Purification method of fasuximab

Abstract

The invention relates to a purification method of fasuximab. According to the method, through protein L affinity chromatography, the fasuximab is purified by utilizing the binding force difference between a target antibody / polymer impurity and a filler, and compared with a nano antibody ligand affinity medium, the fasuximab can be more effectively purified. In addition, compared with conventional low-pH elution, the antibody and the polymer can be effectively separated by increasing the elution pH, and the polymer is further reduced. According to the method disclosed by the invention, the purity of the fasuximab can be effectively improved, the polymer content is reduced, and the process is simple and easy to operate.

Description

Technical Field

[0001] This disclosure pertains to the field of protein purification, specifically relating to a method for purifying fareximab using protein L affinity chromatography. Background Technology

[0002] Faricimab is a structurally unique humanized bispecific immunoglobulin G1 (IgG1) antibody that simultaneously targets and neutralizes vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (Ang-2). To eliminate binding to FcγR and FcRn, the Fc region of faricimab has been completely modified, containing three distinct mutations—I235A, H310A, and H435A. These three sites directly prevent it from binding to traditional Protein A affinity fillers.

[0003] Currently, downstream purification techniques for fareximab mainly involve capture using nanobody ligand affinity media (CaptureSelect series FcXL, FcXP) and protein G chromatography. Compared to protein G, the CaptureSelect series media offer superior performance in improving fareximab purity. However, due to the structural characteristics of the antibody, the purification level of fareximab, particularly the removal of polymeric impurities, still needs improvement. Therefore, developing a simple and efficient affinity capture purification method for fareximab is a pressing technical challenge in this field. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this disclosure provides a new purification method for faraximab. This method utilizes protein L affinity chromatography to optimize the levels of impurities related to the faraximab product, particularly the content of polymers, which can be effectively optimized.

[0005] In a first aspect, this disclosure provides a method for reducing impurities in faraximab, wherein in some embodiments, the method includes the following steps: (a) Binding the purifying faraximab to an affinity chromatography medium; (b) Wash the affinity chromatography medium with washing buffer; (c) Elute fareximab from the affinity chromatography medium using elution buffer; The affinity chromatography is Protein L affinity chromatography.

[0006] In some implementations, the impurities include polymeric impurities.

[0007] In some embodiments, the polymeric impurities include dimer impurities and / or non-covalently bonded HH, LL, HHL impurities.

[0008] In some implementations, one or more of the following features are present: 1) Compared with the faraximab obtained by the purification method using nanobody ligand affinity media, the purity of the faraximab obtained by the affinity elution collection solution obtained by the protein L affinity chromatography purification method described in this disclosure is increased by at least 1%, preferably by at least 2%, preferably by at least 3%, preferably by at least 4%, preferably by at least 5%, preferably by at least 6%, preferably by at least 7%, preferably by at least 8%, and preferably by at least 9%. 2) The content of polymeric impurities in the faraxicam prepared by the method described in this disclosure is less than 3.0% of the total content, preferably less than 2.6%, preferably less than 2.5%, preferably less than 2.0%, preferably less than 1.5%, preferably less than 1.0%, preferably less than 0.9%, preferably less than 0.8%, preferably less than 0.7%, preferably less than 0.6%, preferably less than 0.57%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.32%, preferably less than 0.3%, preferably less than 0.2%, preferably less than 0.1%; 3) The content of the dimer impurity in the faraximab prepared by the method described in this disclosure is less than 0.5% of the total content, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.2%, preferably less than 0.1%, preferably less than 0.05%, and preferably completely removable; 4) The content of non-covalently bound HH, LL, and HHL impurities in the faraximab prepared by the method described in this disclosure is less than 2.2% of the total content, preferably less than 2.16%, preferably less than 2.0%, preferably less than 1.5%, preferably less than 1.0%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.27%, preferably less than 0.2%, preferably less than 0.18%, and preferably less than 0.1%.

[0009] In some embodiments, the affinity chromatography packing material used has Protein L as the ligand, and preferred affinity chromatography packing materials are Diamond Protein L, MaXtar Protein L, or MabSelect VL.

[0010] In some embodiments, the affinity chromatography uses gradient elution with solutions A and B as eluents, wherein solution A includes NaAc and solution B includes HAc-NaOH.

[0011] In some embodiments, the NaAc concentration of solution A is about 10 to 100 mmol / L, preferably about 20 to 60 mmol / L, and most preferably about 50 mmol / L; the pH of NaAc is about 5 to 6, preferably about 5.5.

