A downstream purification process for a monoclonal antibody

Abstract

The application discloses a downstream purification process method of a monoclonal antibody, and the specific steps include: a) deep filtration; b) affinity chromatography; c) low-acid virus inactivation; d) anion chromatography; and e) cation chromatography. The application firstly performs deep filtration on cell culture solution, removes a large amount of cells or cell fragments, host cell proteins (HCPs) and host cell DNA (HCD) quickly, reduces the pressure of subsequent purification chromatography and process analysis, and makes process research and process control more accurate, secondly sets two-step elution in the affinity chromatography to remove a large amount of process impurities such as HCPs and HCD, and finally studies the optimal range of the process through design of experiment (DoE) in the two-step ion chromatography. The application aims to provide a low-cost and more efficient downstream purification process research idea.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and specifically to a downstream purification process for recombinant antibodies. Background Technology

[0002] The background art description is provided only to provide information relevant to the present invention and does not necessarily constitute prior art.

[0003] Antibody drugs mainly include monoclonal antibody drugs, antibody-drug conjugates (ADCs), bispecific antibody drugs, and Fc fusion protein drugs. Among them, monoclonal antibody drugs are widely used in targeted therapy and immunotherapy. Antibodies produced by a single B cell clone that express only a specific antigenic epitope have good specificity and safety. Based on immunogenicity, monoclonal antibody drugs include murine monoclonal antibodies, human-mouse chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.

[0004] According to statistics, nine of the world's top ten best-selling drugs in 2018 were biologics, including seven monoclonal antibody drugs and two fusion protein drugs. These nine biologics generated $76.9 billion in sales, accounting for 88.80% of the total sales of the top ten drugs. With the improvement of human medical standards and increasing demand, the global sales of monoclonal antibody drugs are projected to reach $235.6 billion in 2023 and $328 billion in 2030. However, the domestic monoclonal antibody drug market in China is still in its early stages, and many drugs still rely on imports. Encouragingly, by the end of 2020, a large number of domestically produced biologics had been rapidly included in the national medical insurance catalog. It is estimated that my country's monoclonal antibody drug market will reach $156.5 billion by 2023 and is expected to reach $367.8 billion by 2030.

[0005] The rapid and efficient purification of antibodies from cell culture medium, involving various separation and chromatography techniques, is a pressing technical problem in this field. This invention aims to provide a low-cost and more efficient process research approach.

[0006] Clarification of cell culture medium is the first step in the isolation and purification of monoclonal antibodies. Its purpose is to quickly remove cells and cell debris using simple operations, while improving the efficiency of subsequent chromatographic purification and providing conditions for product process analysis.

[0007] The purpose of affinity chromatography is to specifically capture proteins in cell culture medium. This process requires the simultaneous removal of large amounts of impurities such as HCD (host cell DNA) and HCP (host cell protein). Therefore, two rinsing steps are added before protein elution in this step to rapidly remove large quantities of process impurities. The selection of rinsing conditions is particularly important.

[0008] The target protein was then further purified using ion chromatography, including both anion exchange and cation exchange chromatography, with the DoE design employed to study the optimal process window. Anion exchange chromatography was used to remove acidic substances such as bacterial endotoxins and HCD, while cation exchange chromatography removed aggregates and improved the charge purity of the antibody. Both methods rely on ionic strength interactions to bind and elute the target analyte; therefore, selecting appropriate ionic strength and pH is crucial. Summary of the Invention

[0009] This invention provides a downstream purification process for monoclonal antibodies, which can safely, effectively, and cost-efficiently purify target proteins.

[0010] This disclosure provides a process for downstream purification of monoclonal antibodies, which includes the following steps:

[0011] a) Deep filtration: Deep filtration of the culture medium of cells expressing monoclonal antibodies is performed using a series of deep capsule filters and sterile filters.

[0012] b) Affinity chromatography: Equilibrate the affinity chromatography column with a neutral salt-containing equilibration buffer, load the product obtained in step a) onto the affinity chromatography column, reequilibrate with the equilibration buffer, then elute with two different concentrations of salt-free elution buffer, and finally elute with salt-free elution buffer.

[0013] c) Low acid inactivation of virus: Adjust the pH of the product obtained in step b) to 3.4 ± 0.4 and place at room temperature for 1-4 hours;

[0014] d) Anion exchange chromatography: Neutralize the product obtained in step c) to pH 7.3-7.9 and adjust the conductivity to 2.3-2.8 ms / cm. Load the product onto an anion exchange chromatography column and collect the flow-through peak.

