Antibody purification method

Abstract

Provided is a method for purifying an antibody from a cell culture harvest solution. By means of combining a conventional antibody purification method and octanoic acid or octanoate incubation, the method reduces the content and types of host cell proteins (HCPs), wherein a polysorbate-stabilized antibody composition is obtained by the purification method, the octanoate incubation can further reduce the aggregate content and improve the protein purity, and the process is simple and easy to operate.

Description

A method for purifying antibodies

[0001] This application claims priority to Chinese Patent Application No. 2024117742214, filed on December 4, 2024, entitled "A purification method for controlling the degradation of polysorbate in high-concentration antibody compositions", and Chinese Patent Application No. 2025117839178, filed on November 28, 2025, entitled "A purification method for antibodies", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody purification, and more specifically to a purification method using chromatography-bound reagent incubation, and to the controlled degradation of polysorbate in the antibody composition obtained by this purification method. Background Technology

[0003] Polysorbates (PS) are a class of amphiphilic nonionic surfactants. Due to their good biocompatibility and high hydrolipophilic-lipophilic balance (HLB), especially polysorbate 20 (PS20) and polysorbate 80 (PS80), they are widely used in biopharmaceutical formulations as protein stabilizers to prevent protein denaturation and aggregation. However, polysorbates are easily degraded through oxidation or hydrolysis by certain lipases / esterases, releasing free fatty acids. Degradation of polysorbates can reduce their effectiveness in protecting protein drug components and, over time, form visible or invisible particles, affecting the stability and safety of protein formulations. With the development of high-concentration protein formulations for antibody drugs, the degradation problem of polysorbates has become increasingly prominent. Therefore, regulatory authorities are increasingly scrutinizing polysorbate control strategies to ensure that the polysorbate content remains constant throughout the shelf life of drug products.

[0004] The hydrolysis of polysorbate (PS) is primarily caused by the cleavage of ester bonds by high-risk host cell proteins (HCPs), which include lipases, esterases, and other process-related impurities that may not possess known lipase or esterase activity. Even at low HCP levels in bioproducts, PS hydrolysis still occurs. The hydrolysis of PS increases with time and the concentration of endogenous lipoprotein lipase A2 (rLPLA2) (Troii Hall et al., (2016) Pharmaceutical Biotechnology 105(5)1633-1642). Higher concentrations of rLPLA2 (1 ppm) result in approximately 90% hydrolysis of PS80 after 5 days, while trace amounts of rLPLA2 (0.1 ppm) result in 30% hydrolysis of PS80 and 10%-15% hydrolysis of PS20 after 5 days. In one example of a biologic, the amount of hydrolytic enzymes is extremely small (Tobias Graf et al., (2021) Pharmaceutical Biotechnology 110(11)3558-3567), so that existing detection techniques cannot detect low levels of HCPs (<10 ppb). However, trace amounts of highly active hydrolytic enzymes may be sufficient to affect the stability of PS in the drug during long-term storage. Patent WO2021 / 076620 describes a method to reduce polysorbate degradation in protein formulations by modifying host cells to reduce or eliminate the expression of palmitoyl protein thioesterase 1 (PPT1), lysosomal acid lipase (LAL), lipoprotein lipase (LPL), phospholipase D3 (PLD3), and / or phospholipase A2 (LPLA2).

[0005] Caprylic acid (CFA) is a naturally occurring octanoic acid, an amphiphilic molecule containing a hydrophobic alkyl chain and a hydrophilic carboxylate, and can be used as a surfactant. CFA can bind to specific sites on proteins, inducing protein unfolding and triggering protein denaturation and precipitation. Studies have found that during monoclonal antibody purification, CFA can selectively precipitate non-IgG proteins, effectively removing hemolytic glycosides (HCPs) from the antibody. Sodium caprylate, the sodium salt of CFA, has better solubility and is easier to handle in production than CFA. Patent WO2018 / 047080 describes achieving higher HCP removal rates by adding sodium caprylate and arginine to the protein A washing solution. Two experiments investigated the removal of two difficult-to-remove HCPs, cathepsin A and PLBL-2, after in vitro expression. However, effective methods and data support for controlling polysorbate degradation are still lacking, especially for high-concentration polysorbate degradation in biopharmaceuticals.

[0006] Therefore, in the field of antibodies, mitigating the degradation of polysorbate in antibody compositions, especially high-concentration antibody compositions, is a challenging direction in this field. Summary of the Invention

[0007] The inventors conducted purification process optimization research and adopted a chromatography process combined with reagent incubation to solve the problem of polysorbate degradation in antibody compositions.

[0008] Therefore, in one aspect, this disclosure provides a method for reducing polysorbate degradation in antibody compositions through an improved purification process. The purification process of this disclosure reduces the content and types of residual host cell proteins in the composition, effectively controlling the degradation of polysorbate during the storage of the antibody composition.

[0009] In one aspect, this disclosure provides a method for reducing the content of lipase, lipoprotein lipase and / or phospholipase A2 in an antibody composition, comprising the step of incubation with caprylic acid or caprylate.

[0010] In one aspect, this disclosure provides a purification method for reducing polysorbate degradation in an antibody composition, comprising the steps of incubation with caprylic acid or caprylate.

