Bispecific antibodies and methods for their purification

Abstract

The application provides a kind of bispecific antibody and its purification method, wherein the purification method comprises: a) the sample containing bispecific antibody is loaded into affinity chromatography column, and elution and elution are carried out;The biological macromolecules in the sample are adsorbed on the affinity chromatography column in the stage of elution, and the biological macromolecules include antibodies, host cell proteins and protein aggregates, and the antibodies include bispecific antibodies and homodimers;The antibodies are separated from the affinity chromatography column in the stage of elution, and the eluate in the stage of elution is collected;B) the eluate is loaded into hydrophobic membrane material, and flow-through is carried out, and the flow-through fluid containing purified antibodies is collected;The flow-through fluid used in flow-through contains 0.73-0.77 M (NH4) 2SO4.The problem that it is difficult to efficiently remove homodimers in bispecific antibodies in the prior art can be solved, and it is suitable for the field of antibody purification.

Description

[0001] This application is based on and claims priority to Chinese application CN application number 2025104276894 filed on April 7, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of antibody purification, and more specifically, to a bispecific antibody and its purification method. Background Technology

[0003] Bispecific antibodies (bsAbs) are antibodies capable of binding to two different targets or epitopes simultaneously, and their dual-targeting capability offers greater therapeutic potential. Currently, there is considerable interest in the design and production of bsAbs to achieve enhanced efficacy through novel mechanisms of action. However, the recombinant production of IgG-like bsAbs is often accompanied by increased levels of product-related impurities (byproducts and aggregates) due to the co-expression of up to four different polypeptide chains or involving the assembly of extended chains (in the form of additional IgG). This is caused by homodimerization of heavy chains and intermolecular crosslinking. Since some byproducts are very similar to the target bsAb, their removal poses a significant challenge to downstream processing (https: / / doi: 10.1016 / j.pep.2019.105457).

[0004] Although the knob-in-hole (KiH) strategy is used to promote heterodimerization in the recombinant production of bispecific antibodies, small amounts of homodimers (especially hole-hole homodimers) can still be generated. This byproduct needs to be removed via downstream processes. However, because the homodimers and target bsAbs are often very similar in size, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) may not easily distinguish between the two substances. Therefore, methods other than SEC-HPLC need to be developed to monitor the removal of this byproduct. Currently, analytical hydrophobic interaction chromatography (HIC) is a powerful tool for quantitatively monitoring the removal of hole-hole homodimers during bsAb purification (https: / / doi:10.1016 / j.pep.2019.105457). An orthogonal physicochemical analysis method, including capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) and ultra-high performance chromatography-mass spectrometry (RP-UPLC-MS), is used to monitor and characterize this chain pair impurity for manufacturing process control and product release (https: / / doi:10.1016 / j.xphs.2021.04.010). While the above techniques enable the detection of the dimer, no feasible purification method exists.

[0005] Currently, existing bispecific antibody platforms are complex and diverse, posing significant challenges to the removal of their homodimers, resulting in limited removal efficiency. For example, affinity chromatography captures complex byproducts, ion exchange chromatography has low resolution, and hydrophobic chromatography is difficult, time-consuming, and labor-intensive to develop. These limitations significantly reduce the economic efficiency and robustness of downstream purification processes. Summary of the Invention

[0006] The main objective of this invention is to provide a bispecific antibody and its purification method to solve the problem of the difficulty in efficiently removing homodimers from bispecific antibodies in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for purifying bispecific antibodies is provided, the purification method comprising: a) loading a sample containing bispecific antibodies onto an affinity chromatography column for elution; wherein biomolecules in the sample are adsorbed onto the affinity chromatography column during the elution phase, the biomolecules including antibodies, host cell proteins, and protein aggregates, and the antibodies including bispecific antibodies and homodimers; wherein the antibodies are separated from the affinity chromatography column during the elution phase, and the eluent from the elution phase is collected; b) loading the eluent onto a hydrophobic membrane material for flow-through, and collecting the flow-through containing purified antibodies; wherein the flow-through contains 0.73-0.77 M of (NH4)2SO4.

[0008] Furthermore, the flow-through solution also contains an acetate-sodium acetate buffer system, and the COO in the flow-through solution... - The concentration was 48-52 mM, and the pH of the flow-through solution was 5.4-5.6.

