Eluate collection during antibody chromatography steps

Abstract

The present invention relates to an improved method for peak fractionation and eluate collection during a chromatography step aimed at the purification of human therapeutic antibodies.

Description

[Technical Field]

[0001] The present disclosure relates to protein purification methods. In particular, the present invention relates to an improved method for peak collection during an antibody elution step from a chromatography resin by using an optimized peak cut for initiating eluate collection. This optimized peak cut can be consistently and robustly applied to initiate eluate collection in elution runs with a variety of elution peak widths and heights. More specifically, the present invention relates to a method for purifying a human therapeutic antibody by applying an improved peak cut for initiating eluate collection during a CEX purification step, and to a purified antibody composition obtained by the method disclosed herein. [Background technology]

[0002] For therapeutic administration, every drug substance must meet different quality standards. For example, to ensure the clinical safety and tolerability of a therapeutic monoclonal antibody (mAb), one or more purification steps must follow the mAb manufacturing process to remove unwanted contaminants, such as aggregated and fragmented products, nucleic acids, viruses, host cell proteins (HCPs), residual media components, and cell culture additives. Therefore, the goal of all manufacturing and purification process development is to establish a reliable, reproducible, and robust method that results in a protein product with high purity and yield. Importantly, maintaining consistent product quality from batch to batch is essential in pharmaceutical manufacturing.

[0003] To achieve adequate homogeneity and meet the high quality standards of therapeutic-grade products used in clinical applications, so-called purification steps are required in the purification process stream. Often, these purification steps include ion exchange chromatography (IEX), which is used after the initial antibody affinity chromatography to remove remaining aggregates and impurities.

[0004] Besides the selection of resin type and conditions (e.g., loading density, bind-elute mode or flow-through mode) applied during the purification process, the selection of the fraction collection mode (Figure 1) is another important aspect. "Peak collection" is the most efficient mode of eluate collection and can be used to increase the purity of the collected protein. To do so, appropriate peak collection specifications must be determined to start and end eluate collection during elution. Peak collection ensures that the desired collected fraction contains minimal amounts of material eluted in adjacent fractions (not collected). Peak collection can be performed manually or automatically. In manual mode, the operator decides to switch to the next fraction collection tube. In automated fraction collection mode, defined integration parameters (e.g., absorbance) control the collection. Changes in sample composition (e.g., pH, salt concentration, conductivity, protein concentration, loading density) can lead to variations in peak shape that may not be recognized by the entered integration parameters, so automated peak collection is only recommended if the sample composition does not change between chromatographic runs.

[0005] For industrial-scale production, peak cut parameters are typically specific signals of absorbance at 280 nm (A280), conductivity, or pH, which are predetermined as thresholds for starting and stopping fraction collection. For example, Borg et al. (J Chromatogr A. 2014 Sep 12; 1359:170-81) disclose a pooling design that applies a constant starting collection criterion triggered when the optical density (OD) at 280 nm reaches 0.5 during peak rise. The end cut point is determined by a percentage of the maximum peak value. WO2014140570 relates to controlling the collection of eluate output from a separation process, in which collection is started and stopped when a defined measure of suspended material reaches a distinct threshold. Yigzaw et al. (Curr Pharm Biotechnol. 2009 Jun; 10(4):421-6) describe stopping eluate collection based on absorbance fluctuations at different percentages of the peak maximum as end cut points. Westerberg et al. (Bioprocess Biosyst Eng. 2010 Mar;33(3):375-82) disclose an HIC case study in which model simulation was used to determine the parameter with the greatest impact on the selection of the initial cut point for initiating collection. In this case, the parameter was determined to be the conductivity of the load buffer. A function was then established to estimate the dependence of the absorbance signal at the cut point on the conductivity of the sample load. The maximum value of the function at a particular conductivity was then determined to be the ideal cut point.

[0006] US20160272673 describes a chromatographic method for isolating and purifying DVD-Igs™ from a sample, where the purified DVD-Igs™ is reduced in host cell proteins, aggregates, and viruses compared to the sample. US20160264618 discloses a method for purifying antibodies by cation exchange chromatography, where collection of eluate begins when the UV signal in the chromatogram rises to a predetermined value of 50 mAU, 100 mAU, or more, depending on the elution run.

[0007] In general, the challenge in preparative fractionation lies in determining optimal peak cut points for starting and stopping eluate collection so that product recovery is maximized and impurity concentrations are absent or low and within specification. Determining optimized peak collection criteria (for pooling product eluates) that are insensitive to batch-to-batch variations in sample composition and various process parameters (e.g., pH, load) is crucial for a scale-independent method of purifying antibody proteins. In particular, purification methods that result in compositions with improved yields, high purity, and at low cost are of great value for process development.

[0008] US20130303732 provides a method for controlling contaminants in a biopharmaceutical purification process by using light scattering and UV absorbance as a continuous monitoring system to provide information about the elution peak fraction in real time, rather than traditional pooling methods that rely on a predetermined percentage of UV peak maximum to initiate the pooling process independently of product quality.

[0009] It will be appreciated that the technical problem underlying this application is to provide a means for improving antibody elution during chromatography, along with efficient reduction of aggregates and impurities, by simultaneously maintaining optimal yield of the target antibody independent of the pH of the elution buffer, salt concentration, and loading density. The present invention fulfills these needs by providing a method for specifying a reference absorbance signal to be used together with a planned fluid volume to find an optimized starting point for eluate collection. The advantage is that an ideal eluate collection start is achieved independent of the often unpredictable peak heights and shapes of elution runs under various conditions. Furthermore, a method for peak collection by applying these improved peak start criteria during the purification of therapeutic antibodies is also provided. Summary of the Invention [Means for solving the problem]

[0010] Here, we provide a method for purifying a protein by chromatography, which comprises: a) loading a sample containing protein onto a chromatography resin; b) optionally washing the resin; c) applying an elution buffer to the chromatography resin; and d) starting the collection of eluate, where the collection of eluate begins at a predetermined interval (D0) after the absorption signal of the eluate reaches a predetermined value (A0); Includes:

[0011] The determination of the absorption signal A0, which is used as a reference signal to obtain the start signal for collecting the eluate at predetermined intervals D0 after A0 is reached, is as follows: a) At least two different elution runs containing the protein sample to be purified (ER1, , ER N ) (N=an integer equal to or greater than 2) elution peak chromatograms, wherein the different elution runs vary in pH, loading density, or salt conditions; and b) designating absorbance values A0 in the elution peaks, where A0 is the absorption signal (A) at the peak maximum of each elution peak; MAX 1,...,A MAX N), and ER1, . . ., ER N The steps are the same for each elution peak. Includes:

[0012] The scheduled interval D0 is as follows: a) At least two different elution runs containing the protein sample to be purified (ER1, , ER N receiving an elution peak chromatogram of the chromatogram of the sample, wherein the different elution runs vary in pH, loading density, or salt conditions; b) For each of the elution peaks received in step a), the absorption signals A1,...,A N A step of specifying A1, . . . , A N wherein each of the corresponding eluate fractions has an aggregate / impurity content of less than 5%; c) Absorption signals A1, . . . , A for each elution run in the chromatogram N Fluid volumes C1, . . ., C N determining d) The fluid quantities C1, . . . , C relative to the fluid quantity A0 (which is C0) N The differences between D1, . . ., D N calculating e) the differences D1, . . . , D N Averaging (i.e., averaging) the values of the predetermined fluid volume interval D0. The fluid volume intervals are obtained by a method including:

[0013] Additionally, the present inventors provide a method for purifying an antibody, comprising applying a mixture containing an antibody and aggregates / impurities onto an ion exchange chromatography resin, washing the resin, eluting the antibody from the chromatography resin, and collecting eluate fractions by peak collection using a collection start point determined by the method described herein, which is insensitive to different peak widths and heights.

