Purification method of bispecific antibody with fragment removed

Abstract

The invention discloses a purification method of a bispecific antibody capable of removing fragments, which comprises the following steps: using an eluent containing PEG (polyethylene glycol) and CaCl2 in affinity chromatography, and matching with a high-pH impurity washing solution containing arginine and hydrophobic chromatography for fine purification to obtain the bispecific antibody with few impurities and high purity. In the purified eluent obtained through two-step chromatography, the proportion of the bispecific antibody is increased to 98% or above, the proportion of fragments is reduced to about 0.5%, fragment impurities generated in the preparation process of the bispecific antibody are effectively removed, and the purification method is efficient and has good application prospects.

Description

Technical Field

[0001] This invention relates to a method for purifying antibodies, and more particularly to a method for purifying bispecific antibodies by removing fragments. Background Technology

[0002] Antibody drugs occupy a central position in modern biomedicine. From the initial murine monoclonal antibodies to humanized antibodies, and now to bispecific antibodies and antibody-drug conjugates (ADCs), their structures and functions have become increasingly complex, demanding higher standards for purification processes. Bispecific antibodies, or biantibodies for short, are artificial antibodies that can simultaneously and specifically bind to two antigens or antigenic epitopes. Due to their specificity and dual functionality, they have become a research hotspot in antibody engineering and have broad application prospects in areas such as tumor treatment and autoimmune diseases.

[0003] The biggest structural difference between bispecific antibodies and monoclonal antibodies is that bispecific antibodies can have two different Fab and Fc domains, resulting in different heavy and light chains in the antibody molecule. This leads to product-related impurities that differ from those produced during monoclonal antibody purification. Antibody fragments are often generated due to factors such as disulfide bond reduction and shearing forces. Examples include "half-antibodies" with only one Fc domain and "single-arm fragments" lacking a Fab arm. These fragments are inactive and may even trigger immune responses, thus requiring removal. However, while these types of fragment impurities differ significantly from structurally correct bispecific antibody molecules in terms of pharmacological efficacy and biological activity, they are chemically very similar, making removal difficult. Molecular modification can reduce the formation of fragment impurities during antibody development, but it cannot completely eliminate the generation of mismatch byproducts. Therefore, removing antibody fragment impurities remains a significant challenge in the isolation and purification of bispecific antibodies.

[0004] Purification strategies are often developed based on the differences in structure, affinity, charge properties, and hydrophobicity between the target bispecific antibody molecule and the fragment to remove fragment impurities. Among existing bispecific antibody fragment removal methods, affinity chromatography is mostly used for capture, and complex ion chromatography is used for purification, or hydroxyapatite chromatography is used for separation and purification to remove fragment impurities. However, complex ion chromatography and hydroxyapatite are expensive, and due to the differences in charge and hydrophobicity of complex ion packing materials, it is often difficult to find a balance, often resulting in excessively strong binding forces and low recovery rates. Currently, there is no efficient and economical method for purifying bispecific antibody fragments. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for purifying bispecific antibodies with efficient fragment removal.

[0006] Technical solution: The purification method includes the following steps: Step 1: Crude purification of the bispecific antibody sample is performed using affinity chromatography. The eluent used in the affinity chromatography contains PEG and CaCl2 to obtain a crude purified eluent. Step 2: The crude eluent is purified using hydrophobic chromatography to obtain a purified eluent, which is the purified bispecific antibody.

[0007] Affinity chromatography primarily removes antibody fragments and host cell proteins (HCPs), while hydrophobic chromatography removes impurities such as polymers, degradation fragments, and endotoxins.

