Elution buffer collection during antibody chromatography

Abstract

This invention relates to an improved method for peak fractionation and eluent collection in a chromatographic process for purifying human therapeutic antibodies.

Description

Technical Field

[0001] This disclosure relates to protein purification methods. Specifically, the invention relates to an improved method for peak fractionation during antibody elution from chromatographic resins using optimized peak cutoff for initial eluent collection. This optimized peak cutoff is robustly applied to initial eluent collection in elution cycles with varying peak widths and heights. More specifically, the invention relates to a method for purifying human therapeutic antibodies by applying an improved peak cutoff to initial eluent collection during CEX purification, and to purified antibody compositions obtained by the methods disclosed herein. Background Technology

[0002] For therapeutic administration, each drug must meet different quality standards. To ensure clinical safety and tolerability (e.g., for therapeutic monoclonal antibodies (mAbs), one or more purification steps must be followed during the mAb manufacturing process to remove unwanted contaminants such as aggregated and fragmented products, nucleic acids, viruses, host cell proteins (HCPs), residual culture medium components, and cell culture additives. Therefore, the goal of each manufacturing and purification process development is to establish a reliable, reproducible, and robust method to obtain protein products with high purity and high yield. Importantly, maintaining consistent quality across each batch is absolutely essential in pharmaceutical manufacturing.

[0003] To achieve sufficient homogeneity and meet the high-quality standards of therapeutic-grade products used in clinical applications, so-called purification steps are required during the purification process. These purification steps typically include ion-exchange chromatography (IEX) following initial antibody affinity chromatography to remove residual aggregates and impurities.

[0004] In addition to selecting the type and conditions of resin used during refining (e.g., loading density, binding elution mode, or flow-through mode), the grading mode ( Figure 1 The choice of peak fractionation is another important aspect. Peak fractionation is the most efficient mode of eluent collection and can be used to improve the purity of collected proteins. Therefore, appropriate peak fractionation specifications for starting and stopping eluent collection during elution must be determined. Peak fractionation ensures that the collected target fraction includes only a very small amount of the substance eluted in adjacent fractions (not collected). Peak fractionation can be performed manually or automatically. In manual mode, the operator decides to change to the next fraction collection tube. In automatic fractionation mode, defined integration parameters (e.g., absorbance) control the fractionation. Automatic peak fractionation is recommended only when the sample composition remains unchanged between chromatographic runs, as variations in sample composition (e.g., pH, salt concentration, conductivity, protein concentration, loading density) can result in different peak shapes that may not be recognized by the entered integration parameters.

[0005] In industrial-scale manufacturing, peak cut parameters are typically specific signals of absorbance, conductivity, or pH at 280 nm (A280) and are predefined as thresholds for starting and stopping fraction collection. For example, Borg et al. (J Chromatogr A. 2014 Sep 12; 1359: 170-81) disclosed a pooling design that applies a constant start-collection criterion, triggered when the optical density (OD) at 280 nm reaches 0.5 during peak rise. The final cut point is determined by the percentage of the maximum peak value. WO2014140570 relates to controlling the collection of eluent output during separation, starting and stopping collection when a defined measurement of suspended matter reaches a significant threshold. Yigzaw et al. (Curr Pharm Biotechnol. 2009 Jun; 10(4): 421-6) described the termination of eluent collection based on absorbance changes at different percentages of the maximum peak value as the final cut point. Westerberg et al. (Bioprocess Biosyst Eng. 2010 Mar; 33(3):375-82) disclosed a HIC case study in which a model simulation was used to determine the parameter that has the greatest influence on the selection of the first cut point for the start of collection. In this case, the parameter was determined to be the conductivity of the loading buffer. A function was then set to estimate the relationship between the absorption signal at the cut point and the conductivity of the loaded sample. The maximum value of this function at a given conductivity was determined as the ideal cut point.

[0006] US20160272673 describes the isolation and purification of DVD-lgs from a sample. TM The chromatographic method in which the purified DVD-lgs TM Compared to the sample, it exhibits reduced host cell proteins, aggregates, and viruses. US20160264618 discloses a method for purifying antibodies by cation exchange chromatography. Eluent collection begins when the UV signal in the chromatogram increases to a predetermined value of 50 mAU, 100 mAU, or higher (depending on the elution round).

[0007] Generally, the problem with fractionation lies in determining the optimal peak cutoff points for starting and ending eluent collection, ensuring the highest product recovery, minimal or no impurity concentrations, and within specifications. For scale-independent methods of purifying antibody proteins, it is crucial to determine optimized peak fractionation criteria (for pooling product eluents) that are less sensitive to batch-to-batch interferences from sample composition and different process parameters (such as pH and loading). In particular, purification methods that produce compositions with increased productivity, high purity, and reduced costs are of significant value for process development.

[0008] US20130303732 provides a method for controlling contaminants in biopharmaceutical purification processes by using light scattering and UV absorbance as continuous monitoring systems to provide real-time information on elution peak fractions, rather than conventional pooling methods that do not consider product quality and rely on a predetermined percentage of UV peak maximum values ​​to initiate the pooling process.

[0009] The potential technical problems of this application can be seen from the provided method for improving antibody elution during chromatography by simultaneously maintaining optimal yield of the target antibody, effectively reducing aggregates and impurities, and being unaffected by the pH, salt concentration, and loading density of the elution buffer. This invention addresses these needs by providing a method for finding an optimized starting point for eluent collection by using a specified reference absorbance signal in conjunction with a predetermined flow interval. Its advantage is that it achieves an ideal eluent collection start, independent of the typically unpredictable peak height and shape of elution cycles under different conditions. Furthermore, this invention provides a method for peak fractionation by applying these improved peak initiation criteria during the purification of therapeutic antibodies. Summary of the Invention

[0010] Herein, the inventors provide a method for purifying proteins by chromatography, comprising the following steps:

[0011] a) Load the protein-containing sample onto the chromatographic resin.

[0012] b) Optionally, clean the resin.

[0013] c) Apply elution buffer to the chromatographic resin, and

[0014] d) Start collecting the eluent, wherein after the absorbance signal of the eluent reaches a predetermined value (A0), the collection of the eluent begins at a predetermined interval (D0).

[0015] The determination of the absorbance signal A0, used as a reference signal to obtain the initial signal of the eluent collected at a predetermined interval D0 after reaching A0, includes the following steps:

[0016] a) Receive at least two different elution cycles (ER1, ..., ER2) of the protein sample to be purified. N The elution peak chromatogram (N = an integer greater than 1) shows the elution peaks, where the pH, loading density, or salt conditions differ in different elution cycles.

[0017] b) Specify the absorbance value A0 in the elution peak, where A0 is the absorbance signal at the peak maximum of each elution peak (A... MAX 1, ..., A MAX The range of N) is 10-50%, and in ER1, ..., ER N Each elution peak is identical.

[0018] The predetermined interval D0 is a flow interval obtained through a method including the following steps:

[0019] a) Receive at least two different elution cycles (ER1, ..., ER2) of the protein sample to be purified. N The elution peak chromatograms show that the pH, loading density, or salt conditions varied in different elution rounds.

[0020] b) Designate the absorbance signal of each elution peak received in step a) as A1, ..., A N Where A1, ..., A N The aggregate / impurity content of each of the corresponding eluent fractions is less than 5%.

[0021] c) Measure the absorbance signal A1, ..., A2 for each elution round in the chromatogram. N The flow C1, ..., C N ,

[0022] d) Calculate each of the stated flows C1, ..., C N The differences D1, ..., D from the flow rate of A0 (i.e., C0) are: N ,and

[0023] e) Calculate the differences D1, ..., D2. N The average value (i.e., the average value) is obtained to obtain the predetermined flow interval D0.

[0024] Furthermore, the inventors provide a method for purifying antibodies, comprising applying a mixture containing antibodies and aggregates / impurities onto an ion-exchange chromatography resin, washing the resin, eluting the antibody from the chromatography resin, and collecting the eluent fraction by peak fractionation, the collection starting point being determined by the method described herein. The start of eluent collection is robust for different peak widths and peak heights.

[0025] The method is particularly suitable for purifying antibody samples characterized by chromatographic peaks in which aggregates / impurities have accumulated in the leading part (i.e., the rising part). For example, as... Figure 2 As shown, high molecular weight (HMW) and low molecular weight (LMW) impurities are enriched at the beginning of the sharp increase in the peak of the unrefined antibody sample.