[0014] This method is particularly suited for purifying antibody samples that are characterized by chromatographic peaks with aggregate / impurity accumulation at the very front (i.e., rising) of the peak. For example, as in the case of high molecular weight (HMW) and low molecular weight (LMW) impurities, the impurities are concentrated at the beginning of the rapidly increasing peak in the unpurified antibody sample, as shown in Figure 2.

[0015] Rather than determining a distinct A280 absorption signal (absorbance measured at UV 280 nm) to initiate eluate collection, we determined the A280 absorption signal (herein designated as A0) that indicates the occurrence of a peak. A0 may be the same, for example, in terms of the corresponding fluid volume (unit of measurement: column volume, CV) between the normalized elution peaks of at least two different elution runs, where the different elution runs vary in pH, salt, or loading density conditions. Alternatively, A0 may be determined by the different elution runs E1, ..., E N 50% of the absorption signal of the peak maximum of the elution peak having the lowest height of the elution peaks of the different elution runs E1, ..., E N The elution peak is 10% to 50% of the total peak maximum.

[0016] The absorption signal A0 is measured in fluid volume C0 (unit of measurement: column volume, CV) for different elution runs E1, . . . , E N C0 intersects with the elution peak curve of the different elution runs E1,...,E NThe fluid volume of A0 (which is C0) will then be used along with a predetermined fluid volume interval (referred to herein as a "delay", e.g., 0.6 column volumes) at which the next A280 absorbance signal arrives, together with its corresponding fluid volume, to define the start point for eluate collection.

[0017] In one embodiment, the ion exchange chromatography step is a multimodal cation exchange chromatography step in bind-elute mode (Capto MMC ImpRes, GE Healthcare), where a salt gradient is used to elute the antibody from the column.

[0018] In one embodiment, the antibody to be purified is a monoclonal antibody. The present invention also provides monoclonal antibodies purified by a process using the methods of the present disclosure to separate the monoclonal antibody from aggregates and / or impurities in the process stream. [Brief explanation of the drawings]

[0019] [Figure 1] Schematic chromatograms of preparative mode using (A) quantitative prep and (B) peak prep (modified from AEKTA avant User Manual 29-0351-84 AD, GE Healthcare) are shown. [Figure 2] Representative chromatograms of an elution run during the CEX purification step are shown. The antibody monomer content (left Y-axis) and HMW and LMW aggregates / impurities (right Y-axis) of the eluate fractions (X-axis) are shown. The aggregates / impurities accumulate at the beginning of the elution peak (fractions H7, A8, B8, C8, D8, E8, F8, G8). [Figure 3A-3C]Figure 3 shows the elution peak chromatograms of the three test elution runs ER1, ER2, and ER3 used to determine the fluid volume interval D0. The shaded segments represent the optimal elution pooling area from 2300 to 400 mAU (for ER1, Figure 3A), 1800 to 400 mAU (for ER2, Figure 3B), and 1300 to 400 mAU (for ER3, Figure 3C), respectively. The start of eluate collection for each run is the absorption signal point with the corresponding eluate fraction containing less than 10% aggregates / impurities (compare with Figure 2). [Figures 4A-4G] Elution peak chromatograms for different elution runs #4 to #10 (ER4 to ER10) are shown, where a predetermined fluid volume interval D0 for starting eluate collection was applied and conditions were varied. [Figure 5] Normalized elution peak chromatograms with peak collection start and end points displayed for antibody purification using Capto MMC ImpRes are shown. The illustrated elution runs (ER4-ER10) were performed under seven different conditions, varying pH and loading density (Table 3). (a) Intersection of A0 and normalized C0; b) Planned D0 interval "delay" (here: 0.6 CV); c) Peak fraction collection start (start of eluate collection); d) Peak fraction collection range; e) Peak fraction collection stop (stop of eluate collection) (here: 400 mAU, UV 280 nm); A0: Planned absorbance signal measured at UV 280 nm (here: 700 mAU). [Figure 6] The concept of determining the interval D0 to be used to arrive at the starting point for eluate collection is shown. The elution peaks of two elution runs with varying peak shapes are illustrated schematically. The reference absorbance A0 and the absorbance signals at the maximum peak heights (AMAX1, AMAX2) and for the optimal elution start (A1, A2) are displayed, along with the corresponding fluid volumes C0, C1, and C2. The optimal elution start relates to elution fractions with aggregate content and impurities within specifications. Typically, fractions with less than 4% aggregates / impurities (i.e., HMW and LMW content) (i.e., greater than 96% monomer content) are considered optimal. [Figure 7]The concept of applying a scheduled interval D0 to elution peaks with different shapes (compared to the peak shapes illustrated in Figure 5) is shown. Eluate collection begins at fluid volume interval D0 when absorbance signal AX reaches CX. [Figure 8] 1 shows the phase characteristics of the predefined gradient elution phases in the Method Editor of UNICORN™ 7.1 (Build 7.1.0.378). DETAILED DESCRIPTION OF THE INVENTION

[0020] Protein purification by chromatography In the pharmaceutical industry, the manufacturing process of a target molecule, such as a therapeutic mAb, is typically divided into: i) upstream processing (USP), which includes production of the target protein; ii) downstream processing (DSP), which includes production of the target protein in a pure form by purification; and iii) final processing to achieve product integrity and safety.

[0021] Typically, the first step in the downstream purification process after the production stage involves clarification of the harvested cell culture broth, in which the desired protein is separated from cells, cell debris, and other contaminants using one or more steps of precipitation, flocculation, (depth) filtration, and / or centrifugation. The downstream purification process further includes one or more (orthogonal) chromatographic separation steps, such as affinity chromatography, ion exchange, hydrophobic interaction, hydroxyapatite, chromatofocusing, gel filtration, and reversed phase, to efficiently remove process- and product-related impurities. These contaminants include, but are not limited to, host cell proteins (HCPs), leached protein A, product isoforms, high molecular weight (HMW) species, low molecular weight (LMW) species, and excision or degradation products. Parameters that can lead to HMW species such as dimers and higher order aggregates (multimers) include, but are not limited to, protein concentration, pH, ionic strength, oxygen, temperature, salt concentration, shear forces, and external stresses (e.g., interaction with metal surfaces, exposure to air, freezing and / or thawing). Unwanted post-translational modifications or molecular unfolding can also promote aggregation.

