antibody.

[0300] 42. The method of embodiment 41, wherein the antibody is a humanized monoclonal antibody.

[0301] 43. The method of embodiment 42, wherein the antibody is an IgGl, or IgG2 or IgG3 or IgG4.

[0302] 44. The method of embodiment 43, wherein the antibody is an IgG4.

[0303] 45. The method of embodiment 41, wherein the antibody is an anti-IL-13.

[0304] 46. The method of embodiment 44, wherein the antibody is lebrikizumab.

[0305] 47. The method of embodiment 45, wherein the anti-IL-13 antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 1, CDR-H2 having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 4, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0306] 48. The method of any one of embodiments 45, 46, or 47, wherein the HIC step comprises operating the resin-containing column in flow-through mode, including an equilibration buffer and a wash buffer, wherein the equilibration buffer and the wash buffer each comprise 50 mM sodium acetate, pH 5.0.

[0307] 49. The method of embodiment 48, wherein the flow-through is monitored at an absorbance of 280 nanometers and between 0.5 OD and 1.5 OD of flow-through is collected.

[0308] 50. The method of embodiment 48, wherein up to 8 column volumes of flow-through are collected.

[0309] 51. The method of embodiment 48, further comprising an affinity chromatography step.

[0310] 52. The method of embodiment 51, wherein the affinity chromatography is protein A chromatography.

[0311] 53. The method of embodiment 48, further comprising an ion exchange chromatography step.

[0312] 54. The method of embodiment 53, wherein the ion exchange chromatography is anion exchange chromatography.

[0313] 55. The method of embodiment 48, comprising a first protein A affinity chromatography step and a second anion exchange chromatography step followed by a hydrophobic interaction chromatography (HIC) step.

[0314] 56. The method of embodiment 55, wherein the affinity chromatography step comprises MABSELECT SURE TM resin, the anion exchange chromatography step comprises Q SEPHAROSE TM Fast Flow, and the HIC step comprises PHENYL SEPHAROSE TM6HIC step with POROS® 6 Fast Flow (high sub).

[0315] 57. The method of embodiment 56, wherein

[0316] The affinity chromatography step comprises operating a column containing MABSELECT SURE TM resin in bind-elute mode;

[0317] The anion exchange chromatography step comprises operating a column containing Q SEPHAROSE TM Fast Flow resin in bind-elute mode, and

[0318] The HIC step comprises operating a column containing PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin in flow- through mode.

[0319] 58. The method of embodiment 41, wherein the antibody is an anti-Ab.

[0320] 59. The method of embodiment 44, wherein the antibody is crenezumab.

[0321] 60. The method of embodiment 58, wherein the anti-Ab antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 23, CDR-H2 having the amino acid sequence of SEQ ID NO: 24, and CDR-H3 having the amino acid sequence of SEQ ID NO: 25, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 26, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28.

[0322] 61. The method of any one of embodiments 58, 59, or 60, wherein the HIC step comprises operating a column containing resin in flow-through mode, and the HIC step comprises an equilibration buffer and a wash buffer, wherein the equilibration buffer and the wash buffer each comprise 150 mM sodium acetate pH 5.0.

[0323] 62. The method of embodiment 61, wherein the flow-through is monitored at 280 nanometer absorbance and the flow-through is collected starting at 0.5 OD, with collection continuing for 10 column volumes.

[0324] 63. The method of embodiment 61, further comprising an affinity chromatography step.

[0325] 64. The method of embodiment 63, wherein the affinity chromatography is protein A chromatography.

[0326] 65. The method of embodiment 61, comprising a mixed mode chromatography step.

[0327] 66. The method of embodiment 61, comprising a first protein A affinity chromatography step and a second mixed mode chromatography step and a subsequent hydrophobic interaction chromatography (HIC) step.

[0328] 67. The method of embodiment 66, wherein the affinity chromatography step comprises a MABSELECT SURE TM resin, the mixed mode chromatography step comprises a CAPTO TM Adhere, and the HIC step comprises a PHENYL SEPHAROSE TM 6 Fast Flow (high sub).

[0329] 68. The method of embodiment 67, wherein

[0330] the affinity chromatography step comprises operating a column containing MABSELECT SURE TM resin in a bind-elute mode;

[0331] the mixed mode chromatography step comprises operating a column containing CAPTO TM Adhere resin in a flow-through mode, and

[0332] the HIC step comprises operating a column containing PHENYL SEPHAROSE TM 6 Fast Flow (high sub) resin in a flow-through mode.

[0333] 69. The method of embodiment 36, wherein the amount of hamster PLBL2 is quantified using an immunoassay or a mass spectrometry assay.

[0334] 70. The method of embodiment 69, wherein the immunoassay is a total Chinese hamster ovary protein ELISA or a hamster PLBL2 ELISA.

[0335] 71. The method of embodiment 69, wherein the mass spectrometry assay is LC-MS / MS.

[0336] 72. A composition comprising an anti-Ab monoclonal antibody purified from a Chinese hamster ovary host cell, wherein the composition comprises the anti-Ab antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0337] 73. The composition of embodiment 72, wherein the anti-Ab antibody is crenezumab.

[0338] 74. The composition of embodiment 72, wherein the anti-Ab antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 23, CDR-H2 having the amino acid sequence of SEQ ID NO: 24, and CDR-H3 having the amino acid sequence of SEQ ID NO: 25, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 26, CDR-L2 having the amino acid sequence of SEQ ID NO: 27, and CDR-L3 having the amino acid sequence of SEQ ID NO: 28.

[0339] 75. The composition of embodiment 74, wherein the anti-Ab antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29.

[0340] 76. The composition of embodiment 74, wherein the anti-Ab antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

[0341] 77. The composition of embodiment 74, wherein the anti-Ab antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO: 30.

[0342] 78. The method of embodiment 43, wherein the antibody is an IgGl.

[0343] 79. The method of embodiment 41, wherein the antibody is an anti-IL17A / F.

[0344] 80. The method of embodiment 79, wherein the antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 15, CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and CDR-H3 having the amino acid sequence of SEQ ID NO: 17, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 18, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20.

[0345] 81. The method of embodiment 79 or embodiment 80, wherein the HIC step comprises operating the resin-containing column in flow-through mode, and the HIC step comprises an equilibration buffer and a wash buffer, wherein the equilibration buffer and the wash buffer each comprise 50 mM sodium acetate pH 5.5.

[0346] 82. The method of embodiment 81, wherein the effluent is monitored at an absorbance of 280 nanometers and the effluent is collected starting at 0.5 OD, with collection continuing for 10 column volumes.

[0347] 83. The method of embodiment 81, further comprising an affinity chromatography step.

[0348] 84. The method of embodiment 83, wherein the affinity chromatography is protein A chromatography.

[0349] 85. The method of embodiment 81, further comprising a cation exchange chromatography step.

[0350] 86. The method of embodiment 81, comprising a first protein A affinity chromatography step and a second cation exchange chromatography step and a subsequent hydrophobic interaction chromatography (HIC) step.

[0351] 87. The method of embodiment 86, wherein the affinity chromatography step comprises a MABSELECT SURE TM resin, the cation exchange chromatography step comprises 50 HS resin, and the HIC step comprises a PHENYL SEPHAROSE TM 6 Fast Flow (high sub) resin.

[0352] 88. The method of embodiment 87, wherein

[0353] the affinity chromatography step comprises operating the column containing MABSELECT SURE TM resin in bind-elute mode;

[0354] The cation exchange chromatography step comprises operating a column containing POROS® 50HS resin in bind-elute mode, and 50HS resin in bind-elute mode, and

[0355] The HIC step comprises operating a column containing POROS® 50HS resin in flow- through mode. TM 6 Fast Flow (High Sub) resin in flow-through mode.

[0356] 89. A composition comprising an anti-IL17A / F monoclonal antibody purified from Chinese hamster ovary host cells, wherein the composition comprises the anti-IL17A / F antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

[0357] 90. The composition according to embodiment 89, wherein the anti-IL17A / F antibody comprises three heavy chain CDRs that are CDR-H1 having the amino acid sequence of SEQ ID NO: 15, CDR-H2 having the amino acid sequence of SEQ ID NO: 16, and CDR-H3 having the amino acid sequence of SEQ ID NO: 17, and three light chain CDRs that are CDR-L1 having the amino acid sequence of SEQ ID NO: 18, CDR-L2 having the amino acid sequence of SEQ ID NO: 19, and CDR-L3 having the amino acid sequence of SEQ ID NO: 20.

[0358] 91. The composition according to embodiment 90, wherein the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21.

[0359] 92. The composition according to embodiment 90, wherein the anti-IL17A / F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 22.

[0360] 93. The composition according to embodiment 90, wherein the anti-IL17A / F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22.

[0361] 94. A method of treating an IL-13 mediated condition in a patient, the method comprising administering to the patient a therapeutic composition, wherein the therapeutic composition comprises the therapeutic composition of any one of embodiments 1-8.

[0362] 95. The method of embodiment 94, wherein the immunogenicity of the therapeutic composition to hamster PLBL2 is lower than the immunogenicity of the reference composition to hamster PLBL2 upon administration, wherein the reference composition comprises an anti-IL-13 monoclonal antibody purified from Chinese hamster ovary host cells and a residual amount of hamster PLBL2 greater than 30 ng / mg, or greater than 50 ng / mg, or greater than 100 ng / mg, or greater than 200 ng / mg, or greater than 300 ng / mg.

