Method for increasing antibody yield during ion exchange chromatography - Patent Application 20070122997

Abstract

The present invention relates to a method for increasing antibody yield during antibody purification from a sample by ion exchange chromatography in flow-through mode by preconditioning the sample with Tris without using NaCl to adjust the conductivity.

Description

[Technical Field]

[0001] The present disclosure relates to a method for increasing antibody yield during antibody purification from a sample by ion exchange chromatography. The method includes subjecting an antibody sample pretreated with Tris without using NaCl to adjust conductivity to multimodal anion exchange chromatography in flow-through mode prior to the IEX step. The addition of Tris not only adjusts conductivity but also improves antibody yield in the flow-through with a high monomer content. Furthermore, the present invention relates to a pharmaceutical composition comprising an antibody purified by the method described herein. [Background technology]

[0002] Monoclonal antibodies (mAbs) are utilized in a wide range of immunochemical techniques in basic research as well as therapeutic and diagnostic applications. To be used in pharmaceutical applications, therapeutic antibodies must meet high quality standards. Therefore, to meet these requirements, the goal of all manufacturing and purification process development is to develop a robust, scalable, and reliable process that results in high yields and purity of the target product.

[0003] During purification, target antibodies should be freed from undesirable contaminants such as host cell proteins (HCPs), nucleic acids, viruses, media components (e.g., insulin), cell culture additives (e.g., PEG ethers, antifoaming agents), and aggregated and fragmented products. Antibodies are typically produced by hybridomas or transfected host cells (e.g., CHO, HEK). Therefore, cellular material must be removed from the cell culture at the beginning of the purification process. Subsequently, antibody-containing samples are usually processed through an affinity chromatography step in bind-elute mode ("capture," e.g., by Protein A chromatography for IgG antibodies), followed by viral inactivation, neutralization, and depth filtration. To meet the quality standards for therapeutic-grade products, an additional so-called "final purification" step is included in the purification process. Typically, two or more chromatography steps are performed after the "capture" step to remove residual aggregates and impurities. Impurities typically removed in these steps are process-derived contaminants such as HCPs, nucleic acids, media components, eluted Protein A, and endotoxins. Removal of aggregates and impurities is often achieved by using ion exchange chromatography (IEX) after the initial antibody affinity chromatography. In particular, multimodal chromatography (MMC) media are well suited for post-Protein A purification of mAbs (Pinto IF et al., Pharm. Bioprocess. (2015) 3(3), 263-279). The operating mode also plays an important role in the success of multimodal chromatography. In flow-through (FT) mode, for example, the pH of the sample and buffer can be selected to alter the charge of the antibody or the chromatography media, allowing the antibody to pass through without binding, while leaving most impurities bound to the column. The purity of the antibody found in the flow-through fraction can be improved by optimizing conditions such as protein load, pH, and conductivity.

[0004] For example, International Publication No. 2010071208 discloses an antibody purification method using MMC in flow-through mode, utilizing a loading solution containing 20 mM citric acid, pH 6.2, and specific amino acids selected from arginine, histidine, proline, glutamic acid, and citrulline. U.S. Patent No. 10,023,608 describes the purification of adalimumab using an MMC step in flow-through mode, followed by an HIC step, in which the sample is preconditioned with NaCl and a Tris-based buffer to achieve appropriate sample conductivity (approximately 15-19 mS / cm) and pH (approximately 6.9-7.3) before loading onto the MMC resin. The conductivity is adjusted with a buffer containing NaCl. Various other multistep chromatographic purification methods exist. For example, International Publication No. 2005044856 relates to the removal of high molecular weight aggregates from antibody samples using a hydroxyapatite resin, optionally combined with anion exchange chromatography. In preparation for hydroxyapatite chromatography in flow-through mode, antibody samples are conditioned with a loading buffer containing 0.2-2.5 M NaCl. International Publication No. 2010048183 relates to the removal of HCPs from antibody samples by sequential ion exchange at acidic pH and HIC chromatography. Prior to the IEX step in FT mode, the sample is equilibrated with a buffer containing 20 mM sodium phosphate and 150 mM NaCl or 25 mM trolamine and 40 mM NaCl. International Publication No. 2011090719 describes a method for antibody purification involving multiple chromatography steps in which the low-pH eluate from Protein A chromatography is further purified without the need for pH adjustment. International Publication No. 2012059308 discloses an intermediate final purification step involving either anion exchange (AEX) or cation exchange (CEX) chromatography in flow-through mode.Before entering the AEX and CEX columns, the samples were diluted with demineralized water to a final conductivity of 5 mS or the pH and conductivity were adjusted with 50 mM NaH2PO4, respectively. Summary of the Invention [Problem to be solved by the invention]

[0005] A common drawback of methods using two or more chromatography steps is the loss of target protein after each particulate step, often resulting in a significant decrease in antibody yield. More steps increase purification time, which can be detrimental to protein stability and activity. Therefore, there is a continuing need to develop methods that provide improved yields while still providing satisfactory purity. Such methods would be extremely valuable for the purification of therapeutic and diagnostic compounds.