[0008] In step one, the bispecific antibody sample is preferably filtered before affinity chromatography; preferably, a 0.2–0.45 μm membrane is used to filter the bispecific antibody fermentation broth first; the membrane material is preferably polyethersulfone. The PEG content (mass percentage) is preferably 1.5–3.0%, and the CaCl2 is preferably 100–300 mM. The eluent used for affinity chromatography is preferably 20–50 mM NaAc-HAc, 1.5–3.0% PEG4000, 100–300 mM CaCl2, pH 2.8–3.2. Protein A affinity chromatography packing material is used for affinity chromatography. The matrix of the protein A affinity chromatography packing material is preferably a spherical, narrowly dispersed, highly cross-linked agarose gel matrix. The particle size distribution of the packing material D90 / D10 is ≤2.0, preferably ≤1.5, and more preferably ≤1.2. The degree of cross-linking of the packing material is 4%–10%, preferably 6%–8%, and more preferably 6%. The particle size of the packing material is 30-100 μm, preferably 65 μm. Smaller particle sizes result in better separation and removal of more impurities and proteins. The affinity chromatography is preferably performed using MabPurix A65 Excel chromatography packing material.

[0009] In step two, the packing material used for hydrophobic chromatography is preferably high-rigidity agarose as the matrix, with phenyl groups as hydrophobic groups, and the particle size of the packing material is 30-100 μm, preferably 45 μm. High rigidity means that the gel strength of agarose is ≥1500 g / cm², preferably ≥1800 g / cm², and more preferably ≥2000 g / cm². The hydrophobic chromatography packing material is preferably Agarosix HC45 Phenyl.

[0010] The affinity chromatography and hydrophobic chromatography steps preferably include equilibration, loading, reequilibration, washing, and elution. The washing process preferably uses a solution containing arginine (Arg). Using arginine effectively solves the problem of high HCP residues after affinity chromatography purification using conventional methods. Because HCPs bind to antibodies, they adhere to Protein A packing material through non-specific interactions such as electrostatics, hydrogen bonds, and van der Waals forces, and are eluted along with the antibody. Arginine, through its guanidinium group on its side chain, disrupts the electrostatic, hydrophobic, and hydrogen bond interactions between HCP impurities and antibody molecules, thereby removing the HCP impurities.

[0011] The affinity chromatography preferably uses a high pH (~9.0) arginine-containing solution for washing impurities. The high pH allows some impurities, such as HCP, to dissociate from the packing material, thereby improving the impurity removal rate.

[0012] It is preferred to use two washing buffers in affinity chromatography: the first washing buffer contains 20-50 mM Tris-HCl, 0.3-0.5 M arginine, and pH 9.0-9.2; the second washing buffer contains 20-50 mM NaAc-HAc and pH 5.5-5.8.

[0013] The preferred washing buffer used in hydrophobic chromatography contains 25-50 mM PB, 0.3-0.5 M arginine, and pH 5.5-6.5.

[0014] Mechanism of Invention: This invention removes fragment impurities generated during the preparation of bispecific antibodies through a two-step chromatography process: crude purification and fine purification. In the crude purification step, affinity chromatography utilizes the specific affinity between the bispecific antibody molecules and specific ligands on the chromatographic packing material to separate the bispecific antibody molecules from impurities. The addition of PEG and CaCl2 to the elution buffer serves two purposes: PEG enhances protein retention on the affinity chromatography column, and this effect increases with increasing protein molecule size, thereby improving resolution; CaCl2 effectively improves the resolution of antibody molecules and fragments. Crude purification removes most impurities, effectively reducing the pressure on the subsequent hydrophobic chromatography step. In the fine purification step, based on the significant differences in hydrophobicity between the target antibody molecules and impurities such as aggregates, degradation fragments, and endotoxins, a high-purity bispecific antibody protein solution can be obtained through salt gradient elution.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention increases the proportion of bispecific antibodies in the purified eluent obtained by two-step chromatography to more than 98%, and reduces the proportion of fragments to about 0.5%, effectively removing fragment impurities generated during the preparation of bispecific antibodies. It is an efficient and economical method for purifying bispecific antibody fragments. Attached Figure Description

