Method for increasing antibody yield during ion exchange chromatography

Abstract

To provide a method for increasing an antibody yield during antibody purification.SOLUTION: A method for increasing an antibody yield in a flow-through of ion exchange chromatography during antibody purification includes: a. a step of providing a sample including an antibody; b. a step of adjusting the electric conductivity of the sample; c. a step of processing the adjusted sample by ion exchange chromatography of a flow-through mode; and d. a step of collecting the flow-through including the antibody, where the electric conductivity of the sample on step b) is adjusted to at least 10 mS / cm by using tris, and pH after adjusting electric conductivity is within the range of pH6.5 to 7.5.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Monoclonal antibodies (mAbs) have a wide range of applications in basic research as well as therapeutic and diagnostic applications. They are also used in a wide range of immunochemical techniques. For pharmaceutical use, therapeutic antibodies must be of high quality. Therefore, all manufacturing and refinement processes must be carried out to meet these requirements. The goal of manufacturing process development is to develop a robust, scalable, and reliable process that results in high yield and purity of the target product. The key is to develop a reliable process.

[0003] During purification, the target antibody is purified from host cell proteins (HCPs), nucleic acids, viruses, media components ( insulin), cell culture additives (e.g., PEG ethers, antifoaming agents), and coagulation agents. The antibody should be free from undesired contaminants such as aggregates and fragmented products. Typically, hybridomas or transfected host cells (e.g., CHO, HEK) Therefore, cellular material is removed from the cell culture at the beginning of the purification process. Subsequently, the antibody-containing sample is typically subjected to af- an affinity chromatography step ("capture", e.g., Protein A for IgG antibodies) by chromatography) followed by viral inactivation, neutralization, and depth filtration. To meet the quality standards of a therapeutic grade product, additional so-called "final refinement" is carried out. The purification process usually involves a "preparation" step to remove residual aggregates and impurities. After the "capture" step, two or more chromatography steps are performed. Impurities commonly removed by this step include HCPs, nucleic acids, media components, eluted Protein A, and endogenous Removal of aggregates and impurities is essential for the initial antibody affinity purification. Ion exchange chromatography (IEX) is used after ion exchange chromatography. In particular, multimodal chromatography (MMC) media , well suited for post-protein A purification of mAbs (Pinto IF et al, Pharm.Bioprocess.(2015)3(3),263-279). Operation model The code also plays an important role in the success of multimodal chromatography. In flow-through (FT) mode, for example, select the pH of the sample and buffer, and then add the antibody. Or, the charge of the chromatography medium is altered so that the antibody does not bind, but rather most of the impurities are removed. The pure antibody found in the flow-through fraction is The efficiency can be improved by optimizing conditions such as protein loading, pH, and conductivity.

[0004] For example, International Publication No. 2010071208 describes a method for preparing a soluble collagen containing 20 mM citric acid, p H6.2 and a compound selected from arginine, histidine, proline, glutamic acid, and citrulline The loading solution containing the specific amino acid selected was used for the MMC in FT mode. U.S. Patent No. 10,023,608 discloses a method for purifying antibodies. Before loading the sample into the column, adjust the sample to the appropriate conductivity (approximately 15-19 mS / cm) and pH (approximately 6. To achieve this, the buffer solution was preconditioned with NaCl and Tris base. MMC step in flow-through mode with conditioned samples and The purification of adalimumab using a subsequent HIC step is described, where the conductivity is NaC It is prepared in a buffer containing l. In addition, various multi-step chromatographic purification methods are also available. For example, WO 2005044856 describes a method for using hydroxyapatite resin in combination with ion exchange chromatography; Removal of high molecular weight aggregates from antibody samples. In preparation for silica gel chromatography, antibody samples were prepared in 0.2–2.5 M NaCl. The loading buffer is adjusted to contain 1. The antibody sample was purified by sequential ion exchange and HIC chromatography at acidic pH. Prior to the IEX step in FT mode, the sample was 20 mM sodium phosphate and 150 mM NaCl, or 25 mM trolamine and 40 mM The solution is equilibrated with a buffer containing 100 M NaCl. The kit allows low pH eluates from Protein A chromatography to be purified without the need for pH adjustment. A method for antibody purification comprising multiple chromatography steps for further purification WO 2012059308 pamphlet describes the flow-through mode. Anion exchange chromatography (AEX) or cation exchange chromatography intermediate final purification steps, including either chromatographic (CEX) or column chromatography. The sample was then passed through the AEX and CEX columns to a final conductivity of 5 mS. The solution is diluted with demineralized water or adjusted with 50 mM NaH2PO4 until the pH and conductivity are Each was adjusted. Summary of the Invention [Problem to be solved by the invention]

[0005] A common drawback of methods using two or more chromatography steps is that each particle (p This is the loss of target protein after the (articulate) step, which reduces antibody yield. The more steps, the longer the purification time, which is often due to the increased protein Therefore, while providing improved yields, There is a continuing need to develop methods that result in satisfactory purity. These compounds are of great value for purification processes of pharmaceutical and diagnostic compounds.

[0006] In flow-through mode, appropriate pH and conductivity conditions must be defined to allow the antibody to pass through the dendritic cells without binding. The charge of the target antibody is tailored to allow it to pass through the column, while most of the impurities bind to the column. The advantage of flow-through mode is that higher loads can be used and washing and Fewer elution steps. Purity of antibody in the flow-through can be determined by load, pH, salt Although this can be improved by optimizing conditions such as conductivity, the level of contaminants remains constant. It is difficult to predict the optimum conditions because they vary depending on the strain. There may be differences in the steps, resulting in loading samples of varying composition. Generally, the prior art (GE Healthcare Instructions 28- 9064-05 AA) is a multimodal Capto adhesive in flow-through mode. To obtain the best yield using re, high sample load, low pH, and low conductivity are recommended. For optimal removal of aggregates, the pH should be high and the load should be low. Aggregate removal is often achieved by removing Protein A and HCPs. For optimal removal of Protein A and HCPs, The pH needs to be high and the conductivity low. Therefore, the loading conditions should be set to prioritize yield. The optimum loading conditions are a compromise between the load, pH, and the conditions that prioritize contaminant removal. The balance between conductivity and water content is important. NaCl is a useful salt for adjusting conductivity and is inexpensive. The change in NaCl concentration, and therefore the change in conductivity, is a function of the ion exchanger. In flow-through mode, the binding strength of the charged groups of proteins bound to the A purification step is used to collect weakly bound proteins, thereby improving the yield of the target antibody. The rate may be increased.