[0006] In flow-through mode, the charge of the target antibody must be individually tailored to define appropriate pH and conductivity conditions so that the antibody passes through the resin unbound and most impurities bind to the column. The advantages of flow-through mode are the ability to use higher loads and fewer wash and elution steps. The purity of the antibody in the flow-through can be improved by optimizing conditions such as load, pH, salt, and conductivity. However, because contaminant levels vary depending on the cell line, optimal conditions are difficult to predict. Furthermore, differences in preceding purification steps may result in loaded samples with varying compositions. Generally, prior art (GE Healthcare Instructions 28-9064-05 AA) teaches that high sample loads, low pH, and high conductivity are required to obtain the highest yields using multimodal Capto adhere in flow-through mode. For optimal aggregate removal, high pH and low load and conductivity are required. Aggregate removal is often less sensitive to conductivity than Protein A and HCP removal. For optimal removal of Protein A and HCPs, a high pH and low conductivity are required. Therefore, loading conditions are a compromise between conditions that prioritize yield and conditions that prioritize contaminant removal. The optimal loading conditions are a balance between loading, pH, and conductivity. NaCl is a useful salt for adjusting conductivity and is widely used because it is inexpensive. Changes in NaCl concentration, and therefore conductivity, affect the binding strength of charged groups on proteins bound to the ion exchanger. In flow-through mode, a wash step may be used to collect weakly bound proteins, thereby increasing the yield of the target antibody.

[0007] Further purification steps are disclosed, for example, in EP 2639239, which relates to a method for removing protein aggregates from a sample using CEX. In this method, a feed sample is dialyzed in Tris-HCl buffer, pH 7.5, with a conductivity of 3 mS / cm. WO 2014196780 describes a method for removing impurities by sequentially using CEX, a filter, and AEX without affinity chromatography. Prior to AEX, the sample is treated with Tris-HCl and Bis-Tris to adjust the conductivity to 1.4 mS / cm. WO 2014207763 discloses the purification of adalimumab by affinity and hydrophobic interaction chromatography. None of the prior art protein purification methods describes Tris base as a compound for adjusting conductivity.

[0008] The technical problem underlying this application may be seen in providing a method for increasing antibody yield during multimodal ion exchange chromatography in FT mode while maintaining efficient reduction of aggregates and other impurities in antibody-containing samples. The present invention meets these needs by providing a method characterized by an increase in the conductivity of the antibody sample to be purified by preconditioning the sample with Tris only (i.e., without the use of NaCl or any other salt) before loading the chromatography column in flow-through mode. [Means for solving the problem]

[0009] As described herein, the inventors have discovered that adding Tris at neutral pH to an antibody sample eluate after an initial Protein A capture chromatography and before loading onto a mixed-mode anion exchange chromatography resin in flow-through mode results in surprisingly higher antibody yields compared to performing the step without preconditioning the sample eluate with Tris (i.e., without increasing the conductivity) or adjusting the conductivity with NaCl.

[0010] In certain embodiments, the present disclosure provides a method for purifying an antibody from a composition containing the antibody and aggregates and / or impurities, the method comprising: a) subjecting the composition to capture chromatography to generate a capture chromatography eluate; b) adding 2 M Tris, pH 7.1, in the range of 5-20% (v / v) to the capture eluate; c) subjecting the preconditioned eluate of step b) to mixed-mode (multimodal) anion exchange chromatography in flow-through mode to generate a mixed-mode eluate; d) subjecting the mixed-mode eluate to a second mixed-mode chromatography in a bind-elution mode to generate a second mixed-mode eluate; and e) collecting fractions containing the antibody, wherein the method increases antibody yield.

[0011] In certain embodiments, the present disclosure provides a method for purifying an antibody from a composition containing the antibody and aggregates and / or impurities, the method comprising: a) subjecting the composition to capture chromatography to produce a capture chromatography eluate; b) adding 2 M Tris, pH 7.1, in the range of 5-20% (v / v) to the capture eluate; c) subjecting the preconditioned eluate of step b) to mixed-mode (multimodal) anion exchange chromatography in flow-through mode to produce a mixed-mode eluate; d) subjecting the mixed-mode eluate to a second mixed-mode chromatography in a bind-elution mode to produce a second mixed-mode eluate; and e) collecting fractions containing the antibody, wherein the method reduces the amount of aggregates and / or impurities from the composition.

[0012] In certain embodiments, the present disclosure provides a method for purifying an antibody from a composition containing the antibody and aggregates and / or impurities, the method comprising: a) subjecting the composition to capture chromatography to produce a capture chromatography eluate; b) adding 2 M Tris, pH 7.1, in the range of 5-20% (v / v) to the capture eluate; c) subjecting the preconditioned eluate of step b) to mixed-mode (multimodal) anion exchange chromatography in flow-through mode to produce a mixed-mode eluate; d) subjecting the mixed-mode eluate to a second mixed-mode chromatography in a bind-elution mode to produce a second mixed-mode eluate; and e) collecting fractions containing the antibody, wherein the method increases the yield of the antibody and reduces the amount of aggregates and / or impurities from the composition.

[0013] Certain embodiments of the present invention relate to methods for purifying anti-IL-17C antibodies, or antigen-binding portions thereof, from a sample so that the antibodies are substantially free of host cell proteins (HCPs), eluted Protein A, aggregates, and other impurities.

[0014] In one embodiment, the present disclosure provides a method for purifying an IL-17C antibody, which includes an initial recovery step to remove cells and cellular debris, the recovery step comprising one or more centrifugation or depth filtration steps.

[0015] In certain embodiments, the initial antibody-containing sample is subjected to an affinity chromatography step. Examples include affinity supports containing Protein A, Protein G, or other Fc-binding proteins, and affinity supports containing the antigen against which the antibody of interest was raised. Protein A is particularly useful for affinity purification of IgG antibodies. In one aspect, the Protein A column is equilibrated with an appropriate buffer before loading the sample. An example of a suitable buffer is PBS, pH 7.0-7.3. Following equilibration, the sample can be loaded onto the column. After loading the column, one or more wash steps, for example, using the equilibration buffer, can be applied. Other washes using different buffers can also be used before eluting the column. Elution of the antibody from the affinity column is carried out using an appropriate elution buffer. An example of a suitable elution buffer is 50 mM acetate buffer, pH 3.6. The eluate can be monitored using techniques well known to those skilled in the art. For example, absorbance is measured at OD280. The eluted fraction of interest can then be prepared for further steps, usually involving final purification chromatography.

