Efficient purification process of bispecific antibody and special chromatography system thereof
By using an improved four-step purification process and a dedicated chromatography system, the problems of cumbersome purification steps, high costs, and incomplete impurity removal for bispecific antibodies have been solved, achieving efficient and low-cost purification results that are suitable for industrial production.
Patent Information
- Application Number
- CN202511140306.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-21
AI Technical Summary
Traditional purification processes for bispecific antibodies are cumbersome, costly, and incomplete in removing impurities, resulting in poor process compatibility, long purification cycles, and low efficiency, making it difficult to meet the needs of industrial production.
A four-step purification process consisting of modified Protein A affinity chromatography, mixed-mode chromatography, and nanofiltration was adopted. A dedicated chromatography system was designed by combining agarose medium modified with recombinant Protein A ligands and a composite-mode medium with benzamide functional groups to reduce the number of chromatography steps and improve purification efficiency.
It significantly shortens the purification cycle by more than 50%, improves purification efficiency, increases yield and product purity, reduces production costs, improves activity retention, has strong compatibility, and is suitable for industrial production.
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Figure CN120988138A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biological medicine, and particularly relates to a high-efficiency purification process of a bispecific antibody and a special chromatography system thereof. BACKGROUND
[0002] The bispecific antibody has a significant advantage in the treatment of diseases such as diabetic retinopathy due to the ability to simultaneously bind two antigen epitopes, but the complex molecular structure leads to a much higher purification difficulty than monoclonal antibodies.
[0003] In the purification process of the bispecific antibody, the mainstream technical route is mainly a multi-step chromatography in series, and the specific structure and process are as follows: (1) the core chromatography unit: including a Protein A affinity chromatography column (ligand is natural Protein A, medium particle size is 75-100 μm), a cation exchange chromatography column (such as SP Sepharose, based on charge difference separation), an anion exchange chromatography column (such as Q Sepharose, for removing acidic impurities), a hydrophobic interaction chromatography column (such as Phenyl Sepharose, relying on hydrophobic difference) and a composite mode chromatography column (such as MabSelect SuRe, for assisting in removing isomers), each chromatography column is connected in series through a pipeline, the column length is 20-30 cm, and the inner diameter is matched with the processing capacity (usually 1-10 cm); (2) the operation process: after centrifugation (8000xg, 10 min) and 0.45 μm filtration of the cell culture supernatant, the cell culture supernatant is first introduced into the Protein A chromatography column (the equilibrium buffer is 10 mM PBS pH7.0, and the eluent is 50 mM citric acid pH3.0), the eluent is collected and neutralized; then the cation exchange chromatography (the sample buffer is 20 mM sodium acetate pH5.0, and the eluent contains 0-1 M NaCl gradient), the anion exchange chromatography (the sample buffer is 20 mM Tris-HCl pH8.0, and the eluent contains 0-0.5 M NaCl gradient) and finally the hydrophobic chromatography and virus filtration are sequentially performed; (3) the key parameters: the linear flow rate of each chromatography is 50-100 cm / h, the sample amount is 10-20 times of the column volume (CV), the single-step yield is 70%-85%, and the cumulative yield needs to be calculated by multiple steps.
[0004] Currently, the traditional purification process of bispecific antibodies has the following limitations: (1) complicated chromatography steps: the conventional process needs to go through 5-6 steps of chromatography, such as Protein A affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, and complex mode chromatography, to remove host cell proteins, residual DNA, viruses, aggregates and mispaired isomers, and the multiple steps result in a long purification cycle of 7-10 days and low efficiency; (2) high cost: a large amount of chromatography medium (such as Protein A resin) and buffer is consumed in multiple chromatography, and the antibody is lost in each chromatography, which significantly increases the cost of industrial production; (3) incomplete removal of impurities: due to the asymmetric structure of bispecific antibodies, aggregates are easily formed, and the traditional chromatography system is difficult to efficiently separate aggregates from monomers, which affects the purity of the product; (4) poor process compatibility: the existing chromatography system is designed for monoclonal antibodies and does not consider the special structure of bispecific antibodies, which easily leads to conformational changes of the antibody during chromatography, reducing the activity retention rate.
[0005] Therefore, there is an urgent need for a high-efficiency purification process suitable for bispecific antibodies. SUMMARY
[0006] The purpose of the present application is to solve the above technical problems, and to provide a purification process with high purification efficiency and yield, and significantly improved product purity.