[0015] e) Cation exchange chromatography: Equilibrate the cation exchange chromatography column with a low-salt equilibration buffer, adjust the pH of the product obtained in step d) to 4.6-5.2 and the conductivity to 4.0-5.0 ms / cm, load the product onto the cation exchange chromatography column, reequilibrate with the low-salt equilibration buffer, elute stepwise with two high-salt elution buffers, and combine the elution products to obtain the purified monoclonal antibody.

[0016] In the method disclosed herein, specifically, clarification of the cell culture medium is the primary step in the isolation and purification of monoclonal antibodies, aiming to rapidly remove cells and cell debris using simple operations. This step reduces the burden on subsequent purification chromatography and process analysis, allowing for more precise process research and control. In step a), a deep capsule filter (0.8-0.2 μm) is selected, and a series sterilization filter (0.22 μm) is used for membrane filtration sterilization.

[0017] In step b) of affinity chromatography, the column is first equilibrated with 50 mM Tris-HAc, 150 mM NaCl, pH 7.2, followed by direct deep filtration and sample loading. After sample loading, the column is reequilibrated with 50 mM Tris-HAc, 150 mM NaCl, pH 7.2. Then, a first wash is performed with elution buffer 1: 500 mM Tris-HAc, pH 7.4, followed by a second wash with elution buffer 2: 50-100 mM HAc-NaAc, pH 5.0-5.5. Finally, elution is performed with elution buffer 50-100 mM HAc-NaAc, Gly-HCl, or citrate, pH 3.7.

[0018] Preferably, a second rinsing is performed using rinsing buffer 2: 50mM HAc-NaAc, pH 5.0, and elution is performed using elution buffer 50mM HAc-NaAc, pH 3.7.

[0019] In step c) of low-acid virus inactivation, the product obtained in step b) is adjusted to pH 3.4 ± 0.4 and placed at room temperature for 1-4 hours. Preferably, the obtained affinity chromatography elution collection solution is adjusted to pH 3.4 ± 0.4 with the corresponding acid anion, placed at room temperature (18-26℃) for 1-4 hours, and after low-acid virus inactivation, the solution is neutralized to pH 6.5 ± 0.5 with 1M Tris-HAc buffer, pH 9.0.

[0020] Preferably, the pH is adjusted to 3.7±0.1 using 1M HAc and left to stand at room temperature for 1 hour.

[0021] In the d) anion exchange chromatography operation, flow-through mode is performed. Preferably, 1M Tris-HAc, pH 9.0 is used as the pH adjustment solution, and WFI (water for injection) is used as the diluent. The pH and conductivity of the virus-neutralized low-acid inactivation solution are adjusted to 7.3-7.9, and the conductivity is adjusted to 2.3-2.8 ms / cm. After filtration through a 0.2 μm filter, this solution is used as the loading solution for anion exchange chromatography. The chromatography column is equilibrated with 20mM Tris-HAc, 15.7mM NaCl, pH 7.7. The sample is then loaded with the pH- and conductivity-adjusted solution, with a loading capacity ≤200 mg / ml GEL. The flow-through peak is collected; at this point, the antibody protein is in the flow-through peak, while process-related impurities remain on the chromatography column. Subsequently, the anion exchange chromatography column is regenerated with 20mM Tris-HAc, 1M NaCl, pH 7.7, and then stored at room temperature in 20% ethanol.

[0022] Preferably, 1M Tris-HAc buffer solution is used, pH is adjusted to 7.7±0.2 at pH 9.0, and water for injection is used to adjust the conductivity to 2.6±0.2 ms / cm.

