Bispecific antibodies and methods of purification thereof

Abstract

The invention provides a bispecific antibody and a purification method thereof. The purification method comprises the following steps: a) loading a sample containing a bispecific antibody to a affinity chromatographic column, and leaching and eluting; the biomacromolecules in the sample are adsorbed on the affinity chromatography column in the leaching stage, the biomacromolecules comprise antibodies, host cell proteins and protein aggregates, and the antibodies comprise bispecific antibodies and homodimers; separating the antibody from the affinity chromatography column in the elution stage, and collecting the eluate in the elution stage; b) loading the eluent to a hydrophobic membrane material, carrying out flow-through, and collecting a flow-through liquid containing the purified antibody; and the flow-through liquid used for flow-through contains 0.73 to 0.77 M of (NH4) 2SO4. The method can solve the problem that homodimers in bispecific antibodies are difficult to efficiently remove in the prior art, and is suitable for the field of antibody purification.

Description

Technical Field

[0001] The present invention relates to the field of antibody purification, and in particular, to a bispecific antibody and a purification method thereof. Background Art

[0002] Bispecific antibodies (bsAbs) are antibodies that can simultaneously bind two different targets or epitopes, and their dual-targeting ability provides greater therapeutic potential. Currently, there is a strong interest in the design and production of bsAbs to achieve higher efficacy through new mechanisms of action. However, due to the co-expression of up to four different polypeptide chains or the assembly involving extended chains (in the form of additional IgG), the recombinant production of IgG-like bsAbs is usually accompanied by an increase in the levels of product-related impurities (by-products and aggregates) caused by heavy-chain homodimerization and intermolecular mislinkage. Since some by-products are very similar to the target bsAb, their removal poses a major challenge to downstream processing (https: / / doi:10.1016 / j.pep.2019.105457).

[0003] Although the knob-in-hole (KiH) strategy has been used in the recombinant production of bispecific antibodies to promote heterodimerization, homodimers (especially hole-hole homologous dimers) can still be produced in small amounts. This by-product needs to be removed through downstream processes. However, since the sizes of homodimers and the target bsAb are usually very close, it may not be easy to distinguish these two substances using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Therefore, methods other than SEC-HPLC need to be developed to monitor the removal of this by-product. However, currently, analytical hydrophobic interaction chromatography (HIC) is a powerful tool for quantitatively monitoring the removal of hole-hole homodimers in bsAb purification (https: / / doi:10.1016 / j.pep.2019.105457). A set of orthogonal physicochemical analysis methods, including capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and ultra-performance chromatography mass spectrometry (RP-UPLC MS), are used to monitor and characterize such chain pair impurities for manufacturing process control and product release (https: / / doi:10.1016 / j.xphs.2021.04.010). Although the detection of dimers can be achieved using the above technologies, there is no practicable purification method yet.

[0004] Currently, in the prior art, bispecific antibody platforms are complex and diverse, and there are still great challenges in removing their homodimers, and the removal effect is not obvious. For example, the by-products are complex after affinity chromatography capture, the resolution of ion exchange chromatography is low, and the development of hydrophobic chromatography is difficult, time-consuming and laborious, etc. The limitations of the prior art have greatly reduced the economy and robustness of downstream purification processes. Summary of the Invention

[0005] The main object of the present invention is to provide a bispecific antibody and a purification method thereof, so as to solve the problem in the prior art that it is difficult to efficiently remove the homodimers in the bispecific antibody.

[0006] To achieve the above object, according to the first aspect of the present invention, a purification method of a bispecific antibody is provided. The purification method includes: a) loading a sample containing the bispecific antibody onto an affinity chromatography column, followed by washing and elution; the biological macromolecules in the sample are adsorbed on the affinity chromatography column during the washing stage, 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 during the elution stage, and the eluate of the elution stage is collected; b) loading the eluate onto a hydrophobic membrane material, performing flow-through, and collecting the flow-through liquid containing the purified antibody; the flow-through liquid used for flow-through contains 0.73 - 0.77 M of (NH4)2SO4.