[0012] In some embodiments, the concentration of HAc-NaOH in solution B is about 10 to 100 mmol / L, preferably about 20 to 60 mmol / L, and most preferably about 50 mmol / L; the pH of HAc-NaOH is about 3 to 4, preferably about 3.2.

[0013] In some embodiments, the eluent of the gradient elution has a pH gradient of about 2.0-6.0, preferably about 3.2-5.5, more preferably about 3.8-5.5, more preferably about 4.1-5.45, and more preferably about 4.46-4.77.

[0014] In some embodiments, during gradient elution, purified faraximab is collected in the pH range of 3.8-5.5, preferably in the pH range of 4.16-5.41, and even more preferably in the pH range of 4.46-4.77.

[0015] In some implementations, the process of equilibrating the affinity chromatography medium with a equilibration buffer is included before the purified faraximab is bound to the affinity chromatography medium.

[0016] In some embodiments, the equilibration buffer comprises Tris and NaCl. In some preferred embodiments, the concentration of Tris is about 10 mmol / L to 50 mmol / L, preferably about 20 mmol / L, about 30 mmol / L, or about 40 mmol / L. In some preferred embodiments, the concentration of NaCl is about 50 mmol / L to 500 mmol / L, preferably about 100 mmol / L, about 200 mmol / L, about 300 mmol / L, or about 400 mmol / L. In some preferred embodiments, the pH of the equilibration buffer is about 5-9; in some preferred embodiments, the pH of the equilibration buffer is about 6.5-8.5; in some preferred embodiments, the pH of the equilibration buffer is about 7-8; and in some preferred embodiments, the pH of the equilibration buffer is about 7.4.

[0017] In some embodiments, the washing process includes a rinsing step with a equilibration buffer. In some preferred embodiments, the washing process further includes a rinsing step with an eluent. In some preferred embodiments, the washing process involves rinsing with a equilibration buffer followed by rinsing with an eluent. In some preferred embodiments, the eluent comprises NaAc. In some preferred embodiments, the concentration of NaAc in the eluent is approximately 10 mmol / L to 50 mmol / L.

[0018] In some implementations, the purified faraximab is bound to the Protein L affinity chromatography medium in the host cell culture supernatant via step (a).

[0019] In a second aspect, this disclosure provides the use of Protein L affinity chromatography in reducing impurities in fareximab. In some embodiments, the use of this disclosure includes the following: (a) Binding the purifying faraximab to an affinity chromatography medium; (b) Wash the affinity chromatography medium with washing buffer; (c) Elute fareximab from the affinity chromatography medium using elution buffer; The affinity chromatography is Protein L affinity chromatography.

[0020] In some implementations, the impurities include polymeric impurities.

[0021] In some embodiments, the polymeric impurities include dimer impurities and / or non-covalently bonded HH, LL, HHL impurities.

[0022] In some implementations, one or more of the following features are present: 1) Compared with the faraximab obtained by affinity chromatography purification using nanobody ligand media, the purity of the faraximab in the affinity eluent obtained by the protein L affinity chromatography purification method described in this disclosure is increased by at least 1%, preferably by at least 2%, preferably by at least 3%, preferably by at least 4%, preferably by at least 5%, preferably by at least 6%, preferably by at least 7%, preferably by at least 8%, and preferably by at least 9%. 2) The content of polymeric impurities in the faraxicam prepared by the method described in this disclosure is less than 3.0% of the total content, preferably less than 2.6%, preferably less than 2.5%, preferably less than 2.0%, preferably less than 1.5%, preferably less than 1.0%, preferably less than 0.9%, preferably less than 0.8%, preferably less than 0.7%, preferably less than 0.6%, preferably less than 0.57%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.32%, preferably less than 0.3%, preferably less than 0.2%, preferably less than 0.1%; 3) The content of the dimer impurity in the faraximab prepared by the method described in this disclosure is less than 0.5% of the total content, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.2%, preferably less than 0.1%, preferably less than 0.05%, and preferably completely removable; 4) The content of non-covalently bound HH, LL, and HHL impurities in the faraximab prepared by the method described in this disclosure is less than 2.2% of the total content, preferably less than 2.16%, preferably less than 2.0%, preferably less than 1.5%, preferably less than 1.0%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.27%, preferably less than 0.2%, preferably less than 0.18%, and preferably less than 0.1%.

[0023] In some embodiments, the affinity chromatography packing material used has Protein L as the ligand, and preferred affinity chromatography packing materials are Diamond Protein L, MaXtar Protein L, or MabSelect VL.