[0009] Further, b) includes: after sterilizing and equilibrating the hydrophobic membrane material, loading the eluent onto the hydrophobic membrane material; equilibrating with an equilibration buffer, wherein the equilibration buffer contains (NH4)2SO4, and the concentration of (NH4)2SO4 in the equilibration buffer is equal to the concentration of (NH4)2SO4 in the flow-through solution.

[0010] Furthermore, the bispecific antibody is the KiH bispecific antibody.

[0011] Furthermore, the flow-through process includes: rinsing the hydrophobic film material after sample loading with the flow-through liquid, monitoring the UV absorbance of the flow-through liquid at 280 nm, starting to collect the flow-through liquid when the UV absorbance reaches 100 mAU, and stopping the collection of the flow-through liquid when the UV absorbance drops to 100 mAU.

[0012] Furthermore, the method for preparing a sample containing bispecific antibodies includes: obtaining a bispecific antibody protein solution produced by cell culture, removing cells and cell debris, and obtaining a sample containing bispecific antibodies; preferably, the cells include CHO cells; preferably, the bispecific antibody includes KiH bispecific antibodies.

[0013] Further, a) includes: rinsing, sterilizing, and equilibrating the affinity chromatography column to obtain a treated affinity chromatography column; loading a sample containing bispecific antibodies onto the treated affinity chromatography column for rinsing and elution; the elution buffer used is 50 mM NaAc-HAc buffer, pH 3.8; the volume of the elution buffer used is 5 CV.

[0014] Further, the rinsing includes a first rinse, a second rinse, and a third rinse. The buffer used for the first rinse is 50 mM Tris-HAc, 150 mM NaCl, pH 7.4; the buffer used for the second rinse is 50 mM NaAC-HAc, 0.5 M NaCl, pH 5.5; and the buffer used for the third rinse is 50 mM NaAc-HAc, pH 5.5. Preferably, the volume of buffer used for the first rinse is 5 CV, the volume of buffer used for the second rinse is 3 CV, and the volume of buffer used for the third rinse is 3 CV.

[0015] To achieve the above objective, according to a second aspect of the present invention, a bispecific antibody is provided, wherein the bispecific antibody is a bispecific antibody prepared using the purification method described above.

[0016] Furthermore, the purity of the target protein molecule in the bispecific antibody is ≥99%; the content of homodimers in the bispecific antibody is ≤1%.

[0017] By applying the technical solution of this invention, hydrophobic membrane chromatography is used for the first time in the above purification method to remove homodimers. The flow-through mode is used to separate the homodimers and the target bispecific antibody in the sample, thereby obtaining a high-purity bispecific antibody in a simple and efficient manner. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 The affinity chromatography spectrum during the initial sample preparation according to Example 1 of the present invention is shown.

[0020] Figure 2The hydrophobic membrane chromatography spectrum of Example 1 of the present invention is shown (the flow-through solution used was 50 mM NaAc-HAc, 0.77 M (NH4)2SO4, pH 5.5).

[0021] Figure 3 The hydrophobic membrane chromatography spectrum of Example 1 of the present invention is shown (the flow-through solution used was 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, pH 5.5).

[0022] Figure 4 The hydrophobic membrane chromatography spectrum of Example 1 of the present invention is shown (the flow-through solution used was 50 mM NaAc-HAc, 0.73 M (NH4)2SO4, pH 5.5).

[0023] Figure 5 The hydrophobic membrane chromatography spectrum of Comparative Example 3 according to the present invention is shown (the flow-through solution used was 50 mM NaAc-HAc, 0.5 M (NH4)2SO4, pH 5.5). Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0025] As mentioned in the background section, current bispecific antibody platforms are complex and diverse, lacking methods to remove homodimer impurities from bispecific antibodies. Membrane chromatography, as an emerging downstream purification technology, offers advantages such as high throughput, fast flow rate, and low pressure drop, effectively reducing development time and saving time costs. It is considered a strong competitor to traditional column chromatography (https: / / doi.org / 10.1016 / j.bej.2023.108913; https: / / doi.org / 10.1080 / 01496395.2016.1223133). However, the number and degree of hydrophobic groups in different proteins affect their hydrophobicity to varying degrees. The difference in hydrophobicity between homodimers and bispecific antibodies (Biantibodies) allows for the separation of homodimers. Although there have been some literature reports on the removal of homodimer byproducts from bispecific antibodies (https: / / doi.org / 10.1080 / 19420862.2016.1267090), the downstream purification technology platform for bispecific antibodies is still immature, and a lot of costs are needed to explore technical routes.