[0007] Further purification steps, such as removing protein aggregates from the sample using CEX, The method for producing the same is disclosed in European Patent No. 2639239. The feed sample was dialyzed in Tris-HCl buffer, pH 7.5, conductivity 3 mS / cm. The pamphlet of International Publication No. 2014196780 describes affinity chromatography. By using CEX, Filter and AEX consecutively without using any fees Prior to AEX, the samples were purified using TrisHCl and BisTrisHCl. The resulting solution is treated with HCl to adjust the conductivity to 1.4 mS / cm. Pamphlet No. 3 describes the use of affinity and hydrophobic interaction chromatography The present invention discloses the purification of limumab. None of the prior art protein purification methods involve adjusting the conductivity. No tris base is mentioned as a compound for adjusting the structure.

[0008] The technical problem underlying this application is the multimodal ion exchange chromatography in FT mode. Increases antibody yield during chromatography while simultaneously reducing aggregates and other contaminants in antibody-containing samples. The present invention may be seen as providing a method for maintaining efficient reduction of impurities. The sample was diluted with Tris only (as a control) before being loaded onto the chromatography column in flow-through mode. Preconditioning (i.e., without NaCl or any other salt) and increasing the conductivity of the antibody sample to be purified. These needs are met by: [Means for solving the problem]

[0009] As described herein, the inventors have developed the first Protein A capture chromatograph. After filtration, the eluate was loaded onto a mixed-mode anion exchange chromatography resin in flow-through mode. Adding Tris at neutral pH to the antibody sample eluate before elution can improve the stability of the sample eluate. By not preconditioning with ris (i.e., not increasing conductivity), or NaC By adjusting the conductivity at 1000 kJ / min, surprisingly higher antibody yields were obtained compared to when the process was carried out. It was found to bring about a rate

[0010] In certain embodiments, the present disclosure provides methods for isolating antibodies from compositions comprising antibodies and aggregates and / or impurities. The present invention provides a method for purifying a compound, the method comprising: a) subjecting a compound to capture chromatography; b) subjecting the sample to 2 M Tris to generate a capture chromatography eluate; and b) adding pH 7.1 to the capture eluate in the range of 5-20% (v / v); The preconditioned eluate from step b) was then mixed in flow-through mode (multiplex mode). A mixed-mode anion exchange chromatography was performed to generate a mixed-mode eluate. and d) the mixed-mode eluate is subjected to a second mixed-mode chromatography in a combined elution mode. to generate a second mixed-mode eluate; and e) collecting the antibody-containing fractions. and a step in which the method increases the yield of the antibody.

[0011] In certain embodiments, the present disclosure provides methods for isolating antibodies from compositions comprising antibodies and aggregates and / or impurities. The present invention provides a method for purifying a compound, the method comprising: a) subjecting a compound to capture chromatography; b) subjecting the sample to 2 M Tris to generate a capture chromatography eluate; and b) adding pH 7.1 to the capture eluate in the range of 5-20% (v / v); The preconditioned eluate from step b) was then mixed in flow-through mode (multiplex mode). A mixed-mode anion exchange chromatography was performed to generate a mixed-mode eluate. and d) the mixed-mode eluate is subjected to a second mixed-mode chromatography in a combined elution mode. to generate a second mixed-mode eluate; and e) collecting the antibody-containing fractions. and a step of: Decrease.

[0012] In certain embodiments, the present disclosure provides methods for isolating antibodies from compositions comprising antibodies and aggregates and / or impurities. The present invention provides a method for purifying a compound, the method comprising: a) subjecting a compound to capture chromatography; b) subjecting the sample to 2 M Tris to generate a capture chromatography eluate; and b) adding pH 7.1 to the capture eluate in the range of 5-20% (v / v); The preconditioned eluate from step b) was then mixed in flow-through mode (multiplex mode). A mixed-mode anion exchange chromatography was performed to generate a mixed-mode eluate. and d) the mixed-mode eluate is subjected to a second mixed-mode chromatography in a combined elution mode. to generate a second mixed-mode eluate; and e) collecting the antibody-containing fractions. and a step of: and / or reduce the amount of impurities.

[0013] Certain embodiments of the invention involve isolating anti-IL-17C antibodies or antigen-binding portions thereof from a sample. Purification is performed to remove host cell proteins (HCPs), eluted Protein A, aggregates, and their and a method for making the product substantially free of other impurities.

[0014] In one embodiment, the present disclosure includes an initial recovery step to remove cells and cell debris. The present invention provides a method for purifying an IL-17C antibody, including the step of recovering the antibody by one or more centrifugation steps. A separation or depth filtration step is included.

[0015] In certain embodiments, the initial collection sample containing the antibody is subjected to affinity chromatography. Examples include Protein A, Protein G, and other Fc-binding proteins. An affinity support containing a protein and an antigen against which an antibody of interest has been raised. In particular, Protein A is an affinity support for IgG antibodies. In one embodiment, the Protein A column is used to purify the sample prior to loading. It is equilibrated with a suitable buffer. 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 onto the column, e.g. One or more washing steps using the equilibration buffer may be applied. Other washes using different buffers may also be used before the affinity column is washed. Elution of the antibody from the column is carried out using a suitable elution buffer. Examples of suitable elution buffers include: The eluate is eluted using techniques well known to those skilled in the art. For example, the absorbance can be measured at OD280. can be prepared for further steps, usually involving final purification chromatography.

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

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

[0018] In certain embodiments, a powerful anion exchange chromatograph with multimodal capabilities Capto adhere ImpRes (GE Healthcare) as a cellulose resin e) may be used as a final purification step. In one embodiment, this step comprises: The antibody being purified does not bind to the ion exchange resin, while DNA, RNA, and host cell proteins Major contaminants such as particles, aggregates, and viruses are bound and therefore efficiently separated Under these conditions, the assay is carried out in flow-through mode.

[0019] In one embodiment, an antibody sample (e.g., affinity chromatography eluate, depth layer The filtrate after filtration can be adjusted by adjusting the pH and ionic strength or conductivity of the sample. Prepared for ion exchange chromatography.

[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. The prepared sample is subjected to ion exchange chromatography in flow-through mode. and d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is increased by adding Tris, preferably 2M Tris The pH is adjusted to 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. The prepared sample is subjected to ion exchange chromatography in flow-through mode. and d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is increased by adding Tris, preferably 2M Tris The pH is adjusted to 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. The prepared sample is subjected to ion exchange chromatography in flow-through mode. and d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted to about 10 to about 5 with Tris. The conductivity is adjusted to about 10 to about 30 mS / cm. In certain embodiments, the pH is adjusted to 1.0 in step b). The conductivity of the sample after rinsing should be at least 10 mS / cm, at least 12 mS / cm. m, at least 14 mS / cm, at least 15 mS / cm. In step b), the sample was preconditioned with Tris before loading onto the IEX resin. The conductivity of the antibody sample after filtering ranges from approximately 10 mS / cm to approximately 30 mS / cm. , in the range of about 12 mS / cm to about 28 mS / cm, about 14 mS / cm to about 26 mS / cm The range is about 15 mS / cm to about 25 mS / cm. The important thing is that the conductivity can be adjusted. This is done using only Tris.