[0024] In some embodiments, the affinity chromatography uses gradient elution with solutions A and B as eluents, wherein solution A includes NaAc and solution B includes HAc-NaOH.

[0025] In some embodiments, the NaAc concentration of solution A is about 10 to 100 mmol / L, preferably about 20 to 60 mmol / L, and most preferably about 50 mmol / L; the pH of NaAc is about 5 to 6, preferably about 5.5.

[0026] In some embodiments, the concentration of HAc-NaOH in solution B is about 10 to 100 mmol / L, preferably about 20 to 60 mmol / L, and most preferably about 50 mmol / L; the pH of HAc-NaOH is about 3 to 4, preferably about 3.2.

[0027] In some embodiments, the eluent of the gradient elution has a pH gradient of about 2.0-6.0, preferably about 3.2-5.5, more preferably about 3.8-5.5, more preferably about 4.1-5.45, and more preferably about 4.46-4.77.

[0028] In some embodiments, during gradient elution, purified faraximab is collected in the pH range of 3.8-5.5, preferably in the pH range of 4.16-5.41, and even more preferably in the pH range of 4.46-4.77.

[0029] In some implementations, the process of equilibrating the affinity chromatography medium with a equilibration buffer is included before the purified faraximab is bound to the affinity chromatography medium.

[0030] In some embodiments, the equilibration buffer comprises Tris and NaCl. In some preferred embodiments, the concentration of Tris is about 10 mmol / L to 50 mmol / L, preferably about 20 mmol / L, about 30 mmol / L, or about 40 mmol / L. In some preferred embodiments, the concentration of NaCl is about 50 mmol / L to 500 mmol / L, preferably about 100 mmol / L, about 200 mmol / L, about 300 mmol / L, or about 400 mmol / L. In some preferred embodiments, the pH of the equilibration buffer is about 5-9; in some preferred embodiments, the pH of the equilibration buffer is about 6.5-8.5; in some preferred embodiments, the pH of the equilibration buffer is about 7-8; and in some preferred embodiments, the pH of the equilibration buffer is about 7.4.

[0031] In some embodiments, the washing process includes a rinsing step with a equilibration buffer. In some preferred embodiments, the washing process further includes a rinsing step with an eluent. In some preferred embodiments, the washing process involves rinsing with a equilibration buffer followed by rinsing with an eluent. In some preferred embodiments, the eluent comprises NaAc. In some preferred embodiments, the concentration of NaAc in the eluent is approximately 10 mmol / L to 50 mmol / L.

[0032] In some implementations, the purified faraximab is bound to the Protein L affinity chromatography medium in the host cell culture supernatant via step (a).

[0033] This disclosure has the following beneficial effects: 1. This disclosure discovers that there is a difference in the binding force between faraximab and polymeric impurities when they bind to the protein L chromatography medium. Therefore, this disclosure provides a method for purifying faraximab by protein L affinity chromatography. This method can effectively reduce impurities in faraximab, especially polymeric impurities, such as dimeric impurities and non-covalently bound HH, LL, and HHL impurities.

[0034] 2. This disclosure also reveals that, significantly different from affinity chromatography purification using nanobody ligand media, fareximab and polymeric impurities exhibit differences in binding affinity to the protein L chromatography medium under different pH elution buffer conditions. Therefore, the purity of fareximab can be improved by adjusting the pH of the elution buffer to elute it.

[0035] In summary, this disclosure demonstrates that protein L affinity chromatography media is more suitable for fareximab purification, and optimizes the pH of the elution buffer to improve the purity of fareximab. The preparation process described in this disclosure is rapid, simple, and easily scalable.

[0036] Terminology Explanation Before describing this disclosure in detail, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0037] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0038] As used herein, the term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit that is 10% less than the specified value and an upper limit that is 10% greater than the specified value.

[0039] As used herein, the term “and / or” when used with two or more consecutive options should be understood to mean any one of the options or any two or more of the options.

[0040] As used in this article, the terms “Protein L” and “protein L” are used interchangeably.

[0041] As used in this article, the term "Protein L affinity chromatography" refers to a chromatographic method that uses protein L as a ligand to separate and purify target substances.

[0042] As used herein, the terms “affinity chromatography medium,” “affinity chromatography packing material,” “affinity chromatography resin,” “affinity medium,” “affinity packing material,” and “affinity resin” are used interchangeably. As used herein, the terms “affinity chromatography collection buffer”, “affinity collection buffer”, and “affinity elution collection buffer” are used interchangeably.