[0026] Therefore, in this application, the inventors attempted to develop a method for purifying bispecific antibodies using hydrophobic membrane chromatography, and based on this, proposed a series of protection schemes for this application. The hydrophobic membrane chromatography technology involved in this application is a novel membrane chromatography technology, and in this application, it is used for the first time to remove homodimers through a flow-through mode.

[0027] In a first typical embodiment of this application, a method for purifying bispecific antibodies is provided. The purification method includes: a) loading a sample containing bispecific antibodies onto an affinity chromatography column for elution; during the elution phase, biomolecules in the sample are adsorbed onto the affinity chromatography column, including antibodies, host cell proteins (HCPs), and protein aggregates; the antibodies include bispecific antibodies and homodimers; during the elution phase, the antibodies are separated from the affinity chromatography column, and the eluent from the elution phase is collected; b) loading the eluent onto a hydrophobic membrane material for flow-through, and collecting the flow-through containing purified antibodies; the flow-through contains 0.73-0.77 M of (NH4)2SO4.

[0028] Protein surfaces generally possess both hydrophobic and hydrophilic groups. Hydrophobic chromatography utilizes the hydrophobicity of a portion of the protein surface, allowing it to bind to a hydrophobic carrier at high salt concentrations and flow through at low salt concentrations. The flow-through mode lowers the salt concentration, thus allowing the more hydrophobic components to bind to the hydrophobic membrane while the less hydrophobic components flow through. This method can be used to separate proteins that are difficult to purify using other methods. In the purification method described above, the sample is first initially purified using an affinity chromatography column. Although it is difficult to remove homodimers from bispecific antibodies in step a), affinity chromatography can remove some impurities, such as host cell proteins (HCPs) and protein aggregates, reducing the purification burden on subsequent hydrophobic membrane materials and improving the purity of the target protein in the final flow-through solution.

[0029] Protein aggregates are large complexes formed by the aggregation of protein molecules through non-specific interactions (such as hydrophobic interactions, electrostatic interactions, etc.). These aggregates are usually the result of protein misfolding or the influence of environmental factors (such as pH, temperature, ionic strength, etc.). Homodimers are dimers formed by the binding of two identical proteins through non-covalent or covalent bonds; this binding is specific. It should be noted that in this application, protein aggregates and homodimers are two different types of substances.

[0030] The hydrophobic chromatography used in step b) is a technique that separates proteins using hydrophobic interactions with hydrophobic membrane materials. In hydrophobic membrane chromatography, molecules with stronger hydrophobicity (such as homodimers) bind more strongly to the hydrophobic membrane, while molecules with weaker hydrophobicity (such as the target antibody) bind relatively weakly. By controlling the conditions of the mobile phase, the target antibody can be eluted in flow-through mode, while the stronger hydrophobic homodimer is retained on the membrane, thus achieving separation.

[0031] There are few reports in the prior art on the removal of homodimers by utilizing hydrophobic interactions in column chromatography, and there are no reports on the removal of homodimers by utilizing hydrophobic interactions in membrane chromatography. In the purification method of this application, a hydrophobic membrane chromatography flow-through mode is creatively combined with ammonium sulfate ((NH4)2SO4) at a specific concentration range to remove homodimer impurities from bispecific antibodies, demonstrating unprecedented efficiency and economy. Specifically, this invention controls the ammonium sulfate concentration in the flow-through solution to be between 0.5-1.0 M, preferably 0.73-0.77 M, including but not limited to 0.73, 0.74, 0.75, 0.76, or 0.77 M. Within this concentration range, a significant difference is formed between the hydrophobicity of the target protein (bispecific antibody) and the hydrophobicity of the homodimer, making the latter more easily bind to the membrane material, while the target protein flows through rapidly (i.e., flow-through), achieving highly efficient purification. If the ammonium sulfate concentration is too high (including but not limited to ≥1.0 M), the purification process will be less efficient. If the ammonium sulfate concentration is too low (including but not limited to ≤0.5M), the homodimer will flow through the target protein (bispecific antibody) together, reducing the homodimer removal efficiency.