[0016] In one embodiment, a low pH adjustment step follows Protein A affinity chromatography. In such an embodiment, the antibody-containing Protein A eluate is adjusted to a pH of about 2.5 to about 3.5 using 1 M acetic acid to reduce and / or inactivate pH-sensitive viruses that may be contaminating the sample. In a specific embodiment, the affinity eluate is adjusted to pH 3 using 1 M acetic acid. After a defined incubation time, the solution is then neutralized to a pH of between about 6.5 and about 7.5. In one embodiment, pH neutralization may be achieved using 1 M Tris, pH 9.5 buffer. In one embodiment, viral inactivation (i.e., low pH adjustment) and neutralization are followed by depth filtration.

[0017] In certain embodiments, ion exchange chromatography follows affinity chromatography. In other embodiments, ion exchange follows a low pH adjustment step. In a preferred embodiment, ion exchange follows depth filtration after the viral inactivation step. The ion exchange step can be either cation or anion exchange. This step can be a single ion exchange procedure, or it can include multiple ion exchange steps in a sequential combination, such as cation exchange followed by anion exchange, or vice versa.

[0018] In certain embodiments, Capto adhere ImpRes (GE Healthcare), a strong anion exchange chromatography resin with multimodal capabilities, may be used as the final purification step. In one embodiment, this step is performed in flow-through mode under conditions such that the antibody being purified does not bind to the ion exchange resin, while major contaminants such as DNA, RNA, host cell proteins, aggregates, and viruses do bind and are therefore efficiently separated.

[0019] In one aspect, an antibody sample (e.g., affinity chromatography eluate, filtrate after depth filtration) is prepared for ion exchange chromatography by adjusting the pH and ionic strength or conductivity of the sample.

[0020] In a preferred embodiment, the method for purifying an antibody comprises: a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. subjecting the prepared sample to ion exchange chromatography in flow-through mode; d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted with Tris, preferably 2 M Tris, pH 7.1.

[0021] In another embodiment, the method for increasing antibody yield comprises: a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. subjecting the prepared sample to ion exchange chromatography in flow-through mode; d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted with Tris, preferably 2 M Tris, pH 7.1.

[0022] In one aspect, a method for purifying an antibody comprises: a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. subjecting the prepared sample to ion exchange chromatography in flow-through mode; d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted to a conductivity of about 10 to about 50 mS / cm using Tris. Preferably, the conductivity is adjusted to about 10 to about 30 mS / cm. In certain embodiments, the conductivity of the sample after preconditioning with Tris in step b) is at least 10 mS / cm, at least 12 mS / cm, at least 14 mS / cm, or at least 15 mS / cm. In certain embodiments, the conductivity of the antibody sample after preconditioning with Tris in step b) prior to loading the sample onto the IEX resin is in the range of about 10 mS / cm to about 30 mS / cm, about 12 mS / cm to about 28 mS / cm, about 14 mS / cm to about 26 mS / cm, or about 15 mS / cm to about 25 mS / cm. Importantly, the conductivity adjustment is performed using Tris alone.

[0023] In one aspect, a method for increasing antibody yield comprises: a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. subjecting the prepared sample to ion exchange chromatography in flow-through mode; d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted to a conductivity of about 10 to about 50 mS / cm using Tris. Preferably, the conductivity is adjusted to about 10 to about 30 mS / cm. In certain embodiments, the conductivity of the sample after preconditioning with Tris in step b) is at least 10 mS / cm, at least 12 mS / cm, at least 14 mS / cm, or at least 15 mS / cm. In certain embodiments, the conductivity of the antibody sample after preconditioning with Tris in step b) prior to loading the sample onto the IEX resin is in the range of about 10 mS / cm to about 30 mS / cm, about 12 mS / cm to about 28 mS / cm, about 14 mS / cm to about 26 mS / cm, or about 15 mS / cm to about 25 mS / cm. Importantly, the conductivity adjustment is performed using Tris alone.

[0024] In one embodiment, a provided sample containing antibodies is obtained after Protein A chromatography, viral inactivation, neutralization, and depth filtration, followed by adjustment of the sample's conductivity, and the adjusted sample is then processed through a first final purification step comprising ion exchange chromatography in flow-through mode, followed by a second final purification step comprising a multimodal cation exchange chromatography step in bind-and-elute mode. The second mixed-mode chromatography performed in bind-and-elute mode may apply gradient elution.

[0025] In certain embodiments, the ion-exchanged sample is subjected to an intermediate filtration step, either before the first ion-exchange step, between two ion-exchange steps, or both. In certain aspects, the filtration step comprises capture ultrafiltration / diafiltration ("UF / DF"). Among other things, such filtration facilitates concentration and buffer exchange of antibodies and antigen-binding portions thereof.