[0022] Affinity chromatography refers to the use of compounds that specifically interact with the desired target protein to be purified. Typically, the compound is immobilized on a resin for the purpose of isolating, purifying, or removing the desired target product. For example, for antibody purification, affinity resins include Protein A from Staphylococcus aureus, Protein G from Streptococcus sp., Protein L from Peptostreptococcus magnus, and their recombinant or synthetic versions or peptides. Resins include MAbSelect™ (GE Healthcare) and Prosep A® (Millipore). For laboratory-scale applications, a single-step affinity purification generally achieves satisfactory purity. For example, Protein A chromatography is the most widely used method for capturing antibodies, and affinity purification maintains >95% purity due to its high specificity for the Fc portion of IgG. Other examples of purification methods include thiophilic adsorption, hydrophobic interaction or aromatic adsorption chromatography, metal chelate affinity chromatography, and size exclusion chromatography (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102). Further removal of residual aggregates and / or impurities can be achieved by a combination of one or two additional chromatography steps, which may include hydroxyapatite, hydrophobic interaction (HIC), and ion exchange chromatography (IEX, e.g., cation exchange (CEX), anion exchange (AEX), or mixed-mode exchange). At the manufacturing scale, removal of aggregates and / or impurities is often achieved by the use of ion exchange chromatography (IEX) after initial antibody affinity chromatography.Commercially available multimodal ion exchangers, such as Capto MMC and Capto adhere, as well as Capto MMC ImpRes and Capto adhere ImpRes (all from GE Healthcare), can be used for contaminant removal downstream of initial affinity capture. IEX separates proteins with different surface charges to achieve high-resolution resolution with high sample load capacities. Separation is based on the reversible electrostatic interaction between charged proteins (i.e., charged amino acid side chains) and the oppositely charged chromatographic medium. AEX contains positively charged functional groups (e.g., strong anion exchangers with quaternary amine groups or weak anion exchangers with secondary amine groups). At pH values higher than the isoelectric point (pI) of the target protein, the protein's net charge is negative, which promotes its binding to the positively charged resin. Elution of the target protein is performed using either an increasing salt gradient, a stepwise elution of predetermined pH and salt concentrations, or a gradual decrease in the pH of the elution buffer. In contrast, CEX involves the purification of proteins on resins with negatively charged functional groups (e.g., strong cation exchangers with sulfite groups or weak cation exchangers with carboxylate anions), where the target protein typically binds to the resin in a low-salt buffer solution with a pH lower than the pI of the target protein (i.e., the protein is positively charged). Elution of the target protein is performed either with an increasing salt gradient, by step elution at a predetermined pH and salt concentration, or by gradually increasing the pH of the elution buffer.

[0023] Protein molecules vary widely in their charge characteristics and exhibit varying degrees of interaction with charged chromatographic media depending on their total charge, charge density, and surface charge distribution. For example, monoclonal antibodies contain ionizable groups, such as carboxyl and amino groups. The charge of these groups is pH-dependent. Therefore, depending on the pI of the antibody, the charge of the protein molecule can be manipulated by exposing the bulk product to various pH conditions. Both AEX and CEX have been demonstrated to be effective in removing not only aggregates but also other impurities.

[0024] Preparative type Typically, separation of dimers and other aggregate species from the target product is challenging due to the similar chemical structures of these entities. Therefore, control of the purification process, and in particular the choice of preparative type, is another important aspect beyond the choice of resin (e.g., AEX, CEX, HIC, etc.) and applied conditions (e.g., protein load, bind-elute mode, or flow-through mode). During the "quantitative prep" step, a fraction collector continuously collects the eluate, changing tubes according to a well-defined set volume throughout the elution step. This type of prep is also known as straight prep (Figure 1A). In contrast, "peak prep" can be used to increase the purity of the collected protein peak while minimizing the number of tubes used (Figure 1B). A combination of "quantitative" and "peak" prep can also be applied, allowing fractions collected by "quantitative prep" and "peak prep" to be directed to different collection tubes. During the "peak collection" step, the separation of aggregates and / or impurities from the target protein is indirectly controlled using specific peak start and stop collection criteria. Several parameters are available as peak cut criteria for starting and stopping peak collection during elution. For industrial-scale production, typical peak cut parameters are specific A280 absorption signals (measured at UV 280 nm), conductivity, or pH, which are pre-defined as threshold criteria for starting and stopping eluate collection. The challenge is to determine optimal cut points (threshold criteria) for starting and stopping product eluate collection so that, on the one hand, product recovery is maximized and, on the other hand, aggregate and / or impurity concentrations are absent or very low and within specifications. Furthermore, determining these points is hindered by potential differences in elution peak shapes of samples to be purified under various elution conditions.

[0025] Peak cut criteria can be easily set only when the aggregates / impurities to be removed from the target protein are sufficiently baseline-separated from the target protein, e.g., by a wash step, so that the target protein "elutes" distinctly from the aggregates / impurities as a distinct peak. In such cases, the signal for elution collection is typically set at a low absorbance value, often based on the percentage of the maximum height achieved by the elution peak during its ascending phase. However, for samples where aggregates / impurities accumulate at the front of the peak and no clear boundary exists between the target protein and the aggregates / impurities (e.g., a linear gradient without a clear peak boundary) (Figure 2), determining the optimal collection start point can be difficult. In particular, charge variants and aggregated forms of proteins remain significant challenges in the purification process. In addition, process parameters (e.g., pH of the elution buffer, amount of protein loaded onto the column) typically fluctuate within a certain range and can directly affect peak shape (i.e., width and height). Selecting too low a peak start criterion (e.g., absorbance) results in ineffective impurity removal for higher and / or narrower peaks, whereas selecting too high a peak start criterion results in reduced yield for lower and / or broader peaks. If a clear A280 signal is never reached, the result is that no sample is collected and the entire target protein is lost. Therefore, determining a universally valid starting point for eluate collection that can be effectively used across a variety of conditions that result in eluates of equal or similar quality is a difficult and challenging task. The solution to this problem is reflected in the claims described herein and illustrated in the examples and figures.

[0026] Embodiment The present disclosure relates to an improved method for eluting antibodies from a chromatography resin during a purification process. In particular, the disclosure relates to a method for determining an absorption signal A0, which is used as a reference signal for obtaining a start signal for collecting eluate at predetermined intervals D0 after the absorption signal A0 is reached, the method comprising: a) At least two different elution runs containing the protein sample to be purified (ER1, , ER N receiving an elution peak chromatogram of the sample, wherein the different elution runs vary in pH, loading density, or salt conditions; b) assigning an absorbance value A0 in the elution peak, A0 being in the range of 10-50% of the absorbance signal at the peak maximum of each elution peak, and ER1,...,ER N The step is the same for each elution peak. Includes:

[0027] In another embodiment, the present disclosure relates to a method for determining an absorption signal A0 to be used as a criterion for an improved starting point for collecting eluate fractions in the chromatography of an antibody sample, the method comprising: a) At least two different elution runs containing antibody samples (ER1, , ER N ) determining an elution peak chromatogram of the chromatogram, wherein different elution runs vary in pH, loading density, or salt conditions; b) Absorption signals (A1, . . . , A1) in each of the different elution rank chromatograms N ), i) Absorption signals (A1, , A N The absorption signal (A) at the peak maximum of each elution peak MAX 1. A MAX N) is 300mAU or less, ii) This absorption signal (A1, , A N the corresponding eluate fractions collected in step (a) are characterized by an aggregate / impurity content of less than 10%; c) normalizing the elution peak chromatogram according to the fluid volume of said absorption signal specified in step b); d) overlaying the normalized elution peak chromatograms; and e) determining an absorption signal A0, where A0 is on the ascending part of the elution peak and is the same for corresponding column volumes between the normalized elution peaks; Includes:

[0028] In another embodiment, in step b), the peak maximum value A of each elution peak MAX The difference in absorption signal relative to the absorption signal at is 150mAU or less, 200mAU or less, 250mAU or less, 300mAU or less, 400mAU or less, 500mAU or less, 600mAU or less, and 700mAU or less.