[0363] 96. The method of embodiment 94, wherein the therapeutic composition is administered subcutaneously once every four weeks, or once every eight weeks, once every 12 weeks.

[0364] 97. The method of embodiment 96, wherein the patient is treated once every four weeks for at least one month, or for at least three months, or for at least six months, or for at least nine months, or for at least 12 months, or for at least 18 months, or for at least two years, or for more than two years.

[0365] 98. The method of embodiment 94, wherein the IL-13 mediated condition is selected from the group consisting of asthma, idiopathic pulmonary fibrosis, and atopic dermatitis.

[0366] 99. The method of embodiment 94, wherein the IL-13 mediated condition is selected from the group consisting of allergic asthma, non-allergic asthma, allergic rhinitis, allergic conjunctivitis, eczema, urticaria, food allergies, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma, and osteoporosis.

[0367] Example

[0368] As used in the following examples and elsewhere herein, “PLB2” and “PLBL2” and “PLBD2” are used interchangeably and refer to the enzyme “phospholipase B-like 2” or its synonym, “phospholipase B domain-like 2.”

[0369] Example 1 - General Methods

[0370] Unless otherwise indicated in the examples, the materials and methods for all examples are as described below.

[0371] MAb raw material

[0372] The mAb feedstock used for all examples was selected from commercial, pilot scale or small scale cell culture batches from Genentech (South San Francisco, CA, U.S.A.). After a period of cell culture fermentation, the cells were separated and, in some cases, the clarified liquid (harvested cell culture fluid, HCCF) was purified by protein A chromatography and one or more additional chromatography steps and filtration steps, as shown in the examples below.

[0373] MAb quantitation

[0374] Antibody concentration was determined using UV-visible spectrophotometry (Model 8453 G1103A; Agilent Technologies; Santa Clara, CA, U.S.A.) or NanoDrop Model ND-1000 (Thermo Fisher Scientific; Waltham, MA, USA) at 280 and 320 nm. Other species than antibodies (i.e. impurities) were too low in concentration to have a significant effect on UV absorbance. Samples were diluted as necessary with a suitable non-interfering diluent to be in the range of 0.1-1.0 absorbance units. Sample preparation and UV measurement were performed in duplicate and the average value was recorded. The mAb absorption coefficient ranges from 1.42-1.645 / mg-ml-cm.

[0375] Total CHO host cell protein (CHOP) quantitation

[0376] ELISA was used to quantify the level of total host cell protein, referred to as CHOP. The ELISA used to detect CHO proteins in the product is based on a sandwich ELISA format. Affinity purified polyclonal antibody to CHOP is coated onto a 96 well microplate. Standards, controls and samples are then loaded in duplicate into separate wells. If CHOP is present in the sample, it will bind to the coating antibody (polyclonal anti-CHOP). After an incubation step, a conjugated horseradish peroxidase (HRP) anti-CHOP polyclonal antibody is added to the plate. After a final wash step, CHOP is quantified by the addition of tetramethylbenzidine (TMB) solution, which also serves as a SUREBLUE RESERVE TM obtained from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, Cat. No. 53-00-03, which produces a colorimetric signal when the HRP enzyme acts. The optical density (OD) at 450 nm in each well was measured. A five parameter curve fitting program (XLfit, ID Business Solutions Ltd, 2009) was used to determine the concentration of CHOP in each sample. Pro, Molecular Devices, Sunnyvale, CA) was used to generate a standard curve from which sample CHOP concentrations were calculated. The total CHOP ELISA has a range of determination from 5 to 320 ng / ml. CHOP concentration in ng / mL refers to the amount of CHOP in a sample using CHOP standards as a calibration. CHOP ratio (in ng / mg or ppm) refers to the ratio of the calculated CHOP concentration to the product concentration, which in some cases is the reported value of the test method. The total CHOP ELISA can be used to quantify total CHOP levels in a sample, but does not quantify the concentration of individual proteins.

[0377] Murine monoclonal anti-hamster PLBL2 ELISA assay

[0378] Generation of mouse anti-hamster PLBL2 monoclonal antibodies and development of an ELISA assay using such antibodies for detection and quantitation of PLBL2 in recombinant polypeptide preparations is described in U.S. Provisional Patent Application Nos. 61 / 877,503 and 61 / 991,228. Briefly, the assay is performed as follows.

[0379] Murine monoclonal antibody 19C10 was coated at a concentration of 0.5 μg / mL in carbonate buffer (0.05 M sodium carbonate, pH 9.6) to half areas of 96-well microplates, overnight at 2-8 °C. After coating, the plates were blocked with blocking buffer (0.15 M NaCl, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween® 300 [Sigma-Aldrich]) to prevent non-specific adhesion of proteins. Standards, controls, and samples were diluted in assay diluent (0.15 M NaCl, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween® 300 [Sigma-Aldrich]) and then loaded in duplicate into separate wells and incubated for 2 hours at room temperature (22-27 °C). If PLBL2 is present in the sample, PLBL2 will bind to the coating (also referred to herein as capture) antibody. Following the incubation step described above, unbound material was washed away with wash buffer (0.05 % polysorbate 20 / PBS [Corning cellgro catalog number 99-717-CM]), biotin-conjugated 15G11 anti-PLBL2 murine monoclonal antibody was diluted to a concentration of 0.03125 μg / mL in assay diluent and added to the wells of the microplate.

[0380] ​​Biotinylation conjugation was performed as follows. Biotinylation kit was purchased from Pierce Thermo Scientific, (P / N 20217, E-Z Link NHS-Biotin), and Streptavidin HRP (SA-HRP) was purchased from Jackson ImmunoCat. No. 016-030-084. The instructions in the Pierce kit were followed. Briefly, IgG was dialyzed into PBS, pH 7.4, and biotin was added to the protein, mixed for 1 hour at room temperature. The labeled antibody was then dialyzed against PBS, pH 7.4 to remove excess biotin, filtered, and protein concentration was determined by A280.

[0381] Following the step of incubation with biotinylated 15G11 for 2 hours at room temperature, streptavidin HRP (1 :200,000 dilution in assay diluent) was added to the wells of the microplate. Following the step of final wash with wash buffer (as described above), TMB solution (50 μΐ / well) (SUREBLUE RESERVE TM , purchased from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, Catalog No. 53-00-03) was added, followed by color development (for PLBL2 quantitation) at room temperature for 10-20 minutes. Detection was performed using Molecular Devices SpectraMax M5e to assess the optical density (OD) in each well at 450 nm. A four parameter curve fitting program (SoftMax Pro v5.2 rev C) was used to generate a standard curve, and the PLBL2 concentration of the sample was calculated from the linear range of the standard curve. Values within the linear range of the standard curve were used to calculate the nominal PLBL2 (ng / mg or ppm). The linear range was approximately EC 10 -EC 85 or 1.5-40 ng / mL, with slight variation from plate to plate. The PLBL2 values obtained using this ELISA were comparable to estimates obtained by other methods (e.g., LC-MS / MS, polyclonal PLBL2 ELISA, or total CHOPE ELISA, diluted to the LOQ of the assay).

[0382] Rabbit polyclonal anti-hamster PLBL2 ELISA assay

[0383] Generation of rabbit anti-hamster PLBL2 polyclonal antibody and development of an ELISA assay using this antibody for detection and quantitation of PLBL2 in recombinant polypeptide preparations are described in U.S. Provisional Patent Application Nos. 61 / 877,503 and 61 / 991,228. Briefly, the assay was performed as follows.

[0384] Affinity purified rabbit polyclonal antibody was coated in half areas of 96-well microplates at a concentration of 0.5 pg / mL in carbonate buffer (0.05 M sodium carbonate, pH 9.6) over night at 2-8 °C. After coating, the plates were blocked with blocking buffer (0.15 M NaCI, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween 20, pH 7.4) to prevent non-specific adhesion of proteins. Standards, controls and samples were diluted in assay diluent (0.15 M NaCI, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween 20, pH 7.4) and then loaded in duplicate into separate wells and incubated for 2 hours at room temperature (22-27 °C). If PLBL2 is present in the sample, PLBL2 will bind to the coated (in this context also referred to as captured) antibody. After the above incubation step, unbound material was washed away with wash buffer (0.05 % polysorbate 20 / PBS [Corning cellgro cat. no. 99-717-CM]) and affinity purified rabbit polyclonal antibody conjugated with horseradish peroxidase (HRP) was diluted in assay diluent to a concentration of 40 ng / mL and added to the wells of the microplate. 300 [Sigma-Aldrich]) to prevent non-specific adhesion of proteins. Standards, controls and samples were diluted in assay diluent (0.15 M NaCI, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween 20, pH 7.4) and then loaded in duplicate into separate wells and incubated for 2 hours at room temperature (22-27 °C). If PLBL2 is present in the sample, PLBL2 will bind to the coated (in this context also referred to as captured) antibody. After the above incubation step, unbound material was washed away with wash buffer (0.05 % polysorbate 20 / PBS [Corning cellgro cat. no. 99-717-CM]) and affinity purified rabbit polyclonal antibody conjugated with horseradish peroxidase (HRP) was diluted in assay diluent to a concentration of 40 ng / mL and added to the wells of the microplate. 300 [Sigma-Aldrich]) to prevent non-specific adhesion of proteins. Standards, controls and samples were diluted in assay diluent (0.15 M NaCI, 0.1 M sodium phosphate, 0.1 % fish gelatin, 0.05 % polysorbate 20, 0.05 % Tween 20, pH 7.4) and then loaded in duplicate into separate wells and incubated for 2 hours at room temperature (22-27 °C). If PLBL2 is present in the sample, PLBL2 will bind to the coated (in this context also referred to as captured) antibody. After the above incubation step, unbound material was washed away with wash buffer (0.05 % polysorbate 20 / PBS [Corning cellgro cat. no. 99-717-CM]) and affinity purified rabbit polyclonal antibody conjugated with horseradish peroxidase (HRP) was diluted in assay diluent to a concentration of 40 ng / mL and added to the wells of the microplate.