[0026] Instead of defining a specific A280 absorbance signal (absorbance measured at UV280 nm) for initiating eluent collection, the inventors determined an A280 absorbance signal pointing to the peak initiation (indicated here as A0). A0 is the same at the corresponding flow rate (measured in column volume, CV) between at least two different elution cycles with different pH, salt, or loading density conditions. Alternatively, A0 can be the value of different elution cycles E1, ..., EN The maximum absorbance signal of the lowest-height elution peak is 50% of the maximum value of the elution peak. Preferably, A0 is used in different elution cycles E1, ..., E N The maximum value of all elution peaks is between 10% and 50%.

[0027] Absorbance signal A0 and different elution cycles E1, ..., E N The elution peak curves intersect at the flow rate C0 (measured in column volume, CV). Different elution cycles E1, ..., E N The C0 values ​​between the various elution peaks may differ, depending on the shape of each elution peak. The flow rate of A0 (i.e., C0) is then used with a predetermined flow interval (referred to herein as a “delay,” e.g., 0.6 column volumes) at which the subsequent A280 absorbance signal is reached, which, together with its corresponding flow rate, defines the starting point for eluent collection.

[0028] In one embodiment, the ion exchange chromatography step is a multimode cation exchange chromatography step combined with elution mode (Capto MMC ImpRes, GE Healthcare), in which antibodies are eluted from the column using a salt gradient.

[0029] In one embodiment, the antibody to be purified is a monoclonal antibody. The present invention also provides monoclonal antibodies purified by process flow separation of the monoclonal antibody from aggregates and / or impurities using the methods of this disclosure. Attached Figure Description

[0030] Figure 1 A schematic chromatogram using fractionation modes (A) fixed volume fractionation and (B) peak fractionation (according to...) (Avant User Manual 29-0351-84AD, modified by GE Healthcare).

[0031] Figure 2 Representative chromatograms of elution cycles during CEX purification. The chromatograms show the antibody monomer content (left Y-axis) and HMW and LMW aggregates / impurities (right Y-axis) in the eluent fractions (X-axis). Aggregates / impurities accumulate at the start of the elution peaks (fractions H7, A8, B8, C8, D8, E8, F8, G8).

[0032] Figures 3A-3C Chromatograms of the elution peaks ER1, ER2, and ER3 used to determine the flow interval D0. The shaded areas are labeled 2300-400 mA U (ER1, ER2, ER3). Figure 3A ), 1800-400mAU (ER2, Figure 3B ) and 1300-400mAU(ER3, Figure 3CThe optimal eluent pooling area was determined. Each eluent collection cycle was initiated under absorbance signal conditions, corresponding to eluent fraction aggregates / impurities below 10% (comparison). Figure 2 ).

[0033] Figure 4A-4G Different elution cycles under different conditions #4-#10 (ER4 to ER5) 10 The elution peak chromatogram was obtained, and the eluent was collected starting at a predetermined flow interval D0.

[0034] Figure 5 A normalized elution peak chromatogram for antibody purification using Capto MMC ImpRes, showing the peak fractionation start and end points. Example elution cycles (ER4 to ER5). 10 The peak fractionation was performed under seven different conditions with different pH and loading densities (Table 3) (a: the intersection of A0 and normalized C0, b: the predetermined D0 interval "delay" (here: 0.6 CV); c: the start of peak fractionation (the start of eluent collection); d: the peak fractionation range, e: the stop of peak fractionation (the stop of eluent collection) here at 400 mAU, UV 280 nm) A0: the predetermined absorbance signal, measured at UV 280 nm, here: 700 mAU).

[0035] Figure 6 Determine the concept of the interval D0 used to reach the eluent collection starting point. The figure shows the elution peaks of two elution cycles with different peak shapes. The absorbance signal (A0) indicates the reference absorbance and the maximum peak height. MAX 1, A MAX 2), and the absorbance signals (A1, A2) for the optimal elution start, along with the corresponding flow rates C0, C1, and C2. The optimal elution start is related to the eluent fraction having aggregate and impurity content within the specification range. Generally, an aggregate / impurity ratio (i.e., HMW and LMW content) below 4% (i.e., monomer content above 96%) is considered the optimal choice for the fraction.

[0036] Figure 7 In different (with) Figure 5 The concept of a predetermined interval D0 is applied to the elution peaks, which are illustrated in the example. When the absorbance signal AX reaches CX, eluent collection begins at the flow interval D0.

[0037] Figure 8 .UNICORN TM The Method Editor in version 7.1 (Build 7.1.0.378) predefines the stage properties for the gradient elution stage. Detailed Implementation

[0038] Protein purification by chromatography

[0039] In the pharmaceutical industry, the manufacturing process of target molecules (such as therapeutic mAbs) is generally divided into i) upstream processing (USP), which includes the production of the target protein; ii) downstream processing (DSP), which includes obtaining the target protein in its pure form through purification; and iii) final processing to obtain product integrity and safety.

[0040] Typically, the first step in downstream purification after the production stage involves clarifying the harvested cell culture mixture, where one or more steps, such as precipitation, flocculation, (deep) filtration, and / or centrifugation, can be used to separate the desired proteins from cells, cell debris, and other contaminants. Downstream purification processes often also include one or more (orthogonal) chromatographic separation steps, such as affinity chromatography, ion exchange, hydrophobic interactions, hydroxyapatite, focusing chromatography, gel filtration, and reversed-phase chromatography, to effectively remove process- and product-related impurities. These contaminants include, but are not limited to, host cell proteins (HCPs), leached protein A, product isotypes, high molecular weight (HMW) substances, low molecular weight (LMW) substances, and shear or degradation products. Parameters that can lead to HMW substances (such as dimers and large aggregates (multimers)) include, but are not limited to, protein concentration, pH, ionic strength, oxygen, temperature, salt concentration, shear force, and external stress exposure (such as interactions with metal surfaces, exposure to air, freezing, and / or thawing). Unwanted post-translational modifications or molecular unfolding can also contribute to aggregation.

[0041] Affinity chromatography refers to the use of compounds that specifically interact with the desired target protein to be purified. Typically, compounds are immobilized on a resin to separate, purify, or remove 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 recombinant or synthetic versions or peptides of these proteins. Resins include MAbSelect. TM (GE Healthcare), Prosep (Millipore), etc. For laboratory-scale applications, one-step affinity purification can usually achieve satisfactory purity. For example, Protein A chromatography, as the most widely used affinity purification method for capturing antibodies, supports >95% purity and excellent recovery due to its high specificity for the Fc moiety 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). Residual aggregates and / or impurities can be further removed by combining one or two additional chromatographic steps, which may include hydroxyapatite, hydrophobic interaction (HIC), and ion exchange chromatography (IEX, such as cation exchange (CEX), anion exchange (AEX), or mixed-mode exchange). At the manufacturing scale, aggregates and / or impurities are usually removed by using ion exchange chromatography (IEX) after initial antibody affinity chromatography. Commercial multimodal ion exchangers, such as Capto MMC and Capto-adhere, and Capto MMC ImpRes and Capto adhere ImpRes (both from GE Healthcare), can be used to remove contaminants downstream of initial affinity capture. IEX separates proteins with different surface charges to provide high-resolution separation and high sample loading capacity. This separation is based on reversible electrostatic interactions between charged proteins (i.e., charged amino acid side chains) and the chromatographic medium with opposite charges. AEX involves purifying proteins on resins with positively charged functional groups (e.g., strong anion exchangers with quaternary ammonium groups or weak anion exchangers with secondary ammonium groups). When the pH is above the isoelectric point (pI) of the target protein, the protein has a negative net charge, which favors its binding to the positively charged resin. Target protein elution can be performed by increasing the salt gradient, eluting stepwise at a predefined pH and salt concentration, or by decreasing the pH of the elution buffer. On the other hand, CEX involves purifying proteins on resins with negatively charged functional groups (e.g., strong cation exchangers with sulfite groups, or weak cation exchangers with carboxylate anions). Here, the target protein is typically bound to the resin in a low-salt-concentration buffer solution with a pH lower than the target protein's pI (i.e., the protein is positively charged). Elution of the target protein can be performed by increasing the salt gradient, eluting stepwise at a predefined pH and salt concentration, or by increasing the pH of the elution buffer.

[0042] Protein molecules exhibit highly diverse charge properties and interact with charged chromatographic media to varying degrees 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 will depend on pH. Therefore, based on the antibody's pI, the charge of a protein molecule can be controlled by exposing batches of product to different pH conditions. AEX and CEX have been shown to effectively remove not only aggregates but also other impurities.