[0029] In other embodiments, A1,...,A N and the absorption signal A at the height maximum of the corresponding elution peak. MAX The distances to the target are in the ranges of 0-150mAU, 0-200mAU, 0-250mAU, 0-300mAU, 0-400mAU, 0-500mAU, 0-600mAU, and 0-700mAU, measured at 280nm.

[0030] In another embodiment, the absorption signals (A1, . . . , A N ) are characterized by an aggregate / impurity content of less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In preferred embodiments, the aggregate / impurity content relates to HMW and LMW species.

[0031] In another embodiment, a method is provided for eluting antibodies from a chromatography resin by setting an optimal starting point for eluate collection, along with efficient separation of the target antibody from aggregates and / or impurities, such as high molecular weight multimeric species (dimers, oligomers, aggregates), low molecular weight species (such as clips), and other contaminants.

[0032] The present disclosure also relates to a method for purifying an antibody from a chromatography resin, comprising the steps of: a) loading a sample containing the antibody and aggregates / impurities onto the resin; b) optionally washing the resin with a wash buffer; c) applying an elution buffer; and d) collecting the antibody eluate, wherein collection of the eluate begins at least 0.1 CV fluid volume after a fluid volume C0 of a predetermined absorbance value A0 at an absorption signal AX. In yet another embodiment, collection of eluate begins at least 0.2 CV, at least 0.3 CV, at least 0.4 CV, at least 0.5 CV, at least 0.6 CV, at least 0.7 CV, at least 0.8 CV, at least 0.9 CV, at least 1.0 CV, at least 1.1 CV, at least 1.2, at least 1.3 CV, at least 1.4 CV, at least 1.5, at least 1.6, at least 1.7 CV, at least 1.8 CV, at least 1.9 CV, at least 2.0 CV, at least 2.1 CV, at least 2.2 CV, at least 2.3 CV, at least 2.4 CV, at least 2.5 CV, at least 2.6 CV, at least 2.7 CV, at least 2.8 CV, at least 2.9 CV, or at least 3.0 CV after fluid volume C0 of A0 at absorbance signal AX.

[0033] In other embodiments, the fluid volume interval D0 at which collection of antibody eluate begins is 0.1 CV, 0.2 CV, 0.3 CV, 0.4 CV, 0.5 CV, 0.6 CV, 0.7 CV, 0.8 CV, 0.9 CV, 1.0 CV, 1.1 CV, 1.2 CV, 1.2 CV, 1.4 CV, 1.5 CV, 1.6 CV, 1.7 CV, 1.8 CV, 1.9 CV, 2.0 CV, 2.1 CV, 2.2 CV, 2.3 CV, 2.4 CV, 2.5 CV, 2.6 CV, 2.7 CV, 2.8 CV, 2.9 CV, 3.0 CV after fluid volume C0 of A0 at absorbance signal AX.

[0034] In yet another embodiment, the disclosure provides a method for eluting an antibody from a chromatography resin, wherein the method comprises one or more ion exchange chromatography steps, characterized in that the start of peak collection (i.e., collection of eluate) is 0.1 to 1.8 CV fluid volume after fluid volume C0 of A0 at absorbance signal AX.

[0035] In certain embodiments, the present disclosure provides methods for eluting antibodies, wherein the methods include one or more cation exchange chromatography steps, characterized in that the start of peak collection (i.e., collection of eluate) is 0.1 to 1.8 CV fluid volume after fluid volume C0 of A0 at absorbance signal AX.

[0036] In all embodiments described herein, the absorption signal AX is a signal representing the absorption of the different elution runs ER1, . . . , ER N may vary between the individual elution peaks.

[0037] Also disclosed is a method for eluting an antibody during a chromatography step, wherein the eluate is collected with a delay of at least 0.1 to 3.0 CV fluid volume, preferably 0.4 to 1.2 CV, after a predetermined fluid volume of an absorption signal A0, wherein the absorption signal A0 is the absorbance at the first crossing point of at least two chromatographic elution peaks obtained by at least two different elution runs with different pH or loading density conditions in an overlaid and normalized chromatogram. Preferably, the normalization is according to the fluid volume.

[0038] In another embodiment, the elution runs are performed under different pH and loading density conditions.

[0039] In one embodiment, the present disclosure provides a method for eluting an antibody, wherein the method comprises affinity chromatography followed by a mixed-mode chromatography step. The mixed-mode step can be characterized by either cation or anion exchange, or a combination of both. The step can be based on a single type of ion exchange mixed-mode step, or can include multiple ion exchange mixed-mode steps, such as performing a cation exchange mixed-mode step before an anion exchange mixed-mode step (or vice versa). In one embodiment, the ion exchange mixed-mode step is a one-step process.

[0040] In certain embodiments, the ion exchange mixed-mode step comprises a two-step ion exchange mixed-mode process. Suitable cation exchange columns are those whose stationary phase contains anionic groups. Examples of such columns are Capto MMC™, Capto MMC™ ImpRes (GE Healthcare), and Nuvia™ cPrime™ (Biorad).

[0041] In another embodiment, a suitable anion exchange column is one whose stationary phase contains cationic groups. Examples of such columns are Capto Adhere™ and Capto Adhere™ ImpRes (GE Healthcare).

[0042] In one embodiment, the affinity chromatography step involves subjecting the primary recovery sample to a column containing a suitable affinity chromatography support. Examples of such chromatography supports include, but are not limited to, Protein A, Protein G, Protein L, affinity supports containing the antigen against which the antibody of interest was raised, and affinity supports containing other Fc-binding molecules. Protein A is particularly useful for affinity purification of IgG antibodies. In a specific embodiment, the Protein A is selected from ProSep® Ultra Plus Protein A, MabSelect SuRe™ Protein A, and Amsphere Protein A™ resins. In one embodiment, the Protein A column is equilibrated with a suitable buffer prior to sample loading. An example of a suitable buffer is PBS, pH 7.0-7.3. Following this equilibration, the sample is loaded onto the column. After loading onto the column, the column is washed one or more times, for example, with the equilibration buffer. Additional washes with different buffers may be used prior to elution of the column. The Protein A column can then be eluted using an appropriate elution buffer. An example of a suitable elution buffer comprises a sodium acetate buffer, pH about 3.6.

[0043] In one embodiment, an affinity chromatography eluate is prepared for multimodal CEX by adjusting the pH and ionic strength of the sample buffer. For example, the affinity eluate can be adjusted to a pH of about 4.5 to about 7.0 with a load density of about 10 to about 200 g / L. Preferably, the pH is 4.95 to 5.65, and the load density is 20 to 40 g / L. Before loading the affinity eluate sample onto the multimodal CEX column, the column can be equilibrated with a suitable buffer. An example of a suitable buffer is 20 mM sodium acetate, 20 mM MES, pH 5.5. After equilibration, the affinity eluate is loaded onto the column. After loading, the column is washed one or more times with a suitable buffer. An example of a suitable buffer is the equilibration buffer itself. In another embodiment, the affinity eluate is prepared for a mixed-mode chromatography step under similar conditions.

[0044] One embodiment of the present invention relates to a method for purifying antibodies from a sample such that the resulting antibody eluate is substantially free of process and product-related impurities such as host cell proteins, DNA, leached Protein A, aggregates, HMW species, LMW species and fragments.