[0007] Further, the flow-through liquid further contains an acetic acid - sodium acetate buffer system, and the concentration of COO - in the flow-through liquid is 48 - 52 mM, and the pH of the flow-through liquid is 5.4 - 5.6.

[0008] Further, b) includes: after disinfecting and equilibrating the hydrophobic membrane material, loading the eluate onto the hydrophobic membrane material; performing equilibration 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 liquid.

[0009] Further, the bispecific antibody is a KiH bispecific antibody.

[0010] Further, the flow-through includes: rinsing the hydrophobic membrane material after loading with the flow-through liquid, monitoring the UV absorbance value of the flow-through liquid at 280 nm, starting to collect the flow-through liquid when the UV absorbance value reaches 100 mAU, and stopping collecting the flow-through liquid when the UV absorbance value drops to 100 mAU.

[0011] Further, the preparation method of the sample containing the bispecific antibody includes: obtaining a bispecific antibody protein solution produced by cell culture, removing cells and cell debris, and obtaining a sample containing the bispecific antibody; preferably, the cells include CHO cells; preferably, the bispecific antibody includes a KiH bispecific antibody.

[0012] Further, a) includes: rinsing, disinfecting, and equilibrating an affinity chromatography column to obtain a processed affinity chromatography column; loading a sample containing a bispecific antibody onto the processed affinity chromatography column, followed by washing and elution; the buffer used for elution is 50 mM NaAc-HAc buffer, pH 3.8; the volume of the buffer used for elution is 5 CV.

[0013] Further, the washing includes a first wash, a second wash, and a third wash. The buffer used for the first wash is 50 mM Tris-HAc, 150 mM NaCl, pH 7.4, the buffer used for the second wash is 50 mM NaAC-HAc, 0.5 M NaCl, pH 5.5, and the buffer used for the third wash is 50 mM NaAc-HAc, pH 5.5. Preferably, the volume of the buffer used for the first wash is 5 CV, the volume of the buffer used for the second wash is 3 CV, and the volume of the buffer used for the third wash is 3 CV.

[0014] To achieve the above object, according to the second aspect of the present invention, a bispecific antibody is provided, and the bispecific antibody is a bispecific antibody prepared by using the purification method of the above bispecific antibody.

[0015] Further, the purity of the target protein molecule in the bispecific antibody is ≥99%; the content of the homodimer in the bispecific antibody is ≤1%.

[0016] Applying the technical solution of the present invention, the hydrophobic membrane chromatography technology is first used to remove homodimers in the above purification method, and the flow-through mode is used to separate the homodimers and the target bispecific antibody in the sample, so as to obtain a bispecific antibody with higher purity in a simple and efficient manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0018] Figure 1 Shows the affinity chromatography profile in the preparation of the preliminary sample according to Example 1 of the present invention.

[0019] Figure 2 Shows the hydrophobic membrane chromatography profile according to Example 1 of the present invention (the flow-through solution used is 50 mM NaAc-HAc, 0.77 M (NH4)2SO4, pH 5.5).

[0020] Figure 3Shows the hydrophobic membrane chromatography spectrum according to Embodiment 1 of the present invention (the flow-through solution used is 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, pH 5.5).

[0021] Figure 4 Shows the hydrophobic membrane chromatography spectrum according to Embodiment 1 of the present invention (the flow-through solution used is 50 mM NaAc-HAc, 0.73 M (NH4)2SO4, pH 5.5).

[0022] Figure 5 Shows the hydrophobic membrane chromatography spectrum according to Comparative Example 3 of the present invention (the flow-through solution used is 50 mM NaAc-HAc, 0.5 M (NH4)2SO4, pH 5.5). Detailed implementation manners