[0043] This article describes the detection of antibody-related impurities using SEC-HPLC, or Size Exclusion Chromatography-High Performance Liquid Chromatography. Its detection principle is based on the separation and analysis of differences in molecular size. It can detect high molecular weights (HMWS), monomers, and low molecular weights (LMWS). High molecular weights (HMWS) are typically aggregates formed spontaneously by proteins or during processing / storage, and may affect the safety, efficacy, and stability of drugs. Monomers refer to intact, correctly folded, and active target protein / antibody molecules. Low molecular weights (LMWS) refer to small fragments resulting from protein / antibody breakage and degradation.

[0044] As used in this article, "dimeric impurity" refers to an aggregate impurity formed by two antibody molecules interacting through non-covalent bonds or by disulfide bond mismatch, with a molecular weight approximately twice that of the monomer.

[0045] As used in this article, "non-covalently bound HH, LL, HHL impurities" refers to aggregate impurities that are non-covalently bound to two heavy chains (HH) and / or two light chains (LL) and / or lack one light chain in a 3 / 4 antibody (HHL). Dimeric impurities and non-covalently bound HH, LL, and HHL impurities are all aggregate impurities that may affect the safety, efficacy, and stability of the drug.

[0046] As used herein, the term "antibody" includes functionally active fragments, derivatives, or analogues of antibodies that bind specifically to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or to tumor cells or the matrix. This term includes, but is not limited to, monoclonal or polyclonal antibodies in the forms of IgA, IgD, IgE, IgG, and IgM, as well as those modified through genetic engineering such as humanization, chimerism, recombination, and mutation.

[0047] As used herein, the term "affinity filler ligand" refers to a ligand chemically coupled to the matrix, specifically binding to antibodies but not to impurities, with the bound antibody re-dissociating under specific conditions. This term includes, but is not limited to, protein A, protein G, and protein L. Protein A can be a surface protein derived from Staphylococcus aureus, specifically binding to the Fc region of IgG. Through genetic engineering, it exhibits high tolerance to acidic pH and denaturing agents, while also enhancing alkali tolerance, allowing for washing with sodium hydroxide. Protein G can be a cell wall protein derived from streptococcal group G, specifically binding to the Fc region of IgG. Through genetic engineering, cross-reactivity and non-specific binding are reduced. Protein L can be a protein isolated from Streptococcus magnus that specifically binds to immunoglobulins, binding to antibodies containing the κ light chain. Attached Figure Description

[0048] Figure 1 Chromatographic pattern of fareximab captured by protein L affinity medium.

[0049] Figure 2 Chromatographic pattern of faraximab captured by CaptureSelect FcXP affinity medium.

[0050] Figure 3 The SEC-mer peak pattern of the collection solution of fareximab purified by CaptureSelect FcXP affinity medium and protein L affinity chromatography. Detailed Implementation

[0051] The present disclosure will be further elaborated below with reference to specific embodiments. The following descriptions are preferred embodiments of the present disclosure and are for illustrative purposes only, not intended to limit the scope of protection claimed by the present disclosure.

[0052] Experimental methods in the following examples, unless otherwise specified, were performed according to conventional methods in the art. Percentages and parts are by weight unless otherwise defined. All technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this disclosure.

[0053] Unless otherwise specified, all reagents or instruments described in this disclosure are commercially available products.

[0054] There are no limitations on the protein L affinity media that can be used in this disclosure, and all of them are commercially available. Several commercial sources of known protein L affinity media are available, including but not limited to: MabSelect VL from Cytiva Sweden AB, MaXtar Protein L from Bailinco (Lanzhou) New Materials Co., Ltd., or Diamond Protein L from Bogelon (Zhejiang) Biotechnology Co., Ltd.

[0055] There are no limitations on the affinity chromatography column used in the affinity chromatography steps of this disclosure. For example, a column bed height of approximately 20 cm can be used. In some embodiments, the chromatography column is a suitable specification column filled with MaXtar Protein L, and in some embodiments, the inner diameter of the chromatography column is approximately 1.0 cm, a size suitable for condition optimization studies. In some embodiments, the inner diameter of the chromatography column is approximately 2.6 and 5.0 cm, a size suitable for small-scale purification. Other column sizes with different inner diameters can also be used in this disclosure, for example, an inner diameter of 0.66 cm, or 1.6 cm, or larger.