[0043] Hierarchical types

[0044] Separating dimers and other aggregates from a target product is typically challenging because these entities have similar chemical compositions. Therefore, in addition to the selection of the resin (e.g., AEX, CEX, HIC) and conditions (e.g., protein loading, binding elution mode, or flow-through mode), the control of the purification process, especially the choice of fractionation type, is another important aspect. In a "fixed-volume fractionation" process, the fraction collector continuously collects the eluent and switches tubes according to a defined set volume throughout the elution step. This type of collection is also known as direct collection. Figure 1 A). On the other hand, "peak fractionation" can be used to improve the purity of collected protein peaks and minimize the number of tubes used. Figure 1 B). A combination of "fixed volume" and "peak" collection can also be applied, allowing fractions collected by "fixed volume fractionation" and fractions collected by "peak fractionation" to be introduced into different collection tubes. During "peak fractionation," specific peak start and stop collection criteria are used to indirectly control the separation of aggregates and / or impurities from the target protein. Several parameters can be used as peak cut-off criteria for starting and stopping peak fractionation during elution. In industrial-scale manufacturing, typical peak cut-off parameters are specific A280 absorbance signals (measured at UV 280 nm), conductivity, or pH, and are predefined as threshold criteria for starting and stopping eluent collection. On the one hand, the challenge is determining the optimal cut-off points (threshold criteria) for starting and ending product eluent collection to maximize product recovery and ensure that aggregate and / or impurity concentrations are absent or very low and within specifications. Furthermore, the determination of these points is hampered by the potential variation in elution peak shapes under different elution conditions.

[0045] Peak cutoff criteria can be easily set so that the target protein "elution" is distinguished as a separate peak from the aggregates / impurities only when the aggregates / impurities to be removed from the target protein are well separated from the target protein (e.g., through a washing step). In these cases, the eluent collection signal is typically set to a low absorbance value at the rising portion of the elution peak, usually based on a percentage of the maximum height reached by the elution peak. However, for samples where there is no clean separation zone between the target protein and the aggregates / impurities and where the aggregates / impurities accumulate before the peak (e.g., without a clear linear gradient between peaks)... Figure 2 Determining the optimal starting point for collection is challenging. In particular, the charged variants and aggregate forms of proteins still pose significant challenges to the purification process. Furthermore, process parameters (e.g., pH of the elution buffer, amount of protein loaded on the column) typically vary within a range and can directly affect peak shape (i.e., width and height). Choosing a peak starting criterion that is too low (e.g., absorbance) results in ineffective removal of higher and / or narrower peaks, while choosing a peak starting criterion that is too high leads to reduced yields of lower and / or wider peaks. If the defined A280 signal is not reached at all, no sample will be collected, and the entire target protein will be lost. Therefore, determining a universally effective elution collection starting point that can be used effectively under a range of conditions to produce elution of equivalent or similar quality is a difficult and challenging task. The solution to this problem is reflected in the claims explained herein and illustrated in the examples and figures.

[0046] Implementation

[0047] This invention relates to an improved method for eluting antibodies from chromatographic resin during purification. Specifically, this invention relates to a method for determining an absorbance signal A0, used as a reference signal, to obtain an initial signal for collecting the eluent at predetermined intervals D0 after the absorbance signal A0 is reached, comprising the following steps:

[0048] a) Receive at least two different elution cycles (ER1, ..., ER2) of the protein sample to be purified. N The elution peak chromatogram shows that different elution cycles differ under pH, loading density, or salt conditions.

[0049] b) Specify the absorbance value A0 in the elution peak, where A0 is in the range of 10-50% of the absorbance signal at the maximum peak value of each elution peak, and in ER1, ..., ER N Each elution peak is identical.

[0050] In another embodiment, the present invention relates to a method for determining an absorbance signal A0, which will be used as a reference for an improved starting point for the collection of eluent fractions in antibody sample chromatography, comprising the following steps:

[0051] a) Ensure that the antibody sample has undergone at least two different elution cycles (ER1, ..., ER2). N The elution peak chromatogram shows that different elution cycles exhibit variations under different pH, loading density, or salt conditions.

[0052] b) Specify the absorbance signal (A1, ..., A1) in each of the different elution rounds of the chromatogram. N ),in

[0053] i) Absorbance signal (A1, ..., A1) N ) and each elution peak (A MAX 1, ...A MAXN The numerical difference of the absorbance signal at the maximum peak does not exceed 300 mAU, and among them

[0054] ii) In the absorbance signal (A1, ..., A N The characteristic of the corresponding eluent fraction collected below is that the content of aggregates / impurities is less than 10%.

[0055] c) Calculate the flow rate-normalized elution peak chromatogram based on the absorbance signal specified in step b).

[0056] d) Superimpose the normalized elution peak chromatograms, and

[0057] e) Determine the absorbance signal A0, where A0 is located in the rising portion of the elution peak and is the same at the corresponding column volume between normalized elution peaks.

[0058] In other embodiments, the absorbance signal in step b) is related to the maximum peak value A of each elution peak. MAX The difference in absorbance signals at each location shall not exceed 150 mAU, 200 mAU, 250 mAU, 300 mAU, 400 mAU, 500 mAU, 600 mAU, or 700 mAU.

[0059] In other embodiments, A1, ..., A are measured at 280 nm. N The absorbance signal A at the maximum height of the corresponding elution peak MAX The distance is within the range of 0-150mAU, 0-200mAU, 0-250mAU, 0-300mAU, 0-400mAU, 0-500mAU, 0-600mAU, and 0-700mAU.

[0060] In other embodiments, the absorbance signals (A1, ..., A1) in step b) NThe corresponding eluent fraction is 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 a preferred embodiment, the aggregate / impurity content is related to HMW and LMW substances.

[0061] In another embodiment, a method for eluting antibodies from a chromatographic resin is provided, which effectively separates the target antibody from aggregates and / or impurities (e.g., high molecular weight polymers (dimers, oligomers, aggregates), low molecular weight substances (fragments, etc.) and other contaminants by setting an optimal starting point for eluent collection.

[0062] The present invention also relates to a method for purifying antibodies from a chromatographic resin, comprising the steps of: a) loading the resin with a sample containing antibodies and aggregates / impurities; b) optionally washing the resin with a washing buffer; c) applying an elution buffer; and d) collecting the antibody eluent, wherein the collection of the eluent begins at a flow rate of at least 0.1 CV after a flow rate of C0 at a predetermined absorbance value A0 at the absorbance signal AX. In a further embodiment, the collection of the eluent begins after a flow rate C0 of A0 at absorbance signal AX for 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 CV, at least 1.3 CV, at least 1.4 CV, at least 1.5 CV, at least 1.6 CV, 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.

[0063] In other embodiments, the flow rate interval D0 at which the collection of the antibody eluent begins is 0.1CV, 0.2CV, 0.3CV, 0.4CV, 0.5CV, 0.6CV, 0.7CV, 0.8CV, 0.9CV, 1.0CV, 1.1CV, 1.2CV, 1.2CV, 1.4CV, 1.5CV, 1.6CV, 1.7CV, 1.8CV, 1.9CV, 2.0CV, 2.1CV, 2.2CV, 2.3CV, 2.4CV, 2.5CV, 2.6CV, 2.7CV, 2.8CV, 2.9CV, and 3.0CV, after the flow rate C0 of A0 at the absorbance signal AX.

[0064] In another embodiment, the present invention provides a method for eluting antibodies from a chromatographic resin, wherein the method comprises one or more ion exchange chromatography steps, characterized in that the peak fractionation begins (i.e., eluent collection) at a flow rate of 0.1-1.8 CV after the flow rate C0 of A0 under absorbance signal AX.

[0065] In a particular embodiment, the present invention provides a method for eluting antibodies, wherein the method comprises one or more cation exchange chromatography steps, characterized in that peak fractionation (i.e., eluent collection) begins at a flow rate of 0.1-1.8 CV after the flow rate C0 of A0 under absorbance signal AX.

[0066] In all embodiments described herein, the absorbance signal AX can be displayed in different elution cycles ER1, ..., ER N The different elution peaks are different.

[0067] A method for antibody elution during chromatography is also disclosed, wherein the eluent is collected with a flow rate delay of at least 0.1-3.0 CV, preferably with a delay of 0.4-1.2 CV after a predetermined flow rate of absorbance signal A0, wherein the absorbance signal A0 is the absorbance at the first intersection of at least two chromatographic elution peaks, said at least two chromatographic elution peaks being obtained by different elution cycles under at least two different pH or loading density conditions in overlapping and normalized chromatograms. Preferably, normalization is performed based on flow rate.

[0068] In another embodiment, the elution cycles are performed under different pH and loading density conditions.