[0023] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] As mentioned in the background art, currently in the prior art, bispecific antibody platforms are complex and diverse, and there is a lack of methods for removing homodimer impurities in bispecific antibodies. In the prior art, membrane chromatography, as an emerging downstream purification technology, has advantages such as high throughput, fast flow rate, and small pressure drop, which can effectively reduce the development time and save time costs, showing strong advantages and being regarded as a favorable competitor to replace traditional column chromatography technology (https: / / doi.org / 10.1016 / j.bej.2023.108913; https: / / doi.org / 10.1080 / 01496395.2016.1223133). However, the number of hydrophobic groups of different proteins and the degree of exposure of hydrophobic groups will affect their hydrophobicity to varying degrees. There are differences in hydrophobicity between homodimers and bispecific antibodies (abbreviated as bispecific antibodies), and the hydrophobicity differences can be used to separate homodimers. Although there have been some literature reports on removing homodimer by-products of bispecific antibodies (https: / / doi.org / 10.1080 / 19420862.2016.1267090), the downstream purification technology platform for bispecific antibodies is still immature, and a large amount of cost is required to explore the technical route.

[0025] Therefore, in the present application, the inventors attempted to develop a method for purifying bispecific antibodies using hydrophobic membrane chromatography technology, and based on this, a series of protection schemes of the present application were proposed. The hydrophobic membrane chromatography technology involved in the present application is a type of novel membrane chromatography technology, and for the first time in the present application, this technology is used to remove homodimers through the flow-through mode.

[0026] In the first typical embodiment of the present application, a method for purifying a bispecific antibody is provided. The above purification method includes: a) loading a sample containing the bispecific antibody onto an affinity chromatography column, followed by washing and elution; the biopolymers in the sample are adsorbed onto the affinity chromatography column during the washing stage. The biopolymers include antibodies, host cell proteins (HCPs), and protein aggregates. The antibodies include bispecific antibodies and homodimers; the antibodies are separated from the affinity chromatography column during the elution stage, and the eluate from the elution stage is collected; b) loading the eluate onto a hydrophobic membrane material, performing flow-through, and collecting the flow-through solution containing the purified antibody; the flow-through solution used for flow-through contains 0.73 - 0.77 M of (NH4)2SO4.

[0027] Generally, there are hydrophobic and hydrophilic groups on the surface of proteins. Hydrophobic chromatography utilizes the hydrophobicity of a certain part of the protein surface to bind to a hydrophobic carrier at high salt concentrations and flow through at low salt concentrations. The flow-through mode is to reduce the salt concentration. Therefore, proteins with strong hydrophobicity bind to the hydrophobic membrane, while those with weak hydrophobicity flow through, which can be used to separate proteins that are difficult to purify by other methods. In the above purification method, the sample is first preliminarily purified using an affinity chromatography column. Although it is difficult to remove homodimers in the bispecific antibody in step a), the affinity chromatography column can remove some impurities, such as host cell proteins (HCPs) and protein aggregates, reducing the purification pressure on the subsequent hydrophobic membrane material and improving the purity of the target protein in the final flow-through solution.

[0028] Protein aggregates refer to larger complexes formed by the aggregation of protein molecules through non-specific interactions (such as hydrophobic interactions, electrostatic interactions, etc.). Such aggregates are usually the result of protein misfolding or being affected by environmental factors (such as pH, temperature, ionic strength, etc.). A homodimer is a dimer formed by the binding of two identical proteins through non-covalent or covalent bonds, and this binding is specific. It should be noted that in the present application, protein aggregates and homodimers are two different types of substances.

[0029] The hydrophobic chromatography used in step b) is a technique for separation that utilizes the hydrophobic interaction between proteins and hydrophobic fillers. In hydrophobic membrane chromatography, molecules with stronger hydrophobicity (such as homodimers) have a stronger binding ability to the hydrophobic membrane, while molecules with weaker hydrophobicity (such as the target antibody) have a relatively weaker binding ability. By controlling the conditions of the mobile phase, the target antibody can be eluted in the flow-through mode, while the homodimers with stronger hydrophobicity are retained on the membrane, thus achieving separation.