[0026] In one embodiment, the antibody to be purified is a monoclonal antibody. [Brief explanation of the drawings]

[0027] [Figure 1] Antibody purification may employ one-, two-, or three-step procedures. Numbers indicate steps. Typical yield and purity expectations are shown. AC = affinity chromatography; SEC = size exclusion chromatography; IEX = ion exchange chromatography; CIEX = cation exchange chromatography; AIEX = anion exchange chromatography. [Figure 2] Representative flow-through elution chromatograms of a sample containing an antibody having a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9, preconditioned with or without Tris and purified by multimodal AIEX. Preconditioning: (1) No Tris added, conductivity: 8.2 mS / cm; (2) 5% (v / v) Tris added, conductivity 13.6 mS / cm; (3) 10% (v / v) Tris added, conductivity 17.2 mS / cm; (4) 20% (v / v) Tris added, conductivity 25.5 mS / cm. FT: flow-through. CIP: clean-in-place. DETAILED DESCRIPTION OF THE INVENTION

[0028] Protein purification by chromatography The production of therapeutic antibodies is typically divided into: i) upstream processing (USP), including the production of antibody protein; ii) downstream processing (DSP), including the production of antibody in pure form through purification; and iii) final processing to ensure product integrity and safety. Typically, the first step of downstream purification following the production phase involves clarification of the harvested cell culture mixture, in which the desired antibody can be separated from cells, cell debris, and other contaminants using one or more steps of precipitation, flocculation, (depth) filtration, and / or centrifugation. Downstream purification typically involves one or more (orthogonal) chromatographic separation steps, e.g., based on affinity, ion exchange, hydrophobic interaction, hydroxyapatite, chromatofocusing, gel filtration, and reversed phase, to efficiently remove process- and product-related impurities. These contaminants include, but are not limited to, HCP, eluted Protein A, product isoforms, high molecular weight (HMW), low molecular weight (LMW), and clip or degradation products.

[0029] Affinity chromatography refers to the use of compounds that specifically interact with the desired target protein to be purified. Typically, the compound is immobilized on a resin for the purpose of isolating, purifying, or removing the desired target product. For antibody purification, affinity resins include Protein A from Staphylococcus aureus, Protein G from Streptococcus species, Protein L from Peptostreptococcus magnus, and recombinant or synthetic versions or peptides of these. These resins include MAbSelect™ (GE Healthcare) and Prosep A® (Millipore). For laboratory-scale applications, a single-step affinity purification generally achieves satisfactory purity. For example, Protein A chromatography, due to its high specificity for the Fc portion of IgG, achieves purity exceeding 95% and excellent recovery, making it the most widely used affinity purification method for capturing IgG antibodies. Other examples of well-established purification methods include thiophilic adsorption, hydrophobic interaction or aromatic adsorption chromatography, metal chelate affinity chromatography, and size exclusion chromatography (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102).

[0030] Further removal of aggregates / impurities can be achieved by a combination of one or two additional orthogonal chromatography steps, which may include hydroxyapatite, hydrophobic interaction (HIC), and ion exchange chromatography (IEX, e.g., cation exchange (CEX), anion exchange (AEX), or mixed-mode exchange). At the manufacturing scale, removal of aggregates and impurities is often achieved using IEX after initial antibody affinity chromatography. Commercially available multimodal ion exchangers, such as Capto MMC and Capto adhere, as well as Capto MMC ImpRes and Capto adhere ImpRes (all manufactured by GE Healthcare), can be used for contaminant removal downstream of the initial affinity capture. IEX separates proteins with different surface charges, providing high-resolution separations with high sample loading capacities. This separation is based on reversible electrostatic interactions between charged proteins (i.e., charged amino acid side chains) and the oppositely charged chromatographic medium. AEX involves the purification of proteins on resins with positively charged functional groups (e.g., strong anion exchangers with quaternary amine groups or weak anion exchangers with secondary amine groups). CEX involves the purification of proteins on resins with negatively charged functional groups (e.g., strong cation exchangers with sulfite groups or weak cation exchangers with carboxylate anions). Both AEX and CEX have been demonstrated to be effective in removing not only aggregates but also other impurities in production-scale processes. Each chromatography step, either cation exchange or anion exchange, can be performed in bind-and-elute mode or flow-through mode, depending on the physicochemical properties of the target protein and impurities. Protein molecules vary significantly in their charge characteristics, and their degree of interaction with charged chromatographic media varies depending on differences in overall charge, charge density, and surface charge distribution. For example, monoclonal antibodies contain ionizable groups such as carboxyl and amino groups. The charge of these groups is pH-dependent. Therefore, depending on the isoelectric point (pI) of the antibody, the charge of the protein molecule can be manipulated by exposing the bulk product to different pH conditions.Monoclonal IgG1 antibodies typically have a basic pI of approximately 7-9. In flow-through mode, the charge of the target antibody must be tailored to define appropriate pH and conductivity conditions so that the antibody passes through the resin unbound and the majority of impurities bind to the column. AEX chromatography is often performed in flow-through mode at a neutral to slightly basic pH to remove impurities, such as viruses and DNA, that are expected to bind to the resin while the product is collected in the unbound fraction. Because the separation mode of AEX chromatography resins is based on electrostatic interactions, factors such as conductivity (controlled by salt concentration) also affect the ability of AEX in FT mode to remove DNA, host cell proteins, aggregates, and other impurities.

[0031] The essential core of the present invention is that the conductivity of the sample is adjusted with Tris alone. Surprisingly, it has been found that the addition of Tris, rather than just adjusting the conductivity (e.g., with NaCl), improves the yield of multimodal AEX chromatography in FT mode. In a direct comparison of a sample adjusted with Tris alone to a sample adjusted to the same conductivity with NaCl, the addition of Tris alone was found to result in approximately 5% higher antibody yield after MMC in FT mode, with a slightly lower monomer content, but still within specification.