[0385] HRP conjugation was performed as follows. HRP conjugation kit was purchased from Pierce Thermo Scientific, (P / N 31489, EZ-Link Plus Activated Peroxidase and Kit). The instructions of the Pierce kit were followed. Briefly, IgG was dialyzed into carbonate-bicarbonate buffer, pH 9.4, and EZ-Link Plus Activated Peroxidase was added to the protein, mixed for 1 hour at room temperature. Subsequently, sodium cyanoborohydride and quenching buffer were added to stabilize the conjugation and quench the reaction. The labeled antibody was then dialyzed against PBS, pH 7.4, filtered, and the protein concentration was determined from A280.

[0386] After the step of incubation with the HRP conjugated rabbit polyclonal antibody for 2 hours at room temperature, a final washing step was performed with wash buffer (as described above). Thereafter, TMB solution (50 pi / well) (SUREBLUE RESERVE TM, purchased from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, catalog number 53-00-03), followed by color development (for PLBL2 quantitation) at room temperature for 10-20 minutes. Detection was performed using a Molecular Devices SpectraMax M5e to evaluate the optical density (OD) in each well at 450 nm. A five parameter curve fitting program (SoftMax Pro v5.2 rev C) was used to generate a standard curve, and the PLBL2 concentration of samples was calculated from the linear range of the standard curve. Values within the linear range of the standard curve were used to calculate the nominal PLBL2 (ng / mg or ppm). The quantitative range of the assay was 0.5-50 ng / mL. The PLBL2 values obtained using this ELISA were comparable to estimates obtained by other methods (e.g., murine monoclonal PLBL2 ELISA, LC-MS / MS, or total CHOPE ELISA, diluted to the LOQ of the assay).

[0387] LC-MS / MS assay

[0388] For PLBL2 quantitation by LC-MS / MS, a Waters Acquity H-Class Bio UPLC and an AB Sciex TripleTOF 5600+ mass spectrometer were used. Samples and calibration standards (recombinant PLBL2 spiked into a recombinant humanized monoclonal antibody formulation from a mouse NS0 cell line [the NS0 cell line does not contain hamster PLBL2]) were trypsin reduced and digested. A total of 40 pg of digested sample was injected onto the UPLC using a Waters BEH300 C18 column with a particle size of 1.7 pm. Peptides were eluted using a linear gradient of acetonitrile at a flow rate of 300 mΐ / min and a column temperature of 60 °C.

[0389] Peptides eluted from the UPLC were introduced into the mass spectrometer by electrospray ionization in positive ionization mode. The ion source temperature was set at 400 °C, the ion spray voltage at 5500 v, and the declustering potential at 76 v. Selected peptide ions were fragmented using a collision energy set at 32. The mass spectrometer was operated in multiple reaction monitoring high resolution (MRM HR ) mode using four specific PLBL2 peptides and their fragment ion transitions. The parent ions were selected by the quadrupole mass spectrometer with a mass to charge (M / Z) selection window of 1.2 amu. The fragment ions of each parent ion were separated by the time-of-flight mass spectrometer and selected for data acquisition after quantitation, with a selection window of 0.025 amu.

[0390] The concentration of PLBL2 in a sample was determined by measuring the specific signal response of the four transitions, calibrated from those from standards in the range of 2-500 ppm using linear fit. Table 2 below shows a list of PLBL2 peptides monitored by LC-MS / MS.

[0391] Table 2. List of PLBL2 peptides monitored by LC-MS / MS.

[0392]

[0393] Example 2 - Improved purification process for reducing hamster PLBL2

[0394] A purification process for CHO-produced anti-IL-13 MAb (lebrikizumab) was established to support early phase clinical trials, referred to herein as the "Initial Process". The Initial Process employed the following chromatography steps in sequence: Protein A affinity chromatography (MABSELECT SURE TM ), followed by cation exchange (CAPTO HS), followed by anion exchange (Q SEPHAROSE TM Fast Flow). Additional viral inactivation and filtration steps were included, as well as a final ultrafiltration diafiltration (UFDF) step. The final product (drug substance) was formulated at a concentration of 125 mg / mL in 20 mM histidine acetate, 6% sucrose, 0.03% polysorbate 20, pH 5.7.

[0395] Using the total CHOP ELISA assay (described in Example 1 above), we found that intermediates and drug substance purified according to the Initial Process demonstrated an atypical dilution dependence behavior, resulting in a >20% coefficient of variation between dilutions of a standardized series of samples. This dilution dependence behavior is exemplified by the data presented in Table 3, where each successive two-fold dilution of the anti-IL-13 MAb product resulted in a higher level of CHOP (in ppm) as determined using the total CHOP ELISA. Using a sensitive analytical method, such as LC-MS / MS, we identified a single CHOP species or HCP as the cause of this atypical dilution dependence behavior. In particular, we established that the dilution dependence behavior on the total CHOP ELISA was due to antigen excess. Further investigation allowed us to identify the single HCP as the enzyme, hamster phospholipase B-like 2 (PLBL2). By diluting product samples to the limit of quantitation (LOQ) of the assay, we were able to estimate the levels of PLBL2 in clinical lots of lebrikizumab purified using the Initial Process, determining that levels as high as 300 ppm (300 ng / mg) and above were present.

[0396] Table 3. Product dilution and CHOP levels.

[0397] Dilution factor Total CHOP (ppm) 2 0.58 4 1 8 2 16 4 32 7 64 14 128 26 256 49 512 97 1024 147 2048 228 4096 314 8192 346

[0398] This impurity level (>300 ppm) of a single CHOP species, as we observed, is considered undesirable in MAb products for human clinical and / or therapeutic use, particularly in late stage clinical trials and beyond. For example, such levels can be immunogenic when administered to human subjects, as described in Example 3.

[0399] Accordingly, we investigated various improvements to the initial process, as briefly outlined below. Based on the results of these investigations, we developed an improved purification process, described in detail below, and referred to herein as the "improved process". Use of the improved process resulted in a purified anti-IL-13 MAb (lebrikizumab) product containing substantially reduced levels of PLBL2.

[0400] Efforts to improve the purification process to reduce PLBL2 included processes orthogonal to the initial process, including: precipitation, testing various additives to HCCF, additional column washes, hydrophobic interaction, and mixed mode chromatography. Each improvement investigated was monitored for effectiveness in reducing total CHOP and / or PLBL2 levels by using one or more of the assays described in Example 1. The various improvements investigated are described below.

[0401] Precipitation of CHOP in HCCF and Protein A pool using caprylic acid

[0402] Caprylic acid precipitation has been previously described, including for use in the monoclonal antibody industry (Wang et al., BioPharm International; Downstream Processing 2010, p4-10, Oct 2009; Brodsky et al., Biotechnology and Bioengineering, 109(10):2589, 2012) to selectively precipitate impurities from a protein of interest. Caprylic acid, also known as octanoic acid, is a saturated fatty acid with eight carbon atoms (formula CH3(CH2)6COOH). Studies were conducted using the anti-IL-13 MAb to determine if caprylic acid precipitation of the harvested cell culture fluid (HCCF) or protein A pool would result in CHOP reduction and / or reduction in dilution-dependent behavior in the total CHOP ELISA.

[0403] The anti-IL13 MAb starting material for these studies was 1 KL harvest HCCF and Protein A pool. 1% (v / v) caprylic acid was added to the HCCF and varying concentrations of caprylic acid (0-3% v / v) was added to the Protein A pool at pH 4.5 or pH 5.0. The samples were mixed at ambient temperature for >5 hours, 0.2 μιη filtered, and diluted with Total CHOP ELISA diluent for detection and quantitation using the Total CHOP ELISA. HPLC titer determinations were performed according to standard methods known in the art for determining the titer of the anti-IL-13 MAb in the HCCF before and after caprylic acid treatment.

[0404] Treatment of the HCCF with 1% v / v caprylic acid reduced CHOP by about 5-fold resulting in a 91% yield of anti-IL-13 MAb. When the Protein A pool was treated with varying concentrations of caprylic acid (ranging from 0-3% v / v) we observed a >20% yield loss at pH 5.0 and no loss at pH 4.5. When we evaluated the total CHOP in these caprylic acid treated Protein A pools we observed a 2-fold to 3-fold reduction in CHOP (Figure 1 Figure 1A and B). However, as also shown in Figures 1 Figure 1A and B, the dilution dependence still existed under each of the assay conditions indicating that the caprylic acid precipitation did not effectively address the dilution dependence behavior observed in the Total CHOP ELISA and therefore did not effectively reduce the level of PLBL2 in the product.