[0016] Figure 1This is the affinity chromatography purification chromatogram of Example 1; Figure 2 The hydrophobic chromatography purification spectrum of Example 1; Figure 3 The results of non-reducing SDS-PAGE gel electrophoresis of the bispecific antibody in Example 1 are as follows (lane 1 is the protein marker, lane 2 is the elution of the washing buffer B step of affinity chromatography in step (2) of Example 1, and lanes 3-8 are the crude pure elution buffer in step (2) of Example 1). Figure 4 The results of non-reducing SDS-PAGE gel electrophoresis of the bispecific antibody in Example 1 are shown (lane 1 is the protein marker, lane 2 is the crude pure elution buffer from step (2) of Example 1, and lanes 3-8 are the refined pure elution buffer from step (3) of Example 1). Figure 5 This is the HPLC chromatogram of the bispecific antibody protein filtrate in a specific embodiment; Figure 6 The HPLC chromatogram of the bispecific antibody protein obtained in Example 1 is shown below. Figure 7 The HPLC chromatogram of the bispecific antibody protein obtained in Example 2; Figure 8 The HPLC chromatogram of the bispecific antibody protein obtained in Comparative Example 1 is shown. Figure 9 The HPLC chromatogram of the bispecific antibody protein obtained in Comparative Example 2 is shown. Figure 10 The results of non-reducing SDS-PAGE gel electrophoresis of the bispecific antibody in Comparative Example 1 are shown (lane 1 is the protein marker, lanes 2-12 are the crude pure elution buffer from step (2) of Comparative Example 1). Figure 11 The results of non-reducing SDS-PAGE gel electrophoresis of the bispecific antibody in Comparative Example 3 are as follows (lane 1 is the protein marker, lane 2 is the elution of step B of affinity chromatography in Comparative Example 3 (2), and lanes 3-10 are the crude pure elution in step (2) of Comparative Example 3). Figure 12 The results of non-reducing SDS-PAGE gel electrophoresis of the bispecific antibody in Comparative Example 4 are shown below (lane 1 is the protein marker, lane 2 is the elution of step B of affinity chromatography in Comparative Example 4 (2), and lanes 3-5 are the crude pure elution in step (2) of Comparative Example 4). Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] This invention applies to bispecific antibodies. The samples used in the following examples and comparative examples were naked ADC antibodies with an isoelectric point of 8.5, a molecular weight of 125 kDa, and a bispecific antibody structure consisting of two heavy chains and one light chain. The main impurities were mismatched, free light chain fragments, heavy chain fragments, and partial aggregates. The sample concentration was 3.0 mg / mL, pH 7.2, and conductivity 16 mS / cm. This sample was prepared using conventional genetic engineering antibody techniques. The preparation process included: 1) Sequence determination of the target antigen epitope: obtaining the variable region (VH, VL) sequences of two specific monoclonal antibodies through literature and high-throughput screening; 2) Construction of a bispecific antibody expression vector: sequentially inserting two pairs of VH / VL genes into the same expression framework, using a "CrossMab" heterodimer design to ensure correct pairing of the heavy chain heterodimers; 3) Transfection of host cells: transfecting the constructed expression plasmid into mammalian cell lines and screening for stable cell lines; 4) Cell culture and expression: large-scale culture in a suitable culture medium, controlling cell density and temperature, to obtain the bispecific antibody molecules.

[0019] Example 1

[0020] This embodiment provides a method for purifying bispecific antibody fragments, the steps of which are as follows: (1) Fermentation broth filtration: 100 mL of bispecific antibody sample fermentation broth was filtered through a 0.2 μm filter membrane to remove insoluble impurities and obtain protein filtrate.

[0021] (2) Affinity chromatography crude purification: The steps include equilibration, sample loading, reequilibration, washing, and elution. See the affinity chromatography purification chromatogram. Figure 1 .