[0032] The innovation of the above purification method lies in its ability to overcome the limitations of traditional chromatography techniques in removing such byproducts, significantly simplify the process, reduce operation time, and lower production costs. The hydrophobic membrane material mentioned above includes Sartorius' Sartobind Phenyl, with a phenyl ligand in the chromatographic membrane.

[0033] In a preferred embodiment, the bispecific antibody is a KiH bispecific antibody (Knob-into-Hole bispecific antibody).

[0034] KiH bispecific antibody, short for Knob-into-Hole bispecific antibody technology, is a method for constructing bispecific antibodies. This technology uses genetic engineering to modify the heavy chains of antibodies to promote the correct pairing between different heavy chains, thereby forming antibody molecules with two different antigen-binding sites. Specifically, KiH technology involves mutating the CH3 region of the antibody molecule. In one antibody's heavy chain (Knob chain), the threonine (T) at position 366 of the CH3 region is mutated to tryptophan (Y), forming a prominent "Knobs" structure; while in the other antibody's heavy chain (Hole chain), the tryptophan (Y) at position 366 of the CH3 region is mutated to threonine (T), and the tyrosine (Y) at position 407 is mutated to valine (V), forming a recessed "holes" structure. This design utilizes steric hindrance to ensure correct assembly between the two different heavy chains, significantly reducing mismatches between heavy chains and improving the purity and functionality of the bispecific antibody.

[0035] In a preferred embodiment, the flow-through liquid further contains an acetate-sodium acetate buffer system, and the COO in the flow-through liquid... - The concentration was 48-52 mM, and the pH of the flow-through solution was 5.4-5.6.

[0036] The flow-through solution also contains an acetate-sodium acetate buffer system (NaAc-HAc), which can maintain the pH stability of the flow-through solution even when there is a difference in hydrophobicity between the target protein and the homodimer, thereby further optimizing the purification effect of the bispecific antibody, reducing nonspecific adsorption, and improving the recovery rate of the target protein.

[0037] In a preferred embodiment, b) includes: after sterilizing and equilibrating the hydrophobic membrane material, loading the eluent onto the hydrophobic membrane material; equilibrating with an equilibration buffer, wherein the equilibration buffer contains (NH4)2SO4 and the concentration of (NH4)2SO4 in the equilibration buffer is equal to the concentration of (NH4)2SO4 in the flow-through solution.

[0038] In a preferred embodiment, the flow-through process includes: rinsing the hydrophobic film material after sample loading with a flow-through liquid, monitoring the UV absorbance of the flow-through liquid at 280 nm, starting to collect the flow-through liquid when the UV absorbance reaches 100 mAU, and stopping the collection of the flow-through liquid when the UV absorbance drops to 100 mAU.

[0039] By monitoring UV absorbance in real time, the purification process can be precisely controlled, ensuring efficient recovery of the target protein while avoiding the introduction of impurities. This also prevents the collection of flow-through with excessively low target protein concentrations, which would otherwise necessitate additional concentration work.

[0040] In a preferred embodiment, the method for preparing a sample containing bispecific antibodies includes: obtaining a bispecific antibody protein solution produced by cell culture, removing cells and cell debris, and obtaining a sample containing bispecific antibodies; preferably, the cells include CHO cells.

[0041] Animal cells, including CHO cells, are commonly used expression systems that can stably and efficiently produce bispecific antibodies. By removing cells and cell debris, impurities in subsequent purification steps can be reduced, thus improving purification efficiency.

[0042] In a preferred embodiment, a) includes: rinsing, sterilizing, and equilibrating the affinity chromatography column to obtain a treated affinity chromatography column; loading a sample containing bispecific antibodies onto the treated affinity chromatography column for rinsing and elution; the elution buffer is a 50 mM NaAc-HAc buffer, pH 3.8; the volume of the elution buffer used is 5 CV.