[0405] Additives to HCCF

[0406] Previous work by Sisodiya et al., Biotech J. 7: 1233 (2012) demonstrated that additives such as guanidine or sodium chloride added to the HCCF could reduce CHOP in the subsequent Protein A pool. Arginine was also shown to reduce CHOP when used as a wash reagent on the Protein A column (Millipore Technical Bulletin, Lit. No. TB1024EN00, Rev. A, December, 2005; Millipore Technical Bulletin, Lit. No. 1026EN00, July, 2006, available at www.millipore.com) and we included arginine as an additive to the HCCF. Various salts, chaotropes, and caprylic acid were added to the anti-IL-13 MAb HCCF to evaluate their effectiveness at reducing product and CHOP interactions during capture of the product on the MABSELECT SURE TM (MSS) Protein A chromatography. The additives added to the HCCF tested were: 0.6 M guanidine, 0.6 M arginine, 0.6 M sodium chloride, phosphate buffered saline, and 1% caprylic acid.

[0407] Samples treated with various HCCF additives were loaded onto Protein A chromatography on the MSS. The Protein A pool was adjusted to pH 4.9 and run using the initial method conditions at HS cation exchange chromatography step. The Protein A pool was diluted and HS pool and total Chop ELISA was performed. Adjusted Protein A pools were tested on SEC-HPLC for assessment of % aggregates, % variant species, etc. according to methods known in the art.

[0408] For runs where guanidine or arginine was added to the HCCF, MABSELECT SURE TM Yields were slightly lower on the MSS. Of all the additives tested for the HCCF, guanidine and arginine were most effective at substantially reducing CHOP levels (see Table 4) and presented a reduction in dilution dependence on the Protein A pool (data not shown). However, Further downstream processing of the Protein A pool on the HS showed that the CHOP ELISA dilution dependence remained in the corresponding pool as shown in Figure 2 Thus, the data indicates that the addition of guanidine or arginine to the HCCF was not effective in reducing the PLBL2 levels of the product.

[0409] Table 4. HCCF additives and effect on CHOP.

[0410]

[0411] Wash of Protein A column (MABSELECT SURE TM )

[0412] It was observed that the MABSELECT SURE TMMore dilution dependent CHOP elution on the MSS Protein A chromatography in earlier fractions containing product. This suggests that additional wash steps on the MSS prior to elution can further reduce CHOP / PLBL2. Some washes on the MSS were tested for their ability to reduce CHOP / PLBL2 in the Protein A pool. For this study, purified anti-IL-13 MAb UFDF pool was used as the load material. The UFDF pool was diluted to 1.7 mg / mL (about HCCF titer) and loaded onto the MSS at 29 grams / L of resin. Various washes were tested, for example, 0.5 M arginine pH 8.5, 0.5 M arginine pH 9.5 with and without 1% polysorbate 20, 0.5 M TMAC pH 7.1, 25 mM MOPS pH 7.1, and compared to high salt wash pH 7.0. Product was eluted in acidic conditions (pH 2.8) and pooled, starting at 0.5 OD (A 280) and continuing to a total volume of 2.4 column volumes. Each conditioning pool was diluted and assayed with total CHOP ELISA. The summary of these results is that no wash adequately reduced CHOP / PLBL2 or reduced dilution dependence in total CHOP ELISA. Therefore, it appears that we would not find a Protein A wash condition that effectively reduces the level of PLBL2 in this anti-IL-13 MAb product, so we did not investigate further.

[0413] Wash of cation exchange column (HS)

[0414] Based on theoretical calculations using the amino acid sequences of the anti-IL-13 MAb and PLBL2 impurity, we estimate the pi of PLBL2 to be about 6.0, similar to the anti-IL-13 MAb (pi 6.1). We also estimate that there will be a significant difference in net charge between the anti-IL-13 MAb and PLBL2 at <pH 4 and >pH 10. Therefore, we tested the initial method Various low pH washes on the HS cation exchange step were tested to evaluate if they would effectively and selectively reduce total CHOP and / or PLBL2 and dilution dependence behavior. The following washes were tested at pH 4: (i) acetate gradient, 300 mM - 1000 mM over 20 column volumes (CV); (ii) citrate gradient, 100 mM - 500 mM over 20 CV; (iii) 260 mM citrate wash step; and (iv) arginine gradient 15 mS / cm (conductivity measurement) over 20 CV.

[0415] The results indicate that the anti-IL-13 MAb does not elute with CHOP at pH 4 acetate gradient up to the tested salt concentration of 1 M. Increasing amounts of citrate or acetate resulted in insolubility and precipitation of the product. All of the pH 4 wash conditions resulted in Low yield on HS step, no wash conditions significantly reduced CHOP dilution dependence. Thus, addition of a low pH wash in the cation exchange column did not effectively reduce the level of PLBL2 in the product.

[0416] Hydroxyapatite resins and CAPTO TM Adhere resins

[0417] Ceramic hydroxyapatite (CHT) Macro-Prep®Resin Type I, 40um (BioRad) consists of a repeating hexagonal structure of calcium phosphate (Ca5(PO4)3OH)2. There are two distinct binding sites; the C site has a group of five calcium ion doublets and the P site contains a pair of -OH, each -OH containing three phosphate triplets. This resin has mixed mode characteristics and has been shown to separate challenging impurities such as aggregates (P. Gagnon, New Biotechnology 25(5):287 (2009)).

[0418] To identify initial conditions for running CHT columns, we performed a high throughput robotic screen of Macro-Prep®Resin Type I, 40um, testing a range of pH from 6.5-8.0, and different concentrations of sodium chloride and sodium phosphate for elution. This high throughput robotic screen has been previously described, for example, in Wensel et al., Biotechnol. Bioeng. 100:839 (2008). Samples from these screens were tested in the total CHOP ELISA.

[0419] CAPTO TM Adhere (GE Healthcare) is a mixed mode resin that exhibits both ionic and hydrophobic characteristics. The base matrix is rigid agarose and the ligand is N-benzyl-N-methyl ethanolamine. This resin was first investigated with a high throughput screen, and then evaluated with subsequent column conditions for its ability to reduce total CHOP and / or PLBL2.

[0420] Using a similar high throughput robotic screen method as described above, an initial study was performed to determine the optimal CAPTO TM Adhere column conditions to test the binding of the anti-IL-13 MAb to CAPTO TM Adhere at two loading densities (5 grams / L of resin and 40 g / L of resin). The range of salts and pH were also tested; from 25 mM - 200 mM sodium acetate, pH 4.0 - 6.5. The loading material was the initial method UFDF pool, which contained approximately 200 ppm of total CHOP on the LOQ of the total CHOP ELISA. The CAPTO TM Adhere, and assayed using the total CHOP ELISA.

[0421] The results were as follows. For the CHT chromatography, no test condition substantially reduced total CHOP or PLBL2 or affected the dilution dependent behavior of the assay. In addition, the yield was poor and no clearance of high molecular weight species was achieved. For the CAPTO TM Adhere chromatography, the yield was poor and the assay material showed substantial dilution dependent behavior in the total CHOP ELISA. Therefore, no further development of the CHT and CAPTO TM Adhere resins, as it became apparent that we would not be able to find conditions that would effectively reduce the PLBL2 levels in this anti-IL-13 MAb product using these resins.

[0422] Hydrophobic interaction chromatography resin and membrane

[0423] The HIC membrane adsorber we initially tested was called Sartobind, manufactured by Sartorius. Sartobind consists of a base matrix of regenerated cellulose with covalently attached hydrophobic phenyl ligand groups.

[0424] The membrane tested was a Sartobind HIC 3 mL device (8 mm bed height). We took the flow through from the initial method The HS pool was adjusted to 0.55 M potassium phosphate pH 7.0 and used a flow rate of 10 mL / min. The product was eluted in 0.55 M potassium phosphate pH 7.0 (3 mL fractions were collected in the flow through fraction).

[0425] We observed that the anti-IL-13 MAb became hazy and turbid when the conditions were set to 0.55 M potassium phosphate, requiring an additional 0.2 um filtration step. The results showed that total CHOP was reduced, however, the remaining CHOP still exhibited dilution dependent behavior in the total CHOP ELISA. No further evaluation of this membrane was pursued as it appeared unlikely that effective conditions could be determined to reduce the PLBL2 levels in this product.

[0426] Next, we evaluated several different HIC resins using a high throughput screen. OCTYL- Fast Flow (FF), BUTYL- 4Fast Flow, PHENYL SEPHAROSE TM 6Fast Flow (high sub) and PHENYL SEPHAROSE TM 6Fast Flow (low sub) were obtained from GE Healthcare. These four resins were chosen because they represent a wide range of hydrophobicity (OCTYL- Fast Flow was the least hydrophobic, followed by PHENYL SEPHAROSE TM 6Fast Flow (low sub) and BUTYL- 4Fast Flow, PHENYL SEPHAROSE TM 6Fast Flow (high sub) was the most hydrophobic. We tested several combinations of pH and salt concentration for their effectiveness in reducing PLBL2 in the anti-IL-13 MAb formulation. The anti-IL-13 MAb formulation used for the HIC resin experiments came from a UFDF pool run using the initial method. The anti-IL-13 MAb concentration was 180 mg / mL and the loading density was 40 mg antibody / mL resin. We tested pH 5.5 (25 mM sodium acetate), pH 6.0 (25 mM MES), pH 7.0 (25 mM MOPS), and pH 8.0 (25 mM Tris) and sodium sulfate concentrations between 0 mM and 400 mM. For each condition tested, the flow-through samples were collected, diluted, and tested using the total CHOP ELISA assay.