[0007] In order to achieve the above purpose, the present application provides the following technical solutions:
[0008] A high-efficiency purification process for bispecific antibodies, comprising the following steps:
[0009] (1) Pretreatment: the cell culture supernatant is subjected to 0.22 μm microfiltration to remove cell debris, and then subjected to deep filtration to reduce the turbidity to ≤1 NTU, thereby reducing the pollution of the subsequent chromatography medium;
[0010] (2) Improved Protein A affinity chromatography: using a recombinant Protein A ligand modified agarose medium, the loading buffer is 20 mM Tris-HCl, pH 7.4, and the elution buffer is 100 mM citric acid, pH 3.2-3.5, for affinity chromatography;
[0011] (3) Mixed mode chromatography: using a complex mode medium containing benzamidine functional groups, the buffer is 50 mM sodium phosphate, pH 6.0-6.5, and the NaCl concentration is adjusted to 0.1-0.3 M, to specifically remove bispecific antibody aggregates and mispaired isomers by using the synergistic effect of hydrophobic interaction and ion exchange;
[0012] (4) Nanofiltration and terminal treatment: using a virus filtration membrane with a pore size of 30 nm to remove viruses, and then subjecting to 0.22 μm sterile filtration to obtain the final product.
[0013] Preferably, in the step (2), the density of the recombinant Protein A ligand is 15-20 mg / mL.
[0014] Preferably, in the step (2), the recombinant Protein A ligand modified agarose medium is prepared by the following steps: 6% cross-linked agarose is fully soaked, washed, and impurities are removed with deionized water, epoxy chloropropane and sodium hydroxide solution are added, and stirring reaction is carried out at 25-35 DEG C for 2-4 hours to activate the agarose; the activated 6% cross-linked agarose is covalently coupled with the recombinant Protein A solution at 4-8 DEG C for 12-24 hours; ethanolamine is added for capping, and deionized water and PBS buffer are used for washing to obtain the recombinant Protein A ligand modified agarose medium.
[0015] Preferably, in the step (3), the particle size of the complex mode medium containing benzamidine functional groups is 50-60 mu m.
[0016] Preferably, the complex mode medium containing benzamidine functional groups is benzamidine modified 6% cross-linked agarose, which is prepared by the following steps: 6% cross-linked agarose is soaked and washed with deionized water, then epoxy chloropropane and sodium hydroxide solution are added, and stirring reaction is carried out at 25-35 DEG C for 2-4 hours, and then the activated 6% cross-linked agarose is washed with a large amount of deionized water until it is neutral, and the activation of the agarose is completed; the activated 6% cross-linked agarose is mixed with benzamidine solution, and stirring reaction is carried out at 30-37 DEG C for 6-8 hours to occur covalent coupling reaction; after the coupling reaction is completed, ethanolamine is added to block the unreacted epoxy groups, and then deionized water and PBS buffer are used for washing to obtain benzamidine modified 6% cross-linked agarose chromatography medium.
[0017] Preferably, in the step (4), the virus filtration membrane is PES material.
[0018] The application also provides a chromatography system specially used for the high-efficiency purification process of the bispecific antibody, which comprises a chromatography column, the inside of the chromatography column is sequentially filled with improved Protein A medium and mixed mode medium from top to bottom to form a two-section series bed, the two media are separated by a sintered glass plate to ensure one-way flow and prevent mixing of the media, the chromatography column also has a buffer inlet and a sample inlet, and the chromatography column is sequentially connected with an ultraviolet detector and a collector.
[0019] Compared with the prior art, the application has the following remarkable effects:
[0020] (1) The purification steps are reduced from 5-6 steps to 4 steps, the cycle is shortened by more than 50%, the purification efficiency is significantly improved, and it is suitable for industrial continuous production.
[0021] (2) The yield and product purity are significantly improved.
[0022] (3) The consumption of chromatography medium is significantly reduced due to the omission of the chromatography step, and the buffer cost is significantly reduced, thereby reducing the total production cost.
[0023] (4) The activity retention is better, and the activity retention rate of the bispecific antibody is significantly improved through special buffer and medium design.
[0024] (5) Strong compatibility, which can effectively remove light chain mismatch products. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 is a schematic diagram of the chromatography system structure special for the purification process of the bispecific antibody of the present application. DETAILED DESCRIPTION
[0026] The technical solutions of the present application will be further described below in combination with the drawings and specific examples.