[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. The prepared sample is subjected to ion exchange chromatography in flow-through mode. and d. collecting the flow-through containing the antibody; wherein the conductivity of the sample in step b) is adjusted to about 10 to about 5 with Tris. The conductivity is adjusted to about 10 to about 30 mS / cm. In certain embodiments, the preconditioning step b) is performed using Tris. The conductivity of the sample after rinsing is at least 10 mS / cm, at least 12 mS / cm , at least 14 mS / cm, at least 15 mS / cm. Before loading the sample onto the IEX resin, precondition it with Tris in step b). The conductivity of the antibody sample after rinsing is in the range of about 10 mS / cm to about 30 mS / cm. Range of about 12mS / cm to about 28mS / cm, range of about 14mS / cm to about 26mS / cm The range is about 15mS / cm to about 25mS / cm. This is done using only squirrels.

[0024] In one embodiment, the provided sample containing the antibody is purified by Protein A chromatography. , obtained after viral inactivation, neutralization, and depth filtration, and adjustment of the sample conductivity. Following this, the prepared sample was then subjected to ion exchange chromatography in flow-through mode. A first final purification step containing PEGylation followed by multimodal positive control in bind-elute mode It is processed through a second final purification step which includes an ion exchange chromatography step. The second mixed-mode chromatography, performed in bind-and-elute mode, uses gradient elution. may be applied.

[0025] In certain embodiments, the ion-exchanged sample is subjected to two ion exchange steps prior to the first ion exchange step. Either during the ion exchange step, or both, the filtrate is subjected to an intermediate filtration step. In certain embodiments, 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. do.

[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) antibody protein production; ii) upstream processing, including production of the antibody in pure form by purification (D SP); and iii) final processing to ensure product integrity and safety, typically during production. The first step of downstream purification involves clarification of the harvested cell culture mixture, where , using one or more steps of precipitation, flocculation, (depth) filtration and / or centrifugation The desired antibody can be separated from cells, cell debris, and other contaminants. Typically, for example, affinity, ion exchange, hydrophobic interaction, hydroxyaptamer one or more (orthogonal) methods based on chromatofocusing, gel filtration, and reversed phase Includes a chromatographic separation step to efficiently remove process and product-related impurities These contaminants include, but are not limited to, HCPs, eluted proteins, In A, product isoforms, high molecular weight (HMW), low molecular weight (LMW), and clip or fragment Decomposition products include:

[0029] Affinity chromatography is performed using a protein that specifically interacts with the desired target protein to be purified. Refers to the use of a compound to act. Typically, the compound is used to isolate, purify, or remove a desired target product. For antibody purification, for example, affinity resins are used to From Staphylococcus aureus Protein A obtained from Streptococcus species Protein G, Peptostreptococcus magnus Protein L from Bacillus magnus, and recombinant or synthetic versions thereof These resins include MAbSelect™ (GE He Healthcare) and Prosep A (registered trademark) (Millipore). For laboratory-scale applications, one-step affinity purification generally provides satisfactory purity. For example, Protein A chromatography achieves high affinity for the Fc portion of IgG. High specificity of the antibody allows for greater than 95% purity and excellent recovery, making it ideal for capturing IgG antibodies. It is the most widely used affinity purification method. Examples 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 using hydroxyapatite, hydrophobic interaction (HIC) and ion exchange chromatography (IEX, e.g., cation exchange (CEX), anion exchange (AEX), One or two additional direct exchanges (AEX, or mixed-mode exchanges) This can be achieved by a combination of cross-chromatography steps. Removal of analytes and impurities was performed using IEX after the initial antibody affinity chromatography. This is often achieved by using Capto MMC and Capto adhere. , and Capto MMC ImpRes and Capto adhere ImpRe Commercially available multimodal ion exchangers such as s (all manufactured by GE Healthcare) It can be used to remove contaminants downstream of the initial affinity capture. This separation separates different proteins, providing high resolution separation with a high sample load capacity. Separation occurs when a charged protein (i.e., charged amino acid side chains) is separated from an oppositely charged chromatographic medium. AEX is based on the reversible electrostatic interaction between a resin with positively charged functional groups. Acetone (e.g., a strong anion exchanger with quaternary amine groups, or a weak anion exchanger with secondary amine groups) It involves the purification of proteins on an anion exchanger (CEX). CEX is a method for separating proteins with negatively charged functional groups. Resins (e.g., strong cation exchangers with sulfite groups or weak cation exchangers with carboxylate anions) AEX and CEX involve the purification of proteins on a cation exchanger. The process has proven effective in removing not only aggregates but also other impurities. Each chromatographic step, either cation exchange or anion exchange, The filters can be configured in either a bind and elute mode or a filter depending on the physicochemical properties of the target protein and impurities. Protein molecules vary widely in their charge characteristics and can be analyzed in a low-through mode. Charged chromatographic media vary depending on the specific charge, charge density, and surface charge distribution. For example, monoclonal antibodies have different degrees of interaction with carboxyl and amino groups. The charge of these groups is pH dependent. Depending on the isoelectric point (pI) of the antibody, the charge on the protein molecule determines the bulk product under different pH conditions. Monoclonal IgG1 antibodies typically have a denaturing effect of about 7 It has a basic pI of ~9. In flow-through mode, define appropriate pH and conductivity conditions. The target is then concentrated so that the antibody passes through the resin unbound and most of the impurities bind to the column. The charge of the antibody must be individually adjusted. AEX chromatography is used to separate the product into unbound fractions. impurities such as viruses and DNA that are expected to bind to the resin are collected in minutes. To remove soluble ions, run in flow-through mode at neutral to slightly basic pH. The separation mode of AEX chromatography resin is based on electrostatic interactions. , conductivity (controlled by salt concentration), etc. also affect the AEX in FT mode. Affects ability 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. Importantly, not only is the conductivity adjusted (e.g., by NaCl), but the addition of Tris This may improve the yield of multimodal AEX chromatography in FT mode. The sample adjusted with Tris alone and the sample adjusted with NaCl to the same conductivity were In direct comparison with the sample containing only Tris, the antibody yield after MMC in FT mode was was found to be about 5% higher and the monomer content slightly lower, 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for purifying an antibody, comprising: and conductivity 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for increasing antibody yield, comprising: The pH and conductivity of the solution are 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for purifying an antibody, comprising: and the conductivity 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for purifying an antibody, comprising: The conductivity is adjusted to between 10 and 50 mS / cm using Tris. Typically, 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for increasing antibody yield, comprising: The pH and conductivity of the solution are adjusted using Tris to a conductivity between 10 and 50 mS / cm. Preferably, the conductivity is adjusted to 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for purifying an antibody, comprising: and conductivity is between 10 and 30 mS / cm using Tris and approximately 6.5 to 7.5 Preferably, the conductivity is 15 mS / cm and the pH is adjusted to about 7.1. It will be adjusted.