[0030] In the prior art, there are few reports on removing homodimers by hydrophobic interaction principle in column chromatography, and there is no report on removing homo-oligomers by hydrophobic interaction in membrane chromatography. In the purification method of the present application, the flow-through mode of hydrophobic membrane chromatography is creatively combined with ammonium sulfate ((NH4)2SO4) in a specific concentration range to remove homodimer impurities in bispecific antibodies, showing unprecedented high efficiency and economy. Specifically, the present invention controls the ammonium sulfate concentration in the flow-through liquid 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 that of the homodimer, making the latter more likely to bind to the membrane material, while the target protein flows through quickly (i.e., in a flow-through manner), achieving efficient purification. If the ammonium sulfate concentration is too high (including but not limited to ≥1.0 M), it will cause precipitation of the sample, and it is difficult to elute the bispecific antibody bound to the hydrophobic membrane; if the ammonium sulfate concentration is too low (including but not limited to ≤0.5 M), the homodimer will flow through together with the target protein (bispecific antibody), reducing the removal effect of the homodimer.

[0031] The innovation of the above purification method lies in that it not only breaks the limitations of traditional chromatography techniques in removing such by-products, but also significantly simplifies the process flow, reduces the operation time, and lowers the production cost. The above hydrophobic membrane material includes Sartobind Phenyl of Sartorius, and the ligand of the chromatography membrane is phenyl.

[0032] In a preferred embodiment, the above bispecific antibody is a KiH bispecific antibody (Knob-into-Hole bispecific antibody).

[0033] The KiH bispecific antibody, full name Knob-into-Hole bispecific antibody technology, is a method for constructing bispecific antibodies. This technology modifies the heavy chain of the antibody through genetic engineering technology to promote the correct pairing between different heavy chains, thereby forming an antibody molecule with two different antigen-binding sites. Specifically, the KiH technology involves mutating the CH3 region of the antibody molecule. The threonine (T) at position 366 in the CH3 region of the heavy chain of one antibody (Knob chain) is mutated to tryptophan (Y) to form a protruding "Knobs" structure; while the tryptophan (Y) at position 366 in the CH3 region of the heavy chain of the other antibody (Hole chain) is mutated to threonine (T), and the tyrosine (Y) at position 407 is mutated to valine (V) to form a concave "holes" structure. This design utilizes the steric hindrance effect to enable the correct assembly between two different heavy chains, greatly reducing the mispairing between heavy chains and improving the purity and functionality of the bispecific antibody.

[0034] In a preferred embodiment, the flow-through solution further contains an acetic acid-sodium acetate buffer system, and the concentration of COO - in the flow-through solution is 48-52 mM, and the pH of the flow-through solution is 5.4-5.6.

[0035] The flow-through solution further contains an acetic acid-sodium acetate buffer system (NaAc-HAc), which can maintain the stability of the pH in the flow-through solution on the premise that there are differences in the hydrophobicity between the target protein and the homodimer, further optimize the purification effect of the bispecific antibody, reduce non-specific adsorption, and improve the recovery rate of the target protein.

[0036] In a preferred embodiment, b) includes: after disinfecting 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.

[0037] In a preferred embodiment, flow-through includes: rinsing the hydrophobic membrane material after loading with the flow-through solution, monitoring the UV absorbance value of the flow-through solution at 280 nm, starting to collect the flow-through solution when the UV absorbance value reaches 100 mAU, and stopping collecting the flow-through solution when the above UV absorbance value drops to 100 mAU.

[0038] By real-time monitoring of the UV absorbance value, the purification process can be precisely controlled to ensure the efficient recovery of the target protein, while avoiding the mixing of impurities. And it prevents collecting the flow-through solution with too low a target protein concentration, resulting in a relatively low target protein concentration in the finally collected flow-through solution, thus requiring additional concentration work.

[0039] In a preferred embodiment, the method for preparing a sample containing a bispecific antibody includes: obtaining a bispecific antibody protein solution produced by cell culture, removing cells and cell debris to obtain a sample containing the bispecific antibody; preferably, the cells include CHO cells.

[0040] Animal cells include CHO cells. As a commonly used expression system, they can stably and efficiently produce bispecific antibodies. By removing cells and cell debris, the interference of impurities in subsequent purification steps can be reduced, and the purification efficiency can be improved.