[0069] In one embodiment, this disclosure provides a method for eluting antibodies, wherein the method includes a mixed-mode chromatography step following affinity chromatography. This mixed-mode step may employ cation exchange or anion exchange, or a combination of both. The step may be based on a single type of ion exchanger mixed-mode procedure, or may include multiple ion exchanger mixed-mode steps, for example, performing a cation exchange mixed-mode step first, followed by an anion exchange mixed-mode step, or vice versa. In one embodiment, the ion exchange mixed-mode step is a one-step procedure.

[0070] In a particular embodiment, the ion exchange mixed-mode step involves a two-step ion exchange mixed-mode process. A suitable cation exchange column is one whose stationary phase contains anionic groups. An example of such a column is the Capto MMC. TM Capto MMC TM ImpRes (GE Healthcare), Nuvia TM cPrime TM (Biorad).

[0071] In another embodiment, a suitable anion exchange column is one whose stationary phase contains cationic groups. An example of such a column is the Capto Adhere. TM Capto Adhere TM ImpRes (GE Healthcare).

[0072] In one embodiment, the affinity chromatography step includes placing the primary recovered sample in a column containing a suitable affinity chromatographic carrier. Examples of such chromatographic carriers include, but are not limited to, Protein A, Protein G, Protein L, affinity carriers containing antigens against which target antibodies are generated, and affinity carriers containing other Fc-binding molecules. In particular, Protein A can be used for the affinity purification of IgG antibodies. In some aspects, Protein A is selected from... Ultra PlusProtein A, MabSelect SuRe TM Protein A and Amsphere Protein A TM Resin. On one hand, before loading the sample, equilibrate the Protein A column with a suitable buffer. An example of a suitable buffer is PBS, pH 7.0–7.3. After equilibration, load the sample onto the column. After loading, wash the column once or several times with an equilibration buffer, etc. Other washing solutions using different buffers can be used before eluting the column. The Protein A column can then be eluted with a suitable elution buffer. Examples of suitable elution buffers include sodium acetate buffer with a pH of approximately 3.6.

[0073] In one embodiment, an affinity chromatography eluent is prepared for multimode CEX by adjusting the pH and ionic strength of the sample buffer. For example, the pH of the affinity eluent can be adjusted to about 4.5 to about 7.0, and the loading density to about 10 to about 200 g / L. Preferably, the pH is 4.95 to 5.65, and the loading density is 20 to 40 g / L. The column can be equilibrated using a suitable buffer before loading the affinity eluent sample onto the multimode CEX column. An example of a suitable buffer is 20 mM sodium acetate, 20 mM MMEs, pH 5.5. After equilibration, the affinity eluent is loaded into the column. After loading, the column is washed once or multiple times with a suitable buffer. An example of a suitable buffer is the equilibration buffer itself. In another embodiment, an affinity eluent is prepared for a mixed-mode chromatographic step under similar conditions.

[0074] One embodiment of the present invention relates to a method for purifying antibodies from a sample such that the resulting antibody eluent is substantially free of process- and product-related impurities, including host cell proteins, DNA, leached Protein A, aggregates, HMW substances, LMW substances, and fragments.

[0075] In one embodiment, the present invention provides a method for purifying cell culture-derived antibodies or antibody fragments from a crude mixture (which may include HCP, aggregates, and other impurities besides the target antibody), wherein the method includes an affinity chromatography step and one or more IEX chromatography steps, characterized in that the chromatographic peak cutoff criteria for initiating eluent collection include i) a first flow rate time point C0, at which a first predetermined A280 signal A0 is reached as a reference, and ii) a second column volume time point CX, at which a second A280 signal (AX) is reached, at which target protein eluent collection begins. The difference between CX and C0 corresponds to a predetermined flow rate interval D0.

[0076] In one embodiment, the flow difference between CX and C0 is in the range of 0.1 to 2.5 CV flow. In other embodiments, the flow difference between CX and C0 is approximately 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 flow.

[0077] In one aspect, an elution gradient is used during cation exchange chromatography to characterize the antibody. In one embodiment, the pH gradient ranges from 4.9 to 7.0.

[0078] On the other hand, a linear salt gradient is used for elution. In one embodiment, the salt gradient ranges from 100 to 500 mM.

[0079] In one embodiment of the present invention, the antibody to be purified is a human, humanized, or chimeric antibody.

[0080] In some embodiments of the present invention, the antibody to be purified is an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or an IgM isotype antibody and its variants.

[0081] In a preferred embodiment, the antibody to be purified is an IgG1 antibody.

[0082] In one embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof. In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment comprises:

[0083] The region includes the HCDR1 region containing the amino acid sequence SEQ ID No.:1, the HCDR2 region containing the amino acid sequence SEQ ID No.:2, the HCDR3 region containing the amino acid sequence SEQ ID No.:3, the LCDR1 region containing the amino acid sequence SEQ ID No.:4, the LCDR2 region containing the amino acid sequence SEQ ID No.:5, and the LCDR3 region containing the amino acid sequence SEQ ID No.:6.

[0084] In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment comprises the HCDR1 region of SEQ ID No.:1, the HCDR2 region of SEQ ID No.:2, the HCDR3 region of SEQ ID No.:3, the LCDR1 region of SEQ ID No.:4, the LCDR2 region of SEQ ID No.:5, and the LCDR3 region of SEQ ID No.:6.

[0085] In one embodiment, this disclosure relates to a method for purifying an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain and the variable light chain 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 with the variable heavy chain of SEQ ID No.:8 and the variable light chain of SEQ ID No.:7.

[0086] In another embodiment, this disclosure relates to a method for purifying an antibody comprising a heavy chain and a light chain, the heavy chain and light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID No.:10 and the light chain of SEQ ID No.:9.

[0087] In one embodiment, this 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.

[0088] In another embodiment, this disclosure relates to a method for purifying an antibody comprising the heavy chain of SEQ ID No.:10 and the light chain of SEQ ID No.:9.

[0089] In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment thereof comprises: an HCDR1 region comprising the amino acid sequence SEQ ID No.:1, an HCDR2 region comprising the amino acid sequence SEQ ID No.:2, an HCDR3 region comprising the amino acid sequence SEQ ID No.:3, an LCDR1 region comprising the amino acid sequence SEQ ID No.:4, an LCDR2 region comprising the amino acid sequence SEQ ID No.:5, and an LCDR3 region comprising the amino acid sequence SEQ ID No.:6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID No.:8 and the variable light chain of SEQ ID No.:7.

[0090] In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment thereof comprises: an HCDR1 region comprising the amino acid sequence SEQ ID No.:1, an HCDR2 region comprising the amino acid sequence SEQ ID No.:2, an HCDR3 region comprising the amino acid sequence SEQ ID No.:3, an LCDR1 region comprising the amino acid sequence SEQ ID No.:4, an LCDR2 region comprising the amino acid sequence SEQ ID No.:5, and an LCDR3 region comprising the amino acid sequence SEQ ID No.:6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID No.:10 and the light chain of SEQ ID No.:9.

[0091] In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment thereof comprises the HCDR1 region of SEQ ID No.:1, the HCDR2 region of SEQ ID No.:2, the HCDR3 region of SEQ ID No.:3, the LCDR1 region of SEQ ID No.:4, the LCDR2 region of SEQ ID No.:5, and the LCDR3 region of SEQ ID No.:6, and the variable heavy chain and variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID No.:8 and the variable light chain of SEQ ID No.:7.

[0092] In another embodiment, this disclosure relates to a method for purifying an IL-17C-specific antibody or an antibody fragment thereof, wherein the antibody or antibody fragment thereof comprises the HCDR1 region of SEQ ID No.:1, the HCDR2 region of SEQ ID No.:2, the HCDR3 region of SEQ ID No.:3, the LCDR1 region of SEQ ID No.:4, the LCDR2 region of SEQ ID No.:5, and the LCDR3 region of SEQ ID No.:6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID No.:10 and the light chain of SEQ ID No.:9.

[0093] Antibody formulations to which this invention can be applied may include unpurified or partially purified antibodies from natural, synthetic, or recombinant sources. The mixture may be cell culture material, such as soluble cells and cell culture supernatant. In some embodiments, it is a clarified cell culture harvest. In other embodiments, the antibody formulation is a Protein A chromatographic eluent. In yet another embodiment, the mixture is an eluent obtained by an AIEX chromatographic step. The method of this invention can be used as a purification step for purifying antibodies from any mixture containing antibodies.