[0045] In one embodiment, the present disclosure provides a method for purifying a cell culture-derived antibody or antibody fragment from a crude mixture (which may contain HCPs, aggregates, and other impurities in addition to the target antibody), comprising an affinity chromatography step and one or more IEX chromatography steps, characterized in that the chromatographic peak cut criteria for initiating eluate collection include: i) a first fluid volume time C0 at which a first predetermined A280 signal A0 is reached as a reference; and ii) a second column volume time CX at which a second A280 signal (AX) is reached, at which point target protein eluate collection is initiated. The difference between CX and C0 corresponds to a predetermined fluid volume interval D0.

[0046] In one embodiment, the difference in fluid volume between CX and C0 ranges from 0.1 to 2.5 CV fluid volume, while in other embodiments, the difference between CX and C0 is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 CV fluid volume.

[0047] In one aspect, a pH gradient is used for elution during a cation exchange chromatography step for antibody characterization. In one embodiment, the pH gradient ranges from 4.9 to 7.0.

[0048] In another aspect, a linear salt gradient is used for elution, hi one embodiment, the salt gradient ranges from 100 to 500 mM.

[0049] In one embodiment of the present disclosure, the antibody to be purified is a human, humanized, or chimeric antibody.

[0050] In certain embodiments of the invention, the antibodies to be purified are IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM isotype antibodies and variants thereof.

[0051] In a preferred embodiment, the antibody to be purified is an IgG1 antibody.

[0052] In one embodiment, the present disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C. In another embodiment, the present disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment is selected from the following: an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6 Includes:

[0053] In another embodiment, the present disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment comprises the following: an HCDR1 region of SEQ ID NO: 1, an HCDR2 region of SEQ ID NO: 2, an HCDR3 region of SEQ ID NO: 3, an LCDR1 region of SEQ ID NO: 4, an LCDR2 region of SEQ ID NO: 5, and an LCDR3 region of SEQ ID NO: 6.

[0054] In one embodiment, the present disclosure relates to a method for purifying an antibody comprising a variable heavy chain and a variable light chain, the heavy chain and the light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the variable heavy chain of SEQ ID NO:8 and the variable light chain of SEQ ID NO:7.

[0055] In yet another embodiment, the present disclosure relates to a method for purifying an antibody comprising a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the heavy chain of SEQ ID NO:10 and the light chain of SEQ ID NO:9.

[0056] In one embodiment, the present disclosure relates to a method for purifying an antibody comprising a variable heavy chain of SEQ ID NO:8 and a variable light chain of SEQ ID NO:7.

[0057] In yet another embodiment, the present disclosure relates to a method for purifying an antibody comprising a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO:9.

[0058] In another embodiment, the disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment comprises: an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and variable heavy chains and variable light chains having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0059] In another embodiment, the disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment comprises the following: an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the heavy chain of SEQ ID NO: 10 and the light chain of SEQ ID NO: 9.

[0060] In another embodiment, the disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment comprises a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the following: an HCDR1 region of SEQ ID NO: 1, an HCDR2 region of SEQ ID NO: 2, an HCDR3 region of SEQ ID NO: 3, an LCDR1 region of SEQ ID NO: 4, an LCDR2 region of SEQ ID NO: 5, and an LCDR3 region 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.

[0061] In another embodiment, the disclosure relates to a method for purifying an antibody or antibody fragment specific to IL-17C, wherein the antibody or antibody fragment comprises a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the following: an HCDR1 region of SEQ ID NO: 1, an HCDR2 region of SEQ ID NO: 2, an HCDR3 region of SEQ ID NO: 3, an LCDR1 region of SEQ ID NO: 4, an LCDR2 region of SEQ ID NO: 5, and an LCDR3 region of SEQ ID NO: 6, and a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

[0062] Antibody preparations to which the present invention can be applied can include unpurified or partially purified antibodies from natural, synthetic, or recombinant sources. The mixture can also be cell culture material, such as solubilized cells and cell culture supernatant. In certain embodiments, the mixture is a clarified cell culture harvest. In other embodiments, the antibody preparation is a Protein A chromatography eluate. In yet another embodiment, the mixture is an eluate obtained from an AIEX chromatography step. The methods of the present invention can be used as a purification step to purify antibodies from any mixture containing antibodies.

[0063] Additionally, the present disclosure relates to pharmaceutical compositions comprising one or more antibodies purified by the methods described herein.

[0064] The purity of the antibody of interest in the resulting sample product can be analyzed using methods well known to those skilled in the art, including, for example, size exclusion chromatography, Poros™ A HPLC assay, HCP ELISA, Protein A ELISA, and Western blot analysis.

[0065] In preferred embodiments, the methods provided herein result in purified antibodies having an SEC monomer content of 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or greater. In another embodiment, the purified protein has a 100% SEC monomer content.

[0066] In another embodiment, the methods provided herein result in purified antibody in a yield of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater.

[0067] In a preferred embodiment, the present disclosure relates to a method for purifying antibodies by chromatography, which comprises: a) loading a sample containing an antibody onto a chromatography resin; b) optionally washing the resin; c) applying an elution buffer to the chromatography resin; and d) Starting the collection of the eluate wherein collection of the eluate begins at a predetermined interval (D0) after the absorbance signal of the eluate reaches a predetermined value (A0).

[0068] In another embodiment, the predetermined value (A0) is in the range of 10-50% of the absorbance signal at the peak maximum of the elution peak obtained by different elution runs containing the antibody sample to be purified. The different elution runs vary in pH, loading density, or salt conditions. Preferably, the varied conditions are in the range of pH 5-7 and 5-50 g / L resin load.

[0069] In a preferred embodiment, the predetermined absorbance value (A0) is in the range of 0 to 1500 mAU measured at 280 nm. In another embodiment, the predetermined absorbance value (A0) is about 700 mAU measured at 280 nm. In yet another embodiment, A0 is on the ascending part of the elution peak.

[0070] In another embodiment, the method relates to any preceding method, wherein the scheduled interval (D0) is: a) At least two different elution runs containing the antibody sample to be purified (ER1, , ER N receiving an elution peak chromatogram of the chromatogram of the sample, wherein the different elution runs vary in pH, loading density, or salt conditions; b) For each of the elution peaks received in step a), the absorption signals A1,...,A N A step of specifying A1, . . . , A N wherein each of the corresponding eluate fractions has an aggregate / impurity content of less than 5%; c) Absorption signals A1, . . . , A for each elution run in the chromatogram N Fluid volumes C1, . . ., C N determining d) The fluid quantities C1, . . . , C relative to the fluid quantity C0 of A0 N calculating the difference between each of e) averaging (i.e., averaging) the differences, thereby obtaining a planned fluid volume interval (D). The fluid volume interval is determined by

[0071] In a preferred embodiment, the individual absorption signals A at the maximum height of the corresponding elution peaks are MAX For A1, , A N The distances are in the range of 0 to 100 mAU measured at 280 nm.

[0072] In another embodiment, the predetermined interval value (D0) is between 0.4 and 1.2 CV fluid volume, or the predetermined interval value (D0) is 0.6 CV fluid volume.

[0073] In a preferred embodiment, the chromatography is ion exchange (IEX) chromatography. Preferably, the chromatography is cation exchange (CEX) chromatography. Most preferably, the chromatography is multimodal CEX.

[0074] In a preferred embodiment, the present disclosure relates to a method for purifying antibodies by multimodal cation exchange (CEX) chromatography, comprising: a) loading a sample containing said antibody onto a multimodal CEX chromatography resin; b) optionally washing the resin; c) applying the elution buffer to the multimodal CEX chromatography resin; and d) Starting the collection of the eluate Including, where collection of eluate begins at a predetermined interval (D0) of 0.6 CV fluid volume after the absorbance signal of the eluate reaches a predetermined value (A0) of 700 mAU measured at 280 nm; The antibody comprises a heavy chain of SEQ ID NO:10 and a light chain of SEQ ID NO:9.