[0011] In some embodiments, the final concentration of caprylic acid or caprylate during incubation is 5–50 mmol / L.

[0012] One embodiment of this disclosure further includes the following steps: the pH value during the incubation process is 4.0-6.0, and deep filtration is performed after incubation with octanoic acid or octanoate.

[0013] In some embodiments, the method of this disclosure further includes the following steps:

[0014] Virus inactivation, and / or chromatography.

[0015] In some specific embodiments, the chromatography operation includes one or more of affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and complex mode chromatography.

[0016] In some specific embodiments, the steps in the method are combined in any order: incubation with caprylic acid or caprylate, virus inactivation, and chromatography; preferably, the method includes one or more chromatography steps.

[0017] In some specific embodiments, the method of this disclosure includes the following steps:

[0018] Affinity chromatography: Protein A affinity chromatography is performed on the cell harvest medium containing the antibody, followed by elution with elution buffer, and the antibody collection medium is collected.

[0019] Virus inactivation: Perform low pH virus inactivation;

[0020] Octanoic acid or octanoate incubation: After neutralizing the pH of the low-pH virus-inactivated sample, add a certain concentration of octanoic acid or octanoate and stir for incubation;

[0021] Deep filtration: The incubated sample is subjected to deep filtration, and the clear liquid containing the antibody is collected;

[0022] Ion exchange chromatography: The clarified solution is loaded onto an ion exchange chromatography medium, and the collected solution containing the antibody is collected;

[0023] Hydrophobic chromatography or complex mode chromatography: The collected solution is subjected to hydrophobic interaction chromatography or complex mode chromatography to collect the percolation solution containing the antibody, which is used to prepare a high concentration of antibody composition.

[0024] In some embodiments, the virus inactivation is low-pH virus inactivation, wherein the pH of the low-pH virus inactivation is 3.3 to 3.7.

[0025] In some embodiments, the washing buffer for affinity chromatography contains about 0.1-1 mol / L arginine, 10-30 mmol / L Tirs-HCl, and the buffer pH is 7.3-7.9.

[0026] In some embodiments, the final concentration of octanoic acid or octanoate is 5–50 mmol / L, more preferably 10–40 mmol / L; the stirring and incubation conditions for octanoic acid or octanoate are an incubation time of more than 10 min, preferably 30–180 min, and a stirring speed of 50–300 rpm; preferably, the octanoate is sodium octanoate.

[0027] In some embodiments, the deep filter of the deep filtration is made of cellulose diatomaceous earth, with diatomaceous earth as a filter aid, preferably AlHC or X0HC.

[0028] In some embodiments, the ion exchange chromatography is cation exchange chromatography or anion exchange chromatography, with the anion exchange chromatography medium being Q-FF, Diamond Q, Monomix Mab 60Q, or MaXtar Q; and the cation exchange chromatography medium being Capto SP SPImpRes, Eshmuno CPX, Maxtar SP HR, or NanoGel 50SP HP. Preferably, the equilibration buffer in the cation exchange chromatography contains 10-30 mmol / L acetate at pH 5.0-6.0; and the eluent contains 10-30 mmol / L acetate and 100-200 mmol / L NaCl at pH 5.0-6.0.

[0029] In some embodiments, the hydrophobic chromatography medium is Capto phenyl ImpRes, UniHR phenyl 80L, Diamond Phenyl (HS), or MaXtar Phenyl (HS); the composite mode medium is Capto adhere, NM90 Agrose HAM, or Diamond MIX-A.

[0030] In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80.

[0031] In some embodiments, the antibody purified by the method of this disclosure is used to prepare a high-concentration antibody composition, preferably, the concentration of the high-concentration antibody is about 100-200 mg / mL.

[0032] In some embodiments, the antibodies disclosed herein are selected from dupilumab and gusejinumab.

[0033] In some embodiments, the antibody composition obtained by the method of this disclosure has one or more characteristics selected from the following:

[0034] - Compared with conventional purification processes, the residual amount of HCPs in the purified antibody is reduced by at least 30%, preferably by at least 35%, 45%, 55%, 65%, 75%, or 85%;

[0035] - Compared with conventional purification processes, the antibody composition exhibits reduced polysorbate degradation rate after storage; preferably, after storage at 37°C for 4 weeks, the polysorbate degradation rate is reduced by at least 5%, preferably at least 10%, 20%, 30%, or 40%; after storage at 25°C for 3 months, the polysorbate degradation rate is reduced by at least 20%, preferably at least 30%, 40%, 50%, or 60%.

[0036] - Compared with conventional purification processes, the residual amount of caprylic acid or caprylate in the purified antibody is less than about 17.6 μg / ml;

[0037] - Compared to conventional purification processes, the purified antibody showed a reduction of approximately 0.5% or more in mesmerism.

[0038] The advantage of the process disclosed herein lies in the use of caprylic acid or caprylate incubation during the purification process, and the adjustment of sample pH and caprylic acid / caprylate concentration. By controlling the stirring time and stirring speed, the residual amount of HCP, especially lipase, lipoprotein lipase and phospholipase A2, is effectively reduced, thereby effectively mitigating the degradation of polysorbate in the antibody composition.