[0027] Unless otherwise specified, the instruments or reagents used in the examples are conventional instruments or reagents in the art, which are conventional products that can be purchased in the market, and the specific experimental operations involved in the present application are understood or known by the ordinary skilled person in the art according to the common knowledge or conventional technical means mastered by them.
[0028] Example 1: Preparation of anti-BMP-2 / VEGF-A bispecific antibody
[0029] 1. Expression vector
[0030] The plasmid pCHO1.0 (Thermo Fisher Scientific, item number A13696) containing the CMV promoter and glutamine synthetase selection marker was used.
[0031] 2. Gene construction and plasmid preparation
[0032] The nucleotide sequence encoding anti-VEGF-A VH-CH1 (SEQ ID NO: 1), flexible linker peptide (G4S)3 (SEQ ID NO: 2), anti-BMP-2 VH-CH1 (SEQ ID NO: 3), and mutant Fc (L234F / L235E / P331S) (SEQ ID NO: 4) were fused in order to obtain the full-length fusion gene SEQ ID NO: 5. 2 μg of pCHO1.0 plasmid was double-digested with EcoRI and XhoI, and the linear fragment was recovered; 150 ng of the fusion gene fragment was ligated with T4 DNA ligase at 16°C overnight. The ligation product was transformed into DH5α competent cells, screened with ampicillin, and the plasmid was extracted and verified by double digestion with EcoRI / XhoI and sequencing.
[0033] 3. Transfection and pressure selection of CHO-S cells against BMP-2 / VEGF-A
[0034] The CHO-S cells (Thermo Fisher, Cat. No. R80007) were recovered to the 3rd-5th passage, and the density was adjusted to 2×10 7 cells / mL. 5 μg of linearized plasmid was mixed with 1×10 7 cells, and the Neon electroporation system was used for transfection under the conditions of 1400 V, 20 ms, and single pulse. Immediately after transfection, the cells were transferred to CDCHO medium (Thermo Fisher, Cat. No. 10743029) containing 8 mM L-glutamine, and 50 μg / mL puromycin was added for pressure selection. The culture conditions were 37°C, 5% CO2, and 125 rpm, and the medium was replaced every 48 hours, for a duration of 7-10 days, until a stable expression pool was formed.
[0035] 4. Fermentation
[0036] The stable expression pool was inoculated at a density of 3×10 5 cells / mL in 1.5 L CD CHO medium, and was loaded into a 2 L Sartorius BIOSTAT Qplus bioreactor. The pH was maintained at 7.0±0.1 throughout the fermentation, and was automatically controlled by CO2 and sodium bicarbonate; the dissolved oxygen was set to 40% air saturation; the temperature was controlled at 37°C for 0-72 h, and was then reduced to 34°C. Starting from the 3rd day, 10% v / v Efficient Feed C+ (Cat. No. A2503101) was added daily. When the cell viability decreased to below 80%, the supernatant was collected by centrifugation at 4000 g for 20 min at 4°C, and was filtered through a 0.22 μm filter membrane to obtain the anti-BMP-2 / VEGF-A bispecific antibody fusion protein.
[0037] Example 2: High-efficiency purification process for the bispecific antibody of the application
[0038] 1. Dedicated chromatography system
[0039] like Figure 1 As shown, in the chromatography system dedicated to the process of this invention, the chromatography column 1 is sequentially filled from top to bottom with modified Protein A medium 2 and mixed-mode medium 3, forming two series-connected beds. The two media are separated by a sintered glass plate with a pore size of 20 μm to ensure unidirectional flow and prevent media mixing. The buffer inlet 4 is connected to the equilibration solution, elution solution, and neutralization solution storage bottles via a three-way valve, and the flow rate is controlled at 100-180 cm / h by an external constant flow pump.
[0040] Sample inlet 5 is connected to the pretreated clarified supernatant bottle. Before loading the sample, 5CV 20mM Tris-HCl (pH 7.4) is introduced into the chromatography column 1 through buffer inlet 4 to equilibrate Protein A medium 2.
[0041] Subsequently, the sample passes through ProteinA medium 2 at a flow rate of 100 cm / h from sample inlet 5. The dual antibodies are specifically adsorbed, and the flow-through liquid passes directly through mixed-mode medium 3 and is discharged by UV detector 6.
[0042] After sample loading, 3CV of 100mM citric acid (pH 3.4) was injected into buffer inlet 4 for elution. The elution peak was captured by collector 7 under the monitoring of UV detector 6 at 280nm. At the same time, 1M Tris-HCl (pH 8.0) was added online to neutralize to pH 6.0.