[0075] In another embodiment, the present disclosure relates to a method for purifying antibodies specific to IL-17C by multimodal cation exchange (CEX) chromatography, comprising: a) loading a sample containing said antibody onto a multimodal CEX chromatography resin; b) optionally washing the resin; c) applying the elution buffer to the multimodal CEX chromatography resin; and d) Starting the collection of the eluate Including, where collection of eluate begins at a predetermined interval (D0) of 0.6 CV fluid volume after the absorbance signal of the eluate reaches a predetermined value (A0) of 700 mAU measured at 280 nm; The antibody comprises the following: an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and variable heavy and variable light chains having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the variable heavy chain of SEQ ID NO: 8 and the variable light chain of SEQ ID NO: 7.

[0076] In another embodiment, the present disclosure relates to a method for purifying antibodies specific to IL-17C by multimodal cation exchange (CEX) chromatography, comprising: a) loading a sample containing said antibody onto a multimodal CEX chromatography resin; b) optionally washing the resin; c) applying the elution buffer to the multimodal CEX chromatography resin; and d) Starting the collection of the eluate Including, where collection of eluate begins at a predetermined interval (D0) of 0.6 CV fluid volume after the absorbance signal of the eluate reaches a predetermined value (A0) of 700 mAU measured at 280 nm; The antibody comprises the following: an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2, an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3, an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the heavy chain of SEQ ID NO: 10 and the light chain of SEQ ID NO: 9.

[0077] definition As used herein, the term "protein" refers to a continuous chain of amino acids linked together via peptide bonds. This term is used to refer to an amino acid chain of any length and can refer to a minimal chain containing two amino acids linked together via peptide bonds. As used herein, "peptide," "peptide fragment," "polypeptide," "amino acid chain," "amino acid sequence," or any other term used to refer to one or more chains of two or more amino acids, are collectively included in the definition of "protein." This term also encompasses proteins that have undergone post-translational modifications, such as glycosylation, acetylation, phosphorylation, or amidation. Any protein that can be expressed in a host cell can be expressed and purified in accordance with the present invention. For example, the present disclosure can be used to purify enzymes, receptors, antibodies, antibody fragments, hormones, regulatory factors, cytokine antigens, binding agents, fusion proteins, alternative scaffold proteins, and the like.

[0078] A "buffer" is a solution that is not susceptible to changes in pH due to the action of its acid-base conjugate components. For example, various buffers that can be used depending on the desired pH of the buffer are described in "Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems," Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers that control pH within this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations thereof.

[0079] The term "elution buffer" typically refers to a buffer used to remove (elute) a polypeptide (analyte) from a purification device (e.g., a chromatography resin) to which it has previously been applied. Typically, an elution buffer is selected so that separation of the polypeptide of interest from unwanted aggregates / impurities can be achieved. Often, the concentration of a particular component in the elution buffer, such as a particular salt (e.g., NaCl), is changed (gradient) during the elution step. The gradient may be continuous (linear) or stepwise (interrupted by a retention period).

[0080] The term "linear salt gradient" refers to the salt concentration (ionic strength) of the gradient buffer used during the elution step, which changes over time. Typically, the sample is loaded into a low-salt environment to facilitate interaction with the stationary phase. Commonly used salts are sodium and potassium chloride and acetate. To elute the analytes, an appropriate salt concentration is required to disrupt the stationary phase / analyte interaction. Typical elution concentrations range from 100 to 500 mM.

[0081] The term "isoelectric point (pI)" is the pH at which a particular molecule or surface carries no net charge. The pI of a polypeptide depends on the amino acids that make up the polypeptide. At a pH below its pI, the polypeptide carries a net positive charge. At a pH above its pI, the polypeptide carries a net negative charge. Thus, polypeptides can be separated based on their ionization state at a given pH. The actual pI of a polypeptide can be affected by factors such as post-translational modifications and can be determined by experimental methods such as isoelectric focusing.

[0082] The term "chromatography" refers to any existing or future chromatography-based process that purifies one or more target molecules from a sample, for example, by removing aggregates and / or impurities and / or other non-target molecules. During a chromatography step, solutes of interest in a mixture, e.g., polypeptides, are separated from other solutes in the mixture by differences in the rate at which individual solutes of the mixture migrate from a stationary phase under the influence of a mobile phase or in a binding and solute process. Examples of chromatography include, but are not limited to, affinity chromatography, immobilized metal ion affinity chromatography, flow-through chromatography, ion exchange chromatography, size exclusion chromatography, reversed-phase chromatography, simulated moving bed chromatography, hydrophobic interaction chromatography, gel filtration, and chromatofocusing.

[0083] The term "mixed-mode chromatography" or "multimodal chromatography" refers to a purification process using a mixed-mode sorbent, which results in multiple modes of interactions, such as hydrophobic, cation exchange, and hydrogen-bonding interactions, between the polypeptide of interest and the sorbent ligands. Commercially available mixed-mode chromatography resins include Capto™ MMC, Capto™ MMC ImpRes, Capto Blue, Blue Sepharose™ 6 Fast Flow, Capto™ Adhere, and Capto™ Adhere ImpRes from GE Healthcare Life Sciences, Eshmuno® HCX from EMD Millipore, or Nuvia™ cPrime from Bio-Rad.

[0084] The term "cation exchange resin" or "cation exchange sorbent" refers to a solid phase that carries a negative charge and therefore has free cations for exchange with cations in an aqueous solution passed on or through the solid phase. The negatively charged ligands attached to the solid phase to form the cation exchange resin may be, for example, carboxylates or sulfonates. Commercially available cation exchange resins include carboxy-methyl-cellulose, sulfopropyl (SP) immobilized on agarose (e.g., SP Sepharose™ XL, SP-Sepharose™ Fast Flow, SP Sepharose™ High Performance, CM Sepharose™ Fast Flow, CM Sepharose™ High Performance, Capto™ S, and Capto™ SP ImpRes from GE Healthcare Life Sciences, or Fractogel® EMD SE HiCap, Fractogel® EMD SO3″, Fractogel® EMD COO″, Eshmuno™ S, and Eshmuno™ CPX from EMD Millipore, or UNOsphere™ S and Nuvia™ S from Bio-Rad).

[0085] The terms "anion exchange resin" or "anion exchange sorbent" are used herein to refer to a solid phase that is positively charged and has one or more positively charged ligands, such as, for example, quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE Sepharose™ Fast Flow, Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ DEAE, Capto™ Q, and Capto™ Q ImpRes from GE Healthcare Life Sciences, or Fractogel® EMD TMAE HiCap, Fractogel® EMD DEAE, and Eshmuno Q from EMD Millipore, or U Osphere™ Q and Nuvia™ Q from Bio-Rad.

[0086] The term "chromatogram" refers to a graphical representation of one or more output parameters recorded during at least a portion of a chromatographic purification process. A chromatogram may represent an output parameter as a function of time, cumulative column volume, or any other parameter related to a chromatographic purification. In the present method, each purification is recorded as a chromatogram by monitoring the output parameter during the purification step. The term "output parameter" refers to a recordable parameter that indicates the outcome of a chromatographic purification. Examples of output parameters include, but are not limited to, UV absorbance at one or more wavelengths, conductivity, light scattering detection, fluorescence emission, mass spectrometry, recorded fluid volume, recorded pH, and recorded pressure. The output parameters are preferably measured linearly in the downstream flow path of the chromatographic purification. As used herein, A0, A1,..., A N , AX, and A MAX correspond to the absorption signals. C0, C1, . . ., C N , CX, and C MAX are A0, A1, . . ., A N , AX, and AMAX is the corresponding fluid volume (column volume).