[0032] Embodiment In one embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted with Tris.

[0033] In another embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted with Tris.

[0034] In certain embodiments, the present disclosure provides a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity of at least 10 mS / cm.

[0035] In certain embodiments, the present disclosure provides a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity between 10 and 50 mS / cm. Preferably, the conductivity is adjusted to 15 mS / cm.

[0036] In another embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted to a conductivity between 10 and 50 mS / cm using Tris, preferably to a conductivity of 15 mS / cm.

[0037] In another embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) are adjusted with Tris to a conductivity of between 10 and 30 mS / cm and a third pH of about 6.5 to 7.5. Preferably, the conductivity is adjusted to 15 mS / cm and the pH to about 7.1.

[0038] In another embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and the second pH of the sample to a third pH; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the sample in step c) is adjusted with Tris to a conductivity of between 10 and 30 mS / cm and a third pH of about 6.5 to 7.5. Preferably, the conductivity is adjusted to 15 mS / cm and the pH to about 7.1.

[0039] In a preferred embodiment, the sample in step a) is an affinity chromatography eluate obtained after an affinity chromatography step. Most preferably, the sample in step a) is a Protein A chromatography eluate having a first pH of about 3 to about 4, ideally a first pH of 3.6. Non-limiting examples of affinity chromatography supports include, but are not limited to, affinity supports comprising Protein A, Protein G, Protein L, or antigens against which antibodies are raised. In certain embodiments, the Protein A chromatography resin is selected from ProSep Ultra Plus, MabSelect SuRe™, or Amsphere Protein A™ resins. Prior to loading the sample, the affinity column is equilibrated with an appropriate buffer (e.g., PBS, pH 7.0-7.3). After loading the sample onto the column, the column is washed one or more times with an appropriate wash buffer (e.g., PBS, pH 7.0-7.3). The antibodies bound to the affinity support can then be eluted using an appropriate elution buffer (eg, sodium acetate buffer, pH 3.6).

[0040] In other embodiments, the present disclosure relates to a method according to any of the preceding embodiments, wherein the second pH in step b) is adjusted to a pH of about 5.2 to about 5.6. Preferably, the second pH is adjusted to pH 5.5.

[0041] In preferred embodiments, mixed-mode, mixed-modal, or multimodal ("MM") chromatography may be used as the ion exchange chromatography in step d). This mixed-mode step may feature either cation or anion exchange, or a combination of both. This step may be based on a single type of ion exchanger mixed-mode procedure, or may include multiple ion exchanger mixed-mode steps, such as a cation exchange mixed-mode step followed by an anion exchange mixed-mode step, or vice versa. Chromatography media for MM chromatography include, among others, mixtures of the following: anion exchange media, cation exchange media, hydrophobic interaction media, hydrophilic interaction media, hydrogen bonding, π-π bonding, and metal affinity. In some embodiments, MM chromatography media having at least an ion exchange medium, such as an anion exchange medium or a cation exchange medium, is used in MM chromatography. Suitable cation exchange columns are columns in which the stationary phase contains anionic groups. Examples of such columns are Capto MMC™, Capto MMC™ ImpRes (GE Healthcare), and Nuvia™ cPrime™ (Biorad). In one embodiment, the cation exchange mixed-mode chromatography comprises N-benzyl-n-methylethanolamine. In another aspect, a suitable anion exchange column is one whose stationary phase comprises cationic groups. In one embodiment, the mixed-mode chromatography is Capto™ Adhere chromatography or Capto™ Adhere ImpRes chromatography (GE Healthcare). In one embodiment, the initial mixed-mode chromatography is performed in flow-through mode. Prior to loading the sample (e.g., affinity eluate) onto the mixed-mode column, the column may be equilibrated using an appropriate buffer.

[0042] In other embodiments, the antibody sample (eg, affinity chromatography eluate) is prepared for the mixed-mode step by adjusting the sample load, pH, conductivity, and ionic strength.

[0043] In one embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and second pH of the sample to a third pH and loading density; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step c) is adjusted to a conductivity of 10-30 mS / cm and the third pH is adjusted to about 6.5-7.5 at a loading density of about 10-200 g / L. Preferably, the conductivity is adjusted to 15 mS / cm and the third pH is adjusted to pH 7.1 at a loading density of 20-40 g / L.

[0044] In another embodiment, the present disclosure provides: a. providing a sample containing an antibody and having a first pH; b. adjusting the first pH of the sample to a second pH; c. adjusting the conductivity and second pH of the sample to a third pH and loading density; d. subjecting the conditioned sample to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step c) is adjusted to a conductivity of 10-30 mS / cm and the third pH is adjusted to about 6.5-7.5 at a loading density of about 10-200 g / L. Preferably, the conductivity is adjusted to 15 mS / cm and the third pH is adjusted to pH 7.1 at a loading density of 20-40 g / L.

[0045] In one embodiment, the antibody to be purified is applied to a multimodal anion exchange chromatography resin in a solution having a conductivity greater than 10 mS / cm. In another embodiment, the antibody is applied in a solution having a conductivity ranging from about 10 mS / cm to about 30 mS / cm. In some embodiments, the antibody is applied to a multimodal anion exchange chromatography resin in a solution having a conductivity of about 15 mS / cm.

[0046] In one embodiment, the antibody sample is applied in the multimodal anion exchange chromatography step in a range of about 1-300 g, about 5-200 g, about 10-100 g, about 20-50 g, or 20-40 g per liter of resin material.