[0039] This disclosure reveals that introducing a sodium caprylate incubation step into the dupilumab purification process significantly reduces both the content and types of host cell proteins (HCPs). Simultaneously, the free fatty acid content in antibody composition stability studies shows a significant decrease, indicating a marked improvement in polysorbate degradation. Studies have shown that the sodium caprylate incubation process reduces the content of hydrolytic enzymes such as lipase, lipoprotein lipase, and phospholipase A2, thereby reducing polysorbate degradation in high-concentration antibody compositions. Furthermore, the study found that the sodium caprylate incubation method is not only applicable to improving polysorbate degradation in dupilumab antibody compositions, but also has an improving effect on polysorbate degradation in gusejinumab antibody compositions and IL-33 / TSLP mixed antibody compositions, indicating that the method of this disclosure has universal applicability in reducing the content of polysorbate-related hydrolytic enzymes.

[0040] The preparation process described in this disclosure is characterized by its speed, simplicity, ease of scaling up, and suitability for large-scale production. Attached Figure Description

[0041] Figure 1 shows a comparison of HCP detection results by mass spectrometry in Example 1, where the horizontal axis represents the pI of HCP, the vertical axis represents the molecular weight of HCP, and the size of the circle represents the content of HCP.

[0042] Figure 2 shows the polysorbate detection spectrum of the experimental group in Example 2 after being placed at 25°C for 3 months.

[0043] Figure 3 shows the polysorbate detection spectrum of the control group of Example 2, which was placed at 25°C for 3 months.

[0044] Figure 4 shows a typical chromatogram for detecting residual sodium octanoate standard by gas chromatography.

[0045] Figure 5 shows the detection chromatogram of sodium octanoate residue in the antibody composition of Example 2 by gas chromatography.

[0046] Figure 6 shows a comparison of the detection results of free fatty acids from polysorbate in Example 3.

[0047] Invention Details

[0048] It should be understood that this disclosure is not limited to specific methods, reagents, compounds, compositions, or biological systems, which can of course vary. It should also be understood that the terminology used in this disclosure is for describing specific embodiments only and is not intended to limit the scope of this disclosure.

[0049] The term "host cell protein (HCP)" refers to the protein components of a bioproduct derived from the host cells of the production cell line. It mainly includes host cell structural proteins and secretory proteins, and is a mixture of various proteins, not the target component. These proteins are diverse, and their physicochemical properties, such as isoelectric point, hydrophobicity, and relative molecular weight, often vary significantly.

[0050] The term "polysorbate degradation" refers to the process in which the chemical structure (mainly ester bonds) of polysorbate is disrupted, leading to a decrease in molecular weight, altered properties, and the production of degradation products such as free fatty acids and free ethoxylates. The main degradation pathways are oxidation and hydrolysis (chemically induced or enzyme-mediated). The biological agents involved in this disclosure all have a pH range of 5-6, and the possibility of chemical hydrolysis of polysorbate is expected to be negligible. Therefore, polysorbate degradation in this disclosure mainly refers to enzymatic hydrolysis caused by host cell proteins.

[0051] The term "high-risk HCP" refers to HCPs that can cause polysorbate ester bond cleavage. Since HCPs are a mixture of various proteins, high-risk HCPs not only include HCPs that have been clearly reported to cause polysorbate ester bond cleavage, such as esterases like phospholipase B-like protein 2 (PLBL2), lipoprotein lipase (LPL), lysosomal phospholipase A2 (LPLA2), and hepatic carboxylesterase, but also other host cell protein impurities that may not have known lipase or esterase activity.

[0052] The high-concentration antibody composition in this disclosure refers to a biological agent with an antibody protein concentration of ≥70 mg / mL, including but not limited to protein concentrations of ≥70 mg / mL, ≥100 mg / mL, ≥125 mg / mL, ≥150 mg / mL, and ≥200 mg / mL. The high-concentration antibody composition mentioned in this disclosure also includes a high-concentration stock solution; this high-concentration antibody composition is an injectable solution, and lyophilized powder formulations are not within the scope of this disclosure.

[0053] The antibodies in this disclosure include functionally active fragments, derivatives, or analogs of antibodies that specifically bind to target cells (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies that bind to tumor cells or the matrix.

[0054] The degradation rate referred to herein is measured by determining the polysorbate content in the composition after storage at a specific temperature for a specific time. The less the polysorbate content in the composition decreases compared to before storage, the lower the degradation rate of polysorbate, and thus the higher the stability of polysorbate. The antibody compositions produced by the method of this disclosure have improved polysorbate stability, differing from conventional production methods of the control group only in the presence or absence of the caprylic acid / caprylate usage step of this disclosure.

[0055] HCP Residue Determination Method

[0056] ELISA assay: The assay is performed using a known ELISA method in the art, namely the Universal CHO Host Cell Protein Detection Kit (Cygnus Technologies, Inc., Catalog #F550-1) or a similar product, to determine the total amount of immunogenic HCP in the sample. The assay method is described in the kit instructions.