[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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for increasing antibody yield, comprising: The pH and conductivity of the solution were adjusted to between 10 and 30 mS / cm with Tris and approximately 6.5 The third pH is adjusted to about 7.5. Preferably, the conductivity is 15 mS / cm and the pH is about 7. It will be adjusted to .1.

[0039] In a preferred embodiment, the sample in step a) is purified by affinity chromatography. The most preferred affinity chromatography eluate is the one obtained after the first step. Preferably, the sample in step a) has a first pH of about 3 to about 4, and ideally a first pH of about 3 to about 4. Protein A chromatography eluate with H of 3.6. Non-limiting examples of lithography supports include, but are not limited to, protein Protein A, Protein G, Protein L, and the antigens against which antibodies were raised. In certain embodiments, Protein A chromatography resins include , ProSep Ultra Plus, MabSelect SuRe™, or Amsphere Protein A™ resin. Before filtration, the affinity column should be filled with an appropriate buffer (e.g., PBS, pH 7.0–7.3). After loading the sample onto the column, the column is equilibrated with an appropriate wash buffer (e.g. The cells are washed one or more times with PBS (pH 7.0-7.3). The antibody bound to the affinity support can be eluted with an appropriate elution buffer (e.g., sodium acetate buffer, It can be eluted using a 5000 kJ / ml sorbent (pH 3.6).

[0040] In other embodiments, the present disclosure provides a method for preparing a pharmaceutical composition comprising the steps of: Preferably, the second The pH is adjusted to pH 5.5.

[0041] In a preferred embodiment, mixed mode or mixed modal or multimodal ("MM") Chromatography is used as ion exchange chromatography in step d). This mixed-mode step may involve either cation or anion exchange, or both. This step may be characterized by a combination of a single type of ion exchanger or a mixed mode ion exchanger. Based on the procedure, a cation exchange mixed mode step followed by an anion exchange mixed mode step can be performed. The method may include multiple ion exchanger mixed mode steps, such as a mixed mode step, or vice versa. Chromatographic media for MM chromatography include, inter alia, mixtures of: Examples include: anion exchange media, cation exchange media, hydrophobic interaction media, and hydrophilic phases. Interaction mediators, hydrogen bonds, pi-pi bonds, and metal affinities. In the present invention, the present invention has at least an ion exchange medium, such as an anion exchange medium or a cation exchange medium. MM chromatography media are used in MM chromatography. Suitable cations An exchange column is a column in which the stationary phase contains anionic groups. An example of such a column is a C apto MMC(TM), Capto MMC(TM) ImpRes(GE Heal (BioCare), Nuvia™ cPrime™ (Biorad). In an embodiment, the cation exchange mixed mode chromatography is carried out using N-benzyl-n-methyl In another aspect, a suitable anion exchange column has a stationary phase comprising: In one embodiment, the mixed-mode chromatography is performed using a column containing cationic groups. apto™ Adhere Chromatography or Capto™ Adhere ImpRes Chromatography (GE Healthcare). In this example, the first mixed-mode chromatography is performed in flow-through mode. Before loading the sample (e.g., affinity eluate) onto the mode column, the column Equilibration may be carried out using a suitable buffer.

[0042] In other embodiments, the antibody sample (e.g., affinity chromatography solution) The extraction solution can be controlled by adjusting the sample load, pH, conductivity, and ionic strength. Prepare for the merge step.

[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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for purifying an antibody, comprising: The pH is adjusted to a conductivity of 10 to 30 mS / cm, and the third pH is adjusted to a pH of about 10 to about 200 g / cm. The load density is adjusted to about 6.5 to 7.5 L. Preferably, the conductivity is 15 mS / cm. 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. Treat the prepared sample with ion exchange chromatography in flow-through mode. and e. collecting the flow-through containing the antibody; a method for increasing antibody yield, comprising: The conductivity of the solution is adjusted to a conductivity of 10 to 30 mS / cm, and the pH of the third solution is adjusted to a pH of about 10 to about 2. The conductivity is adjusted to about 6.5-7.5 at a loading density of 0.00 g / L. Preferably, the conductivity is 15 mS. / cm, and the third pH was adjusted to pH 7.1 at a loading density of 20-40 g / L. do.

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

[0046] In one aspect, the antibody sample is subjected to a multimodal anion exchange chromatography step. Approximately 1 to 300g, approximately 5 to 200g, approximately 10 to 100g per liter of resin material , about 20 to 50 g, 20 to 40 g range is applied.

[0047] In one embodiment, the present disclosure provides: a. Affinity chromatography with a first pH ( AC) providing an eluate; 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the anti-IL-17C antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific to IL-17C by immunohistochemistry, In step c), the pH and conductivity of the eluate are adjusted to 5% (v / v) with 2 M Tris, 10 %(v / v), 15%(v / v), 20%(v / v), or 5%(v / v)~20%( v / v) in which the antibody is fused to the amino acid sequence of SEQ ID NO: 1. an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 2; an HCDR2 region comprising the amino acid sequence of SEQ ID NO: HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4 a region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; a LCDR3 region comprising a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, and 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 less It comprises a variable heavy chain and a variable light chain, both of which share 99% sequence identity.

[0048] In another embodiment, the present disclosure provides: a. An affinity chromatograph having a first pH of about 3 to about 4, comprising an anti-IL-17C antibody; providing an analyte chromatography (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific to IL-17C by immunohistochemistry, In step c), the pH and conductivity of the eluate are adjusted to 5% (v / v) with 2 M Tris, 10 %(v / v), 15%(v / v), 20%(v / v), or 5%(v / v)~20%( v / v) in which the antibody is fused to the amino acid sequence of SEQ ID NO: 1. an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 2; an HCDR2 region comprising the amino acid sequence of SEQ ID NO: HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4 a region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; a LCDR3 region comprising a variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, and 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 less It comprises a variable heavy chain and a variable light chain, both of which share 99% sequence identity.

[0049] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , in which the pH and conductivity of the eluate in step c) are adjusted to 5% (v / v) with 2 M Tris. , 10%(v / v), 15%(v / v), 20%(v / v), or 5%(v / v)~2 0% (v / v) Tris concentration range, in which the antibody is an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 2; an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2; HCDR3 region containing the amino acid sequence of SEQ ID NO: 3, LC containing the amino acid sequence of SEQ ID NO: 4 DR1 region, comprising the amino acid sequence of SEQ ID NO: 5; LCDR2 region, comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the sequence of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9, and 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 less It comprises a heavy chain and a light chain, both of which share 99% sequence identity.