[0022] The solutions include: Equilibrium buffer A: 20 mM Tris-HCl, 150 mM NaCl, pH 7.2. Wash buffer B: 20 ​​mM Tris-HCl, 0.5 M Arg, pH 9.0. Wash buffer C: 50 mM NaAc-HAc, pH 5.5. Elution buffer D: 50 mM NaAc-HAc, 2% PEG4000, 150 mM CaCl2, pH 3.0.

[0023] Affinity chromatography column: Cefen Technology MabPurix A65 Excel affinity chromatography packing column, 15*100 mm.

[0024] Equilibrate the affinity chromatography column to 5 column volumes using equilibration buffer A at a flow rate of 1.57 mL / min and a residence time of 5 min. Load 39.27 mL of the protein filtrate obtained in the previous step into the affinity chromatography column, and equilibrate again to 5 column volumes using equilibration buffer A (flow rate 1.57 mL / min). Wash the column with 5 column volumes using wash buffer B (flow rate 1.57 mL / min), followed by 5 column volumes using wash buffer C (flow rate 1.57 mL / min) to lower the pH and prevent loss of the dual antibodies. Finally, elute linearly using wash buffer C and eluent D, with a linearity of 0%–100% of eluent D, a pH gradient of 5.5–3.0, an elution volume of 10 CV, and a flow rate of 1.57 mL / min. Acquire chromatographic signals of 50 mAu–50 mAu and collect the eluent in separate tubes to obtain the crude pure eluent.

[0025] (3) Hydrophobic chromatography purification: The hydrophobic chromatography purification chromatogram is shown in [reference needed]. Figure 2 .

[0026] The solutions included: Equilibrium buffer E: 25 mM PB, 0.8 M (NH4)2SO4, pH 6.0; Wash buffer F: 25 mM PB, 0.5 M Arg, 0.8 M (NH4)2SO4, pH 6.0; Elution buffer G: 25 mM PB, 0.5 M Arg, pH 6.0.

[0027] Hydrophobic chromatography column: Agarosix HC45 Phenyl hydrophobic chromatography packing column, 6.6*200 mm.

[0028] The hydrophobic chromatography column was equilibrated with equilibration buffer E at a volume of 6.158 mL, a flow rate of 1.232 mL / min, and a residence time of 5 min. The high salt content of equilibration buffer E facilitated the binding of the target protein to the packing material. The affinity chromatography eluent obtained in step (2) was adjusted to a concentration of 0.8 M with ammonium sulfate, and 6.61 mL was loaded onto the hydrophobic chromatography column. The column was then equilibrated with equilibration buffer E for 5 column volumes (flow rate 1.232 mL / min), followed by elution with washing buffer F for 5 column volumes (flow rate 1.232 mL / min) to replace the environment and improve the resolution of the fragments and target proteins. Finally, linear elution was performed using washing buffer F and elution buffer G, with a linearity of 0%-100% of elution buffer G, an elution volume of 15 CV, and a flow rate of 1.232 mL / min. The chromatographic signal was collected at 50 mAu-50 mAu, and the eluent was collected in separate tubes to obtain the purified eluent, which is the purified bispecific antibody.

[0029] Example 2

[0030] This embodiment provides a method for purifying bispecific antibody fragments. The sample and method are the same as in Example 1, with the difference being the solutions used in the two-step chromatography. The solutions used in this embodiment are as follows.

[0031] Step (2) Affinity chromatography crude purity: Equilibration buffer A: 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. Wash buffer B: 50 mM Tris-HCl, 0.7 M Arg, pH 9.5. Wash buffer C: 50 mM NaAc-HAc, pH 5.5. Eluent D: 50 mM NaAc-HAc, 2.5% PEG4000, 250 mM CaCl2, pH 3.0.

[0032] Step (3) Hydrophobic chromatography purification: Equilibration buffer E: 25 mM PB, 1.0 M (NH4)2SO4, pH 7.0. Wash buffer F: 25 mM PB, 0.5 M Arg, 1.0 M (NH4)2SO4, pH 7.0. Eluent G: 25 mM PB, 0.5 M Arg, pH 7.0.