[0057] Mass spectrometry determination: Detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The sample was replaced with enzyme digestion buffer, and non-denaturing enzyme digestion was performed using trypsin. The digested sample was then heated to reduce its concentration, centrifuged, and the supernatant was used for separation using a C18 or similar reversed-phase column. High-resolution mass spectrometry was then used for detection.

[0058] Antibody purity determination method (SEC-HPLC method)

[0059] Size exclusion chromatography (SEC-HPLC) was used to determine the molecular size heterogeneity of the samples. Separation was performed using a TSKgel G3000 SWXL (TOSOH, catalog number 0008541) or a similar column. Refer to Section 0514, "Size Exclusion Chromatography," Part III, 2025 edition of the Chinese Pharmacopoeia. The content of high molecular weight substances, monomers, and low molecular weight substances in the samples was calculated using the area normalization method.

[0060] Sodium octanoate detection method

[0061] The residual amount of sodium octanoate in the intermediate during the purification process was detected by gas chromatography. Octanoate reference standard was prepared and serially diluted with chloroform; the test sample was extracted with chloroform; the treated reference standard and test sample were then subjected to gas chromatography to detect sodium octanoate residues. The operation of the gas chromatography method can be found in General Chapter 3111 of the 2025 edition of the Chinese Pharmacopoeia.

[0062] Polysorbate component distribution analysis

[0063] The detection was performed using liquid chromatography-mass spectrometry (LC-MS). Acetonitrile or a similar organic solvent was used to precipitate the protein in the sample, and the supernatant was used for separation using a C4 or similar reversed-phase column. High-resolution mass spectrometry was then used for detection.

[0064] Free fatty acid detection

[0065] The detection was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Acetonitrile or a similar organic solvent was used to precipitate the protein in the sample, and the supernatant was used for separation using a C18 or similar reversed-phase column. Multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry was used for detection.

[0066] Quantitative analysis of esterases

[0067] The esterases were detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The sample was transferred to enzyme digestion buffer and digested with trypsin in a non-denaturing manner. The digested sample was then heated to reduce its concentration, centrifuged, and the supernatant was used for separation on a C18 or similar reversed-phase column. Multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry was then used for detection. Detailed Implementation

[0068] 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 and are not intended to limit the scope of protection claimed by the present disclosure. It should be noted that any modifications and variations made by those skilled in the art based on this inventive concept are within the scope of protection of the present disclosure.

[0069] 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.

[0070] Unless otherwise specified, all reagents or instruments described in this disclosure are commercially available conventional products. The sodium caprylate used in this disclosure was purchased from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd. or Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.

[0071] Example 1: Removal of HCP by incubation with sodium octanoate

[0072] HCPs (host cell proteins) are a mixture of a vast number and types, the exact number depending on the type of host cell and production conditions. The variety of HCPs varies considerably, ranging from hundreds to thousands, depending on the host cell and culture conditions. HCPs are inherently highly heterogeneous, differing in molecular weight, isoelectric point, and hydrophobicity, some of which can be difficult to remove. Affinity chromatography, with its high selectivity, is an effective method for removing HCPs; this step alone can remove over 90% of HCPs from the clarified harvest. However, in many cases, reducing HCPs in the final drug product to acceptable levels remains a challenging task. Further effective removal of HCPs can be achieved by incubating the affinity chromatography eluted sample with sodium caprylate.

[0073] The experimental group's process is as follows:

[0074] 1) Affinity chromatography and virus inactivation: The protein A affinity chromatography column was washed with affinity equilibration buffer, followed by loading cell culture harvest medium containing dupilumab antibody. After washing with washing buffer, the column was eluted with elution buffer with a pH range of 3.3–3.7. The antibody was collected under UV light at 280 nm. The pH was then adjusted to 3.3–3.7 with 1 mol / L citric acid, and the column was incubated at room temperature for 60–120 min to inactivate the virus.

[0075] 2) Sodium octanoate incubation and deep filtration: Take the virus-inactivated sample, add 2 mol / L Tris to adjust the pH to 4-6, and slowly add sodium octanoate solution while stirring until the final concentration is 5-50 mmol / L. Control the stirring speed to 100-250 rpm and stir at room temperature for ≥30 min. Filter using a deep membrane bag, collect the filtrate, and perform sterile filtration to obtain the filtered clear liquid.

[0076] The control group samples were not incubated with sodium octanoate. After affinity chromatography and virus inactivation, the samples were taken, and 2 mol / L Tris was added to adjust the pH to 5-6. The samples were then filtered using a deep membrane bag, the filtrate was collected, and sterile filtration was performed to obtain the filtered clear liquid.

[0077] Analysis of experimental results:

[0078] HCP residues were detected in the affinity elution collection, the clarified solution of the control group, and the clarified solution of the experimental group. Mass spectrometry was used for HCP residue detection, which, compared to ELISA, can cover a wider range of HCP species. The results are shown in Table 1: Compared to the samples without sodium octanoate incubation (control group), the HCP content in the experimental group samples decreased by 83.4%, and the number of HCP species decreased by 79.1%. Detailed data are shown in Table 1 and Figure 1.