[0043] In step a), the target protein is first adsorbed using an affinity chromatography column (which also adsorbs homodimers), and impurities that are not adsorbed are removed by rinsing; then elution is performed to elute the target protein adsorbed on the affinity chromatography column and proceed to the subsequent purification steps.

[0044] In a preferred embodiment, the rinsing includes a first rinse, a second rinse, and a third rinse. The buffer used for the first rinse is 50 mM Tris-HAc, 150 mM NaCl, pH 7.4; the buffer used for the second rinse is 50 mM NaAC-HAc, 0.5 M NaCl, pH 5.5; and the buffer used for the third rinse is 50 mM NaAc-HAc, pH 5.5. Preferably, the volume of buffer used for the first rinse is 5 CV, the volume of buffer used for the second rinse is 3 CV, and the volume of buffer used for the third rinse is 3 CV.

[0045] Staged elution allows for the gradual removal of different types of impurities, ensuring high purity of the target protein. This elution strategy effectively improves the bioactivity and stability of antibody drugs and is suitable for the purification needs of various complex samples.

[0046] In a second typical embodiment of this application, a bispecific antibody is provided, which is a bispecific antibody prepared using the purification method described above.

[0047] In a preferred embodiment, the purity of the target protein molecule in the bispecific antibody is ≥99%; the content of homodimer in the bispecific antibody is ≤1%.

[0048] The bispecific antibodies prepared using the above purification method have a purity of 99% or higher, with the content of knob-knob and hole-hole type homodimers ≤1%.

[0049] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0050] Example 1

[0051] I. Preliminary Sample Preparation

[0052] The bispecific antibody protein solution produced from CHO cell culture is subjected to two-stage deep filtration or centrifugation to remove cells or cell debris, obtaining a clear harvest solution. Protein samples are then obtained through affinity sample preparation.

[0053] The specific steps are as follows:

[0054] Affinity chromatography was used to capture target monoclonal antibody proteins. The packing material used was AT Protein ADiamond Plus affinity packing material. The specific experimental procedure is as follows: First, the chromatography column was pretreated, including rinsing 1, pre-sterilization, and equilibration. The sample loaded was the clarified harvest solution, and the loading volume was set to 40 g / L. After loading, rinsing 1, rinsing 2, and rinsing 3 were performed to wash the chromatography column. This process was a binding-elution mode, using isocratic elution to elute the bound protein. The elution volume was 3 column volumes. UV absorbance was monitored at 280 nm, and collection began when the UV absorbance reached 100 mAU and stopped when the UV absorbance decreased to 100 mAU. After elution, regeneration was performed with 3 column volumes of 120 mM acetic acid. Then, rinsing 2, post-sterilization, and storage were performed. Specific information on the affinity chromatography process and buffer solutions is shown in Table 1. The collected fractions were used for yield analysis and purity determination.

[0055] Table 1. Affinity chromatography process and buffer information

[0056]

[0057] Note: "N / A" in Table 1 "Indicates that it is not applicable."

[0058] Protein concentration was detected using a NanoDrop spectrophotometer, and antibody monomer purity was determined using RP-UPLC. Elution buffer samples were collected based on UV280 absorbance, and quality was assessed. Affinity chromatography chromatograms are shown below. Figure 1 The purity data are shown in Table 2. The content of homodimer HH in the eluent is 11.0%, and the content of KK component is 0.2%.

[0059] II. Hydrophobic membrane chromatography (flow-through mode)

[0060] (1) Equipment: AKTA avant 150.

[0061] (2) Membrane chromatography material: Sartobind Phenyl.

[0062] (3) Volume of membrane chromatography material: 3 mL.

[0063] (4) Retention time: 0.3 min.

[0064] (5) Experimental Procedure: First, the membrane material should be sterilized and equilibrated. This hydrophobic membrane chromatography process is flow-through mode. During sample loading and rinsing, the UV absorbance value is monitored at 280 nm. Collection begins when the UV absorbance value reaches 100 mAU and stops when the UV absorbance value drops to 100 mAU. The collected fraction is used for yield analysis and quality testing. This example involves three sets of experiments under different conditions. Specific information on the hydrophobic membrane chromatography process and buffer solution is shown in Table 2.