[0047] In one embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the anti-IL-17C antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0048] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL-17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0049] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and The antibody comprises a CDR2 region, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID NO: 10 and the light chain of SEQ ID NO: 9.

[0050] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an anti-(snti-)IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and a DR2 region, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0051] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and a DR2 region, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0052] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, and a HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3. an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 3; an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6; and heavy and light chains having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID NO: 10 and the light chain of SEQ ID NO: 9.

[0053] In a preferred embodiment, adjusting the pH and conductivity of the antibody sample in step c) with Tris results in an increased yield of antibody in the flow-through after multimodal anion exchange chromatography. In one embodiment, adjustment to a 5% (v / v) concentration with 2 M Tris, pH 7.1, results in an antibody yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or greater after the multimodal anion exchange chromatography step. In another embodiment, adjustment to a 10% (v / v) concentration with 2 M Tris, pH 7.1 results in an antibody yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or greater after the multimodal anion exchange chromatography step. In another embodiment, adjustment to a 15% (v / v) concentration with 2 M Tris, pH 7.1 results in an antibody yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or greater after the multimodal anion exchange chromatography step. In another embodiment, adjustment to a 20% (v / v) concentration with 2 M Tris, pH 7.1 results in an antibody yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or greater after the multimodal anion exchange chromatography step.

[0054] In one embodiment, the antibody or antibody fragment to be purified is a human, humanized, or chimeric antibody or antibody fragment.

[0055] In certain embodiments, the antibodies to be purified are IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype antibodies.

[0056] In a preferred embodiment, the antibody to be purified is of the IgG isotype or a variant thereof. More preferably, the antibody is an IgG1 antibody.

[0057] In one embodiment, the disclosure refers to the purification of antibodies specific for IL-17C. In other embodiments, the purified mAb shares 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to the CDRs compared to the CDRs of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0058] Antibody preparations to which the present invention can be applied include unpurified or partially purified antibodies from natural, synthetic, or recombinant sources. Antibody samples can also be cell culture materials, such as solubilized cells and cell culture supernatants. In certain embodiments, they are harvests of clarified cell cultures. The methods of the present invention can be used as a final purification step to purify antibodies from any mixture containing antibodies. For example, such a mixture can be a Protein A eluate.

[0059] Additionally, the present invention relates to pharmaceutical compositions comprising one or more antibodies purified by the methods described herein.

[0060] The purity of the antibody of interest in the resulting sample product can be analyzed using methods well known to those skilled in the art, such as, for example, size exclusion chromatography, Poros™ A HPLC assay, HCP ELISA, Protein A ELISA, and Western blot analysis.

[0061] In preferred embodiments, the methods provided herein result in purified antibodies having an SEC monomer content of 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater. In another embodiment, the purified protein has a 100% SEC monomer content.

[0062] In another embodiment, the methods provided herein result in purified antibody in a yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater.

[0063] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0064] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0065] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0066] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0067] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0068] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris, and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0069] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; 10. The present invention relates to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising: adjusting the pH and conductivity of the eluate in step c) with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0070] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; 10. The present invention relates to a method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising: adjusting the pH and conductivity of the eluate in step c) with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0071] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; 10. A method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising: adjusting the pH and conductivity of the eluate in step c) with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0072] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH, the AC eluate comprising an antibody; b. adjusting the first pH of the AC eluate to a second pH; c. adjusting the conductivity and the second pH of the eluate to a third pH; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0073] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7.

[0074] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) are adjusted with Tris in the absence of NaCl; and wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0075] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0076] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate containing an antibody and having a first pH of about 3 to about 4; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) is adjusted with 2 M Tris to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v), or a Tris concentration range of 5% (v / v) to 20% (v / v), and wherein the antibody has a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0077] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; 1. A method for increasing the yield of an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising: adjusting the pH and conductivity of the eluate of step c) with Tris in the absence of NaCl; and wherein the antibody has a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0078] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; 1. A method for purifying an antibody specific for IL-17C during purification by multimodal anion exchange (MM-AIEX) chromatography in flow-through mode, comprising:

[0079] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; and (c) adjusting the pH and conductivity of the eluate in step c) to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris in the absence of NaCl, or to a Tris concentration range of 5% (v / v) to 20% (v / v), wherein the antibody has a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0080] In another embodiment, the present disclosure provides: a. providing an affinity chromatography (AC) eluate having a first pH of about 3 to about 4, the AC eluate comprising an anti-IL17C antibody; b. adjusting the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5; c. adjusting the conductivity of the eluate to a conductivity of 10-30 mS / cm, preferably 15 mS / cm, and adjusting the second pH to a third pH of about 7.1; d. subjecting the conditioned eluate to ion exchange chromatography in flow-through mode; e. collecting the flow-through containing the antibody; wherein the pH and conductivity of the eluate in step c) is adjusted to 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) with 2 M Tris in the absence of NaCl, or to a Tris concentration range of 5% (v / v) to 20% (v / v), and wherein the antibody has a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0081] In other embodiments, the conductivity is adjusted to at least 10 mS / cm, in the range of 10-50 mS / cm, 10-30 mS / cm, 11-30 mS / cm, 12-30 mS / cm, 13-30 mS / cm, 10-29 mS / cm, 10-28 mS / cm, 10-27 mS / cm, 10-26 mS / cm, 11-29 mS / cm, 11-28 mS / cm, 11-27 mS / cm, 11-26 mS / cm, 12-29 mS / cm, 12-28 mS / cm, 12-27 mS / cm, 12-26 mS / cm, 13-29 mS / cm, 13-28 mS / cm, 13-27 mS / cm, 13-26 mS / cm, or 13-25 mS / cm.