[0079] Table 1. HCP content and types in samples

[0080] Example 2: Purification method of dupilumab

[0081] Sample source

[0082] First, the dupilumab gene sequence was synthesized and cloned into an expression vector to obtain the recombinant expression plasmid pCGS3.2-HC-LC. The expression system was introduced into CHO cell lines through stable transfection. Finally, the cells were revived and expanded in a culture vessel for 16 days to obtain cell culture harvest medium containing dupilumab antibody molecules.

[0083] Experimental group: Optimization of purification process

[0084] 1) Affinity chromatography and virus inactivation: AT Diamond ProteinA Plus (Borglon (Zhejiang) Biotechnology Co., Ltd.) protein A affinity chromatography medium was used. Cell culture harvest medium containing antibodies was equilibrated with 20 mmol / L Tirs-HCl, 0.2 mol / L NaCl, and pH 7.4. After loading, the medium was equilibrated again with 20 mmol / L Tirs-HCl, 0.2 mol / L NaCl, and pH 7.4, followed by washing with 20 mmol / L Tirs-HCl, 0.5 mol / L Arg-HCl, and pH 7.4. After washing with 20 mmol / L Tirs-HCl and pH 7.4, elution was performed with elution buffer (20 mmol / L acetate buffer) with a pH range of 3.3–3.7. The antibodies were collected under UV light at 280 nm. The pH was adjusted to 3.3–3.7 with 1 mol / L citric acid, and the mixture was incubated at room temperature for 60–120 min to inactivate the virus.

[0085] 2) Sodium octanoate incubation and deep filtration: Take the virus-inactivated sample, add 2 mol / L Tris to adjust the pH to 4.5-5.5, and slowly add 0.5 mol / L sodium octanoate solution while stirring until the final concentration is 10-30 mmol / L. Control the stirring speed to 100-250 rpm and stir at room temperature for ≥30 min. Filter using a Merck A1HC deep membrane pack, collect the filtrate, and perform sterile filtration to obtain the filtered clear liquid.

[0086] 3) Cation exchange chromatography: The pH of the clarifying solution was adjusted using 2 mol / L Tris, loaded onto the cation exchange medium, and after sample loading, washed with 20 mmol / L acetate equilibration buffer at pH 5-6. Then, it was washed with a lower concentration of salt. After washing, it was eluted with 20 mmol / L acetate and 100-160 mmol / L NaCl at pH 5-6. The collected solution containing the antibody was obtained by UV spectroscopy at 280 nm.

[0087] 4) Hydrophobic interaction chromatography: The conductivity of the cation exchange chromatography collection solution is adjusted to 15-25 mS / cm, and the pH is adjusted to pH 7-8. The solution is then loaded into the hydrophobic interaction medium. The eluent containing the antibody is collected under 280 nm UV light. After loading, the solution is washed with 20 mmol / L Tris, 150-250 mmol / L NaCl, and pH 7-8 buffer. The eluent containing the antibody is then collected again under 280 nm UV light.

[0088] 5) Preparation of high-concentration antibody composition samples: Using a Pellicon3 (30kD) ultrafiltration membrane, the sample after column chromatography was ultrafiltered / washed. After the sample was concentrated, the buffer was replaced. The washing buffer was replaced 6-10 times the volume of the concentrated sample. Then, it was concentrated again to the target concentration. Polysorbate 80 was added and sterilized before aliquoting to obtain a high-concentration antibody composition.

[0089] Control group: conventional purification process

[0090] 1) Affinity chromatography and virus inactivation: AT Diamond ProteinA Plus (Borglon (Zhejiang) Biotechnology Co., Ltd.) protein A affinity chromatography medium was used. Cell culture harvest medium containing antibodies was equilibrated with 20 mmol / L Tirs-HCl, 0.2 mol / L NaCl, and pH 7.4. After loading, the medium was equilibrated again with 20 mmol / L Tirs-HCl, 0.2 mol / L NaCl, and pH 7.4, followed by washing with 20 mmol / L Tirs-HCl, 0.5 mol / L Arg-HCl, and pH 7.4. After washing with 20 mmol / L Tirs-HCl and pH 7.4, elution was performed with elution buffer (20 mmol / L acetate buffer) with a pH range of 3.3–3.7. The antibodies were collected under UV light at 280 nm. The pH was adjusted to 3.3–3.7 with 1 mol / L citric acid, and the mixture was incubated at room temperature for 60–120 min to inactivate the virus.

[0091] 2) Deep filtration: The virus-inactivated sample was neutralized to pH 5-6 using 2 mol / L Tris, filtered using a Merck A1HC deep membrane, the filtrate was collected and sterilized to obtain the filtered clear liquid.

[0092] 3) Cation exchange chromatography: The clarified solution is loaded onto the cation exchange medium. After loading, the solution is washed with equilibration buffer (20 mmol / L acetate, pH 5-6), followed by washing with a lower concentration of salt. After washing, the solution is eluted with 20 mmol / L acetate and 100-160 mmol / L NaCl, pH 5-6. The collected solution containing the antibody is obtained by UV spectroscopy at 280 nm.