[0094] Furthermore, this disclosure relates to pharmaceutical compositions comprising one or more antibodies purified by the methods described herein.

[0095] Methods well known to those skilled in the art (e.g., size exclusion chromatography, Porosimetry) can be used. TM The purity of the target antibody in the obtained sample product was analyzed by HPLC, HCPELISA, Protein A ELISA and Western blot analysis.

[0096] In a preferred embodiment, the method provided herein produces purified antibodies with SEC monomer content greater than or equal to 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 greater than or equal to 99.9%. In another embodiment, the purified protein has a 100% SEC monomer content.

[0097] In another embodiment, the method provided herein produces antibodies with a productivity greater than or equal to 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 greater than or equal to 99%.

[0098] In a preferred embodiment, this disclosure relates to a method for purifying antibodies by chromatography, comprising the following steps:

[0099] a) Load the antibody-containing sample onto the chromatographic resin.

[0100] b) Optionally, clean the resin.

[0101] c) Apply elution buffer to the chromatographic resin, and

[0102] d) Begin collecting the eluent.

[0103] The eluent is collected at a predetermined interval (D0) after the absorbance signal of the eluent reaches a predetermined value (A0).

[0104] In another embodiment, the predetermined value (A0) is in the range of 10-50% of the absorbance signal obtained at the maximum peak of the elution peak in different elution cycles of the antibody sample to be purified. The different elution cycles have different pH, loading density, or salt conditions. Preferably, the different conditions are in the range of pH 5 to pH 7, and in the range of 5-50 g / L resin loading.

[0105] In a preferred embodiment, the predetermined absorbance value (A0) measured at 280 nm is in the range of 0 to 1500 mAU. In another embodiment, the predetermined absorbance value (A0) measured at 280 nm is approximately 700 mAU. In yet another embodiment, A0 is located in the rising portion of the elution peak.

[0106] In another embodiment, this disclosure relates to a method according to any of the foregoing methods, wherein the predetermined interval (D0) is a flow interval determined by the following steps:

[0107] a) Receive at least two different elution cycles (ER1, ..., ER2) of the antibody to be purified. N The elution peak chromatogram of the different elution cycles, where the pH, loading density, or salt conditions differ,

[0108] b) Specify the absorbance signal of each elution peak received in a) as A1, ..., A N Where A1, ..., A N The aggregate / impurity content of each of the corresponding elution portions is less than 5%.

[0109] c) Determine the absorbance signal A1, ..., A2 for each elution cycle in the chromatogram. N The flow C1, ..., C N ,

[0110] d) Calculate each of the stated flows C1, ..., C N The difference between the flow rate C0 and A0,

[0111] e) Calculate the average of the differences (i.e. obtain the average value) to obtain the predetermined flow interval (D).

[0112] In a preferred embodiment, the A1, ..., A1 values ​​measured at 280 nm are... N The absorbance signal A at the maximum height of the corresponding elution peak MAX The distance is in the range of 0-100mAU.

[0113] In another embodiment, the predetermined interval (D0) is between 0.4 and 1.2 CV flow rate, or the predetermined interval (D0) is 0.6 CV flow rate.

[0114] In a preferred embodiment, the chromatography is ion exchange (IEX) chromatography. Preferably, the chromatography is cation exchange (CEX) chromatography. Most preferably, the chromatography is multimode CEX.

[0115] In a preferred embodiment, this disclosure relates to a method for purifying antibodies by multimode cation exchange (CEX) chromatography, comprising the following steps:

[0116] a) Load the sample containing the antibody onto a multimode CEX chromatography resin.

[0117] b) Optionally, clean the resin.

[0118] c) Apply elution buffer to the multimode CEX chromatography resin, and

[0119] d) Begin collecting the eluent.

[0120] The eluent is collected at a predetermined interval (D0) of 0.6 CV flow rate after the absorbance signal of the eluent reaches a predetermined value (A0) of 700 mAU measured at 280 nm.

[0121] The antibody described therein comprises the heavy chain of SEQ ID NO:10 and the light chain of SEQ ID NO:9.

[0122] In another embodiment, this disclosure relates to a method for purifying IL-17C-specific antibodies by multimode cation exchange (CEX) chromatography, comprising the following steps:

[0123] a) Load the sample containing the antibody onto a multimode CEX chromatography resin.

[0124] b) Optionally, clean the resin.

[0125] c) Apply elution buffer to the multimode CEX chromatography resin, and

[0126] d) Begin collecting the eluent.

[0127] Specifically, eluent collection begins at a predetermined interval (D0) of 0.6 CV flow rate after the absorbance signal of the eluent reaches a predetermined value (A0) of 700 mAU measured at 280 nm.

[0128] The antibody comprises: an HCDR1 region comprising the amino acid sequence SEQ ID NO.:1, an HCDR2 region comprising the amino acid sequence SEQ ID NO.:2, an HCDR3 region comprising the amino acid sequence SEQ ID NO.:3, an LCDR1 region comprising the amino acid sequence SEQ ID NO.:4, an LCDR2 region comprising the amino acid sequence SEQ ID NO.:5, and an LCDR3 region comprising the amino acid sequence SEQ ID NO.:6, and a variable heavy chain and a variable light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the variable heavy chain of SEQ ID NO.:8 and the variable light chain of SEQ ID NO.:7.

[0129] In another embodiment, this disclosure relates to a method for purifying IL-17C-specific antibodies by multimode cation exchange (CEX) chromatography, comprising the following steps:

[0130] a) Load the sample containing the antibody onto a multimode CEX chromatography resin.

[0131] b) Optionally, clean the resin.

[0132] c) Apply elution buffer to the multimode CEX chromatography resin, and

[0133] d) Begin collecting the eluent.

[0134] Specifically, eluent collection begins at a predetermined interval (D0) of 0.6 CV flow rate after the absorbance signal of the eluent reaches a predetermined value (A0) of 700 mAU measured at 280 nm.

[0135] The antibody comprises: an HCDR1 region containing the amino acid sequence SEQ ID No.:1, an HCDR2 region containing the amino acid sequence SEQ ID No.:2, an HCDR3 region containing the amino acid sequence SEQ ID No.:3, an LCDR1 region containing the amino acid sequence SEQ ID No.:4, an LCDR2 region containing the amino acid sequence SEQ ID No.:5, and an LCDR3 region containing the amino acid sequence SEQ ID No.:6, and a heavy chain and a light chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the heavy chain of SEQ ID No.:10 and the light chain of SEQ ID No.:9.

[0136] definition

[0137] As used herein, the term "protein" refers to a chain of amino acid sequences linked together by peptide bonds. This term is used to refer to an amino acid chain of any length and may refer to the smallest chain containing two amino acids linked together by peptide bonds. As used herein, "peptide," "peptide fragment," "polypeptide," "amino acid chain," "amino acid sequence," or any other term used to refer to two or more amino acid chains is generally included in the definition of "protein." This term also includes proteins that have undergone post-translational modifications (e.g., glycosylation, acetylation, phosphorylation, or amidation). Any protein that can be expressed in a host cell can be expressed and purified according to this invention. For example, this invention can be used to purify enzymes, receptors, antibodies, antibody fragments, hormones, regulatory factors, cytokine antigens, binding agents, fusion proteins, alternative scaffold proteins, etc.

[0138] A "buffer solution" is a solution that resists pH changes through the action of its acid-base conjugate components. Various buffer solutions are described in *Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems*, Gueffroy, D., ed., Calbiochem Corporation (1975), which can be used according to the required pH. Non-limiting examples of buffer solutions that control pH within this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these buffers.

[0139] The term "elution buffer" refers to a buffer solution typically used to remove (elute) peptides (analytes) from a purification apparatus (e.g., a chromatographic resin) to which it has been previously applied. Typically, an elution buffer is chosen that facilitates the separation of the target peptide from unwanted aggregates / impurities. Typically, the concentration of a specific component (e.g., a specific salt (e.g., NaCl)) in the elution buffer changes during the elution process (gradient). This gradient can be continuous (linear) or gradual (interrupted by a hold period).

[0140] The term "linear salt gradient" refers to the change in salt concentration (ionic strength) over time during the elution process using a gradient buffer. Typically, the sample is loaded in a low-salt environment to promote interaction with the stationary phase. Commonly used salts include sodium chloride, potassium chloride, and acetate. Sufficient salt concentration is required to disrupt the stationary phase / analyte interaction in order to elute the analyte. Typical elution concentrations are in the range of 100–500 mg / L.

[0141] 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 it up. When the pH is below its pI, the polypeptide carries a net positive charge. When the pH is above its pI, the polypeptide carries a net negative charge. Therefore, 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 experimentally using methods such as isoelectric focusing.