[0427] The results are shown in Figure 3A -D. As the salt increased, we observed less total CHOP in the flow-through for each resin. OCTYL- Fast Flow resin Figure 3A ) showed the highest levels of total CHOP, while PHENYL SEPHAROSE TM 6Fast Flow (high sub) resin reduced total CHOP to very low levels, even with lower amounts of salt Figure 3D ) and PHENYL SEPHAROSE TM 6Fast Flow (low sub) and BUTYL- Fast Flow resin showed moderate levels of total CHOP. Interestingly, pH also had the least impact on CHOP removal using each resin, except for PHENYL SEPHAROSE TM 6Fast Flow (high sub) Figure 3D ). For this resin, low salt conditions, higher pH resulted in higher CHOP Figure 3D ) in the flow-through fraction. Based on these results, PHENYL SEPHAROSE TM 6Fast Flow (high sub) appeared promising and was selected for further study, including running columns in bind-elute or flow-through mode.

[0428] Using PHENYL SEPHAROSE TM 6Fast Flow (high sub) resin in bind-elute mode operation of HIC requires salt adjustment of the anti-IL-13 MAb load to allow the antibody to bind to the resin. Increasing the salt increases the dynamic binding capacity (mg of anti-IL-13 per mL of resin) of the product to the resin. However, as the salt concentration of the product is increased, we observed increased haze and formation of high molecular weight species (HMW), particularly when lower pH is combined.

[0429] As described above, PHENYL SEPHAROSE TM 6Fast Flow (high sub) can also be operated in flow- through mode, which requires less salt conditions in the load. From a product quality and product stability perspective, e.g., less haze, less HMW in the product, less salt conditions are desired. Therefore, we continued to optimize the flow using PHENYL SEPHAROSE TM 6Fast Flow (high sub) resin in flow-through mode.

[0430] To optimize the flow, we investigated a number of parameters for running the HIC column, including load concentration, load pH, load salt molarity, load density on the resin, bed height, flow rate, temperature, equilibration buffer pH and molarity. For these experiments, we monitored total CHOP using the total CHOP ELISA and also monitored PLBL2 by LC-MS / MS. Some exemplary data is shown in Table 5. The data in Table 5 indicates that running the HIC column in flow-through mode under the specified conditions effectively reduced the PLBL2 levels substantially from the high levels detected in the protein A pool. The PLBL2 levels were reduced several hundred-fold after HIC compared to the levels in the protein A pool.

[0431] Table 5. Total CHOP and PLBL2 levels under different HIC column conditions.

[0432]

[0433] Using the PLBL2 LC-MS / MS assay and other typical product quality assays (e.g., SE-HPLC, CE-SDS, iCIEF) to guide process parameter selection, we determined the following conditions as desirable for running the HIC column, as evaluated by product quality attributes and reduction of PLBL2: equilibration and wash buffers: 50 mM sodium acetate, pH 5.0; target loading density: 100 g / L, flow rate: 150 cm / hr, 22°C ± 3°C. Some small variations from these conditions can also be desirable, e.g., 25°C ± 3°C or 27°C ± 3°C. Optical density (OD) was monitored using absorbance at 280 nm (A280) between 0.5 OD and 1.5 OD or pools (i.e., flowthrough) were collected after 8 column volumes of wash.

[0434] As mentioned above, the initial process was: Protein A affinity chromatography (MABSELECT SURE TM ), followed by cation exchange (CAPTO HS), followed by anion exchange (Q SEPHAROSE TM Fast Flow). After developing a process that reduced PLBL2 levels as described above, we next sought to implement changes to the process in a convenient manner. Thus, we investigated adding a HIC column to the initial process, resulting in a four column process, and replacing either the CEX column or the AEX column with a HIC column, and finally we explored the order of the columns. We found that a three column process, Protein A affinity chromatography (MABSELECT SURE TM ), followed by anion exchange (Q SEPHAROSE TM Fast Flow), followed by HIC (PHENYL SEPHAROSE TM 6Fast Flow (high sub)) operated in flowthrough mode provided the most convenient process that most effectively reduced PLBL2 in the final drug product. This three column process is described in detail below.

[0435] The first affinity chromatography step was a bind-and-elute process using MABSELECT SURE TM resin. After equilibration of the column (25 mM sodium chloride, 25 mM Tris pH 7.7), the HCCF was loaded onto the column and washed with equilibration buffer and a wash buffer of high salt, pH 7.0. The anti-IL-13 MAb was eluted from the column under acidic conditions (pH 2.8).

[0436] The second anion exchange chromatography step used Q SEPHAROSE TMFast Flow (QSFF) resin was run in bind-elute mode. After equilibration of the column (50 mM Tris, pH 8.0), the anti-IL13 pool from the MABSELECT SURE TM The column was equilibrated to pH 8.0 5 and the anti-IL13 MAb was loaded onto the column. The column was washed (50 mM Tris, pH 8.0) and the anti-IL-13 MAb was eluted from the column with 85 mM sodium chloride, 50 mM Tris pH 8.0.

[0437] The third and final hydrophobic interaction chromatography step used PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin was run in flow-through mode. After equilibration of the column (50 mM sodium acetate pH 5.0), the anti-IL13 pool from the QSFF column was adjusted to pH 5.0 and loaded onto the column. The anti-IL-13 MAb was flow-through and the column was washed with equilibration buffer (50 mM sodium acetate pH 5.0). The anti-IL-13 MAb pool was pooled between 0.5 and 1.5 OD or up to 8 column volumes based on A280 start and stop.

[0438] As with the initial process, additional viral inactivation and filtration steps were included, as well as a final ultrafiltration diafiltration (UFDF) step. The final product (drug product) was formulated in 20 mM histidine acetate, 6% sucrose, 0.03% polysorbate 20, pH 5.7 at a concentration of 125 mg / mL.

[0439] A comparison of the initial process with the improved process in terms of total CHOP and PLBL2 as measured by the total CHOP ELISA and the monoclonal PLBL2 ELISA, respectively, is provided in Tables 6 (initial process) and 7 (improved process). The data in Table 6 clearly indicate that the initial process resulted in a purified product (UFDF pool) containing high levels of total CHOP (179, 310, and 189 ng / mg in three different runs) and high levels of PLBL2 (242, 328, and 273 ng / mg in three different runs), while the data in Table 7 clearly indicate that the improved process was quite effective in producing a purified product with substantially reduced levels of total CHOP (1.1, <0.9, 2.8, and 3.4 ng / mg in four different runs) and substantially reduced levels of PLBL2 (0.21, 0.42, 0.35, and 0.24 ng / mg in four different runs). Consistent with the data presented above, the data in Table 7 indicate that running the HIC column under the conditions described above was particularly effective in reducing the levels of total CHOP and PLBL2 in the anti-IL-13 MAb preparation.

[0440] Table 6. Total CHOP and PLBL2 levels at various stages of purification of anti-IL-13 MAbs using the initial method.

[0441]

[0442] Table 7. Total CHOP and PLBL2 levels at various stages of purification of anti-IL-13 MAbs using the improved method.

[0443]

[0444] In summary, in the face of the problem of assay non-linear dilution behavior caused by high levels of a single CHOP species in purified anti-IL-13 MAb preparations, we first identified the CHOP species as hamster PLBL2, an impurity in a recombinant protein preparation produced by CHO cells that had not been previously described. Next we identified purification conditions that effectively reduced the levels of PLBL2 in anti-IL-13 MAb preparations. Finally, we integrated these purification conditions into an overall purification method that resulted in an improvement over the existing anti-IL-13 MAb purification method. This improved method employed a HIC column run in flow- through mode to reduce PLBL2 levels, which was combined with an affinity chromatography step and a run of anion exchange chromatography step. We showed that the improved method robustly and effectively substantially reduced hamster PLBL2 levels in anti-IL-13 MAb preparations. We showed that the improved method reproducibly reduced PLBL2 levels by about 1000-fold compared to the initial method. This reduction in PLBL2 levels was important for producing a purified anti-IL 13 MAb product suitable for use in late stage clinical trials and for later patient treatment uses.

[0445] Method for reducing hamster PLBL2 in anti-Abeta antibody formulations

[0446] Next, we sought to evaluate whether the above purification method, particularly the use of a HIC column for the final chromatography step, would also be effective for reducing PLBL2 levels in other antibody preparations. For this experiment, we selected an anti-Ab antibody, which is produced in CHO cells. Exemplary anti-Ab antibodies and methods of producing such antibodies have been previously described, for example, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408. The anti-Ab antibody used in these particular experiments was called crenezumab. As described for the anti-IL-13 MAbs, we explored various resins and buffers for the second column after the protein A affinity column, and we explored various buffers and running conditions for the HIC column to determine those that were optimal for anti-Ab product quality and stability characteristics and for removal of hamster PLBL2.