[0087] The term "upward part of an elution peak" refers to the signal above the baseline before the maximum peak height is reached. Correspondingly, downward part of an elution peak refers to the signal above the baseline after the maximum peak height.

[0088] The term "antibody" refers to glycosylated and non-glycosylated immunoglobulins of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), as well as combinations and variants thereof. As used herein, the term encompasses antibodies from any species (e.g., human, murine, canine, feline, equine, bovine, chicken, etc.), as well as combinations or variants thereof (e.g., humanized, chimeric antibodies). The term refers to monoclonal and polyclonal antibodies, as well as monospecific and multispecific antibodies (e.g., bispecific antibodies). As used herein, the term also encompasses fusion proteins containing antigenic determinants, as well as any other modified immunoglobulin molecule containing an antigen recognition site. As used herein, the term "antibody" includes intact immunoglobulins as well as antibody fragments, which refer to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fd, Fv, and dAb fragments (Ward et al., (1989) Nature 341:544-546), and single-chain Fv (scFv) (e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Any naturally occurring, enzymatically obtainable, synthetic, alternative scaffold, or genetically engineered polypeptide that specifically binds to an antigen to form a complex is also intended to be encompassed by the term "antibody" as used herein.

[0089] The terms "contaminant" and "impurity" are used interchangeably herein and refer to any unwanted molecule, including biopolymers such as DNA, RNA, one or more host cell proteins, endotoxin, lipids, and one or more additives, that may be present in a sample containing a target protein to be separated from one or more foreign or unwanted molecules using the processes of the present invention. In addition, such contaminants may include any reagents used in steps that may occur prior to the purification process.

[0090] "High molecular weight (HMW) species" includes species having a molecular weight higher than the target protein mass, such as multimers. Multimers include anything other than a target protein monomer. For example, an IgG antibody monomer encompasses a conventional tetrameric antibody composition comprising two heavy and light chains. Multimers include species having a molecular mass higher than the target protein mass, such as dimers (two identical proteins linked covalently or non-covalently), and aggregates (covalently or non-covalently linked complete and / or partial proteins).

[0091] "Low molecular weight (LMW) species" includes species with a molecular weight lower than the target protein mass, such as clips and degradation products.

[0092] As used herein, the term "purification" refers to a downstream processing step after the initial (affinity) capture step, which aims to remove residual amounts of aggregates and / or impurities. The aggregates / impurities removed during the purification process typically have a higher similarity to the product than the impurities removed during the capture step.

[0093] Methods for determining the yield and purity of a polypeptide are well known to those skilled in the art. The yield and purity of a polypeptide can be determined by any suitable analytical method (e.g., band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, ELISA, HPLC, etc.). An exemplary method is size-exclusion chromatography (SEC) or high-performance liquid chromatography (HPLC). Purity can be determined, for example, using a relative "area under the curve" (AUC) value, which is typically obtained for a peak in a chromatogram, e.g., an HPLC chromatogram. Optionally, purity is determined by chromatography or other means using a standard curve generated using a reference material of known purity. Purity can also be determined by weight ratio.

[0094] The term "bind and elute mode" refers to a product separation technique in which at least one product (e.g., an Fc region-containing protein, an antibody) contained in a sample binds to a chromatographic resin or medium and is subsequently eluted.

[0095] The term "absorption" refers to the physical process of absorbing light, whereas "absorbance" is a mathematical measure of the amount of light absorbed by a sample per sample length at a given wavelength λ. This is also known as optical density (OD) or extinction. Many substances absorb ultraviolet (UV) or visible (VIS) light due to their chemical composition. The UV range is 190-380 nm, and VIS is 380-770 nm. In proteins, for example, peptide bonds absorb light at 215 nm, and aromatic groups on certain amino acids absorb at 280 nm. The absorption of light by substances has been used to detect the presence and measure their concentration. A typical absorbance unit is called the "absorbance unit" (AU) and is dimensionless. Absorbance is calculated based on either the amount of light reflected or scattered by the sample or the amount transmitted through the sample. If all the light passes through a sample, none is absorbed, so the absorbance is zero and the transmission is 100%. Conversely, if no light is transmitted through a sample, the absorbance is infinite and the percent transmission is zero. The Beer-Lambert law, A=e×b×c, is used to calculate the absorbance at a given wavelength, where A is the absorbance (dimensionless, A=log 10 where P is the intensity of the incident light, P is the intensity of the transmitted light, e is the molar extinction coefficient or molar attenuation coefficient (M cm), b is the path length of the sample (e.g., the length of the cuvette in cm), and c is the molar concentration of the solute in the solution (mol / L).

[0096] The absorbance units used herein are determined at a wavelength of UV 280 nm and a path length of 0.2 cm.

[0097] The term "predetermined absorbance value A0" refers to a predetermined, predetermined fixed absorption signal in the elution peak, which is determined by the different elution runs ER1, . . . , ER2 of a given antibody sample. N Equal between.

[0098] C0 is the elution run (ER1, . . . , ER N ) is the corresponding fluid volume (unit of measurement is "column volume", CV) of absorbance value A for each of the individual elution peaks in the column (C is the volume of the column containing the absorbance value A for each of the individual elution peaks in the column). N (This may vary between

[0099] A1, , A N are the elution runs ER1, ER2, ER3, ER4, ER5, ER6, ER7, ER8, ER9, ER10, ER11, ER12, ER13, ER14, ER15, ER16, ER17, ER18, ER19 ... N wherein the fractions show no aggregates / impurities, or show less than 10% aggregate / impurity content, or show aggregate / impurity content within specification.

[0100] AX is the absorbance value at which eluate collection effectively begins (AX is the absorbance value for the different elution runs ER1, . . . , ER N Preferably, AX is greater than A0 (AX>A0).

[0101] CX is the fluid volume at which eluate collection effectively begins (CX may vary between different elution runs). CX is reached a predetermined fluid volume interval D0 after C0 and can be calculated by the formula CX = C0 + D0.

[0102] The term "scheduled interval (D0)" refers to a scheduled fluid volume interval. Preferably, D0 is a time interval between fluid volumes C1, . . . , C2 relative to the fluid volume of A0 (which is C0). N It is calculated by averaging (i.e., finding the average value) the differences between C1,...,C N at least two different elution runs (ER1, , ER2) containing the protein sample to be purified. N ) absorption signals A1, , A in the elution peak chromatogram N where different elution runs vary in pH, loading density, or salt conditions, A,...,A NEach of the corresponding eluate fractions has an aggregate / impurity content of less than 10%, less than 7.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, or less than 3%. Alternatively, the median can be used to represent the middle of the set of differences (e.g., if the set is an outlier). DO can also be defined as a predetermined interval without calculation.