[0050] In another embodiment, the present disclosure provides: a. An affinity chromatograph having a first pH, comprising an anti-(snti-)IL17C antibody providing an analyte chromatography (AC) eluate; 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 , in which the pH and conductivity of the eluate in step c) are adjusted to 5% ( v / v), 10%(v / v), 15%(v / v), 20%(v / v), or 5%(v / v) v) Tris concentration is adjusted to a range of 20% (v / v), in which the antibody is HCDR1 region containing the amino acid sequence of SEQ ID NO: 1, HCDR containing the amino acid sequence of SEQ ID NO: 2 2 region, an HCDR3 region containing the amino acid sequence of SEQ ID NO: 3, and an HCDR4 region containing the amino acid sequence of SEQ ID NO: 4 an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; The LCDR3 region contains the amino acid sequence of the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7. and 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 9 It comprises a variable heavy chain and a variable light chain having 8% or at least 99% sequence identity.

[0051] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 , in which the pH and conductivity of the eluate in step c) are adjusted to 5% ( v / v), 10%(v / v), 15%(v / v), 20%(v / v), or 5%(v / v) v) Tris concentration is adjusted to a range of 20% (v / v), in which the antibody is HCDR1 region containing the amino acid sequence of SEQ ID NO: 1, HCDR containing the amino acid sequence of SEQ ID NO: 2 2 region, an HCDR3 region containing the amino acid sequence of SEQ ID NO: 3, and an HCDR4 region containing the amino acid sequence of SEQ ID NO: 4 an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; The LCDR3 region contains the amino acid sequence of the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7. and 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 9 It comprises a variable heavy chain and a variable light chain having 8% or at least 99% sequence identity.

[0052] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 , in which the pH and conductivity of the eluate in step c) are adjusted to 5% ( v / v), 10%(v / v), 15%(v / v), 20%(v / v), or 5%(v / v) v) Tris concentration is adjusted to a range of 20% (v / v), in which the antibody is HCDR1 region containing the amino acid sequence of SEQ ID NO: 1, HCDR containing the amino acid sequence of SEQ ID NO: 2 2 region, an HCDR3 region containing the amino acid sequence of SEQ ID NO: 3, and an HCDR4 region containing the amino acid sequence of SEQ ID NO: 4 an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; The LCDR3 region contains the amino acid sequence of SEQ ID NO: 10, the heavy chain of SEQ ID NO: 9, and the light chain of SEQ ID NO: 9. 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 comprises a heavy chain and a light chain, which have at least 99% sequence identity.

[0053] In a preferred embodiment, the pH and Conductivity adjustment is performed on the flow-through after multimodal anion exchange chromatography. In one embodiment, a 5% COOH / 2 O2 elution using 2 M Tris, pH 7.1 results in increased antibody yield. Adjustment to % (v / v) concentration is performed using a multimodal anion exchange chromatography step. Later, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 9%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 In another embodiment, 2M Tris, pH 7.1 is used to obtain antibody yields of 9%, 90%, or greater. The adjustment to a 10% (v / v) concentration used was performed using multimodal anion exchange chromatography. -After steps, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, In another embodiment, the antibody yield is 88%, 89%, 90% or more. Adjustment to a 15% (v / v) concentration with H7.1 was performed on the multimodal anion exchange chromatograph. After the tography step, 70%, 71%, 72%, 73%, 74%, 75%, and 76% ,77%,78%,79%,80%,81%,82%,83%,84%,85%,86% In another embodiment, the antibody yield is 2M, 87%, 88%, 89%, 90% or greater. Adjustment to a 20% (v / v) concentration with Tris, pH 7.1, allows for the preparation of multimodal anions. After the exchange chromatography step, the following were obtained: 70%, 71%, 72%, 73%, 74%, 75% %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, and 90% or higher antibody yields.

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

[0055] In certain embodiments, the antibodies to be purified are IgA1, IgA2, IgD, IgE, Ig G1, 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 present disclosure refers to the purification of antibodies specific for IL-17C. In embodiments, the mAb to be purified is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, 80%, 85%, 90% in the CDR compared to the CDRs of SEQ ID NO: 5 and SEQ ID NO: 6 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more Share an identity.

[0058] Antibody preparations to which the present invention can be applied include crude antibody preparations from natural, synthetic, or recombinant sources. Antibody samples include, for example, solubilized cells and cellular In certain embodiments, it may be a cell culture material such as a clarified The method of the present invention is to extract antibodies from any mixture containing antibodies. For example, such a mixture can be used as a final purification step to purify proteins. The resulting solution may be an eluate.

[0059] Additionally, the present invention includes one or more antibodies purified by the methods described herein. The present invention relates to a pharmaceutical composition comprising:

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

[0061] In preferred embodiments, the methods provided herein provide a method for detecting 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 more SEC monomer content. In this embodiment, the purified protein has 100% SEC monomer content.

[0062] In another embodiment, the methods provided herein provide a method for treating 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Yields of purified antibody are obtained in excess of 94%, 95%, 96%, 97%, 98%, and 99%.

[0063] In another embodiment, the present disclosure provides: a. Affinity chromatography with a first pH ( AC) providing an eluate; 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 in which the pH and conductivity of the eluate in step c) are adjusted using Tris wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3 a region comprising the amino acid sequence of SEQ ID NO: 4; a LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8; a variable heavy chain and a variable light chain of SEQ ID NO: 7, and 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, It contains a variable heavy chain and a variable light chain.

[0064] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 in which the pH and conductivity of the eluate in step c) are adjusted using Tris wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3 a region comprising the amino acid sequence of SEQ ID NO: 4; a LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8; a variable heavy chain and a variable light chain of SEQ ID NO: 7, and 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, It contains a variable heavy chain and a variable light chain.

[0065] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 in which the pH and conductivity of the eluate in step c) are adjusted using Tris wherein the antibody comprises an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3 a region comprising the amino acid sequence of SEQ ID NO: 4; a LCDR1 region comprising the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 10; and a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 10, %, at least 93%, at least 94%, at least 95%, at least 96%, at least heavy chain and a heavy chain having at least 97%, at least 98%, or at least 99% sequence identity Contains light chains.