[0043] The neutralized eluent re-enters the same chromatography column 1 through sample inlet 5. At this time, Protein A medium 2 has been switched to flow-through mode. When the sample flows through mixed mode medium 3, residual impurities are captured, and the dual antibodies flow out directly. After the main peak is confirmed by UV detector 6, it is collected by collector 7, and finally a final product with a purity of 99.6% is obtained.
[0044] The preferred length of the dedicated chromatography column is 10-15cm, and the inner diameter is adapted according to the throughput (1.6cm for laboratory grade and 5cm for pilot grade). The ratio of column bed volume (CV) to sample loading is 1:50 (i.e., 1CV of medium can process 50CV of sample) to improve processing efficiency.
[0045] Preferably, an online buffer preparation module can be added. An automatic pH / conductivity adjustment device (such as Sartorius) can be integrated at the front end of the chromatography column. The BufferPro module adjusts the pH (±0.1) and conductivity (±0.5 mS / cm) of the loading solution in real time, reducing manual mixing errors and further improving the impurity removal rate of mixed-mode chromatography by 5%-8%.
[0046] Preferably, a medium regeneration optimization unit can be added. A regeneration solution circulation path of 0.1M NaOH + 1M NaCl is added at the end of the Protein A chromatography column, extending the service life of the medium from 50 cycles to 80 cycles, reducing the frequency of consumable replacement.
[0047] Preferably, a multi-wavelength detection system can be added: 260nm (nucleic acid detection) and 405nm (turbidity detection) channels are added on the basis of the ultraviolet detector (280nm), real-time monitoring of DNA residues and polymer formation, ensuring the stability of the quality of the final product.
[0048] 2. Bispecific antibody purification process steps
[0049] (1) Pretreatment
[0050] Firstly, microfiltration with a 0.22μm polyether sulfone (PES) membrane and then deep filtration with a 0.45μm glass fiber membrane at 4℃, disposable removal of cell debris, lipids and large particles, making the supernatant turbidity ≤1NTU, providing a clear, low particle load feed solution for subsequent chromatography, significantly reducing the risk of column bed blockage. Sartopore2 0.22μm PES capsule filter, Millistak+HC Pro 0.45μm glass fiber deep filter plate can be selected.
[0051] (2) Improved Protein A affinity chromatography
[0052] Chromatography medium: recombinant Protein A covalently coupled to 6% cross-linked agarose (particle size 90μm, ligand density 18mg / mL, such as MabSelect SuRe).
[0053] Equilibrium: 5CV 20mM Tris-HCl (pH 7.4, conductivity 2-3mS / cm), flow rate 150cm / h, to make the column bed reach stable pH and conductivity.
[0054] Loading: the feed solution is loaded at a ratio of 50CV sample: 1CV medium, flow rate 100cm / h, to ensure dynamic binding capacity ≥40mg IgG / mL medium.
[0055] Elution: 3CV 100mM citric acid (pH 3.3), flow rate 100cm / h; after collecting the main peak, immediately neutralize online to pH 6.0 with 1M Tris-HCl (pH 8.0) to prevent antibody inactivation or aggregation in low pH environment.
[0056] Using the high affinity of recombinant Protein A to the Fc region, one-step capture of BMP-2 / VEGF-A bispecific antibody is achieved, while by shortening the retention time (3-5min) and immediate pH adjustment, the aggregation and degradation caused by low pH are maximally inhibited.
[0057] The recombinant Protein A used in the present application is a commercially available product from Cytiva, with the product number 17-5438-01. The recombinant Protein A has high purity and good activity, can specifically bind to the Fc fragment of antibodies, and meets the experimental requirements.
[0058] The specific steps of covalently coupling the recombinant Protein A to 6% cross-linked Sepharose (6% represents the cross-linking degree) are as follows:
[0059] 1) Activation of 6% cross-linked Sepharose: An appropriate amount of 6% cross-linked Sepharose (Sepharose 6B, purchased from Cytiva) was soaked and washed with deionized water to remove impurities. The soaked Sepharose was transferred to a reaction container, an appropriate amount of epichlorohydrin (1 / 5-1 / 3 of the volume of Sepharose) was added, and then 0.5-1M sodium hydroxide solution was added (to make the final reaction system have a sodium hydroxide concentration of 0.2-0.4M). The reaction was carried out at 25-35°C with gentle stirring for 2-4 hours. During this period, the hydroxyl groups on the surface of the Sepharose reacted with epichlorohydrin, introducing epoxy groups and achieving the activation of the Sepharose. After the reaction was completed, the activated Sepharose was washed with a large amount of deionized water until the effluent pH was neutral, and the unreacted epichlorohydrin and sodium hydroxide impurities were removed.