[0065] Table 2 Hydrophobic membrane chromatography process and buffer information

[0066]

[0067] Note: "N / A" in Table 2 "Indicates that it is not applicable."

[0068] (6) Analysis and detection: Protein concentration was detected by NanoDrop spectrophotometer. Monomer purity was detected by SEC-HPLC.

[0069] (7) Results: The flow-through liquid samples were collected and their mass was determined based on the UV280 absorbance. The hydrophobic membrane chromatography spectrum is shown in [reference needed]. Figure 2 , Figure 3 and Figure 4 The analytical results are summarized in Table 3. The results show that, under different ammonium sulfate concentrations, hydrophobic membrane chromatography reduced the homodimer content in the target product. Specifically, hydrophobic membrane chromatography at 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, and pH 5.5 was able to remove most of the homodimer impurities, reducing the HH ratio from 11.1% to less than 0.7%, and increasing the purity of the target protein molecule to over 99.1%.

[0070] Table 3 Quality Results of Examples

[0071]

[0072] Note: RP purity represents Reversed-phase high performance liquid chromatography; HH stands for Hole-hole dimers, HK for Hole-knob dimers, and KK for Knob-knob dimers. The "N / A" in Table 3... "Indicates that it is not applicable."

[0073] Comparative Example 1

[0074] This comparative example was subjected to hydrophobic column chromatography under the conditions of 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, and pH 5.5 to remove antibody homodimer impurities.

[0075] (1) Equipment: AKTA avant 150.

[0076] (2) Column chromatography packing material: Capto Phenyl ImpRes.

[0077] (3) Column volume: 6.3 mL.

[0078] (4) Retention time: 5 min.

[0079] (5) Experimental procedure: Similar to the method in Example 1. First, the packing material was pre-sterilized and equilibrated. The equilibration buffer was 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, pH 5.5. The chromatography process was flow-through mode. During sample loading, the UV absorbance was monitored at 280 nm. Collection began when the UV absorbance reached 100 mAU and stopped when the UV absorbance dropped to 100 mAU. The collected fraction was used for yield analysis and quality testing. The eluted fraction was collected based on the UV280 absorbance and subjected to quality testing. The hydrophobic column chromatography results showed that under 0.75 M (NH4)2SO4 conditions, the sample bound to the packing material of the column chromatography. Therefore, under the same conditions, the flow-through mode of hydrophobic column chromatography could not remove homodimers.

[0080] Comparative Example 2

[0081] The purification method was the same as in Example 1, except that the concentration of (NH4)2SO4 in the flow-through buffer was 1.0 M. First, the packing material underwent pre-sterilization and equilibration. The equilibration buffer consisted of 50 mM NaAc-HAc, 1.0 M (NH4)2SO4, and pH 5.5. The chromatography process was performed in flow-through mode. During sample loading, the UV absorbance was monitored at 280 nm. Collection began when the UV absorbance reached 100 mAU and stopped when the UV absorbance decreased to 100 mAU. This collected fraction was used for yield analysis and quality control. The eluted fraction was collected based on the UV280 absorbance and subjected to quality control.

[0082] Results: At a concentration of 1.0 M ammonium sulfate, approximately 80% of the sample precipitated. After filtration, all the sample was bound to the hydrophobic membrane. Therefore, under the condition of 1.0 M ammonium sulfate, the sample was unstable and bound to the hydrophobic membrane, and could not achieve the effect of removing homopolymers in the flow-through mode.

[0083] Comparative Example 3

[0084] The purification method was the same as in Example 1, except that the concentration of (NH4)2SO4 in the flow-through buffer was 0.5 M. First, the packing material was pre-sterilized and equilibrated. The equilibration buffer consisted of 50 mM NaAc-HAc, 0.5 M (NH4)2SO4, and pH 5.5. The chromatography process was in flow-through mode. During sample loading, the UV absorbance was monitored at 280 nm. Collection began when the UV absorbance reached 100 mAU and stopped when it decreased to 100 mAU. The collected fraction was used for yield analysis and quality control. The eluted fraction was collected based on the UV280 absorbance and subjected to quality control. The hydrophobic membrane chromatography spectrum is shown below. Figure 5 As shown, the analysis results are summarized in Table 4.