[0023] In this process, e) cation exchange chromatography serves as a purification step to remove trace amounts of process-related impurities. Preferably, 1M HAc is used as the pH adjustment solution and 1M NaCl as the conductivity adjustment solution to adjust the pH and conductivity of the anion exchange chromatography flow-through collection solution, adjusting the pH to 4.6-5.2 and the conductivity to 4.0-5.0 ms / cm, which is then used as the loading solution for cation exchange chromatography. After equilibration of the chromatography column with 25mM NaAc-HAc, 28mM NaCl, pH 4.9, the sample is loaded using the pH- and conductivity-adjusted solution. After loading, the chromatography column is reequilibrated with 25mM NaAc-HAc, 28mM NaCl, pH 4.9. Subsequently, 150-330mM NaAc-HAc, pH 5.5 is used as the first-step elution buffer 1 for collecting the elution fraction 1. Next, using 230-300 mM NaAc-HAc, pH 5.5 as the elution buffer 2 for the second step, the eluted fraction 2 was collected. Finally, the column was regenerated with 20 mM Tris-HAc, 1 M NaCl, pH 7.7, and stored at room temperature in 20% ethanol.

[0024] Preferably, the pH is adjusted to 4.9±0.2 using 1M HAc, the conductivity is adjusted to 4.5±0.4 ms / cm using 1M NaCl, the first elution is performed using elution buffer 1: 250-330mM NaAc-HAc, pH 5.5, and the second elution is performed using elution buffer 2: 260-300mM NaAc-HAc, pH 5.5.

[0025] More preferably, the first elution step is performed using elution buffer 1: 200mM NaAc-HAc, pH 5.5, and the second elution step is performed using elution buffer 2: 300mM NaAc-HAc, pH 5.5.

[0026] The present invention has the following benefits and effects:

[0027] 1. First, the culture medium is subjected to deep filtration. This step can quickly remove a large number of cellular impurities, reducing the pressure on subsequent purification chromatography and process analysis.

[0028] 2. Use two buffer solutions of high and low concentrations for rinsing in affinity chromatography. This step can quickly remove large amounts of HCP and HCD from the feed solution.

[0029] 3. Flow-through mode is used in anion exchange chromatography, a step that can remove a large amount of acidic substances, such as bacterial endotoxins, viruses, and nucleic acids;

[0030] 4. In cation chromatography, linear elution with high salt or a combination of several high-salt solutions can be used to obtain better purity and yield, and can effectively reduce the surface charge heterogeneity of the target product. Attached Figure Description

[0031] Figure 1 This is a spectrum of pH linear gradient elution in Example 2 of the present invention.

[0032] Figure 2 This illustrates the effect of different feed solution pH and conductivity on HCP removal in Example 4 of the present invention.

[0033] Figure 3 This demonstrates the effect of different feed solution pH and protein loading amount on HCP removal in Example 4 of the present invention.

[0034] Figure 4 This demonstrates the effect of different feed conductivity and protein loading amount on HCP removal in Example 4 of the present invention.

[0035] Figures 5A-5F This is a Sweet Spot analysis diagram in Embodiment 4 of the present invention, using HCP and Protein A as response values.

[0036] in Figure 5A The electrical conductivity is 2.3 mS / cm; Figure 5B The electrical conductivity is 2.4 mS / cm; Figure 5C The electrical conductivity is 2.5 mS / cm; Figure 5D The electrical conductivity is 2.6 mS / cm; Figure 5E The electrical conductivity is 2.7 mS / cm; Figure 5F The conductivity is 2.8 mS / cm.

[0037] Figure 6 This is a contour plot of pH value and conductivity versus the dynamic loading of NanoGel 50SP in Example 5 of the present invention. Detailed Implementation

[0038] Under normal physiological conditions, CLDN18.2 is expressed only in differentiated epithelial cells of the gastric mucosa, and not in other healthy tissues. However, CLDN18.2 is highly expressed in various tumors such as gastric cancer, esophageal cancer, and pancreatic cancer. The occurrence of malignant tumors leads to the disruption of tight junctions, exposing the CLDN18.2 epitope on the surface of tumor cells, making it a specific target.

[0039] The detailed technical content of this invention will now be elaborated with an example (anti-CLDN18.2 monoclonal antibody). Optimal process windows were studied by designing experiments (DoE) to optimize process parameters, considering response values ​​such as molecular purity SEC (size exclusion chromatography), molecular purity CE-SDS (capillary electrophoresis), charge purity CEX (cation exchange chromatography), process impurities (host cell DNA HCD), host cell protein HCP, and affinity chromatography ligand Protein A (where HCD is obtained through quantitative polymerase chain reaction qPCR, and HCP and Protein A are obtained through enzyme-linked immunosorbent assay).