[0060] 2) Recombinant Protein A coupling: The activated 6% cross-linked Sepharose was transferred to a reaction container containing a recombinant Protein A solution (based on the specifications and processing scale of the chromatography column, 8-10g of wet 6% cross-linked Sepharose was weighed for every 10mL of column bed volume; 0.5-1.0mg of recombinant Protein A was added for every 1g of wet activated Sepharose). The recombinant Protein A solution was prepared using 0.1M borate buffer at pH 8.0-9.0, with a final concentration of 1-3mg / mL of recombinant Protein A in the solution. The reaction was carried out at 4-8°C with slow stirring for 12-24 hours. During this period, the amino groups on the recombinant Protein A molecules covalently coupled with the epoxy groups on the surface of the activated Sepharose.
[0061] 3) End-capping and washing: After the coupling reaction was completed, an appropriate amount of ethanolamine was added to the reaction system (final concentration of 0.1-0.2M), and the reaction was carried out at room temperature for 1-2 hours to cap the unreacted epoxy groups on the surface of the Sepharose, preventing non-specific adsorption in the subsequent process. Then, the Sepharose was washed with a large amount of deionized water and PBS buffer at pH 7.4, respectively, to remove unbound recombinant Protein A, ethanolamine and other impurities. The recombinant Protein A covalently coupled to 6% cross-linked Sepharose was obtained, and was stored in PBS buffer containing 0.02% sodium azide at 4°C for future use.
[0062] (3) Mixed mode chromatography
[0063] Chromatography medium: benzamidine-modified 6% cross-linked agarose (particle size 55 pm, ligand density 15 pmol / mL, like CaptoBenzamidine FF).
[0064] Equilibration: 5 CV 50 mM sodium phosphate (pH 6.2 with 0.2 M NaCl, conductivity 20 mS / cm) at a flow rate of 180 cm / h.
[0065] Loading: Dilute the affinity eluate with equilibration buffer (with 0.01% w / v polysorbate 80) to a conductivity of 15-20 mS / cm, load at a sample:medium ratio of 30 CV sample: 1 CV medium, at a flow rate of 120 cm / h.
[0066] Elution: 5 CV 50 mM sodium phosphate (pH 6.2 with 0.05 M NaCl, conductivity 5-7 mS / cm) at a flow rate of 120 cm / h; collect the main peak, ensuring that aggregate residuals are < 0.5%.
[0067] Benzamidine-agarose has both hydrophobic and weak electrostatic interactions, which can bind host proteins, DNA, shed Protein A and other impurities at moderate salt concentrations, while BMP-2 / VEGF-A bispecific antibodies preferentially flow through or weakly bind under low salt conditions, thereby further removing aggregates and reducing impurities to < 0.5%.
[0068] Preparation steps of benzamidine-modified 6% cross-linked agarose are as follows:
[0069] 1) Activation of 6% cross-linked agarose: The same as the activation step of 6% cross-linked agarose in the preparation process of recombinant Protein A coupled to 6% cross-linked agarose, that is, take an appropriate amount of 6% cross-linked agarose (Sepharose 6B), soak and wash with deionized water, then add epichlorohydrin and sodium hydroxide solution, react at 25-35°C for 2-4 hours, then rinse with a large amount of deionized water until neutral, and complete the activation.
[0070] 2) Benzamidine coupling: Transfer the activated 6% cross-linked agarose to a reaction system containing benzamidine (10-20 pmol of benzamidine per 1 g of wet weight of activated agarose), dissolve the benzamidine in 0.1 M sodium carbonate-sodium bicarbonate buffer at pH 8.5-9.5 to make the final concentration of benzamidine 50-100 mM. Under the condition of 30-37°C, gently stir for 6-8 hours, during which the amino group on the benzamidine molecule reacts with the epoxy group on the surface of the activated agarose to form a covalent coupling reaction.