[0093] 4) Hydrophobic interaction chromatography: The conductivity of the cation exchange chromatography collection solution is adjusted to 15-25 mS / cm, and the pH is adjusted to pH 7-8. The solution is then loaded into the hydrophobic interaction medium. The eluent containing the antibody is collected under 280 nm UV light. After loading, the solution is washed with 20 mmol / L Tris, 150-250 mmol / L NaCl, and pH 7-8 buffer. The eluent containing the antibody is then collected again under 280 nm UV light.

[0094] 5) Preparation of high-concentration antibody composition samples: Using a Pellicon3 (30kD) ultrafiltration membrane, the sample after column chromatography was ultrafiltered / washed. After the sample was concentrated, the buffer was replaced. The washing buffer was replaced 6-10 times the volume of the concentrated sample. Then, it was concentrated again to the target concentration. Polysorbate 80 was added and sterilized before aliquoting to obtain a high-concentration antibody composition.

[0095] Analysis of experimental results:

[0096] HCP residue detection and polysorbate stability studies were conducted on the antibody compositions of the experimental and control groups, respectively, to investigate the degradation of polysorbate in the antibody compositions at 25°C. HCP residue was determined using a known ELISA method, and polysorbate content was determined using a known high-performance liquid chromatography-fluorescence detection method. The results are shown in Table 2: Compared with the control group, the HCP content in the experimental group composition decreased by 95%, and after 3 months at 25°C, the degradation rate of polysorbate 80 in the experimental group decreased by 65.3%.

[0097] Table 2 HCP Residue Detection and Polysorbate Stability Study

[0098] The polysorbate 80 content changes between the optimized and conventional process samples can be observed from the liquid chromatography chromatograms in Figures 2 and 3. Sodium caprylate residue was determined in the experimental group's deep-filtration samples, cation exchange chromatography samples, and antibody compositions using gas chromatography. The results are shown in Table 3: Sodium caprylate residue was not detected in the experimental group's cation exchange chromatography samples (limit of quantification was 17.6 μg / ml). Figure 4 shows a typical chromatogram for sodium caprylate residue testing, and Figure 5 shows the chromatogram for sodium caprylate residue detection in the experimental group's antibody compositions.

[0099] Table 3 Results of sodium caprylate residues

[0100] Clarified samples from both the experimental and control groups after deep filtration were analyzed for purity using SEC-HPLC. The results are shown in Table 4: compared with the control group, the experimental group samples had lower polymer content.

[0101] Table 4 SEC-HPLC Purity Results

[0102] Example 3: Sodium caprylate incubation method for reducing polysorbate hydrolytic enzyme activity

[0103] Previous studies have shown that adding just one step of sodium caprylate incubation can significantly reduce the types and contents of HCPs, while also reducing the degradation rate of polysorbate in the stability study of dupilumab antibody compositions. Therefore, we analyzed the polysorbate component distribution of antibody compositions in the control and experimental groups placed at 25°C. The results showed that, compared with the 0°C condition, the degradation of polysorbate in both the control and experimental groups was mainly due to hydrolysis. Further analysis of free fatty acids in the control and experimental antibody compositions placed at 25°C for different times revealed that, compared with the control group, the increase in free fatty acid content in the experimental group was significantly slower, indicating that the sodium caprylate incubation step significantly improved polysorbate hydrolysis. The results are detailed in Figure 6.

[0104] Lipase, lipoprotein lipase, and Group XV phospholipase A2 (PLA2) have been reported to cause polysorbate degradation in protein formulations. These high-risk HCP enzymes often bind to antibodies and are difficult to remove during downstream processing. This study tested the ability of sodium caprylate incubation to scavenge these three HCP enzymes. Mass spectrometry was used to quantify lipase, lipoprotein lipase, and phospholipase A2. In this study, the clearance factor was the ratio of the initial impurity content to the final impurity content. The clearance factor of the sodium caprylate incubation step varied for different HCP enzymes. Lipase showed the highest clearance factor, reaching 39-fold, which was 10 times that of the control process (3.9-fold). Lipoprotein lipase showed a clearance factor of over 13-fold, and phospholipase A2 showed a clearance factor of over 3-fold, which were 5-fold and 2-fold higher than the control process (2.7-fold and 1.5-fold, respectively). This indicates that the content of polysorbate-related hydrolytic enzymes is significantly reduced after the sodium caprylate incubation process.

[0105] Table 5. Results of determination of lipase, lipoprotein lipase and phospholipase A2 content

[0106] Example 4: Purification method of Guselkumab

[0107] The above examples, using dupilumab as a model product, demonstrated that the sodium caprylate incubation process significantly improved polysorbate degradation in the antibody composition by efficiently reducing HCP enzymes that cause polysorbate degradation. To verify the general applicability of the sodium caprylate incubation process in improving polysorbate degradation in antibody compositions, other antibodies were selected for further testing and research.

[0108] Sample source

[0109] First, the ancient Seckiyo monoclonal antibody gene sequence was synthesized and cloned into an expression vector. The recombinant expression vector pDGV-LC-HC system was constructed, and the expression system was introduced into CHO cell lines through stable transformation. Finally, the cells were revived and expanded, and cultured in a tank for 14 days to obtain cell culture harvest medium containing ancient Seckiyo antibody molecules.