[0142] The term "chromatography" refers to any current or future chromatographic-based process for purifying one or more target molecules from a sample, e.g., by removing aggregates and / or impurities and / or other non-target molecules. During chromatography, a target solute (e.g., a peptide) in a mixture is separated from other solutes in the mixture due to the different rates at which individual solutes migrate through the stationary medium under the influence of the mobile phase or during binding and elution. 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 chromatographic focusing.

[0143] The terms "mixed-mode chromatography" or "multi-mode chromatography" refer to purification processes using mixed-mode adsorbents, which offer multiple interaction modes, such as hydrophobic, cation exchange, and hydrogen bonding interactions between the target peptide and the adsorbent ligand. Commercially available mixed-mode chromatography resins include Capto from GE Healthcare Life Sciences. TM MMC, Capto TM MMCImpRes, Capto Blue, Blue Sepharose TM 6Fast Flow, Capto TM Adhere and Capto TM AdhereImpRes, or from EMD Millipore HCX, or Nuvia from Bio-Rad TM cPrime.

[0144] The terms "cation exchange resin" or "cation exchange adsorbent" refer to a negatively charged solid phase that thus possesses free cations to exchange with cations in an aqueous solution passing through or across the solid phase. The negatively charged ligands attached to the solid phase to form the cation exchange resin can be, for example, carboxylates or sulfonates. Commercially available cation exchange resins include carboxymethyl cellulose immobilized on agarose, sulfopropyl (e.g., SP Sepharose from GE Healthcare Life Sciences), etc. TM XL, SP-Sepharose TM Fast Flow, SP Sepharose TM High Performance, CM Sepharose TM Fast Flow, CM Sepharose TM High Performance, Capto TM S and Capto TMSP ImpRes, or from EMD Millipore EMD SE HiCap, EMD SO3” EMD COO, Eshmuno TM S, and Eshmuno TM CPX, or UNOsphere from Bio-Rad TM S and Nuvia TM S).

[0145] The terms "anion exchange resin" or "anion exchange adsorbent" are used herein to refer to a positively charged solid phase, for example, to which one or more positively charged ligands, such as quaternary amino groups, are attached. Commercially available anion exchange resins include DEAE-Sepharose from GE Healthcare Life Sciences. TM Fast Flow, Q Sepharose TM Fast Flow, QSepharose TM High Performance, Q Sepharose TM XL, Capto TM DEAE, Capto TM Q, and Capto TM QImpRes, or from EMD Millipore EMD TMAE HiCap, EMD DEAE, and Eshmuno Q, or UOspher from Bio-Rad TM Q and Nuvia TM Q.

[0146] The term "chromatogram" refers to a graphical representation of one or more output parameters recorded during at least a portion of the chromatographic purification process. Chromatograms may present output parameters as a function of time, cumulative column volume, or any other parameter relevant to the chromatographic purification process. In this method, each purification is recorded as a chromatogram by monitoring the output parameters during the purification process. The term "output parameter" refers to a recordable parameter that indicates the result of the chromatographic purification. Examples of output parameters include, but are not limited to: UV absorption at one or more wavelengths, conductivity, light scattering detection, fluorescence emission, mass spectrometry, recorded flow rate, recorded pH, and recorded pressure. Output parameters are appropriately measured in downstream processes of the chromatographic purification. A0, A1, ..., A... are used herein. N AX and A MAX Corresponding to the absorbance signal. C0, C1, ..., C N CX and CMAX They are A0, A1, ..., A N The corresponding flow rate (cylinder volume) of AX.

[0147] The term "rising portion of the elution peak" refers to the signal above the baseline before reaching its maximum peak height. Correspondingly, the falling portion of the elution peak refers to the signal above the baseline after reaching its maximum peak height.

[0148] The term "antibody" refers to glycosylated and non-glycosylated immunoglobulins of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and combinations and variants thereof. As used herein, the term includes antibodies (e.g., humanized chimeric antibodies) and combinations or variants thereof from any species (e.g., human, mouse, dog, cat, horse, cattle, chicken, etc.). 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 includes fusion proteins containing antigen-determining moieties and any other modified immunoglobulin molecules containing antigen recognition sites. As used herein, the term "antibody" includes complete immunoglobulins as well as antibody fragments involving one or more portions of the antibody's ability to maintain specific interactions with antigens (e.g., through binding, steric hindrance, or stable spatial distribution). Examples of binding fragments include, but are not limited to, Fab, Fab', F(ab')2, 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 available, synthetic, alternative scaffold, or genetically engineered polypeptide that specifically binds to an antigen to form a complex is also intended to be included in the term "antibody" as used herein.

[0149] The terms “contaminant” and “impurity” are used interchangeably herein and refer to any harmful molecule, including biomacromolecules that may be present in a sample containing a target protein, such as DNA, RNA, one or more host cell proteins, endotoxins, lipids, and one or more additives, from which the target protein has been isolated using the methods of the present invention from one or more foreign or harmful molecules. Furthermore, such contaminants may include any reagents that may be used in steps prior to the purification process.

[0150] "High molecular weight (HMW) substances" include substances with a molecular weight greater than that of the target protein, such as polymers. Polymers include all substances except the target protein monomer. For example, the monomer of an IgG antibody comprises a conventional tetrameric antibody composition containing two heavy chains and a light chain. Polymers include substances with a molecular weight greater than that of the target protein, such as dimers (two identical proteins covalently or nonvalently bound) and aggregates (all or part of a protein covalently or nonvalently bound).

[0151] "Low molecular weight (LMW) substances" include substances with a molecular weight lower than that of the target protein, such as fragments and degradation products.

[0152] As used herein, the term "refining" refers to a downstream processing step following the initial (affinity) capture step, which aims to remove residual aggregates and / or impurities. Aggregates / impurities removed during refining are typically more product-like than impurities removed in the capture step.

[0153] Methods for determining peptide yield and purity are known to those skilled in the art. Peptide yield and purity can be determined by any suitable analytical method (e.g., band intensity on a silver-stained gel, polyacrylamide gel electrophoresis, ELISA, HPLC, etc.). Exemplary methods include size exclusion chromatography (SEC) and high-performance liquid chromatography (HPLC). For example, purity can be determined using the relative "area under the curve" (AUC) value, which is typically obtained from peaks in a chromatogram (such as an HPLC chromatogram). Alternatively, purity can be determined by chromatography or other methods using a standard curve generated using a reference substance of known purity. Purity can also be determined by weight ratio.

[0154] The term “binding and elution mode” refers to a product separation technique in which at least one product contained in a sample (e.g., an Fc region containing a protein, an antibody) binds to a chromatographic resin or medium and is subsequently eluted.

[0155] The term "absorption" refers to the physical process of absorbing light, while "absorbance" is a mathematical measure of the amount of light absorbed per unit length of a sample at a given wavelength λ. It is also known as optical density (OD) or extinction. Many substances absorb ultraviolet (UV) or visible light (VIS) due to their chemical composition. The UV range covers 190–380 nm, and the VIS range covers 380–770 nm. For example, in proteins, peptide bonds absorb light at 215 nm, and aromatic groups on certain amino acids absorb light at 280 nm. The absorption of light by a substance has been used to detect the presence of such substances and to measure their concentration. The typical unit of absorbance is called the "absorbance unit," "AU," which is dimensionless. Absorbance is calculated based on the amount of light reflected or scattered by the sample, or the amount of light transmitted through the sample. If all the light passes through the sample, no light is absorbed, so the absorbance is zero, and the transmittance is 100%. On the other hand, if no light passes through the sample, the absorbance is infinite, and the transmittance is zero. Beer-Lambert's law, A = e × b × c, is used to calculate absorbance at a given wavelength, where A is absorbance (dimensionless, A = log0). 10 P0 / P), where P0 is the intensity of the incident light, P is the intensity of the transmitted light, e is the molar absorptivity or molar attenuation coefficient (M-1cm-1), b is the path length of the sample (e.g., the length of the cuvette (cm)), and c is the molar concentration of the solute in the solution (mol / L).

[0156] The absorbance units used in this paper were measured at a wavelength of 280 nm and a path length of 0.2 cm.

[0157] The term "predetermined absorbance value A0" refers to a preset, predetermined fixed absorbance signal in the elution peak, which is defined for different elution cycles ER1, ..., ER2 of the antibody sample. N They are equal.

[0158] C0 is the elution round (ER1, ..., ER) in each chromatogram. N (Measurement unit is "column volume", CV) The corresponding flow rate (different elution cycles ER1, ..., ER) for each individual elution peak absorbance value A0. N The C0 values ​​may differ between them.