[0040] The present invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention. Test methods in the embodiments of the present invention that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0041] Example 1. Deep Filtration

[0042] Clarification of cell culture medium is the first step in monoclonal antibody isolation and purification, aiming to quickly remove cells and cell debris using simple operations. This step reduces the burden on subsequent purification chromatography and process analysis, allowing for more precise process research and control. In this step, a deep capsule filter (0.8-0.2 μm) and a series sterilization filter (0.22 μm) were selected for membrane filtration for sterilization. The results are shown in Table 1; turbidity decreased significantly before and after filtration, protein adsorption was low, and recovery rate was high.

[0043] Table 1. Deep filtration test results

[0044] 117.3 ＞1000 ＜10 94.4

[0045] Example 2. Affinity Chromatography

[0046] Based on the screening results in Example 2, the preferred specific process steps for affinity chromatography studies are as follows: AT Protein A Diamond affinity packing material (BorgLone) was selected. After first equilibrating the column with 50 mM Tris-HAc, 150 mM NaCl, pH 7.2, the sample was directly loaded after deep filtration. After loading, the column was reequilibrated with 50 mM Tris-HAc, 150 mM NaCl, pH 7.2. Then, a first elution was performed with 500 mM Tris-HAc, pH 7.4, followed by a second elution with 50 mM HAc-NaAc, pH 5.0. Finally, elution was performed with 50 mM HAc-NaAc, pH 3.7.

[0047] 1) Screening of eluent

[0048] In affinity chromatography without a rinsing step, the eluent pH was 4.23 and the conductivity was 2.354 mS / cm. However, when the eluent obtained without a rinsing step was neutralized to pH 5.28 using 1M Tris-HCl (pH 9.0), significant turbidity appeared in the eluent, presumably due to host cell proteins (HCPs). To confirm this hypothesis, a full-load rinsing process was added to remove a large amount of HCPs, thereby reducing the turbidity of the affinity chromatography eluent during neutralization. The results showed that no turbidity occurred after adding two rinsing steps (results of rinsing buffer 1 are shown in Table 2, and rinsing buffer 2 are shown in Table 3). Therefore, it is believed that the turbidity was indeed caused by a large amount of HCPs, and adding a rinsing step can effectively remove HCPs.

[0049] First, eight elution buffers were screened, as detailed in Table 2.

[0050] Table 2. Screening and detection results of affinity chromatography elution buffer 1

[0051] 1 50mM NaAc-HAc, 500mM imidazole, pH 5.5 84.8 98.4 210.0 2 50mM NaAc-HAc, 800mM imidazole, pH 5.5 81.0 98.3 212.7 3 50mM Tris-HCl, 1M NaCl, 10mM EDTA, pH7.4 86.6 98.3 629.0 4 0.2M Na citrate, 1% Tween 20, pH 5.0 83.2 98.6 249.9 5 0.2M Na citrate, 10% PEG1000, pH 5.0 89.8 98.7 672.2 6 50mM Tris-HCl, 1M NaCl, pH7.4 86.3 98.4 575.6 7 50mM Tris-HAc, 150mM NaCl, pH7.2 90.9 98.6 518.1 8 500mM Tris-HAc, pH 7.4 93.1 98.4 1041.5

[0052] As shown in Table 2, the SEC purity of the eight eluents was comparable, and the HCP was within acceptable limits. Based on the yield results, buffer 8, which had the highest yield, was selected as eluent 1 for subsequent experiments.

[0053] After rinsing with 500mM Tris-HAc at pH 7.4, HCP remained at a high level. To further remove HCP, four rinsing buffers were selected (see Table 3 for details).

[0054] Table 3. Screening results of affinity chromatography elution buffer 2

[0055] 1 100mM NaAc-HAc, pH 5.5 97.7 98.2 323.1 2 100mM NaAc-HAc, pH 5.0 97.5 98.2 286.1 3 50mM NaAc-HAc, pH 5.0 93.2 98.3 234.9 4 50mM NaAc-HAc, pH 5.5 94.6 97.9 253.0

[0056] As shown in Table 3, the four elution buffers 2 were comparable in terms of yield, SEC removal, and HCP removal. Based on the HCP results, 50 mM NaAc-HAc with pH 5.0, which had a higher HCP removal capacity, was selected as the elution buffer 2 for subsequent experiments.