[0447] We found that a three column approach, Protein A affinity chromatography (MABSELECT SURE TM ), followed by mixed mode chromatography (CAPTO TM Adhere), followed by HIC operated in flow-through mode (PHENYL SEPHAROSE TM 6 Fast Flow (high sub)) conveniently and effectively reduced PLBL2 in the final drug product. This three column approach is described in detail below.

[0448] The first affinity chromatography step was a bind-elute process using MABSELECT SURE TM resin similar to that described above for the anti-IL-13 MAb.

[0449] The second mixed mode chromatography step used CAPTO TM Adhere resin operated in flow-through mode. After equilibration of the column (20 mM MES, 150 mM sodium acetate, pH 6.25), the anti-Ab pool from the MABSELECT SURE TM column was adjusted to pH 6.25 and loaded onto the column. Anti-Ab MAb pool began to pool at 0.5 OD during loading. After loading was complete, the column was washed with 5 column volumes (CV) of equilibration buffer (20 mM MES, 150 mM sodium acetate, pH 6.25) and the entire 5 CV was also collected.

[0450] The third and final hydrophobic interaction chromatography step used PHENYL SEPHAROSE TM 6 Fast Flow (High Sub) resin operated in flow-through mode. After equilibration of the column (150 mM sodium acetate pH 5.0), the anti-Ab pool from the CAPTO TM Adhere column was adjusted to pH 5.0 and loaded onto the column. Anti-Ab MAbs flowed through and the column was also washed with equilibration buffer (150 mM sodium acetate pH 5.0). Anti-Ab MAb pool began to pool at 0.5 OD based on A280 during loading. The column was washed with 10 CV of equilibration buffer (150 mM sodium acetate pH 5.0) and the entire 10 CV was also collected. As with the anti-IL-13 MAbs, additional viral inactivation and filtration steps were included, as well as a final ultrafiltration diafiltration (UFDF) step.

[0451] Results using the above described process in four different purification runs are shown in Table 8 below.

[0452] Table 8. PLBL2 levels at various stages of purification of anti-Ab MAbs using HIC.

[0453]

[0454] The results shown in Table 8 demonstrate that the use of a HIC resin as a final chromatography step effectively reduced the level of residual PLBL2 in the anti-Abeta MAb preparation to an amount similar to that seen in the anti-IL-13 MAb. While a loading density of 300 g / L produced the desired results from the perspective of product recovery and PLBL2 reduction, a further reduction in residual PLBL2 was observed when the loading density of the HIC column was reduced from 300 g / L to 100 g / L.

[0455] We also investigated two other conditions of the HIC chromatography step, loading pH and loading sulfate molarity. For these experiments, we used CAPTO Q ImpRes® resin with a loading density of 60 g / L and a flow rate of 100 cm / hr. We tested each of the loading pH values shown in Table 9 under conditions of low sulfate molarity (0 mM) and high sulfate molarity (240 mM). Each of the loading pH values shown in Table 9 was tested at a loading density of 60 g / L. As shown in the results presented in Table 9, reducing the loading pH to pH 4 or pH 5 or increasing the molarity of the loading sulfate (to 240 mM sulfate) was effective in reducing the level of PLBL2 in the final HIC pool, respectively. The combination of pH 4.0 and 240 mM sulfate in the loading was particularly effective in minimizing the amount of residual PLBL2 in the HIC pool. TM Adhere pool start. We used 0 mM sodium sulfate or 800 mM sodium sulfate stock solutions at different pH values to adjust the loading pH and loading sulfate molarity to the values shown in Table 9. We tested each of the loading pH values shown in Table 9 under conditions of low sulfate molarity (0 mM) and high sulfate molarity (240 mM). Each of the loading pH values shown in Table 9 was tested at a loading density of 60 g / L. As shown in the results presented in Table 9, reducing the loading pH to pH 4 or pH 5 or increasing the molarity of the loading sulfate (to 240 mM sulfate) was effective in reducing the level of PLBL2 in the final HIC pool, respectively. The combination of pH 4.0 and 240 mM sulfate in the loading was particularly effective in minimizing the amount of residual PLBL2 in the HIC pool.

[0456] Table 9. PLBL2 levels observed in the HIC pool at a range of loading pH and sulfate molarity.

[0457]

[0458] Thus, the use of a HIC resin as a final chromatography step for the purification of a CHO-produced polypeptide, such as the anti-IL-13 MAb and anti-Abeta MAb described herein, effectively reduces the amount of residual hamster PLBL2 to very low levels, e.g., 1 ng / mg or less in the HIC pool.

[0459] Method for reducing hamster PLBL2 in IgGl antibody formulations

[0460] Next, we evaluated whether the described methods for purifying anti-IL-13 and anti-Ab IgG4 antibody preparations (particularly the use of HIC columns for the final chromatography step) would also effectively reduce PLBL2 levels in IgGl antibody preparations. For these experiments, we first selected an anti-IL17 A / F antibody, which is an IgGl antibody and is produced in CHO cells. Exemplary anti-IL17 A / F antibodies and methods of producing such antibodies have been previously described, e.g., WO 2009136286 and U.S. Patent No. 8,715,669. As described for the anti-IL-13 and anti-Ab MAbs, we explored various resins (specifically, PHENYL SEPHAROSE TM FF [low sub] and PHENYL SEPHAROSE TM FF [high sub] and buffer conditions (specifically, 50 mM sodium acetate, pH 5.5 and 50 mM Tris, 85 mM sodium acetate, pH 8.0) for the HIC columns to determine those for optimal product quality and stability characteristics for anti-IL17 A / F and for removal of hamster PLBL2.

[0461] We found that a three-column process, protein A affinity chromatography (MABSELECT SURE TM ), followed by cation exchange chromatography (CAPTO 50H S) operated in bind-elute mode, and HIC (PHENYL SEPHAROSE TM 6 Fast Flow (high sub)) operated in flow-through mode, conveniently and effectively reduced PLBL2 in the final drug product. This three-column process is described in detail below.

[0462] The first affinity chromatography step was a bind-elute process using MABSELECT SURE TM resin similar to that described above for the anti-IL13 and anti-Ab MAbs. The second cation exchange chromatography step used CAPTO 50H S resin operated in bind-elute mode. After equilibration of the column (40 mM sodium acetate, pH 5.5), the pH-adjusted anti-IL17 A / F MABSELECT SURE TM pool (pH 5.0) was loaded onto the column. The column was washed (40 mM sodium acetate, pH 5.5) and then the anti-IL17 A / F antibody was eluted from the column with a conductivity gradient created using 40 and 400 mM sodium acetate, pH 5.5. Elution during the gradient was pooled based on A280, starting at > 0.5 OD and ending at < 2.0 OD.

[0463] The third and final hydrophobic interaction chromatography step used PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin was operated in flow-through mode. After equilibration of the column (50 mM sodium acetate pH 5.5), the anti-IL17A / F pool from the previous step was loaded directly onto the column without adjusting the pH. Anti-IL17A / F MAbs flowed through. Anti-IL17A / F MAbs were pooled based on A280 during loading, starting at > 0.5 OD. The column was washed with 10 CV of equilibration buffer (50 mM sodium acetate, pH 5.5), and pooling was ended at < 1.0 OD during the wash. 50 HS column. Anti-IL17A / F MAbs were pooled based on A280 during loading, starting at > 0.5 OD. The column was washed with 10 CV of equilibration buffer (50 mM sodium acetate, pH 5.5), and pooling was ended at < 1.0 OD during the wash.

[0464] Results using the above method during one purification run are shown in Table 10 below.

[0465] Table 10. PLBL2 levels at various stages of HIC purification of anti-IL17A / F MAbs.

[0466]

[0467] The results shown in Table 10 indicate that using a HIC resin as a final chromatography step effectively reduced the level of residual PLBL2 in anti-IL17A / F MAb (IgGl) preparations, in amounts similar to those observed for anti-IL13 and anti-Ab MAbs (IgG4).

[0468] Anti-CMV Antibodies

[0469] In addition to testing anti-IL17A / F, we tested another IgGl MAb, an anti-CMV-MSL antibody, which was also produced in CHO cells. Exemplary anti-CMV antibodies, including anti-CMV-MSL, and methods of producing such antibodies have been previously described, e.g., WO 2012047732.

[0470] Again, we found that a three-column method, Protein A affinity chromatography (MABSELECT SURE TM ), followed by cation exchange chromatography (CAPTO S 50 HS) operated in bind-elute mode, and HIC (PHENYL SEPHAROSE TM 6Fast Flow (high sub)) operated in flow-through mode, conveniently and effectively reduced PLBL2 in the final drug product. This three-column method is described in detail below.

[0471] The first affinity chromatography step was using MABSELECT SURE TMResin binding-elution method. The second cation exchange chromatography step used 50HS resin and operated in a bind-elute mode. After equilibration of the column (40 mM sodium acetate, pH 5.5), the pH adjusted CMV-MSL MAbSELECT SURE TM pool (pH 5.0) was loaded onto the column. The column was washed (40 mM sodium acetate, pH 5.5) and then the CMV-MSL antibody was eluted from the column with a conductivity gradient created using 40 and 400 mM sodium acetate, pH 5.5. Pooling was based on A280 during gradient elution, starting at > 0.5 OD and ending at < 1.0 OD.