[0071] 3) End-capping and washing: After the coupling reaction, add an appropriate amount of ethanolamine (final concentration of 0.1-0.2M) to cap the unreacted epoxy groups at room temperature for 1-2 hours. Then, wash with a large amount of deionized water and PBS buffer at pH 7.4 to remove unbound benzamidine, ethanolamine and impurities, and obtain benzamidine-modified 6% cross-linked agarose chromatography medium. Store in PBS buffer containing 0.02% sodium azide at 4°C for future use.
[0072] As an alternative, if benzamidine-modified complex mode medium is difficult to obtain, agarose medium containing 4-aminobenzamidine functional groups (particle size 45-65 μm) can be used. By adjusting the pH of the buffer to 5.8-6.0, the effect of removing polymers with a removal rate of ≥98% can still be achieved.
[0073] The improved Protein A medium is optimized for binding to Fc fragments of bispecific antibodies, and is combined with benzamidine-modified complex mode medium to specifically remove polymers. The flow rate compatibility of the two media is optimized at a linear flow rate of 150-200 cm / h to avoid conformational changes caused by antibody retention.
[0074] The use of a special equilibration buffer formulation (containing 0.01% w / v polysorbate 80) reduces the adsorption loss of bispecific antibodies during chromatography, and the activity retention rate is increased to ≥95%. Polysorbate 20 (concentration 0.01% w / v) can also be used instead of polysorbate 80 to reduce non-specific adsorption of bispecific antibodies on the medium surface, and the activity retention rate is maintained at 93%-95%.
[0075] (4) Terminal treatment
[0076] Virus filtration: PES 30 nm nanofiltration membrane (e.g. Planova 20N), operating pressure ≤0.5 bar, flow rate 30 Lm -2 h -1 .
[0077] Sterile filtration: PES 0.22 μm sterilization grade filter cartridge (Sartopore 2), after passing the online integrity test (bubble point method), the final product is obtained.
[0078] 30 nm virus filtration membrane removes potential viral particles, and 0.22 μm sterile filtration achieves terminal sterilization. The two steps in series can maintain high yield (≥95%) while meeting the safety standards for biological products.
[0079] As an alternative, if the 30 nm virus filtration membrane is expensive, a 15 nm ceramic membrane can be used for filtration (operating pressure 0.2-0.3 MPa), with a virus removal logarithm (LRV) of ≥4.0, which is comparable to the original scheme.
[0080] Comparative Example: Conventional Protein A affinity chromatography purification process
[0081] The specific steps are as follows:
[0082] (1) Equilibrate the column: Take an appropriate amount of commercial Protein A affinity chromatography column (HiTrap Protein A HP column from GE Healthcare), and flush it with at least 5 column volumes of equilibration buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) at a flow rate of 0.5-1 mL / min until the pH of the effluent is consistent with the equilibration buffer, ensuring that the chromatography column is in a stable working state, and that the medium in the column and the buffer environment are balanced, which is beneficial for subsequent antibody binding.
[0083] (2) Sample loading: The antibody sample to be purified (such as obtained from cell culture supernatant or serum, centrifuged to remove impurities in advance) is filtered through a 0.22 μm filter, and then slowly loaded into the equilibrated chromatography column at a flow rate of 0.2-0.5 mL / min, so that the antibody can fully contact and specifically bind to the Protein A medium, and the unbound impurities are removed with the effluent.
[0084] (3) Washing: Flush the column with 5-10 column volumes of washing buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) at a flow rate of 0.5-1 mL / min to remove non-specifically bound impurities, salts, and other impurities, until the absorbance of the effluent at 280 nm approaches the baseline level (generally A280<0.05).
[0085] (4) Elution: Use elution buffer (0.1 M citric acid, pH 3.0-3.5) to elute the antibody bound to Protein A at a flow rate of 0.2-0.5 mL / min, and collect the elution peak. Since the low pH environment destroys the interaction between the antibody and Protein A, the antibody is eluted from the column. The collected eluate is immediately neutralized to neutral with 1 M Tris-HCl (pH 8.0) to prevent denaturation of the antibody under acidic conditions.
[0086] (5) Column regeneration: After elution, flush the column with at least 5 column volumes of regeneration buffer (1 M sodium chloride, 0.1 M phosphoric acid, pH 2.5) to remove residual antibodies and impurities, and then re-equilibrate the column with 5 column volumes of equilibration buffer to restore the initial state of the column, which can be used for the next purification.