[0066] In another embodiment, the present disclosure provides: a. An affinity chromatography (AC) eluate having a first pH, comprising an antibody. providing a 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific to IL-17C by immunohistochemistry, wherein the pH and conductivity of the eluate of step c) is adjusted with Tris, 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 HCDR2 region comprising the amino acid sequence of SEQ ID NO: 3; an HCDR3 region comprising the amino acid sequence of SEQ ID NO: LCDR1 region comprising the amino acid sequence of SEQ ID NO:4, LCDR2 region comprising the amino acid sequence of SEQ ID NO:5 a LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a sequence of SEQ ID NO: 8. Variable light chain of sequence number 7 and at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, or at least 99% sequence identity between the variable heavy chain and the variable Contains light chains.

[0067] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific to IL-17C by immunohistochemistry, wherein the pH and conductivity of the eluate of step c) is adjusted with Tris, 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 HCDR2 region comprising the amino acid sequence of SEQ ID NO: 3; an HCDR3 region comprising the amino acid sequence of SEQ ID NO: LCDR1 region comprising the amino acid sequence of SEQ ID NO:4, LCDR2 region comprising the amino acid sequence of SEQ ID NO:5 a LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a variable heavy chain and a sequence of SEQ ID NO: 8. Variable light chain of sequence number 7 and at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 9 7%, at least 98%, or at least 99% sequence identity between the variable heavy chain and the variable Contains light chains.

[0068] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific to IL-17C by immunohistochemistry, wherein the pH and conductivity of the eluate of step c) is adjusted with Tris, 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 HCDR2 region comprising the amino acid sequence of SEQ ID NO: 3; an HCDR3 region comprising the amino acid sequence of SEQ ID NO: LCDR1 region comprising the amino acid sequence of SEQ ID NO:4, LCDR2 region comprising the amino acid sequence of SEQ ID NO:5 a heavy chain and a sequence of SEQ ID NO: 10; a LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6; 9 light chain and at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It comprises a heavy chain and a light chain having at least 98% or at least 99% sequence identity.

[0069] In another embodiment, the present disclosure provides: a. An affinity chromatography (AC) eluate having a first pH, comprising an antibody. providing a 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 wherein the pH and conductivity of the eluate in step c) are Tris; in which the antibody is prepared using H comprising the amino acid sequence of SEQ ID NO: 1. CDR1 region, comprising the amino acid sequence of SEQ ID NO: 2; HCDR2 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 LCDR1 region comprising the amino acid sequence of SEQ ID NO: 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 of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, at least 90%, at least 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% It comprises a variable heavy chain and a variable light chain that have sequence identity.

[0070] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 wherein the pH and conductivity of the eluate in step c) are Tris; in which the antibody is prepared using H comprising the amino acid sequence of SEQ ID NO: 1. CDR1 region, comprising the amino acid sequence of SEQ ID NO: 2; HCDR2 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 LCDR1 region comprising the amino acid sequence of SEQ ID NO: 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 of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, at least 90%, at least 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% It comprises a variable heavy chain and a variable light chain that have sequence identity.

[0071] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 wherein the pH and conductivity of the eluate in step c) are Tris; in which the antibody is prepared using H comprising the amino acid sequence of SEQ ID NO: 1. CDR1 region, comprising the amino acid sequence of SEQ ID NO: 2; HCDR2 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 LCDR1 region comprising the amino acid sequence of SEQ ID NO: 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 of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9, and 1%, 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 It comprises heavy and light chains having the same molecular weight.

[0072] In another embodiment, the present disclosure provides: a. An affinity chromatography (AC) eluate having a first pH, comprising an antibody. providing a 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. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , wherein the pH and conductivity of the eluate of step c) are determined by adding Tris in the absence of NaCl. wherein the antibody is prepared using an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1. a region containing the amino acid sequence of SEQ ID NO: 2; an HCDR2 region containing the amino acid sequence of SEQ ID NO: 3; an HCDR3 region containing the amino acid sequence of SEQ ID NO: 4; an LCDR1 region containing the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6; A variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, with at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98% or at least 99% sequence identity The variable heavy chain and the variable light chain have the following structure:

[0073] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , wherein the pH and conductivity of the eluate of step c) are determined by adding Tris in the absence of NaCl. wherein the antibody is prepared using an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1. a region containing the amino acid sequence of SEQ ID NO: 2; an HCDR2 region containing the amino acid sequence of SEQ ID NO: 3; an HCDR3 region containing the amino acid sequence of SEQ ID NO: 4; an LCDR1 region containing the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6; A variable heavy chain of SEQ ID NO: 8 and a variable light chain of SEQ ID NO: 7, with at least 90%, at least 91% %, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98% or at least 99% sequence identity The variable heavy chain and the variable light chain have the following structure:

[0074] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , wherein the pH and conductivity of the eluate of step c) are determined by adding Tris in the absence of NaCl. wherein the antibody is prepared using an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1. a region containing the amino acid sequence of SEQ ID NO: 2; an HCDR2 region containing the amino acid sequence of SEQ ID NO: 3; an HCDR3 region containing the amino acid sequence of SEQ ID NO: 4; an LCDR1 region containing the amino acid sequence of SEQ ID NO: 5; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6; A heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9, with 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 It comprises a heavy chain and a light chain.

[0075] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for increasing antibody yield during purification by chromatography, which method comprises: The pH and conductivity of the eluate in step c) are adjusted to 5% (v / v), 10% (v / v) with 2 M Tris. v), 15%(v / v), 20%(v / v), or 5%(v / v)~20%(v / v) and a range of Tris concentrations in which the antibody has a heavy chain of SEQ ID NO: 10 and a sequence It has a light chain number 9.

[0076] In another embodiment, the present disclosure provides: a. Affinity chromatography with an antibody having a first pH of about 3 to about 4 ( AC) providing an eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies by centrifugation, in which step c) The pH and conductivity of the exudate were adjusted to 5% (v / v), 10% (v / v), and 15% (v / v) with 2M Tris. / v), 20% (v / v), or in the range of Tris concentrations from 5% (v / v) to 20% (v / v) The antibody is adjusted to a range of SEQ ID NO: 10 heavy chain and SEQ ID NO: 9 light chain. do.

[0077] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 wherein the pH and conductivity of the eluate in step c) are Tris; in which the antibody has a heavy chain of SEQ ID NO: 10 and a heavy chain of SEQ ID NO: 9 It has a light chain of

[0078] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , wherein the pH and conductivity of the eluate of step c) are determined by adding Tris in the absence of NaCl. wherein the antibody comprises a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9. Has.