[0110] Experimental group: Optimized process

[0111] 1) Affinity chromatography and virus inactivation: The cell culture harvest medium containing antibodies was subjected to protein A affinity chromatography, eluted with 20 mmol / L acetate, pH 3.3-3.7 elution buffer, and the antibody was collected under 280 nm UV light. Then, the pH was adjusted to 3.3-3.7 with 1 mol / L citric acid, and the virus was inactivated by standing at room temperature for 90-120 min.

[0112] 2) Sodium octanoate incubation and deep filtration: Take the virus-inactivated sample, add 2 mol / L Tris to neutralize the pH to 4.5-5.5, add 0.5 mol / L sodium octanoate solution to the sample while stirring, slowly add sodium octanoate to a final concentration of 15-40 mmol / L, control the stirring speed to 100-250 rpm, stir at room temperature for ≥30 min, filter using Merck A1HC deep membrane, collect the filtrate, and perform sterile filtration to obtain the filtered clear liquid;

[0113] 3) Cation exchange chromatography: The pH of the clarified solution was adjusted to 5-6 using 2 mol / L Tris, then loaded into the cation exchange medium and eluted with 150-200 mol / L NaCl. The collected solution containing the antibody was obtained by UV spectroscopy at 280 nm.

[0114] 4) Composite mode chromatography: The sample conductivity of the collection solution from cation exchange chromatography is adjusted to 15-25 mS / cm, loaded into the composite mode medium, and the exudate containing the antibody is collected under 280 nm ultraviolet light.

[0115] 5) Preparation of high-concentration antibody composition samples: Using a Pellicon3 (30kD) ultrafiltration membrane, the sample runoff after column chromatography was ultrafiltered / washed. After the sample was concentrated, the buffer was replaced 6 to 10 times the volume of the concentrated solution. Then, it was concentrated again to the target concentration. Polysorbate 80 was added and sterilized before aliquoting to obtain a high-concentration antibody composition.

[0116] Compared to the experimental group, the control group was prepared using a process that did not include the sodium octanoate incubation step.

[0117] Analysis of experimental results:

[0118] HCP residue detection and polysorbate stability studies were conducted on antibody compositions from both the experimental and control groups to investigate the degradation of polysorbate in the antibody compositions at 37°C. The results of this example were similar to those of Example 2, as shown in Table 6: Compared to the control group, the HCP content in the antibody compositions of the experimental group decreased by 39%, and after 4 weeks at 37°C, the degradation rate of polysorbate 80 in the antibody compositions of the experimental group decreased from 17% to 10%.

[0119] Table 6 HCP Residue Detection and Polysorbate Stability Study

[0120] Clarified samples from both the experimental and control groups after deep filtration were subjected to SEC-HPLC purity testing. The results are shown in Table 7: Compared with the control group, the polymer content in the experimental group samples was lower.

[0121] Table 7 SEC-HPLC Purity Results

[0122] Example 5: IL33 / TSLP antibody purification method

[0123] Sample source

[0124] The sequence and structure of the IL33 / TSLP mixed antibody are detailed in molecules QB11465 and QB11718 in WO2025117485A1. First, the IL33 antibody gene sequence was synthesized and inserted into a blank vector to obtain a recombinant expression plasmid. The TSLP antibody gene sequence was synthesized and inserted into a blank vector to obtain a recombinant expression plasmid. The recombinant expression vector was co-transfected into CHO cell lines using a stable transfection method. Finally, the cells were revived and expanded in culture for 14 days to obtain a cell culture harvest medium containing IL33 / TSLP antibody molecules.

[0125] Experimental group: Optimized process

[0126] 1) Affinity chromatography and virus inactivation: Protein A affinity chromatography was performed on the cell culture harvest medium. Elution was carried out using 20 mmol / L acetate, pH 3.4-3.8 elution buffer. The antibody was collected under 280 nm UV light. Then, the pH was adjusted to 3.4-3.8 with 1 mol / L citric acid, and the virus was inactivated by standing at room temperature for 90-120 min.

[0127] 2) Sodium octanoate incubation and deep filtration: Take the virus-inactivated sample, add 2 mol / L Tris to neutralize the pH to 4.5-5.5, add 0.5 mol / L sodium octanoate solution to the sample while stirring, slowly add sodium octanoate to a final concentration of 20-40 mmol / L, control the stirring speed to 100-250 rpm, stir at room temperature for ≥60 min, filter using Merck X0HC deep membrane pack, collect the filtrate, and perform sterile filtration to obtain the filtered clear liquid;

[0128] 3) Anion exchange chromatography: The pH of the clarified solution was adjusted to 6-7 using 2 mol / L Tris, loaded onto anion exchange medium, and the collected solution containing the antibody was collected under 280 nm ultraviolet light.

[0129] 4) Composite mode chromatography: Adjust the conductivity of the anion exchange chromatography collection solution to 70-90 mS / cm, adjust the pH to 7-8, load it into the composite mode medium, elute with a pH 7-8 salt-free buffer, and collect the elution solution containing the antibody under 280 nm UV light.

[0130] 5) Preparation of high-concentration antibody composition samples: Using a Pellicon3 (30kD) ultrafiltration membrane, the sample runoff after column chromatography was ultrafiltered / washed. After the sample was concentrated, the buffer was replaced 6 to 10 times the volume of the concentrated solution. Then, it was concentrated again to the target concentration. Polysorbate 80 was added and sterilized before aliquoting to obtain a high-concentration antibody composition.