[0033] Comparative Example 1 This comparative example provides a purification method for bispecific antibody fragments. The sample is the same as in Example 1, and the method is basically the same as in Example 1. The difference is that the elution buffer D in the affinity chromatography crude purity step (2) is replaced with 50 mM NaAc-HAc, pH 3.0.

[0034] Comparative Example 2 This comparative example provides a purification method for bispecific antibody fragments. The sample is the same as that in Example 1, and the method is basically the same as that in Example 1. The difference is that the elution buffer D in the affinity chromatography crude purity step (2) is replaced with 50mM NaAc-HAc, 2% PEG4000, pH3.0.

[0035] Comparative Example 3 This comparative example provides a purification method for bispecific antibody fragments. The sample is the same as that in Example 1, and the method is basically the same as that in Example 1. The difference is that the elution buffer D in the affinity chromatography crude purity step (2) is replaced with 50mM NaAc-HAc, 150mM CaCl2, pH 3.0.

[0036] Comparative Example 4 This comparative example provides a purification method for bispecific antibody fragments. The sample and method are the same as those in Example 1, except that: the elution buffer D is 50 mM NaAc-HAc, 5% PEG4000, 500 mM CaCl2, pH 3.0; and hydrophobic chromatography was not performed subsequently.

[0037] Non-reducing SDS-PAGE electrophoresis and size exclusion chromatography were used to detect the eluent and elution buffer obtained in Example 1, the crude pure elution buffer obtained in Comparative Example 1, and the eluent and crude pure elution buffer obtained in Comparative Examples 3 and 4. Size exclusion purity was determined for the purified bispecific antibodies and original protein filtrates from Examples 1-2 and Comparative Examples 1-2.

[0038] I. SDS-Page Electrophoresis Detection Materials: GenScript SurePAGE precast gel, Bis-Tris, 10×8, 15%; Electrophoresis buffer: Tris-Mops; SDS sample buffer: Sangon 5X Native Protein Loading Dye.

[0039] Method: The washing buffer and elution buffer were mixed with SDS sample buffer at a volume ratio of 4:1. The precast gel was then inserted into the electrophoresis tank, and Tris-Mops electrophoresis buffer was poured in until it completely covered the surface of the precast gel to ensure no leakage. The comb was then removed, and the mixed solution was added to the channels of the precast gel. Electrophoresis was started by setting the voltage and current. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel. The precast gel was then removed and stained with Coomassie Brilliant Blue for 30-60 min, and then destained with water until the background was clear.

[0040] Result: As Figure 3-4 As shown, when PEG and CaCl2 are added simultaneously to the affinity elution buffer, electrophoresis analysis of the affinity chromatography eluent reveals that aggregates cannot be removed, but most fragment impurities can be removed. Electrophoresis analysis of the hydrophobic chromatography eluent shows that aggregates can be completely removed, and a small number of fragments that cannot be removed in affinity chromatography can be removed well in hydrophobic chromatography. The two-step chromatography process of this invention can remove fragments and aggregates from double antibody samples.

[0041] contrast Figure 10 It can be seen that without the addition of PEG and CaCl2 to the affinity chromatography eluent, the fragments are almost impossible to remove in the affinity chromatography step. (Comparison) Figure 11 When only CaCl2 is added to the affinity chromatography eluent, it can effectively remove fragments of medium molecular weight, such as 45-75 kDa; however, it is not effective in removing small and large molecular weight fragments. (Comparison) Figure 12 When excessive amounts of PEG and CaCl2 are added to the affinity chromatography elution buffer (e.g., 5% PEG4000, 500mM CaCl2), the target protein precipitates and aggregates, making it impossible to perform the hydrophobic purification chromatography step. This indicates that the content of PEG and CaCl2 is also very important for the fragment removal effect.