[0079] In another embodiment, the present disclosure provides: a. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including for increasing the yield of antibodies specific for IL-17C during purification by immunohistochemistry - Patent Application 20070122997 wherein the pH and conductivity of the eluate in step c) are 5%(v / v), 10%(v / v), 15%(v / v), 20%(v / v) in 2M Tris or adjusted to a Tris concentration range of 5% (v / v) to 20% (v / v), among which , 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. An affinity chromatography column having a first pH of about 3 to about 4 containing an anti-IL17C antibody providing an chromatographic (AC) eluate; b. Adjust the first pH of the AC eluate to a second pH of about 5.2 to about 5.6, preferably 5.5. and adjusting the c. Adjust the conductivity of the eluate to 10 to 30 mS / cm, preferably 15 mS / cm. and adjusting the second pH to a third pH of about 7.1; d. Subject the conditioned eluate to ion exchange chromatography in flow-through mode. Steps and; e. collecting the flow-through containing the antibody; Multimodal anion exchange (MM-AIEX) chromatograph in flow-through mode, including The present invention relates to a method for purifying antibodies specific for IL-17C during purification by chromatographic techniques. , wherein the pH and conductivity of the eluate of step c) is adjusted to 2M Tris(2M HCl) in the absence of NaCl. 5% (v / v), 10% (v / v), 15% (v / v), 20% (v / v) or The Tris concentration is adjusted to a range of 5% (v / v) to 20% (v / v), in which the above-mentioned antibody 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 at least 10 mS / cm, between 10 and 50 mS / cm. m range, 10-30mS / cm, 11-30mS / cm, 12-30mS / cm, 13 ~30mS / cm, 10~29mS / cm, 10~28mS / cm, 10~27mS / c m, 10~26mS / cm, 11~29mS / cm, 11~28mS / cm, 11~27 mS / cm, 11~26mS / cm, 12~29mS / cm, 12~28mS / cm, 1 2~27mS / cm, 12~26mS / cm, 13~29mS / cm, 13~28mS / cm, 13-27mS / cm, 13-26mS / cm, or 13-25mS / cm range is adjusted to.

[0082] definition The term "protein" as used herein refers to a molecule or molecules that are linked together through peptide bonds. The term refers to a continuous chain of amino acids. The term can be used to refer to an amino acid chain of any length. However, those skilled in the art will appreciate that the term is not limited to long chains, but includes two peptides linked together via a peptide bond. It will be understood that the term "amino acid" can refer to a minimal chain containing at least one amino acid. "peptide," "peptide fragment," "polypeptide," "amino acid chain," "amino acid sequence," "string," or any other term used to refer to two or more amino acid chains or groups of amino acid chains The terms are used in general terms, although each of these terms may have a more specific meaning. The term "protein" is used in place of these terms. The term may be used interchangeably with or synonymously with either of these terms. undergo post-translational or post-synthetic modifications such as glycosylation, acetylation, phosphorylation, or amidation The protein further comprises:

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

[0084] "Tris" or tris(hydroxymethyl)aminomethane has the formula (HOCH2)3CN It is an organic compound containing H2. Synonyms include TRIS, Tris, Tris base, Tris buffer, Trizma, Trisamine, THAM, tromethamine, tromethamine 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)" refers to 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. A polypeptide carries a net positive charge at a pH below its pI. At a given pH, a polypeptide carries a net negative charge. The actual pI of a polypeptide can be determined based on its ionization state. The actual pI can be determined by experimental methods such as isoelectric focusing. It can be done.

[0086] The term "chromatography" refers to the removal of, for example, impurities and / or other non-target molecules. Current or future chromatographic methods for purifying one or more target molecules from a sample by removing refers to a chromatography-based process. During chromatography, e.g., polypeptides The solutes of interest in a mixture of two or more molecules are transported through a stationary medium under the influence of the mobile phase. It is separated from other solutes in a mixture as a result of differences in the rate at which they move or in a binding and elution process. Examples of liquid chromatography purification include, but are not limited to: These include: affinity chromatography, immobilized metal ion affinity -chromatography, flow-through chromatography, ion-exchange chromatography -, size exclusion chromatography, reversed phase chromatography, simulated moving bed chromatography Fee, hydrophobic interaction chromatography, gel filtration, chromatofocusing.

[0087] "Mixed-mode chromatography" or "multimodal chromatography" The term refers to the interaction of hydrophobicity, cation exchange, and hydrogen bonding between the polypeptide of interest and the adsorbent ligands. This refers to a purification process using a mixed-mode adsorbent that provides multiple interaction modes such as Commercially available mixed-mode chromatography resins include GE Healthcare Life Capto™ MMC, Capto™ MMC I from e Sciences mpRes, Capto Blue, Blue Sepharose(TM)6 Fas t Flow, Capto™ Adhere, and Capto™ Adhere ImpRes; or Eshmuno® H from EMD Millipore CX; or Nuvia™ cPrime from Bio-Rad.

[0088] "Cation exchange resin," "cation exchange adsorbent," or "cation exchange matrix" The term refers to the free cations that are exchanged with cations in the aqueous solution on or through the solid phase. The term refers to a negatively charged solid phase having a negatively charged ligand attached to the solid phase to form a cation exchange resin. The cation exchanger may be, for example, a carboxylate or a sulfonate. Resins include carboxymethylcellulose and sulfopropyl immobilized on agarose. (SP) (e.g., S from GE Healthcare Life Sciences) P Sepharose(TM) XL, SP-Sepharose(TM) Fast F low, SP Sepharose™ High Performance, CM Sepharose(TM) Fast Flow, CM Sepharose(TM) H igh Performance, Capto(TM) S, and Capto(TM) SP ImpRes; or Fractogel® from EMD Millipore ) EMD SE HiCap, Fractogel® EMD SO3”, Fr actogel® EMD COO”, Eshmuno™ S, and Esh muno™ CPX; or UNOsphere™ S from Bio-Rad and Nuvia (trademark) S).

[0089] "Anion exchange resin," "anion exchange adsorbent," or "anion exchange matrix" The term refers to a molecule having one or more positively charged ligands attached thereto, such as, for example, a quaternary amino group. It is used herein to refer to a positively charged solid phase on which a commercially available anion exchange The resin was DEA from GE Healthcare Life Sciences. E Sepharose(TM) Fast Flow, Q Sepharose(TM) Fast Flow, Q Sepharose™ High Performance e, Q Sepharose(TM) XL, Capto(TM) DEAE, Capto( ™ Q, and Capto™ Q ImpRes; or EMD Millipore Fractogel® EMD TMAE HiCap from Fractog el® EMD DEAE, and Eshmuno Q; or from Bio-Rad Examples of such products include U Osphere™ Q and Nuvia™ Q from Pharmacy.