[0472] In this particular run, a Viresolve Pro was used as a viral filtration and a Fluorodyne UEDF filter was used as a pre-filtration between the cation exchange and hydrophobic interaction chromatography steps.

[0473] The third and final hydrophobic interaction chromatography step used a PHENYL SEPHAROSE TM 6Fast Flow (High Sub) resin operated in flow-through mode. After equilibration of the column (50 mM sodium acetate pH 5.5), the anti-CMV-MSL pool from the 50HS column was loaded directly onto the column without pH adjustment. Anti-CMV-MSL MAb flowed through. Anti-CMV-MSL MAb was pooled based on A280 during loading, starting at > 0.5 OD. The column was washed with 10 CV of equilibration buffer (50 mM sodium acetate, pH 5.5) and pooling was ended during the wash at < 0.5 OD.

[0474] The results of using the above method during one purification run are shown in Table 11 below.

[0475] Table 11. PLBL2 levels in various stages of HIC purification of anti-CMV-MSL MAb.

[0476]

[0477] The results shown in Table 11 demonstrate that the use of HIC resin as a final chromatography step effectively reduces the level of residual PLBL2 in the anti-CMV-MSL MAbs formulation, in amounts similar to that seen in anti-IL-13, anti-Abeta, and anti-IL17 A / F Mabs. Thus, the use of HIC resin as a final chromatography step in the purification of CHO-produced polypeptides, such as anti-IL-13 Mabs and other Mabs described herein, effectively reduces the residual amount of hamster PLBL2 to very low levels, e.g., less than 1 ng / mg in the HIC pool. Thus, we demonstrate that the use of a HIC chromatography step as described herein to reduce PLBL2 levels is effective for IgGl and IgG4 Mabs, showing the general applicability of the method for reducing hamster PLBL2 levels in recombinant polypeptide preparations.

[0478] Example 3 - Evaluation of human anti-hamster PLBL2 responses in patients administered anti-IL13 MAb compositions containing different amounts of hamster PLBL2

[0479] To evaluate the potential impact of CHO PLBL2 impurities, we developed an ELISA assay (bridging ELISA assay) to detect antibodies against hamster PLBL2 in human subjects receiving an anti-IL-13 MAb (lebrikizumab). Serum samples from patients participating in various clinical studies of lebrikizumab as well as placebo- receiving subjects were analyzed for evidence of anti-hamster PLBL2 antibodies pre- and post-dosing. Details of the clinical studies have been previously described (WO 2012 / 083132, Corren et al., N Engl J Med 365: 1088-98 (2011)), only the most relevant details of these studies are provided below.

[0480] An antibody bridging ELISA assay to detect anti hamster PLBL2 antibodies in human serum was developed and validated using two conjugated reagents to capture all isotypes of anti hamster PLBL2 antibodies: purified biotin conjugated hamster PLBL2 (Biotin-PLBL2) and purified digoxigenin conjugated hamster PLBL2 (DIG-PLBL2). Production and purification of hamster PLBL2 was performed using standard methods known to those skilled in the art, which are also disclosed in U.S. Provisional Application Nos. 61 / 877,503 and 61 / 991,228, conjugation to biotin or digoxigenin was performed using standard methods known to those skilled in the art. In this semi-homogeneous antibody bridging ELISA assay, 75 μL / well of conjugate solution containing 3 μg / mL of each Biotin-PLBL2 and DIG-PLBL2 in assay diluent (PBS / 0.5% BSA / 0.05% polysorbate 20 / 0.05% Proclin 300, pH 7.4 ± 0.1) was incubated with 75 μL / well of 1:20 diluted serum and control samples in assay diluent overnight (16-24 hours) at ambient temperature in polypropylene microtubes (National Scientific Supply Co.; Claremont, CA). After incubation, 100 μL / well of the mixture was transferred from the microtubes to streptavidin coated 96 well microplates (StreptaWell 96-Well Plates, GIBCO®, Invitrogen Corp.; Carlsbad, CA) and incubated for 1 hour at ambient temperature. After the incubation, the plates were washed 3 times with 200 μL / well of wash buffer (PBS / 0.05% polysorbate 20 / 0.05% Proclin 300, pH 7.4 ± 0.1) and 100 μL / well of 1:10,000 diluted anti-human IgG-HRP conjugate (Southern Biotech, Birmingham, AL) was added to each well. The plates were incubated for 1 hour at ambient temperature. After the incubation, the plates were washed 3 times with 200 μL / well of wash buffer and 100 μL / well of TMB substrate (KPL, Gaithersburg, MD) was added to each well. The plates were incubated for 10 minutes at ambient temperature. The reaction was stopped by adding 100 μL / well of 1 N H2SO4to each well. The plates were read at 450 nm using a SpectraMax® M5 plate reader (Molecular Devices, Sunnyvale, CA). The results were analyzed using SoftMax® Pro 5.2 software (Molecular Devices, Sunnyvale, CA). The results of the antibody bridging ELISA assay are shown in Table 1. TMHigh Bind; Roche Diagnostics; Indianapolis, IN), the microplate was washed 3 times with 400 μL / well of wash buffer (PBS / 0.05% polysorbate 20) in an automated plate washer instrument (BioTek ELx405) and incubated at ambient temperature for 2 hours ± 10 minutes. The plate was washed 4 times with 400 μL / well wash buffer in the plate washer, followed by detection with 100 μL / well of 400 ng / mL horseradish peroxidase (HRP)-conjugated mouse anti-digoxin antibody (Jackson ImmunoResearch Cat. 200-032-156) and incubated at ambient temperature for 2 hours ± 10 minutes. After the plate was washed 4 times with 400 μL / well wash buffer in the plate washer, a 1 : 1 mixture of peroxidase substrate (tetramethylbenzidine) (0.4 g / L TMB) and peroxidase solution B (0.02% hydrogen peroxide) (KPL Cat. 50-76-03) was added at 100 μL / well and incubated at ambient temperature for 18-28 minutes for color development, which was stopped by adding 100 μL / well of 1 M phosphoric acid. The plate was read at 450 nm to detect absorbance and at 630 nm for reference absorbance. The positive control for this assay was a monoclonal antibody construct consisting of murine anti- hamster PLBL2-specific complementarity determining regions (CDRs) on a human IgGl framework. The relative sensitivity of the assay using this antibody was determined to be 25 ng / mL. Assay drug tolerance experiments using this antibody indicated that up to 50 μg / mL of lebrikizumab or 1 μg / mL of hamster PLBL2 in serum did not cause interference or cross-reactivity in the assay.

[0481] To perform the assay, serum samples were first screened at a minimum dilution of 1 / 20 in the assay. Positive samples screened were then confirmed for hamster PLBL2 specificity using a competition confirmatory assay. If a sample was confirmed positive, the sample was serially diluted to obtain a titer value. Positive reactions were reported in titer units, which is the logio of the dilution factor at which the sample signal equals the signal of the assay cutpoint (threshold for determining positivity).

[0482] Four clinical studies of patient samples analyzed using the anti-hamster PLBL2 ELISA as described above are briefly described below. Study 1 was a randomized, double-blind, placebo-controlled, proof-of-concept Phase II study to evaluate the effect of lebrikizumab in asthmatic patients with inadequate disease control during long-term treatment with inhaled corticosteroids (ICS). A total of 219 patients were randomized (grouped), of which 106 received at least one subcutaneous (SC) dose of 250 mg lebrikizumab and 92 received six monthly doses.

[0483] Study 2 was a Phase II, randomized, double-blind, placebo-controlled, dose-ranging study in asthmatic patients who were not receiving ICS therapy. Patients received one of three doses (500, 250, or 125 mg) of lebrikizumab or placebo by SC administration. Study drug was administered four times during a 12-week treatment period. A total of 158 patients were exposed to at least one dose of lebrikizumab, and 145 patients received all four doses.

[0484] Study 3 was a Phase I PK study of lebrikizumab in healthy Japanese and Caucasian volunteers. Twenty healthy Japanese and Caucasian subjects (10 subjects in each ethnic group) were randomized in three discrete groups in a 7:3 ratio to lebrikizumab (125, 250, and 375 mg SC) and placebo. Subjects received one dose on Day 1 and were followed for 120 days. A total of 42 subjects each received one dose of lebrikizumab.

[0485] In Studies 1-3, a total of 306 subjects, of which 264 were asthmatic patients, each received at least one dose of hamster PLBL2-containing material. Exposure to hamster PLBL2 was variable, depending on the dose of lebrikizumab received.

[0486] Study 4 was a Phase lib, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of lebrikizumab in uncontrolled asthmatic patients using ICS and a second controller medication. Patients received one of three doses (250, 125, or 37.5 mg) of lebrikizumab or placebo monthly by SC administration. In Study 4, a total of 463 patients were randomized (grouped), of which 347 received at least one dose of lebrikizumab. Exposure to hamster PLBL2 was variable, depending on the dose of lebrikizumab received.

[0487] Table 12 below provides a summary of each of Studies 1-4, which indicates the range of levels of hamster PLBL2 exposure and the dose of lebrikizumab for the subjects.

[0488] Table 12. Hamster PLBL2 Exposure in Lebrikizumab Clinical Trials.

[0489]

[0490] a Range from four different batches of clinical material.