[0041] In a preferred embodiment, a) includes: rinsing, disinfecting, and equilibrating an affinity chromatography column to obtain a treated affinity chromatography column; loading a sample containing the bispecific antibody onto the treated affinity chromatography column, followed by rinsing and elution; the buffer used for elution is 50 mM NaAc-HAc buffer, pH 3.8; the volume of the buffer used for elution is 5 CV.

[0042] In step a), first, use an affinity chromatography column to adsorb the target protein (while also adsorbing homodimers), and wash away the impurities that are not adsorbed; then perform elution to elute the target protein adsorbed on the affinity chromatography column and enter the subsequent purification steps.

[0043] In a preferred embodiment, the washing includes a first wash, a second wash, and a third wash. The buffer used for the first wash is 50 mM Tris-HAc, 150 mM NaCl, pH 7.4, the buffer used for the second wash is 50 mM NaAC-HAc, 0.5 M NaCl, pH 5.5, and the buffer used for the third wash is 50 mM NaAc-HAc, pH 5.5. Preferably, the volume of the buffer used for the first wash is 5 CV, the volume of the buffer used for the second wash is 3 CV, and the volume of the buffer used for the third wash is 3 CV.

[0044] By performing staged washing, different types of impurities can be gradually removed to ensure the high purity of the target protein. This washing strategy can effectively improve the biological activity and stability of the product in the purification of antibody drugs and is applicable to the purification requirements of various complex samples.

[0045] In the second typical embodiment of the present application, a bispecific antibody is provided, which is a bispecific antibody prepared by using the above purification method of the bispecific antibody.

[0046] In a preferred embodiment, the purity of the target protein molecules in the bispecific antibody is ≥ 99%; the content of homodimers in the bispecific antibody is ≤ 1%.

[0047] The bispecific antibody prepared by using the above purification method can reach a purity of 99% or more, and the content of homodimers of the knob-knob and hole-hole types is ≤ 1%.

[0048] Next, specific examples will be used to further explain the beneficial effects of the present application in detail.

[0049] Example 1

[0050] I. Preparation of the sample in the early stage

[0051] Perform two-stage depth filtration or centrifugation on the bispecific antibody protein solution produced by CHO cell culture to remove cells or cell debris and obtain a clarified harvest solution. Obtain a protein sample through affinity sample preparation.

[0052] The specific operation is as follows:

[0053] The target monoclonal antibody protein was captured using an affinity chromatography process. The packing material used was the AT Protein A Diamond Plus affinity packing material. The specific experimental procedure is as follows: First, the chromatography column was pretreated, including steps such as rinsing 1, pre-sterilization, and equilibration. The sample loaded was the clarified harvest fluid, and the loading capacity was set at 40 g / L. After the loading was completed, steps such as washing 1, washing 2, and washing 3 were carried out to rinse the chromatography column. This process was in the binding-elution mode, and an isocratic elution method was used to elute the bound protein. The elution volume was 3 column volumes, and the UV absorbance value was monitored at 280 nm. Collection started when the UV absorbance value reached 100 mAU and stopped when the UV absorbance value dropped to 100 mAU. After the elution was completed, it was regenerated with 3 column volumes of 120 mM acetic acid. Then steps such as rinsing 2, post-sterilization, and storage were carried out. The specific information on the affinity chromatography process and buffer is shown in Table 1. The collected fractions were used for yield analysis and purity detection.

[0054] Table 1 Information on the Affinity Chromatography Process and Buffer

[0055]

[0056]

[0057] Note: "N / A*" in Table 1 indicates not applicable.

[0058] The protein concentration was detected using a NanoDrop spectrophotometer, and the purity of the antibody monomer was detected using the RP-UPLC method. Based on the UV280 absorbance value, the eluate samples were collected for quality detection. The affinity chromatography spectrum is shown in Figure 1 , and the purity data is shown in Table 2. The content of the homodimer HH in the eluate was 11.0%, and the content of the KK component was 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 the Membrane Chromatography Material: 3 mL.