[0159] A1, ..., A N For each elution cycle (ER1, ..., ER) N The absorbance value of the elution peak is determined, and the corresponding eluent fraction shows no aggregates / impurities, or shows that the aggregate / impurity content is less than 10%, or shows that the aggregate / impurity content is within the specified range.

[0160] AX is the absorbance value at the effective start of eluent collection (for different elution cycles ER1, ..., ER...).N (The values ​​of AX may differ between them). Preferably, the value of AX is greater than A0 (AX>A0).

[0161] CX is the flow rate at which effective eluent collection begins (CX may vary between different elution cycles). CX is reached at a predetermined flow interval D0 after C0 and can be calculated using the formula CX = C0 + D0.

[0162] The term "predetermined interval (D0)" refers to a predetermined flow interval. Preferably, the flow rates C1, ..., C are obtained by averaging (i.e., obtaining the average value). N Calculate D0 from the difference between the flow rate of A0 and the flow rate of C0 (i.e., C0). C1, ..., C N It refers to at least two different elution cycles (ER1, ..., ER2) performed on the protein sample to be purified. N The absorbance signals A1, ..., A1 in the elution peak chromatogram of the elution peaks are... N The flow rate varies depending on the pH, loading density, or salt conditions of different elution cycles, where A1, ..., A N The aggregate / impurity content of each of the corresponding eluent fractions is less than 10%, less than 7.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, and less than 3%. Alternatively, the median can be used to describe the middle value of the set of differences (e.g., if the set has outliers). D0 can also be defined as a predetermined interval without any calculation.

[0163] The amino acid and coding nucleic acid sequences in Table 1 are examples of IL-17C antibodies and their portions.

[0164] Table 1 Example IL-17C antibody sequence

[0165]

[0166]

[0167]

[0168]

[0169] Working Example

[0170] Example 1. Determine the interval D0.

[0171] Clarified cell supernatant obtained from mammalian cell cultures expressing recombinant IgG1 in a 3000-liter bioreactor was loaded onto a MabSelect SuRe (GE Healthcare) Protein A column. IgG present in the harvest selectively binds to Protein A. After loading, several washing steps were performed. Antibodies were then eluted from the column using a gradient elution with approximately 5 column volumes of elution buffer. The elution chamber contained antibodies and residual aggregates and impurities, comprising approximately 2% a mixture of dimers, multimers, and aggregates, and approximately 1% low molecular weight (LMW) contaminants. Following virus inactivation, deep filtration, and AIEX purification steps, IgG samples were loaded into the binding-elution mode. The sample was processed on a multi-mode CEX column (Capto MMC ImpRes, GE Healthcare) of the Avant system, followed by a washing step and elution with a linear salt gradient. Individual elution cycles ER1 to ER3 were performed under different pH and loading density conditions, as shown in Table 2. To remove residual aggregates / impurities while maintaining a high yield of the target protein, peak fractionation needs to be initiated shortly before or after the peak maximum, depending on the process conditions and peak shape. Table 2 lists the absorption signals A1 to A3 at the start of optimal eluent collection for each elution cycle and the corresponding flow rates. Chromatograms of elution cycles ER1 to ER3 are shown below. Figures 3A-3C As shown.

[0172] Table 2. The pH and loading volume of gradient buffers for three purification experiments of IgG antibodies on Capto MMC ImpRes from ER1 to ER3.

[0173]

[0174] A MAX A0 is the absorbance signal at the maximum height of the elution peak. A0 is the predetermined reference absorbance signal. C0 is the corresponding flow rate of A0. In this embodiment, C0 may be different for each individual elution peak. The absorbance at the start of eluent collection represents the optimal individual absorbance signals A1, A2, and A3 at the start of eluent collection. C1, C2, and C3 are the corresponding flow rates at the start of eluent collection. D1, D2, and D3 are the individual differences between C1, C2, and C3 and their corresponding C0 (“intervals”). The predetermined interval D0 is obtained by averaging the differences D1, D2, and D3. In this embodiment, the average value is (0.7 + 0.6 + 0.6) / 3 = 0.63.

[0175] Example 2. Use D0 to start eluent collection.

[0176] Depending on the pH of the gradient buffer and the protein loading per resin volume, the resulting elution peaks vary substantially in width and height, thus the optimal absorption signals for the start of eluent collection differ substantially (Example 1). To apply a consistently robust start to eluent collection, eluent collection is initiated using the A280 absorbance signal (AX) at a predetermined interval (D0) relative to the reference A280 signal (A0). The predetermined A280 signal A0 is set to 700 mA, and the optimal A280 signal (AX) is achieved using the 0.6 CV flow interval (D0) determined in Example 1, serving as the starting point for eluent collection.

[0177] The absorption signal AX, which is reached after the flow interval D0 and at which eluent collection begins, is indicated by the corresponding flow rate for each elution cycle in Table 3.

[0178] Table 3 The CX flow rate reached after the predetermined flow rate D0 and the corresponding absorption signal AX value indicate the elution cycle ER4 to ER5. 10 .

[0179]

[0180] Figure 4A-4G The data shows the elution cycles from ER4 to ER5. 10 The chromatograms show the peak collection standards of this invention. The analytical results (yield, FIMW, LMW, monomer) are shown in Figure 4A-G below.

[0181] Table 4A. Analysis results of round 4 (109DDA14) * 1)( Figure 4A )

[0182]

[0183] Table 4B. Analysis results of round 5 (109DDA14) * 2)( Figure 4B )

[0184]

[0185] Table 4C. Analysis results of round 6 (109DDA14) * 3)( Figure 4C )

[0186]

[0187] Table 4D. Analysis results of round 7 (109DDA14) * 4)( Figure 4D )

[0188]

[0189] Table 4E. Analysis results of round 8 (101DGE05) * 1)( Figure 4E )

[0190]

[0191] Table 4F. Analysis results of round 9 (101DGE05) * 2)( Figure 4F )

[0192]

[0193] Table 4G. Analysis results of round 10 (101DGE05) * 3)( Figure 4G )

[0194]

[0195] Figure 5 Displaying different elution cycles ER4 to ER 10 Standardized elution peaks (based on flow rate) were observed, and eluent collection began at predetermined intervals. The endpoint for each eluent collection was set at 400 mAU. Table 5 summarizes the eluent yields and SEC monomer fractions obtained at AX / D0 in seven purification cycles.

[0196] Table 5. Seven purification rounds of antibody on Capto MMC ImpRes, ER4 to ER5 10 The process parameters (pH, loading amount) and the yield and SEC monomer content in the eluent collected at a predetermined time interval D0 were obtained.

[0197]

[0198] Example 3: Control settings and stage attributes.

[0199] In the control software UNICORN TM In the Method Editor of 7.1 (Build 7.1.0.378, GE Healthcare), it is impossible to integrate the "delayed" peak gradation start during linear gradient elution within the mask in the gradient elution phase. Figure 8 Therefore, a workaround for computational commands was developed. (In UNICORN) TM Manually edit UNICORN using text commands in the Method EditorTM The software's predefined elution phase begins with a desired 0.6 CV delay peak fractionation after a predefined fixed UV280 absorbance signal (here: 700 mAU). Therefore, collection initiation is independent of the maximum peak height. By adjusting the predefined elution phase instructions within Unicorn, optimal initiation of eluent collection, independent of process conditions and the resulting different elution peak shapes, was achieved. Table 6 shows the UNICORN... TM The standard predefined text instructions for the linear elution gradient stage are provided, with peak grading starting at 50 mAU. Table 7 shows the manual editing instructions for the linear elution gradient stage, with peak grading delayed by 0.6 CV after the predetermined and preset UV280 absorbance signal of 700 mAU.

[0200] Table 6. UNICORN TM The predefined linear elution gradient phase in 7.1 (see...) Figure 8 The standard text instructions specify that peak gradation should begin at 50 mAU. The bold highlighted line indicates the portion where the UV280 signal is collected starting at 50 mAU.

[0201]

[0202] Table 7. Manually edited text input UNICORN TM 7.1 For the linear elution gradient phase, after a predetermined UV280 signal of 700 mAU, the delay peak gradation begins at 0.6 CV. The bold-highlighted lines indicate the text description of the start of collection.