[0491] Anti-Hamster PLBL2 Antibody Assay. Anti-Hamster PLBL2 antibody was detected using the anti-hamster PLBL2 antibody assay described above in a retrospective analysis of selected time points from Study 1. Samples from placebo and dosed subjects were analyzed to determine pre-existing reactivity levels and the occurrence of antibody responses to lebrikizumab dosing. There were 113 placebo subjects and 106 dosed subjects who received at least one dose of lebrikizumab. The time points selected for analysis were Day 0, 29, 85, 141, 225, and early termination. Samples were taken prior to the next dose; therefore, the Day 29 sample was drawn prior to the second dose administration. The percentage of subjects positive for anti-hamster PLBL2 antibody at each time point was calculated by taking the number of subjects positive at each time point and dividing by the total number of subjects tested at each time point. This data is shown in Table 13.

[0492] Table 13. Results of anti-hamster PLBL2 antibody in Study 1.

[0493]

[0494] aOf the 8 lebrikizumab subjects who did not continue in the drug study early, only 3 reported adverse events as the reason for discontinuation of the drug study.

[0495] Six placebo subjects in Study 1 who were positive at Day 0 prior to dosing remained positive throughout the study. Samples from these subjects were confirmed positive in the confirmatory competition assay with titers ranging from 1.6 to 2.9 titer units at Day 0. Titers obtained at later visits were similar to those obtained at Day 0. Some other placebo subjects had low levels of positive responses during the study.

[0496] Of the subjects in Study 1 who received lebrikizumab, 98% (104 / 106) had a positive antibody response after dosing and remained positive through the end of the study, with the majority of subjects becoming positive after receiving at least two doses of lebrikizumab. Post-dose titers ranged from 1.35 to 4.76 titer units, with titers generally increasing over time. The clinical significance of the development of anti-hamster PLBL2 antibody is unclear. No clinically important safety signals were identified in this study that could be related to the high incidence of hamster PLBL2 antibody.

[0497] Interim analysis was also performed on samples collected in Study 4. Samples from placebo and dosed subjects were analyzed to determine the pre-existing level of response and the occurrence of anti-hamster PLBL2 antibody responses to lebrikizumab dosing. There were 116 placebo subjects and 347 dosed subjects who received at least one dose of lebrikizumab. Samples from 92 placebo subjects and 268 dosed subjects were represented in this data set. Results are shown in Table 14.

[0498] Table 14. Anti-hamster PLBL2 antibody results from Study 4 for subjects who had no prior exposure to Lebrikizumab.

[0499]

[0500] aOf the 12 lebrikizumab subjects who did not continue in the drug study early, only 4 reported adverse events as the reason for discontinuation of the study drug.

[0501] The four placebo subjects in Study 4 who were positive on Day 0 prior to dosing had low levels of positive responses, just above the limit of detection of the assay. Low levels of response were detectable at some, but not all, subsequent time points.

[0502] The 15 subjects in Study 4 who received lebrikizumab who were positive on Day 0 prior to dosing remained positive at subsequent time points, with increasing titers after multiple doses. In addition, 10 subjects in Study 4 had previously received lebrikizumab in Study 1. Of these subjects, 9 were subsequently dosed with lebrikizumab in Study 4, while 1 received placebo. All 10 subjects were positive on Day 0 prior to dosing in Study 4 and remained positive at subsequent time points. The data for these 10 subjects are excluded from Table 14 because of their prior exposure to lebrikizumab.

[0503] There appears to be a difference in the rate of positivity between dose groups among the subjects who received lebrikizumab in Study 4. However, since these data are incomplete, no conclusions can be made at this time point regarding the significance of these differences. Similar to the data from Study 1, the majority of subjects became positive after receiving at least two doses of lebrikizumab. Post-dose titers ranged from 1.68 to 4.55 titer units, with titers generally increasing over time. Because this is an incomplete data set, the percent positive and the range of titers can change as additional data accumulate.

[0504] The interim safety assessment of Study 4 showed similar safety profile to the earlier completed studies with no clinically significant safety signals, including no reports of anaphylaxis or serious hypersensitivity reactions. Notably, of the 9 patients who received lebrikizumab in Study 1 and were subsequently re-dosed with lebrikizumab in Study 4, 6 of them reported no adverse events at the time point of the interim analysis and only 1 patient reported any local injection site reactions. No clinical sequelae of this anti- hamster PLBL2 antibody response have been identified to date in the clinical trials.

[0505] We also evaluated the 125 mg dose group from Study 2, and those results are shown in Table 15.

[0506] Table 15. Results of anti-hamster PLBL2 antibody in Study 2.

[0507]

[0508] Of the 2 subjects who did not continue in the drug study early, neither reported an adverse reaction that was a reason for discontinuation of the study drug.

[0509] The 2 subjects from Study 2 who were positive on Day 0 prior to dosing remained positive at all subsequent time points with increasing titers after multiple doses. Of the Study 2 subjects who received 125 mg of lebrikizumab, 87% (46 / 53) were positive for the antibody response after dosing and remained positive through the end of the study, with the majority of subjects becoming positive after receiving at least two doses of lebrikizumab. The post-dose titers ranged from 1.51 to 4.09 titer units, with titers generally increasing over time.

[0510] Conclusions

[0511] To assess the potential impact of CHO PLBL2 impurities, an assay was developed to detect anti-hamster PLBL2 antibody in subjects who received lebrikizumab formulations containing significant levels of hamster PLBL2. Based on the full data set from Study 1 and the 125 mg dose group from Study 2 and the partial data set from Study 4, the hamster PLBL2 present in the lebrikizumab formulation generated an immune response in the majority of subjects exposed to hamster PLBL2.

[0512] A number of placebo and lebrikizumab dose group subjects had pre-existing immune reactivity in the anti-hamster PLBL2 antibody assay. The cause of this pre-existing reactivity is unknown; antibody reactivity to CHO host cell proteins has been previously characterized and confirmed in normal human serum samples that had no known prior exposure to CHO-derived biologies (Xue et al., The AAPS Journal 12(1):98-106 (2010)). However, there are no published data specific to PLBL2.

[0513] For subjects with pre-existing immune reactivity in the anti-hamster PLBL2 antibody assay at the start of the study, there was a sustained rise in antibody titers after repeat dosing of lebrikizumab. For subjects who were antibody negative at the start of the study, a majority of subjects in all four studies became positive after at least two administrations of lebrikizumab and remained positive at all subsequent time points.

[0514] The clinical significance of the development of anti-hamster PLBL2 antibodies is unclear. Although the incidence of hamster PLBL2 antibodies was high in the study subjects, there was no apparent correlation with safety events. Importantly, no safety signals were identified in these completed or ongoing studies, particularly no anaphylactic, anaphylactoid, or serious hypersensitivity reactions were reported. Nonetheless, there is concern that long-term exposure to repeat dosing can increase the potential for undesirable effects, such as anaphylaxis, hypersensitivity, and immune complex deposition, particularly in the asthma patient population and other patient populations that are allergic or hypersensitive. Therefore, it is important to administer IL-13 MAb (e.g., lebrikizumab) formulations containing substantially reduced levels of hamster PLBL2 in later clinical studies and thereafter (where there can be a long period of such repeat dosing) to patients in order to minimize immunogenicity.

[0515] Other antibody sequences are provided in Table 16 below.

[0516] Table 16. Anti-IL17 A / F antibody amino acid sequences (SEQ ID NOS: 15-22) and anti-Abeta antibody amino acid sequences (SEQ ID NOS: 23-30).

[0517]

[0518] ​​​​​​​

Claims

1. A composition comprising an anti-IL-13 monoclonal antibody purified from Chinese hamster ovary host cells, wherein the composition comprises the anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

2. A purified anti-IL-13 monoclonal antibody preparation isolated from Chinese hamster ovary host cells, wherein the preparation is purified by a method comprising a hydrophobic interaction chromatography (HIC) step, thereby producing a purified preparation, wherein the purified preparation comprises the anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

3. A purified anti-IL-13 monoclonal antibody preparation isolated from Chinese hamster ovary cells, wherein the antibody preparation is purified by a method comprising a first protein A affinity chromatography step, a second anion exchange chromatography step, and a third hydrophobic interaction chromatography (HIC) step, thereby producing a purified preparation, wherein the purified preparation comprises the anti-IL-13 antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

4. A method of purifying a recombinant polypeptide produced in Chinese hamster ovary host cells, wherein the method provides a purified preparation comprising the recombinant polypeptide and a residual amount of hamster PLBL2.

5. A composition comprising an anti-Aβ monoclonal antibody purified from Chinese hamster ovary host cells, wherein the composition comprises the anti-Aβ antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

6. A composition comprising an anti-IL17A / F monoclonal antibody purified from Chinese hamster ovary host cells, wherein the composition comprises the anti-IL17A / F antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng / mg, or less than 15 ng / mg, or less than 10 ng / mg, or less than 8 ng / mg, or less than 5 ng / mg, or less than 3 ng / mg, or less than 2 ng / mg, or less than 1 ng / mg, or less than 0.5 ng / mg.

7. A method of treating an IL-13 mediated condition in a patient, the method comprising administering to the patient a therapeutic composition, wherein the therapeutic composition comprises the therapeutic composition of claim 1.

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