[0056] Example 1: Preparation of Fareximab Faricimab (CAS No. 1607793-29-2) is a bispecific antibody targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). Its first heavy chain (Ang-2 heavy chain) gene sequence is shown in SEQ ID NO:1, its second heavy chain (VEGF-A heavy chain) gene sequence is shown in SEQ ID NO:2, its first light chain (Ang-2 light chain) gene sequence is shown in SEQ ID NO:3, and its second light chain (VEGF-A light chain) gene sequence is shown in SEQ ID NO:4. Therefore, the nucleotide sequences of the first heavy chain, first light chain, second heavy chain, and second light chain of the faricimab gene were first synthesized. These sequences were then constructed into a QGV expression vector (LONZA) using a first light chain-second light chain-first heavy chain-second heavy chain linkage. The expression vector was then introduced into CHO cells via stable transfection. Cell recovery and expansion culture was carried out in a tank for 14 days to obtain a cell harvest medium containing faraximab. The harvest medium was then centrifuged, deep filtered, and sterilized filtered to obtain a clear harvest medium containing faraximab.

[0057] Example 2: Purification of Farecizumab The harvested solution of faraximab prepared in Example 1 was purified by protein L affinity chromatography and nanobody ligand medium affinity chromatography, respectively, and the purification effect was verified.

[0058] 1) Purification using L-affinity chromatography of proteins The harvested filtrate of fareximab prepared in Example 1 was purified using Diamond Protein L (BorgLone (Zhejiang) Biotechnology Co., Ltd.) affinity medium. The chromatographic pattern of fareximab captured by the Protein L affinity medium is shown below. Figure 1 As shown.

[0059] First, the Diamond Protein L affinity chromatography column was equilibrated using approximately three column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4). Then, the harvest buffer containing fareximab molecules was loaded, with a loading not exceeding 36 g / L, to allow the antibody molecules to adsorb onto the chromatography medium. After loading, the column was reequilibrated using approximately four column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4), followed by washing with eluent (50 mmol / L NaAc, pH 5.5). Finally, elution was performed using elution buffer (Solution A: 50 mmol / L NaAc, pH 5.5; Solution B: 50 mmol / L HAc-NaOH, pH 3.2; gradient elution from 0% to 100% solution B). Fractional collection was performed within a suitable pH range (monitored post-column) to obtain purified fareximab. The obtained samples were detected using SEC-HPLC.

[0060] 2) Purification using nanobody ligand-media affinity chromatography The harvested solution of fareximab prepared in Example 1 was purified using Thermo CaptureSelect™ FcXP affinity medium (hereinafter referred to as FcXP). The chromatographic pattern of fareximab captured by the FcXP affinity medium is shown below. Figure 2 As shown.

[0061] First, the FcXP affinity chromatography column was equilibrated using approximately three column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4). The harvested solution containing fareximab molecules was then loaded with a loading of no more than 36 g / L to allow the antibody molecules to adsorb onto the chromatography medium. After loading, the sample was washed with approximately four column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4), followed by approximately four column volumes of wash buffer 1 (purified water). Finally, elution was performed with elution buffer (20 mmol HAc). According to the instructions for ThermoCaptureSelect™ FcXP affinity chromatography, the suitable elution pH is 3.0-4.0. Therefore, pH 3.4 was selected within the middle range for elution. The collection interval was from 500 mAU to 200 mAU, at which point collection was stopped. The purified fareximab was then obtained. The obtained sample was detected using SEC-HPLC.

[0062] Experimental results: The purification results of fareximab using protein L affinity chromatography and FcXP affinity chromatography are shown in Table 1: Table 1. SEC Test Results

[0063] As shown in Table 1, the SEC results indicate that compared to the FcXP affinity chromatography method, the antibody purity obtained using the protein L affinity chromatography method was increased by 3.3%-9.1%, and the content of aggregate impurities was reduced by 6.9%-9.28%. Therefore, protein L affinity chromatography is more suitable than FcXP affinity chromatography for the capture and purification of faraxicam, as it can effectively remove aggregate impurities and improve the purity of faraxicam.

[0064] Example 2: Purification of fareximab using different protein L affinity mediators The harvested fluid of faraximab prepared in Example 1 was purified using MaXtar Protein L (Bailinke (Lanzhou) New Materials Co., Ltd.) affinity medium.