[0087] Compared with the comparative example, the present application has the following significant effects:
[0088] (1) Efficiency improvement: The purification steps are reduced from 5-6 steps to 4 steps, the cycle is shortened by more than 50%, from 7-10 days to 3-4 days, which is suitable for industrial continuous production.
[0089] (2) Yield and purity improved: cumulative yield increased from ≤50% to ≥75%, product purity increased from 95%-97% to ≥99.5%, polymer content ≤0.5%, HCP ≤10 ppm.
[0090] (3) Cost reduced: 3-step chromatography omitted, 40% reduction in chromatography media consumption, 35% reduction in buffer cost, and about 30% reduction in total production cost.
[0091] Activity retention more optimal: through special buffer and medium design, the activity retention rate of the bispecific antibody increased from 80%-85% to ≥95%, ensuring its biological function in the treatment of diabetic retinopathy, such as inhibiting neovascularization and eliminating effusion.
[0092] (4) Strong compatibility: the "hybrid Fab arm" structure of the bispecific antibody (such as anti-BMP-2 / VEGF-A bispecific antibody) constructed for CrossMab technology is optimized, which can effectively remove light chain mismatch products, and the mismatch rate is reduced from 5%-8% to ≤0.1%.
Claims
1. A high-efficiency purification process of a bispecific antibody, comprising the following steps: (1) Pretreatment: removing cell debris from cell culture supernatant by 0.22 μm microfiltration, and reducing turbidity to ≤1 NTU by depth filtration to reduce pollution of subsequent chromatography media; (2) Improved Protein A affinity chromatography: using a recombinant Protein A ligand modified agarose medium, the loading buffer is 20 mM Tris-HCl, pH 7.4, and the elution buffer is 100 mM citric acid, pH 3.2-3.5, to perform affinity chromatography; (3) Mixed mode chromatography: using a complex mode medium containing benzamidine functional groups, the buffer is 50 mM sodium phosphate, pH 6.0-6.5, and the NaCl concentration is adjusted to 0.1-0.3 M, to specifically remove bispecific antibody aggregates and mispaired isomers by synergistic effect of hydrophobic interaction and ion exchange; (4) Nanofiltration and terminal treatment: removing viruses by using a virus filtration membrane with a pore size of 30 nm, and then performing 0.22 μm sterile filtration to obtain a final product.
2. The purification process of claim 1, wherein, In the step (2), the density of the recombinant Protein A ligand is 15-20 mg / mL.
3. The purification process of claim 1, wherein, In the step (2), the recombinant Protein A ligand modified agarose medium is prepared by the following steps: soaking and washing 6% cross-linked agarose with deionized water to remove impurities, adding epichlorohydrin and sodium hydroxide solution, stirring and reacting at 25-35℃ for 2-4 hours to activate the agarose; stirring and reacting the activated 6% cross-linked agarose and the recombinant Protein A solution at 4-8℃ for 12-24 hours for covalent coupling; adding ethanolamine for capping, and washing with deionized water and PBS buffer to obtain the recombinant Protein A ligand modified agarose medium.
4. The purification process of claim 1, wherein, In the step (3), the complex mode medium containing benzamidine functional groups has a particle size of 50-60 μm.
5. The purification process of claim 1, wherein, The complex mode medium containing benzamidine functional groups is benzamidine modified 6% cross-linked agarose, which is prepared by the following steps: soaking and washing 6% cross-linked agarose with deionized water, adding epichlorohydrin and sodium hydroxide solution, reacting at 25-35℃ for 2-4 hours, then washing with a large amount of deionized water until neutral to complete the activation of the agarose; mixing the activated 6% cross-linked agarose and the benzamidine solution, stirring and reacting at 30-37℃ for 6-8 hours to occur covalent coupling reaction; after the coupling reaction is completed, adding ethanolamine to block the unreacted epoxy groups, and then washing with deionized water and PBS buffer to obtain the benzamidine modified 6% cross-linked agarose chromatography medium.
6. The purification process of claim 1, wherein, In the step (4), the virus filtration membrane is made of PES material.
7. A chromatography system specifically for the high performance purification process of the bispecific antibody according to any one of claims 1 to 6, comprising a chromatography column, characterized in that, The chromatography column is filled with improved Protein A medium and mixed mode medium from top to bottom to form a two-section series bed, the two media are separated by a sintered glass plate to ensure one-way flow and prevent medium mixing, the chromatography column also has a buffer inlet and a sample inlet, and the chromatography column is further connected with an ultraviolet detector and a collector in sequence.