[0042] II. Size exclusion chromatography detection Chromatographic column: SRT SEC-300 Excel (5 μm particle size, 300 Å pore size, 7.8 mm × 300 mm). Detection mobile phase: 50 mM potassium phosphate buffer + 250 mM potassium chloride, pH 6.2. Detection flow rate: 1 mL / min. Injection volume: 20 uL. Injected samples: purified bispecific antibodies from Examples 1-2 and Comparative Examples 1-2, and original protein filtrate, respectively. Chromatography system: Agilent 1260 Infinity II HPLC system. Run time: 15 min. Detection wavelength: 280 nm.

[0043] Results: As shown in Table 1. Figure 5-9 As shown.

[0044] Table 1 Purity of Bispecific Antibody Protein Solution Aggregates% Main Peak Fragment% As is 7.833 75.200 17.050 Example 1 1.063 98.461 0.476 Example 2 0.601 98.729 0.670 Comparative Example 1 3.536 82.333 14.131 Comparative Example 2 4.981 85.977 9.042 As shown in Table 1, in Examples 1 and 2 of this invention, affinity chromatography was used as the crude purity. The eluent D contained PEG and CaCl2. After purification, the HPLC purity of the bispecific antibody solution was greater than 98%, and the fragment percentage decreased from 17.05% to less than 0.7% (see Table 1). Figure 6 and Figure 7 In Comparative Example 1, the eluent D was replaced with a solution that did not contain PEG and CaCl2 (see...). Figure 8 Compared to Examples 1-2, the obtained bispecific antibody solution was less effective at removing fragment impurities. In Comparative Example 2, elution buffer D was replaced with a solution without CaCl2 (see...). Figure 9 The bispecific antibody solution obtained in Example 1 showed slightly better removal of fragment impurities than that obtained in Comparative Example 1, but it was still not as effective as that in Examples 1-2. Therefore, the purification method provided by this invention can effectively remove fragment impurities generated during the preparation of bispecific antibodies.

Claims

1. A purification method of a fragmented bispecific antibody, characterized by, The method comprises the following steps: Step 1: crude purification of the bispecific antibody sample by affinity chromatography, wherein the eluent used in the affinity chromatography contains PEG and CaCl2, to obtain a crude eluent; Step 2: fine purification of the crude eluent by hydrophobic chromatography, to obtain a fine eluent, i.e. the purified bispecific antibody.

2. The purification method of claim 1, wherein, In step 1, the content of PEG is 1.5-3.0%, and the content of CaCl2 is 100-300 mM.

3. The purification method of claim 1, wherein, In step 1, the eluent used in the affinity chromatography is 20-50 mM NaAc-HAc, 1.5-3.0% PEG4000, 100-300 mM CaCl2, and pH 2.8-3.

2.

4. The purification method of claim 1, wherein, In step 1, the affinity chromatography uses protein A affinity chromatography filler.

5. The purification method of claim 1, wherein, In step 2, the filler used in the hydrophobic chromatography uses high-rigidity agarose as the matrix and phenyl as the hydrophobic group.

6. The purification method of claim 1, wherein, The steps of the affinity chromatography or the hydrophobic chromatography comprise equilibration, sample loading, re-equilibration, impurity washing and elution.

7. The purification method of claim 6, wherein, The impurity washing uses an arginine-containing solution.

8. The purification method of claim 6, wherein, In the affinity chromatography, two impurity washing solutions are used, wherein the first impurity washing solution contains 20-50 mM Tris-HCl, 0.3-0.5 M arginine, and pH 9.0-9.2, and the second impurity washing solution contains 20-50 mM NaAc-HAc, and pH 5.5-5.

8.

9. The purification method of claim 6, wherein, In the hydrophobic chromatography, the impurity washing solution contains 25-50 mM PB, 0.3-0.5 M arginine, and pH 5.5-6.

5.

10. The purification method of claim 1, wherein, In step 1, the bispecific antibody sample is filtered before affinity chromatography.

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