[0082] definition As used herein, the term "protein" refers to a continuous chain of amino acids linked together via peptide bonds. While the term is used to refer to amino acid chains of any length, those skilled in the art will understand that the term is not limited to long chains and can refer to a minimal chain comprising two amino acids linked together via peptide bonds. As used herein, "peptide," "peptide fragment," "polypeptide," "amino acid chain," "amino acid sequence," or any other term used to refer to two or more amino acid chains or groups of amino acid chains are generally included in the definition of "protein," even though each of these terms may have a more specific meaning. The term "protein" may be used in place of or synonymously with any of these terms. The term further includes proteins that have undergone post-translational or post-synthetic modifications, such as glycosylation, acetylation, phosphorylation, or amidation.

[0083] A "buffer" is a solution that resists changes in pH through the action of acid-base conjugate components. For example, various buffers that can be employed depending on the desired pH of the buffer are described in "Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems," Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers that control pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof.

[0084] "Tris" or tris(hydroxymethyl)aminomethane is an organic compound with the formula (HOCH2)3CNH2. Synonyms include TRIS, Tris, Tris base, Tris buffer, Trizma, Trisamine, THAM, tromethamine, trometamol, tromethane, and trisaminol. The preferred IUPAC name is 2-amino-2-(hydroxymethyl)propane-1,3-diol. CAS Registry Number: 77-86-1.

[0085] The term "isoelectric point (pI)" is the pH at which a particular molecule or surface carries no net charge. The pI of a polypeptide depends on the amino acids that make up the polypeptide. At a pH below its pI, a polypeptide carries a net positive charge. At a pH above its pI, a polypeptide carries a net negative charge. Thus, polypeptides can be distinguished based on their ionization state at a given pH. The actual pI of a polypeptide can be affected by factors such as post-translational modifications. The actual pI can be determined by experimental methods such as isoelectric focusing.

[0086] The term "chromatography" refers to current or future chromatography-based processes that purify one or more target molecules from a sample, e.g., by removing impurities and / or other non-target molecules. During chromatography, solutes of interest in a mixture, such as polypeptides, are separated from other solutes in the mixture as a result of differences in the rates at which individual solutes of the mixture move through a stationary medium under the influence of a mobile phase, or in a binding and elution process. Examples of liquid chromatography purification include, but are not limited to, affinity chromatography, immobilized metal ion affinity chromatography, flow-through chromatography, ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, simulated moving bed chromatography, hydrophobic interaction chromatography, gel filtration, and chromatofocusing.

[0087] The term "mixed-mode chromatography" or "multimodal chromatography" refers to a purification process using mixed-mode sorbents that offer multiple modes of interaction between the polypeptide of interest and the sorbent ligand, such as hydrophobicity, cation exchange, hydrogen bonding, etc. Commercially available mixed-mode chromatography resins include Capto™ MMC, Capto™ MMC ImpRes, Capto Blue, Blue Sepharose™ 6 Fast Flow, Capto™ Adhere, and Capto™ Adhere ImpRes from GE Healthcare Life Sciences; or Eshmuno® HCX from EMD Millipore; or Nuvia™ cPrime from Bio-Rad.

[0088] The terms "cation exchange resin," "cation exchange adsorbent," or "cation exchange matrix" refer to a negatively charged solid phase having free cations that are exchanged for cations on or in an aqueous solution passed through 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 sulfopropyl (SP) immobilized on carboxymethylcellulose, agarose (e.g., SP Sepharose™ XL, SP-Sepharose™ Fast Flow, SP Sepharose™ High Performance, CM Sepharose™ Fast Flow, CM Sepharose™ High Performance, Capto™ S, and Capto™ SP ImpRes from GE Healthcare Life Sciences; or Fractogel® EMD SE HiCap, Fractogel® EMD SO3″, Fractogel® EMD COO″, Eshmuno™ S, and Eshmuno™ CPX from EMD Millipore; or UNOsphere™ S and Nuvia™ S from Bio-Rad).

[0089] The terms "anion exchange resin," "anion exchange sorbent," or "anion exchange matrix" are used herein to refer to a positively charged solid phase having one or more positively charged ligands, such as, for example, quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE Sepharose™ Fast Flow, Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ DEAE, Capto™ Q, and Capto™ Q ImpRes from GE Healthcare Life Sciences; or Fractogel™ EMD TMAE HiCap, Fractogel™ EMD DEAE, and Eshmuno Q from EMD Millipore; or U Osphere™ Q and Nuvia™ Q from Bio-Rad.

[0090] The term "antibody" refers to glycosylated and non-glycosylated immunoglobulins of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and combinations and variants thereof. As used herein, the term encompasses antibodies from any species (e.g., murine, canine, feline, IgY, etc.), and combinations thereof, such as human, humanized, and chimeric antibodies. The term refers to monoclonal and polyclonal antibodies, as well as monospecific and multispecific antibodies (such as bispecific antibodies). As used herein, the term also encompasses fusion proteins containing antigenic determinants and any other modified immunoglobulin molecules containing an antigen recognition site. As used herein, the term "antibody" includes intact immunoglobulins as well as antibody fragments, which refer to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilization of spatial distribution). Examples of binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fd, Fv, and dAb fragments (Ward et al., (1989) Nature 341:544-546), single-chain Fv (scFv) (e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Any polypeptide, whether naturally occurring, enzymatically obtainable, synthetic, alternative scaffold, or genetically engineered, that specifically binds to an antigen is intended to be encompassed by the term "antibody" as used herein.