[0131] Compared to the experimental group, the control group was prepared using a process that did not include the sodium octanoate incubation step.

[0132] Analysis of experimental results:

[0133] High-concentration antibody compositions from both the experimental and control groups were tested for residual HCP content and polysorbate stability to investigate the degradation of polysorbate in the antibody compositions at 37°C. The results are shown in Table 8: Compared to the control group, the HCP content in the experimental group's antibody compositions decreased by 46%. After 4 weeks at 37°C, the degradation rate of polysorbate 80 in the experimental group's antibody compositions decreased from 74% to 35%.

[0134] Table 8 HCP Residue Detection and Polysorbate Stability Study

[0135] in conclusion

[0136] This study found that introducing a sodium caprylate incubation step into the dupilumab purification process significantly reduced both the content and types of host cell proteins (HCPs). Simultaneously, the free fatty acid content decreased significantly in the antibody composition stability study, indicating a marked improvement in polysorbate degradation. The study showed that sodium caprylate incubation improves polysorbate degradation in antibody compositions by reducing the content of hydrolytic enzymes such as lipase, lipoprotein lipase, and phospholipase A2. Furthermore, the study found that the sodium caprylate incubation method is not only applicable to improving polysorbate degradation in dupilumab antibody compositions, but also has an improving effect on polysorbate degradation in gusejinumab antibody compositions and IL-33 / TSLP mixed antibody compositions, indicating that this method has general applicability in reducing the content of polysorbate-related hydrolytic enzymes.

[0137] The above description is merely a preferred embodiment of this disclosure and is not intended to limit the scope of the substantive technical content of this disclosure. The substantive technical content of this disclosure is broadly defined within the scope of the claims of this application. Any technical entity or method completed by others that is the same as the scope defined by the claims of this application or is an equivalent modification shall be deemed to be covered within the scope of the claims of this application.

Claims

A method for reducing the content of lipase, lipoprotein lipase and / or phospholipase A2 in an antibody composition, wherein the method comprises the following steps: octanoic acid or octanoate incubation, wherein the final concentration of octanoic acid or octanoate during the incubation is 5-50 mmol / L. A method for reducing the degradation of polysorbate in an antibody composition, wherein the method comprises the following steps: octanoic acid or octanoate incubation, wherein the final concentration of octanoic acid or octanoate during the incubation is 5-50 mmol / L. The method according to claim 1 or 2, wherein the method comprises: the pH during the incubation is 4.0-6.0, and deep filtration is performed after the octanoic acid or octanoate incubation; preferably, the deep filter of the deep filtration is cellulose material, and diatomite is the filter aid. The method according to claim 1 or 2, wherein the method further comprises the following steps: virus inactivation, and / or chromatography operation; preferably, the chromatography operation comprises one or more of affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and complex mode chromatography. The method according to claim 4, wherein the following steps in the method are combined in any order: octanoic acid or octanoate incubation, virus inactivation, and chromatography operation; preferably, the method comprises one or more steps of chromatography operation. The method according to any one of claims 1-5, wherein the conditions of the octanoic acid or octanoate stirring and incubation are that the incubation time is greater than 10 min, and the stirring speed is 50-300 rpm; preferably, the octanoate is sodium octanoate. The method according to claim 4, wherein the virus inactivation is low-pH virus inactivation, and the pH used in the low-pH virus inactivation is 3.3-3.7; preferably, wherein the washing buffer of the affinity chromatography comprises about 0.1-1 mol / L arginine, 10-30 mmol / L Tirs-HCl, and the pH of the washing buffer is 7.3-7.9; preferably, wherein the ion exchange chromatography is cation exchange chromatography or anion exchange chromatography; more preferably, the equilibrium solution in the cation exchange chromatography contains 10-30 mmol / L acetate, pH 5.0-6.0, and the eluent contains 10-30 mmol / L acetate, 100-200 mmol / L NaCl, pH 5.0-6.0, the anion exchange chromatography medium is Q-FF, Diamond Q, Monomix Mab 60Q, MaXtar Q, the cation exchange chromatography medium is Capto SP ImpRes, Eshmuno CPX, Maxtar SP HR, NanoGel 50SP HP, and still preferably, wherein the hydrophobic chromatography medium is Capto phenyl ImpRes, UniHR phenyl 80L, Diamond Phenyl(HS), MaXtar Phenyl(HS), and the complex mode medium is Capto adhere, NM90 Agarose HAM, Diamond MIX-A. According to any one of claims 1-7, the residual caprylic acid or caprylate in the antibody solution purified by the method is less than about 17.6 μg / ml. The method according to any one of claims 1-8, wherein the HCP content in the antibody solution purified by the method is reduced by at least 30% compared to a purification process that does not involve incubation with caprylic acid or caprylate; preferably, the polysorbate degradation rate in the antibody composition purified by the method is reduced by at least 5%, more preferably by at least 10%, 20%, 30%, or 40%. The method according to any one of claims 1-9, wherein the antibody is selected from dupilumab and gusejinumab.

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