[0103] The amino acid and encoding nucleic acid sequences shown in Table 1 are examples of IL-17C antibodies, and portions thereof.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] [Table 4] [Example]

[0108] Example 1. Determination of the interval D0 Clarified cell supernatant obtained from a mammalian cell culture expressing recombinant IgG1 in a 3000 L bioreactor was loaded onto a MabSelect SuRe (GE Healthcare) Protein A column. The IgG in the harvest is selectively bound to Protein A. Following loading, several wash steps were performed. The antibody was then eluted from the column with a gradient elution containing approximately 5 column volumes of elution buffer. The elution pool contained the antibody and residual aggregates and impurities, of which approximately 2% was a mixture of dimers, multimers, and aggregates, and approximately 1% was low molecular weight (LMW) contaminants. After viral inactivation, depth filtration, and AIEX purification steps, the IgG sample was loaded onto a multimodal CEX column (Capto MMC ImpRes, GE Healthcare) equipped with an AEKTA avant system in bind-elute mode, followed by a wash step and elution with a linear salt gradient. Separate test elution runs ER1–ER3 were performed under different pH and loading density conditions, as shown in Table 2. To obtain a high-purity collected antibody eluate, "peak collection" was used. To remove residual aggregates / impurities while maintaining a high yield of target protein, peak collection should begin at the peak maximum, or just before or just after the maximum, depending on the process conditions and peak shape. The absorbance signals A1-A3 and corresponding fluid volumes for the optimal start of eluate collection for individual test elution runs are listed in Table 2. Chromatograms for elution runs ER1-ER3 are shown in Figures 3A-3C, respectively.

[0109] [Table 5]

[0110] A MAXis the absorption signal at the maximum height of the elution peak. A0 is the predetermined reference absorption signal. C0 is the corresponding fluid volume of A0. C0 can be different for each individual elution peak, as in this example. The absorption at elution collection indicates the optimal individual absorption signals A1, A2, and A3, respectively, for the start of elution collection. C1, C2, and C3 are the corresponding fluid volumes at elution collection. D1, D2, and D3 are the individual differences ("intervals") between C1, C2, and C3 and the corresponding C0. The predetermined interval D0 is obtained by averaging the differences D1, D2, and D3. In this example, this average value is (0.7 + 0.6 + 0.6) / 3 = 0.63.

[0111] Example 2. Using D0 to initiate eluate collection Depending on the pH of the gradient buffer and the protein load per resin volume, the resulting elution peaks vary substantially in width and height, and therefore the individual absorbance signals for the optimal start of eluate collection also vary (Example 1). To consistently and robustly apply eluate collection initiation, the A280 absorbance signal (AX) at a predetermined interval (D0) relative to the reference A280 signal (A0) was used to initiate eluate collection. The predetermined A280 signal A0 was set to 700 mAU, and the 0.6 CV fluid volume interval (D0) determined in Example 1 was used to arrive at the optimal A280 signal (AX) as the start point for eluate collection.

[0112] The absorbance signal AX reached after the fluid volume interval D0, and at which eluate collection began, is shown in Table 3 along with the corresponding fluid volume for each elution run.

[0113] [Table 6]

[0114] Elution runs ER4 to ER5 show the peak collection criteria of the present invention. 10 The chromatograms of the above are shown in Figures 4A to 4G. The analytical results (yield, HMW, LMW, monomer) are shown in the figures and in Tables 4A to 4G below.

[0115] [Table 7]

[0116] [Table 8]

[0117] [Table 9]

[0118] [Table 10]

[0119] [Table 11]

[0120] [Table 12]

[0121] [Table 13]

[0122] Different elution runs ER4, ER5, ER6, ER7, ER8, ER9, ER10, ER11, ER12, ER13, ER14, ER15, ER16, ER17, ER18, ER19, ER20, ER21, ER22, ER23, ER24, ER25, ER26, ER27, ER28, ER2 10 The normalized elution peaks (according to the fluid volume) are shown in Figure 5. The end point of each eluate collection was set at 400 mAU. The yields and SEC monomer fractions of the eluates obtained at AX / D0 for the seven purification runs are summarized in Table 5.

[0123] [Table 14]

[0124] Example 3. Control Settings and Phase Characteristics The Method Editor of the control software UNICORN™ 7.1 (Build 7.1.0.378, GE Healthcare) does not offer the possibility of integrating a "delayed" peak collection start during the linear gradient elution step within the gradient elution phase mask (Figure 8). Therefore, a calculation command workaround was created. The default elution phase of the UNICORN™ software was manually edited by text instructions in the UNICORN™ Method Editor to set the desired 0.6 CV delayed peak collection start after a default, fixed UV280 absorbance signal (here: 700 mAU). The start of collection is therefore independent of the maximum peak height. This modification of the default elution phase instructions in Unicorn™ ensured an optimal start of eluate collection, independent of process conditions and the resulting different elution peak shapes. Table 6 shows the text instructions for the standard default linear elution gradient phase in UNICORN™, including the peak collection start at 50 mAU. Table 7 shows the manually edited instructions for the linear elution gradient phase, including a scheduled and preset 0.6 CV delayed peak collection start after a UV280 absorbance signal of 700 mAU.

[0125] [Table 15]

[0126] [Table 16]

Claims

1. 1. A method for purifying an antibody by chromatography, comprising: Loading the antibody-containing sample onto the chromatography resin; Optionally, washing the chromatography resin; applying an elution buffer to the chromatography resin; Start collecting the eluate wherein collection of the eluate begins at a predetermined interval (D0) after the absorption signal of the eluate reaches a predetermined value (A0); the predetermined value (A0) is 10-50% of the absorption signal at the peak maximum of an elution peak obtained by a different elution run containing the sample to be purified; The scheduled interval (D0) is as follows: a) receiving chromatograms of at least two different elution runs (ER 1 , . . . , ER N ) comprising the sample to be purified, the different elution runs varying in conditions with respect to pH, loading density or salt; b) designating absorption signals A 1 , ..., A N for each of said chromatograms received in step a), wherein each of the corresponding eluate fractions of A 1 , ..., A N has an aggregate / impurity content of less than 4%; c) determining the fluid volumes C 1 , . . . , C N of the absorption signals A 1 , . . . , A N of each of the different elution runs in the chromatogram; d) calculating the difference between each of said fluid quantities C 1 , . . . , C N and the fluid quantity C 0 of A 0 ; and e) averaging said differences, thereby obtaining said scheduled interval (D0). The method of claim 1, wherein the fluid volume interval is determined by

2. 2. The method of claim 1, wherein the conditions are within the range of pH 5 to pH 7 and within the range of 5 to 50 g / L resin load.

3. 3. The method according to claim 1 or 2, wherein said predetermined value (A0) is in the range of 0 to 1500 mAU measured at 280 nm.

4. 4. The method of claim 3, wherein the predetermined value (A0) is 700 mAU measured at 280 nm.

5. The absorption signal A at the maximum height of the corresponding elution peak ＭＡＸ A against １ , ..., A Ｎ The method according to any one of claims 1 to 4, wherein the distance is in the range of 0 to 300 mAU measured at 280 nm.

6. 6. The method of claim 5, wherein said predetermined interval (D0) is between 0.4 and 1.2 CV fluid volume.

7. 7. The method of claim 6, wherein the scheduled interval (D0) is 0.6 CV fluid volume.

8. The method according to any one of claims 1 to 7, wherein the chromatography is ion exchange (IEX) chromatography.

9. 9. The method of claim 8, wherein the chromatography is cation exchange (CEX) chromatography.

10. The method of any one of claims 1 to 9, wherein the antibody comprises a VH of SEQ ID NO: 8 and a VL of SEQ ID NO:

7.

11. The method of claim 10, wherein the antibody comprises a heavy chain of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9.

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