[0091] The terms "contaminant" and "impurity" are used interchangeably herein and refer to any undesired molecule, including biopolymers such as DNA, RNA, one or more host cell proteins, endotoxin, lipids, and one or more additives, that may be present in a sample containing a target protein that has been separated from one or more foreign or undesired molecules using the methods of the present invention. Additionally, such contaminants may include any reagents used in steps that may occur prior to the purification process.

[0092] "High molecular weight (HMW) species" includes species with a molecular weight higher than the target protein mass, such as multimers. Multimers include all but a monomer of the target protein. For example, an IgG antibody monomer encompasses the traditional tetrameric antibody composition containing two heavy and light chains. Multimers include species with a molecular weight higher than the target protein mass, such as dimers (two identical proteins bound together by covalent or non-covalent bonds) and aggregates (whole proteins and / or protein portions bound together by covalent or non-covalent bonds).

[0093] "Low molecular weight (LMW) species" include species with a molecular weight lower than that of the target protein, such as clips, and degradation products.

[0094] As used herein, the term "final purification" refers to downstream processing steps that occur after the initial (affinity) capture step and are intended to remove residual amounts of impurities that are present in the product stream and that typically have a greater similarity to the product than the impurities removed during the capture step.

[0095] Methods for determining the yield or purity of a polypeptide are known to those skilled in the art. The yield or purity of a polypeptide may be determined by any suitable analytical method (e.g., band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, ELISA, HPLC, etc.). An exemplary method is size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC). Purity may be determined using a relative "area under the curve" (AUC) value, which may typically be obtained for a peak in a chromatogram, such as an HPLC chromatogram.

[0096] The term "bind and elute mode" refers to a product separation technique in which at least one product contained in a sample (e.g., an Fc region containing protein) binds to a chromatographic resin or medium and is subsequently eluted.

[0097] The term "flow-through mode" refers to conditions in which contaminants bind to the chromatographic support while the target protein passes through.

[0098] The amino acid and encoding nucleic acid sequences in Table 1 are examples of IL-17C antibodies, and portions thereof.

[0099] [Table 1]

[0100] [Table 2]

[0101] [Table 3] [Example]

[0102] Example 1: Four separate purification runs were performed to examine the effect of adding Tris (i.e., increasing the Tris concentration) to a sample containing an antibody having a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO:9 on yield and purity before loading the flow-through onto a Capto adhere ImpRes column (GE Healthcare). One run was performed without Tris, and three runs were performed with 2 M Tris pH 7.1 added to the sample eluate at 5, 10, and 20% (v / v). The resulting flow-through was analyzed for antibody yield and purity (SEC monomer). The results are listed in Table 2, and the corresponding chromatograms are shown as an overlay in Figure 2. Adding 5% (v / v) Tris increased yield by 20%, with a slight 0.4% decrease in the SEC monomer fraction of the resulting pool. Increasing the amount of Tris added to the sample resulted in a further increase in yield, but to a lesser extent (approximately 4% and 7% increases for 5% Tris compared to 10% and 20% Tris, respectively), and a further slight decrease in the monomer fraction.

[0103] [Table 4]

[0104] Example 2: To determine whether adding Tris to antibody samples as in Example 1 not only adjusts conductivity but also improves antibody yield in the flow-through after Capto adhere ImpRes chromatography, a direct comparison of preconditioning samples with Tris (Run 1) and NaCl (Run 2) was performed. For sample preparation, conductivity was adjusted with 2 M Tris pH 7.1 in Run 1 and 5 M NaCl in Run 2 to a target conductivity of 15 mS / cm (Table 3).

[0105] [Table 5]

[0106] Both loads had the same conductivity but different buffer matrices. pH and conductivity measurements were performed at ambient temperature of 20° C.±2° C. Purification results and QC data are shown in Table 4.

[0107] [Table 6]

[0108] Without Tris (Run 2), the yield was approximately 5% lower compared to Run 1, where the conductivity was adjusted to 15 mS / cm with 2 M Tris, pH 7.1.

Claims

1. a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. subjecting the prepared sample to ion exchange chromatography in flow-through mode; d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted to 13-30 mS / cm with Tris, and the pH after adjusting the conductivity is in the range of pH 6.5-7.5, the antibody-containing sample is an affinity chromatography eluate, and the conductivity of the sample in step b) is adjusted with Tris in the absence of NaCl. A method for increasing the yield of antibody in the flow-through of ion exchange chromatography during antibody purification.

2. 2. The method of claim 1, wherein the affinity chromatography eluate is a Protein A chromatography eluate having a pH of about 3 to about 4.

3. 3. The method of claim 2, wherein the pH of the sample of about 3 to about 4 is adjusted to a pH of about 5.2 to about 5.6, preferably a pH of 5.

5.

4. 10. The method of claim 1, wherein the conductivity is adjusted to 15 mS / cm.

5. The method according to any one of claims 1 to 4, wherein the ion exchange chromatography is multimodal anion exchange chromatography.

6. The method according to any one of claims 1 to 5, wherein the monoclonal antibody to be purified is a monoclonal antibody.

7. The method of claim 6, wherein the purified monoclonal antibody is an anti-IL17c antibody.

8. 8. The method of claim 7, wherein the purified monoclonal anti-IL17C antibody comprises a VH of SEQ ID NO:8 and a VL of SEQ ID NO:

7.

9. 9. The method of claim 8, wherein the purified monoclonal anti-IL17C antibody consists of a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO:

9.

10. The method of any one of claims 1 to 9, wherein the yield of purified antibody in the flow-through is greater than 75%.

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