[0090] The term "antibody" refers to the five major classes (isotypes) of immunoglobulins: IgA , IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), and combinations thereof It refers to any glycosylated and non-glycosylated immunoglobulin variant. In usage, the term refers to antibodies from any species (e.g., murine, canine, feline, IgY, etc.). This includes antibodies, human antibodies, humanized antibodies, chimeric antibodies, and combinations thereof, for example. The terms monoclonal and polyclonal antibodies, as well as monospecific and polyspecific As used herein, the term also refers to an antibody (such as a bispecific antibody). fusion proteins containing the antigen recognition site, and any other modified immunoglobulin molecule containing the antigen recognition site As used herein, the term "antibody" includes intact immunoglobulins as well as These include antibody fragments, which are one or more portions of an antibody that retain the ability to specifically interact with an antigen. refers to multiple moieties (e.g., by binding, steric hindrance, stabilizing spatial distribution). Examples of such fragments include Fab, Fab', F(ab')2, Fd, Fv, and dAb fragments (Wa rd et al., (1989) Nature 341:544-546), single-stranded F v(scFv) (e.g. Bird et al., (1988) Science 24 2:423-426; and Huston et al., (1988) Proc. Nat. l. Acad. Sci. 85:5879-5883), but are not limited to It is not a naturally occurring, enzymatically accessible, synthetic, or alternative skin antigen that specifically binds to an antigen. Any polypeptide, whether synthetic or engineered, is referred to as a "polypeptide" as used herein. Antibodies are intended to be encompassed by the term "antibody."

[0091] The terms "contaminant" and "impurity" are used interchangeably herein and are not intended to be limiting unless otherwise specified. The method is used to separate a target protein from one or more foreign or unwanted molecules. DNA, RNA, one or more host cells that may be present in the protein-containing sample. including biopolymers such as cellular proteins, endotoxins, lipids, and one or more additives. Furthermore, such contaminants are removed prior to the purification step. The reagents may include any reagents used in the steps that may be used.

[0092] "High molecular weight (HMW) species" refers to multimers and other molecules with a molecular weight higher than the mass of the target protein. Multimers include all but the monomers of the target protein. For example, an IgG antibody monomer is a conventional tetrameric antibody composition containing two heavy chains and a light chain. The multimers include dimers (two identical molecules bound by covalent or non-covalent bonds). proteins) and aggregates (covalently or non-covalently bound whole proteins and / or These include species with a higher molecular weight than the target protein mass, such as a protein moiety.

[0093] "Low molecular weight (LMW) species" are those with molecular weights lower than the target protein such as clips. These include lower species and degradation products.

[0094] As used herein, the term "final purification" refers to the first (affinity) capture step. It occurs after the capture step and is present in the product stream and is typically removed during the capture step. The intent is to remove residual amounts of impurities that are more similar to the product than the impurities introduced. Refers to downstream processing steps.

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

[0096] The term "binding and elution mode" refers to the binding of at least one product contained in a sample. (e.g., Fc region containing proteins) bound to a chromatography resin or medium , followed by elution, refers to a product separation technique.

[0097] The term "flow-through mode" refers to the mode in which contaminants bind to the chromatographic support. It refers to the conditions under which 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. do.

[0099] [Table 1]

[0100] [Table 2]

[0101] [Table 3] [Example]

[0102] Example 1: A Capto adhere ImpRes column (GE Healthcare) was used. Prior to loading in the flow-through, the antibody was prepared by adding a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9. The effect of adding Tris to a sample containing PEG (i.e., increasing the Tris concentration) on the yield and purity was investigated. To investigate, four different purification runs were performed: one without Tris, one with sample eluate, and one with HCl. Three runs were performed with 2M Tris pH 7.1 added at 5, 10, and 20% (v / v) to the sample. The collected flow-through was analyzed for antibody yield and purity (SEC monomer). The results are listed in Table 2. The corresponding chromatograms are shown as an overlay in Figure 2. Addition of 5% (v / v) Tris The addition of HCl increased the yield by 20%, while the SEC monomer fraction of the resulting pool decreased by only 0.4%. Increasing the amount of Tris added to the sample resulted in a further increase in yield, but to a lesser extent. Lower (5% Tris addition compared with 10% and 20% Tris addition) by approximately 4% and 5%, respectively (approximately 7% increase) with a further slight decrease in the monomeric portion.

[0103] [Table 4]

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

[0105] [Table 5]

[0106] Both loads had the same conductivity, but the buffer matrices were different. The yield measurements were carried out at an ambient temperature of 20° C.±2° C. The purification results and QC data are shown in Table 4.

[0107] [Table 6]

[0108] For the Tris-free run (Run 2), the conductivity was adjusted to 15 mS / cm in 2 M Tris, pH 7.1. Compared to run 1, the yield was approximately 5% lower.

Claims

1. a. providing a sample containing an antibody; b. adjusting the conductivity of the sample; c. The prepared sample was subjected to ion exchange chromatography in flow-through mode. processing; d. collecting the flow-through containing the antibody; and wherein the conductivity of the sample in step b) is at least 10 mS / cm, and the pH after adjusting for conductivity is in the range of pH 6.5 to 7.

5. Increase antibody yield in ion exchange chromatography flow-through during purification How to do this.

2. The antibody-containing sample is an affinity chromatography eluate.

1. The method according to claim 1.

3. The affinity chromatography eluate has a pH of about 3 to about 4.

3. The method of claim 2, wherein the eluate is a methionine A chromatography eluate.

4. The pH of the sample of about 3 to about 4 is changed to a pH of about 5.2 to about 5.6, preferably 5.

5. The method of claim 3, wherein the pH is adjusted.

5. 10. The method of claim 1, wherein the conductivity of the sample is adjusted to a conductivity between 10 and 50 mS / cm. The method according to any one of claims 1 to 4.

6. The method of claim 5, wherein the conductivity is adjusted to a range of 13 to 30 mS / cm.

7. 6. The method of claim 5, wherein the conductivity is adjusted to 15 mS / cm.

8. The ion exchange chromatography is a multimodal anion exchange chromatography. The method according to any one of claims 1 to 7, wherein

9. The method according to any one of claims 1 to 8, wherein the monoclonal antibody to be purified is a monoclonal antibody. The method according to any one of claims 1 to 5.

10. 10. The method of claim 9, wherein the purified monoclonal antibody is an anti-IL17c antibody. 。

11. The purified monoclonal anti-IL17C antibody comprises a VH of SEQ ID NO: 8 and a VH of SEQ ID NO: 7 The method of claim 10, comprising the VL of

12. The purified monoclonal anti-IL17C antibody comprises a heavy chain of SEQ ID NO: 10 and a heavy chain of SEQ ID NO:

12. The method of claim 11, wherein the antibody comprises 9 light chains.

13. 13. Any of claims 1 to 12, wherein the yield of the purified antibody in the flow-through is greater than 75%. The method according to any one of claims 1 to 4.

14. The conductivity of the sample of step b) is adjusted with Tris in the absence of NaCl. The method according to any one of claims 1 to 13,

15. A pharmaceutical composition obtainable by the method according to any one of claims 1 to 14.

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