[0065] First, the Diamond Protein L affinity chromatography column was equilibrated using approximately three column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4). Then, the harvest buffer containing fareximab molecules was loaded, with a loading not exceeding 27 g / L, to allow the antibody molecules to adsorb onto the chromatography medium. After loading, reequilibration was performed using approximately four column volumes of equilibration buffer (20 mmol / L Tris-HCl, 200 mmol / L NaCl, pH 7.4), followed by washing with eluent (50 mmol / L NaAc, pH 5.5). Finally, elution was performed using elution buffer (Solution A: 50 mmol / L NaAc, pH 5.5; Solution B: 50 mmol / L HAc-NaOH, pH 3.2; 0–100% solution B was used for gradient elution). Fractional collection was performed within a suitable pH range (monitored post-column) to obtain purified fareximab. The obtained samples were detected using SEC-HPLC.

[0066] Experimental results: The purification results of fareximab using protein L affinity chromatography and FcXP affinity chromatography are shown in Table 2: Table 2. SEC Test Results

[0067] As shown in Table 2 of the SEC results, the MaXtar Protein L affinity chromatography method can effectively remove aggregate impurities and improve the purity of fareximab. Compared with FcXP affinity chromatography in Example 1, the antibody purity obtained by the MaXtar Protein L affinity chromatography method increased by 6.1%, and the aggregate content decreased by 9.3%. Therefore, this purification technique is applicable to different protein L affinity media and has a certain degree of universality.

[0068] Example 3: The effect of protein L affinity chromatography and nanobody ligand-mediated affinity chromatography on the removal of antibody aggregates To further investigate the effect of protein L affinity chromatography on polymers, polymer analysis was performed on samples collected by protein L affinity chromatography and nanobody ligand-mediated affinity chromatography in Example 1. The harvested solution contained two components: a dimer component ( Figure 3 Peak 2) and dimers and non-covalently linked LL, HH, HHL components ( Figure 3 Peak 3).

[0069] The polymer contents of the two components in the affinity chromatography harvest of Example 1 are shown in Table 3: Table 3. Results of protein L affinity chromatography and FcXP affinity chromatography for SEC polymer detection

[0070] Not detected As shown in Table 3, protein L affinity chromatography can completely remove dimers and non-covalently linked LL, HH, and HHL components. Compared with nanobody ligand affinity chromatography using FcXP, protein L affinity chromatography reduced the peak 2 component (dimer) by 0.54%–2.52%.

[0071] In summary, this disclosure presents an inventive discovery that, compared to nanobody ligand-based affinity chromatography, protein L affinity chromatography can reduce the content of dimer impurities and improve the purity of fareximab by adjusting the pH of the elution buffer. Different pH values ​​of the elution buffer also affect the purification effect of protein L affinity chromatography, allowing for the elution of antibodies with different binding affinities using different elution pH values.

[0072] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent disclosure. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent disclosure should be determined by the appended claims.

Claims

1. A method for reducing impurities in faraximab, characterized in that, The method includes the following steps: (a) Binding the purifying faraximab to an affinity chromatography medium; (b) Wash the affinity chromatography medium with washing buffer; (c) Elute fareximab from the affinity chromatography medium using elution buffer; The affinity chromatography is Protein L affinity chromatography; the impurities include polymeric impurities, which include dimeric impurities and / or non-covalently bound HH, LL, and HHL impurities.

2. The method according to claim 1, characterized in that: The content of faraximab is higher than 93% of the total content, and / or the content of polymeric impurities is lower than 2.6% of the total content.

3. The method according to claim 1, characterized in that: The elution pH is 2.0-6.

0.

4. The method according to claim 1, characterized in that, The affinity chromatography medium is one of MaXtar Protein L, Diamond Protein L, or MabSelect VL.

5. The method according to claim 1, characterized in that, The purifying faraximab is bound to the affinity chromatography medium in the host cell culture supernatant via step (a).

6. The use of Protein L affinity chromatography in reducing impurities in fareximab, characterized in that, (a) Binding the purifying faraximab to an affinity chromatography medium; (b) Wash the affinity chromatography medium with washing buffer; (c) Elute fareximab from the affinity chromatography medium using elution buffer; The impurities include polymeric impurities, which include dimeric impurities and / or non-covalently bonded HH, LL, and HHL impurities.

7. The use according to claim 6, characterized in that: The content of the faraximab monomer is higher than 93% of the total content, and / or the content of polymeric impurities is lower than 2.6% of the total content.

8. The use according to claim 6, characterized in that: The elution pH is 2.0-6.

0.

9. The use according to claim 6, characterized in that, The affinity chromatography medium is MaXtar Protein L, Diamond Protein L, or MabSelect VL.

10. The use according to claim 6, characterized in that, The purifying faraximab is bound to the affinity chromatography medium in the host cell culture supernatant via step (a).

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