[0203] sequence list <110> Morpheus <120> Elution buffer collection during antibody chromatography <130> MS292 / PCT <140> <141> <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 1 Asp Tyr Ala Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 2 Tyr Ile Gly Gly Val Gly Glu Gly Thr Gln Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 3 Gly Phe Ala Ile Arg Tyr Tyr Gly Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 4 Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Tyr 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 5 Gln Asp Ser Lys Arg Pro Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic peptides <400> 6 Gln Val Phe Thr Phe Pro Leu Val Thr Thr 1 5 10 <210> 7 <211> 110 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic polypeptides <400> 7 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Phe Thr Phe Pro Leu Val Thr 85 90 95 Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 <210> 8 <211> 120 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic polypeptides <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Gly Gly Val Gly Glu Gly Thr Gln Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Ala Ile Arg Tyr Tyr Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 9 <211> 214 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic polypeptides <400> 9 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Phe Thr Phe Pro Leu Val Thr 85 90 95 Thr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 10 <211> 450 <212> PRT <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic polypeptides <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Gly Gly Val Gly Glu Gly Thr Gln Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Ala Ile Arg Tyr Tyr Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 11 <211> 330 <212> DNA <213> Artificial sequence <220> <221> source <223> Note: Description of artificial sequences: synthetic polynucleotides <400> 11 tcctacgagc tgacccagcc cccctccgtg tccgtgtctc ctggccagac cgcctccatc 60 tcctacgagc tgacccagcc cccctccgtg tccgtgtctc ctggccagac cgcctccatc 60 acctgttccg gcgacaagct gggcgataag tacgcctact ggtatcagca gaagcccggc 120 acctgttccg gcgacaagct gggcgataag tacgcctact ggtatcagca gaagcccggc 120 cagtcccccg tgctggtcat ctaccaggac tccaagcggc cctccggcat ccctgagcgg 180 cagtcccccg tgctggtcat ctaccaggac tccaagcggc cctccggcat ccctgagcgg 180 ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccgag 240 ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccgag 240 gacgaggccg actactactg ccaggtgttc accttccccc tggtcaccac cgtgttcggc 300 gacgaggccg actactactg ccaggtgttc accttccccc tggtcaccac cgtgttcggc 300 ggaggcacca agctgaccgt gctgggccag 330 ggaggcacca agctgaccgt gctgggccag 330 <210> 12<210> 12 <211> 360<211> 360 <212> DNA<212> DNA <213> Artificial sequence<213> Artificial sequence <220><220> <221> Source <221> Source <223> / Note="Description of artificial sequence: synthetic polynucleotide" <223> / Note="Description of artificial sequence: synthetic polynucleotide" <400> 12 <400> 12 gaggtgcagc tgctggaatc cggcggagga ctggtgcagc ctggcggctc cctgagactg 60 gaggtgcagc tgctggaatc cggcggagga ctggtgcagc ctggcggctc cctgagactg 60 tcttgcgccg cctccggctt caccgtgtcc gactacgcta tgcactgggt ccgacaggcc 120 tcttgcgccg cctccggctt caccgtgtcc gactacgcta tgcactgggt ccgacaggcc 120 cctggcaagg gcctggaatg ggtgtcctat atcggcggcg tgggcgaggg cacccagtac 180 cctggcaagg gcctggaatg ggtgtcctat atcggcggcg tgggcgaggg cacccagtac 180 gctgagtctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 gctgagtctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc cagaggcttc 300 gccatccggt actacggctt cgactactgg ggccagggca ccctggtcac cgtgtctagc 360 <210> 13 <211> 642 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic polynucleotide" <400> 13 tcctacgagc tgacccagcc cccctccgtg tccgtgtctc ctggccagac cgcctccatc 60 acctgttccg gcgacaagct gggcgataag tacgcctact ggtatcagca gaagcccggc 120 cagtcccccg tgctggtcat ctaccaggac tccaagcggc cctccggcat ccctgagcgg 180 ttctccggct ccaactccgg caacaccgcc accctgacca tctccggcac ccaggccgag 240 gacgaggccg actactactg ccaggtgttc accttccccc tggtcaccac cgtgttcggc 300 ggaggcacca agctgaccgt gctgggccag cctaaggccg ctccctccgt gaccctgttc 360 cccccatcct ccgaggaact gcaggccaac aaggccaccc tggtctgcct gatctccgac 420 ttctaccctg gcgccgtgac cgtggcctgg aaggccgaca gctctcctgt gaaggccggc 480 gtggaaacca ccaccccctc caagcagtcc aacaacaaat acgccgcctc ctcctacctg 540 tccctgaccc ccgagcagtg gaagtcccac cggtcctaca gctgccaggt cacacacgag 600 ggctccaccg tggaaaagac cgtggcccct accgagtgct cc 642 <210> 14 <211> 1350 <212> DNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic polynucleotide" <400> 14 gaggtgcagc tgctggaatc cggcggagga ctggtgcagc ctggcggctc cctgagactg 60 tcttgcgccg cctccggctt caccgtgtcc gactacgcta tgcactgggt ccgacaggcc 120 cctggcaagg gcctggaatg ggtgtcctat atcggcggcg tgggcgaggg cacccagtac 180 gctgagtctg tgaagggccg gttcaccatc tcccgggaca actccaagaa caccctgtac 240 ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc cagaggcttc 300 gccatccggt actacggctt cgactactgg ggccagggca ccctggtcac cgtgtctagc 360 gcctccacca agggcccctc cgtgttccct ctggccccct ccagcaagtc cacctctggc 420 ggcaccgctg ccctgggctg cctggtcaag gactacttcc ccgagcccgt gaccgtgtcc 480 tggaactctg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540 ggcctgtact ccctgtcctc cgtcgtgacc gtgccctcca gctctctggg cacccagacc 600 tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg ggtggaaccc 660 aagtcctgcg acaagaccca cacctgtccc ccctgccctg cccctgaact gctgggcgga 720 ccttccgtgt tcctgttccc cccaaagccc aaggacaccc tgatgatctc ccggaccccc 780 gaagtgacct gcgtggtggt ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac 900 tccacctacc gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 960 gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 1020 aaggccaagg gccagccccg cgagccccag gtgtacacac tgccccctag ccgggaagag 1080 atgaccaaga accaggtgtc cctgacctgt ctggtcaagg gcttctaccc ctccgacatt 1140 gccgtggaat gggagtccaa cggccagccc gagaacaact acaagaccac cccccctgtg 1200 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 1260 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1320 cagaagtccc tgtccctgag ccccggcaag 1350

Claims

1. A method for purifying antibodies by chromatography, comprising the following steps: a) Load the antibody-containing sample onto the chromatographic resin. b) Optionally, clean the resin. c) Apply elution buffer to the chromatographic resin, and d) Begin collecting the eluent. Wherein, after the absorbance signal of the elution buffer reaches a predetermined value A0, the collection of the elution buffer begins at a predetermined flow interval D0, and wherein the predetermined value A0 is in the range of 10-50% of the absorbance signal obtained at the maximum peak value of the elution peak in different elution cycles of the antibody sample to be purified. The predetermined flow interval D0 is a flow interval determined by the following steps: a) receiving at least two different elution runs ERI,..., ERn of a sample of an antibody to be purified, wherein the different elution runs differ in pH, loading density or salt conditions, N elution peak chromatograms, wherein the different elution runs differ in pH, loading density or salt conditions, b) assigning to each elution peak received in step a) an absorbance signal A1,..., An N wherein the aggregate / impurity content of each of the corresponding eluate fractions A1,..., An N is below 4%, c) Measure the absorbance signal A1, ..., A2 for each elution cycle in the chromatogram. N The flow C1, ..., C N , d) Calculate each of the stated flows C1, ..., C N The difference between the flow rate C0 and A0, e) Calculate the average of the differences, wherein the average is a predetermined flow interval D0.

2. The method according to claim 1, wherein the conditions are in the range of pH 5 to pH 7 and in the range of resin loading from 5 g / L to 50 g / L.

3. The method of claim 2, wherein the predetermined value A0 is measured at 280 nm and is in the range of 0 mAU to 1500 mAU.

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

5. The method according to claim 1, wherein, The A1, ..., A measured at 280 nm N The absorbance signal A at the maximum height of the corresponding elution peak MAX The distance is in the range of 0 mAU to 300 mAU.

6. The method of claim 5, wherein the predetermined flow interval D0 is between 0.4 CV flow and 1.2 CV flow.

7. The method of claim 6, wherein the predetermined flow interval D0 is 0.6 CV flow.

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

9. The method according to claim 8, wherein the chromatography is cation exchange chromatography.

10. The method according to any one of claims 1-7, wherein the antibody is a monoclonal antibody comprising VH of SEQ ID NO: 8 and VL of SEQ ID NO:

7.

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

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