[0085] Results: When the ammonium sulfate concentration in the sample was 0.5M, the purity of RP was not significantly improved, and the removal effect of homologous mismatch was lower than that in Example 1.

[0086] Table 4 Comparative Sample Quality Results

[0087]

[0088] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: Compared with existing technologies, such as ion chromatography and mixed-mode chromatography, it is difficult to completely remove homodimers in a single step. However, the purification method of this application can effectively remove homodimers through a hydrophobic flow-through mode, increasing the monomer purity of the target protein molecule to over 99.1%, which is superior to hydrophobic column chromatography under the same conditions. Moreover, the purification method is simple, has fewer steps, and is suitable for industrial production.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for purifying a bispecific antibody, characterized in that, The purification method includes: a) Load the sample containing bispecific antibodies onto an affinity chromatography column for elution and washing; The biomacromolecules in the sample are adsorbed onto the affinity chromatography column during the elution phase. The biomacromolecules include antibodies, host cell proteins, and protein aggregates. The antibodies include the bispecific antibody and homodimers. The antibody is separated from the affinity chromatography column during the elution phase, and the eluent from the elution phase is collected. b) Load the eluent onto a hydrophobic membrane material, perform flow-through, and collect the flow-through containing the purified antibody; The flow-through solution used in the flow-through contains 0.73-0.77 M of (NH4)2SO4.

2. The purification method according to claim 1, characterized in that, The flow-through fluid also contains an acetic acid-sodium acetate buffer system, and the COO in the flow-through fluid... - The concentration of the solution is 48-52 mM, and the pH of the flow-through solution is 5.4-5.

6.

3. The purification method according to claim 2, characterized in that, b) includes: After sterilizing and equilibrating the hydrophobic membrane material, the eluent is loaded onto the hydrophobic membrane material. The equilibration is performed using an equilibration buffer containing (NH4)2SO4, and the concentration of (NH4)2SO4 in the equilibration buffer is equal to the concentration of (NH4)2SO4 in the flow-through solution.

4. The purification method according to claim 1, characterized in that, The bispecific antibody is a KiH bispecific antibody.

5. The purification method according to any one of claims 2-4, characterized in that, The flow passage includes: The hydrophobic film material after sample loading is rinsed with the flow-through liquid, and the UV absorbance of the flow-through liquid at 280 nm is monitored. When the UV absorbance reaches 100 mAU, the flow-through liquid is collected and the collection is stopped when the UV absorbance drops to 100 mAU.

6. The purification method according to claim 1, characterized in that, The method for preparing the sample containing bispecific antibodies includes: A bispecific antibody protein solution produced by cell culture is obtained, cells and cell debris are removed, and a sample containing the bispecific antibody is obtained. Preferably, the cells comprise CHO cells; Preferably, the bispecific antibody includes KiH bispecific antibody.

7. The purification method according to claim 1, characterized in that, a) includes: The affinity chromatography column is rinsed, sterilized, and equilibrated to obtain the treated affinity chromatography column. The sample containing the bispecific antibody is loaded onto the treated affinity chromatography column for elution and washing. The elution buffer used was a 50 mM NaAc-HAc buffer, pH 3.8; The volume of buffer solution used for elution is 5 CV.

8. The purification method according to claim 1 or 7, characterized in that, The rinsing includes a first rinsing, a second rinsing, and a third rinsing. The buffer solution used for the first elution was 50 mM Tris-HAc, 150 mM NaCl, pH 7.

4. The buffer solution used for the second elution was 50 mM NaAC-HAc, 0.5 M NaCl, pH 5.

5. The buffer solution used for the third elution was 50 mM NaAc-HAc, pH 5.

5. Preferably, the volume of buffer solution used for the first rinsing is 5 cV. The volume of buffer solution used for the second rinsing is 3 cV. The volume of buffer solution used for the third rinse is 3 CV.

9. A bispecific antibody, characterized in that, The bispecific antibody is a bispecific antibody prepared using the purification method of any one of claims 1-8.

10. The bispecific antibody according to claim 9, characterized in that, The purity of the target protein molecule in the bispecific antibody is ≥99%; the content of homodimer in the bispecific antibody is ≤1%.

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