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

[0064] (5) Experimental operation procedure: First, steps such as pre-sterilization and equilibration of the membrane material should be carried out. The hydrophobic membrane chromatography process is in the flow-through mode. During the loading and washing processes, the UV absorbance is monitored at 280 nm. Collection starts when the UV absorbance reaches 100 mAU and stops when it drops to 100 mAU. The collected fractions are used for yield analysis and quality detection. In this example, three groups of experiments under different conditions are involved. The specific information of the hydrophobic membrane chromatography process and the buffer is shown in Table 2.

[0065] Table 2 Information on the hydrophobic membrane chromatography process and the buffer

[0066]

[0067] Note: "N / A*" in Table 2 indicates not applicable.

[0068] (6) Analysis and detection: The protein concentration is detected by a NanoDrop spectrophotometer. The monomer purity is detected by SEC-HPLC.

[0069] (7) Results: The flow-through liquid samples are collected based on the UV280 absorbance value and their quality is detected. The hydrophobic membrane chromatography spectra are shown in Figure 2 、 Figure 3 and Figure 4 . The analysis results are summarized in Table 3. The results show that under different ammonium sulfate concentration conditions, after the treatment of the target product by hydrophobic membrane chromatography, the content of homologous dimers in the sample decreases. Among them, under the conditions of 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, and pH 5.5, hydrophobic membrane chromatography can remove most of the homologous dimer impurity components, reducing the HH ratio from 11.1% to less than 0.7%, and improving the purity of the target protein molecule to more than 99.1%.

[0070] Table 3 Quality results of the example

[0071]

[0072] Note: RP purity represents Reversed-phase high performance liquid chromatography, HH represents Hole-hole dimers, HK represents Hole-knob dimers, KK represents Knob-knob dimers, and "N / A*" in Table 3 indicates not applicable.

[0073] Comparative Example 1

[0074] In this comparative example, hydrophobic column chromatography is carried out under the conditions of 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, and pH 5.5 to remove antibody homologous dimer 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 operation process: Similar to the method of Example 1. First, perform steps such as pre-sterilization and equilibration on the packing material. Among them, the equilibration buffer is 50 mM NaAc-HAc, 0.75 M (NH4)2SO4, pH 5.5. This chromatography process is in flow-through mode. During the sample loading process, monitor the UV absorbance at 280 nm. Start collecting when the UV absorbance reaches 100 mAU, and stop collecting when the UV absorbance drops to 100 mAU. Use the collected fraction for yield analysis and quality detection. Collect the elution fraction based on the UV280 absorbance and perform quality detection on it. It can be seen from the results of hydrophobic column chromatography that under the condition of 0.75 M (NH4)2SO4, the sample binds to the packing material of the column chromatography. Therefore, under the same conditions, the flow-through mode of hydrophobic column chromatography cannot achieve the effect of removing homodimers.

[0080] Comparative Example 2

[0081] The purification method is the same as that of Example 1, except that the concentration of (NH4)2SO4 in the flow-through solution is 1.0 M. First, perform steps such as pre-sterilization and equilibration on the packing material. Among them, the equilibration buffer is 50 mM NaAc-HAc, 1.0 M (NH4)2SO4, pH 5.5. This chromatography process is in flow-through mode. During the sample loading process, monitor the UV absorbance at 280 nm. Start collecting when the UV absorbance reaches 100 mAU, and stop collecting when the UV absorbance drops to 100 mAU. Use the collected fraction for yield analysis and quality detection. Collect the elution fraction based on the UV280 absorbance and perform quality detection on it.

[0082] Result: Under the concentration of 1.0 M ammonium sulfate in the loaded sample, about 80% of the sample precipitates, and all the samples bind to the hydrophobic membrane after filtration. Therefore, under the condition of 1.0 M ammonium sulfate, the sample is unstable and binds to the hydrophobic membrane, and cannot achieve the effect of removing homologous aggregates in the flow-through mode.

[0083] Comparative Example 3

[0084] The purification method was the same as that in Example 1, except that the concentration of (NH4)2SO4 in the flow-through solution was 0.5 M. First, steps such as pre-sterilization and equilibration of the packing material were carried out. Among them, the equilibration buffer was 50 mM NaAc-HAc, 0.5 M (NH4)2SO4, pH 5.5. This chromatography process was in the flow-through mode. During the sample loading process, the UV absorbance was monitored at 280 nm. Collection was started 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 detection. The elution fractions were collected based on the UV280 absorbance and their quality was detected. The hydrophobic membrane chromatography profile was as shown in Figure 5 shown, and the analysis results are summarized in Table 4 as shown.

[0085] Results: When the ammonium sulfate concentration in the sample was 0.5 M, there was no significant improvement in RP purity, and the effect of removing homologous mismatches was lower than that in Example 1.

[0086] Table 4 Quality results of the comparative example

[0087]

[0088] From the above description, it can be seen that the above embodiments of the present invention achieved the following technical effects: Compared with the prior art, for example, it is difficult for ion chromatography and mixed-mode chromatography techniques to completely remove homologous dimers in one-step chromatography. However, by using the purification method of the present application, homologous dimers can be effectively removed through the hydrophobic flow-through mode, and the monomer purity of the target protein molecule can be increased to more than 99.1%, which is superior to the hydrophobic column chromatography process under the same conditions. Moreover, the purification method is simple and has few steps, and is suitable for industrial production.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A purification method for a bispecific antibody, characterized in that, The purification method includes: a) Loading a sample containing a bispecific antibody onto an affinity chromatography column, followed by washing and elution; Biomacromolecules in the sample are adsorbed onto the affinity chromatography column during the washing stage. The biomacromolecules include antibodies, host cell proteins, and protein aggregates, and the antibodies include the bispecific antibody and homodimers; The antibodies are separated from the affinity chromatography column during the elution stage, and the eluate from the elution stage is collected; b) Loading the eluate onto a hydrophobic membrane material, performing flow-through, and collecting the flow-through liquid containing the purified antibody; The flow-through liquid used for flow-through contains 0.73 - 0.77 M (NH4)2SO4.

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

6.

3. The purification method according to claim 2, characterized in that, The step b) includes: After disinfecting and equilibrating the hydrophobic membrane material, loading the eluate onto the hydrophobic membrane material; The equilibration is carried out using 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 liquid.

4. The purification method according to claim 1, wherein 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-through includes: Washing the loaded hydrophobic membrane material with the flow-through liquid, monitoring the UV absorbance value of the flow-through liquid at 280 nm, starting to collect the flow-through liquid when the UV absorbance value reaches 100 mAU, and stopping collecting the flow-through liquid when the above UV absorbance value drops to 100 mAU.

6. The purification method according to claim 1, characterized in that The preparation method of the sample containing a bispecific antibody includes: Obtaining a bispecific antibody protein solution produced by cell culture, removing cells and cell debris, and obtaining the sample containing the bispecific antibody; Preferably, the cells include CHO cells; Preferably, the bispecific antibody includes a KiH bispecific antibody.

7. The purification method according to claim 1, wherein The step a) includes: Rinsing, disinfecting, and equilibrating the affinity chromatography column to obtain the treated affinity chromatography column; Loading the sample containing the bispecific antibody onto the treated affinity chromatography column, and performing the washing and the elution; The buffer used for elution is 50 mM NaAc - HAc buffer, pH 3.8; The usage volume of the buffer used for elution is 5 CV.

8. The purification method according to claim 1 or 7, wherein The washing includes a first wash, a second wash, and a third wash, The buffer used for the first wash is 50 mM Tris - HAc, 150 mM NaCl, pH 7.4, The buffer used for the second wash is 50 mM NaAC - HAc, 0.5 M NaCl, pH 5.5, The buffer used for the third wash is 50 mM NaAc - HAc, pH 5.5, Preferably, the usage volume of the buffer used for the first wash is 5 CV, The usage volume of the buffer used for the second wash is 3 CV, The usage volume of the buffer used for the third wash is 3 CV.

9. A bispecific antibody, characterized in that, The bispecific antibody is a bispecific antibody prepared by using the purification method of the bispecific antibody according to 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 the homodimer in the bispecific antibody is ≤1%.

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