[0057] 2) Elution buffer

[0058] First, the pH of the elution buffer was screened. AT Protein A Diamond affinity chromatography column (Borglon) was selected. The column was first equilibrated with 50 mM Tris-HAC, 150 mM NaCl, pH 7.2, followed by deep filtration before loading the sample. After loading, the column was reequilibrated with 50 mM Tris-HAC, 150 mM NaCl, pH 7.2, then eluted with 50 mM NaAc-HAc, 500 mM imidazole, pH 5.5, followed by equilibration with 50 mM citrate, pH 6.0. Finally, a gradient elution (0-100%) was performed using 50 mM citrate, pH 2.4. See the chromatogram for details. Figure 1 .

[0059] Depend on Figure 1 It can be seen that during the linear pH gradient elution process, the target protein started eluting at pH 4.2 and continued until pH 3.7, indicating that the target protein was basically completely eluted. Thus, pH 3.7 was determined to be the preferred pH value for the elution buffer.

[0060] Next, the elution buffer salts were screened. Six elution buffers, consisting of two concentrations each from three systems (citric acid, glycine, and acetic acid), were selected for screening and testing, as detailed in Table 4. The results shown in Table 4 indicate that these three buffers were comparable in terms of yield, purity, Protein A residue, and HCP removal.

[0061] Table 4. Screening results of affinity chromatography elution buffer.

[0062]

[0063] To facilitate the subsequent anion exchange chromatography, we further investigated the volume and conductivity of the elution buffers after elution and neutralization for these three types of elution buffers. The results are shown in Table 5. The results indicate that when the pH of the elution collection buffer is neutralized to around 7.1 (the loading pH for anion exchange chromatography is typically above 7), these three types of elution buffers exhibit certain differences. The glycine-hydrochloric acid buffer had the largest elution volume, the lowest neutralization conductivity, and the largest elution volume-to-column volume ratio. The citrate buffer had the smallest elution volume, the highest neutralization conductivity, and the smallest elution volume-to-column volume ratio. The acetate-sodium acetate buffer had a moderate elution volume, moderate neutralization conductivity, and a moderate elution volume-to-column volume ratio. Based on these results, 50 mM NaAc-HAc at pH 3.7 was selected as the elution buffer for subsequent experiments.

[0064] Table 5 Comparison of volume and conductivity results after elution and neutralization of different types of elution buffers.

[0065]

[0066] Example 3. Low-acid inactivation of virus

[0067] Based on the screening results in Example 3, the preferred specific process steps for the low-acid inactivated virus research experiment are as follows: The obtained affinity chromatography elution collection solution is adjusted to pH 3.7±0.1 with 1M HAc and incubated at room temperature (18-26℃) for 1 hour. After low-acid virus inactivation, the solution is neutralized to pH 6.5±0.5 with 1M Tris-HAc buffer, pH 9.0.

[0068] Process screening: The affinity chromatography eluent was placed at different pH values ​​for different times, and its purity changes (SEC, CE-SDS, R) were observed. The results are detailed in Table 6.

[0069] Table 6. Screening and detection results of low-acid inactivated virus

[0070]

[0071]

[0072] As shown in Table 6, there was no significant difference in results when the virus was incubated at room temperature for 1–4 hours at pH 3.4 ± 0.4. Since prolonged low-acid treatment may lead to an increase in aggregates or cause proteins to aggregate, a low-acid inactivation condition of pH 3.7 ± 0.1 for 1 hour was selected for subsequent experiments.

[0073] Example 4. Anion Exchange Chromatography

[0074] Based on the screening results in Example 4, the specific process steps for the anion exchange chromatography study are as follows: UniGel-80Q chromatography packing material (Suzhou Nanomicro) was selected, and the anion exchange chromatography operation was performed in flow-through mode. Using 1M Tris-HAc at pH 9.0 as the pH adjustment solution and WFI (water for injection) as the diluent, the pH and conductivity of the virus-neutralized solution were adjusted to 7.7±0.2 and 2.6±0.2 ms / cm. After 0.2 μm filtration, this solution was used as the loading solution for anion exchange chromatography. The chromatography column was equilibrated with 20mM Tris-HAc and 15.7mM NaCl at pH 7.7. The sample was then loaded using the pH- and conductivity-adjusted solution. The flow-through peak was collected; at this point, the antibody protein was in the flow-through peak, while process-related impurities remained on the chromatography column. The anion exchange chromatography column was then regenerated with 20 mM Tris-HAc, 1 M NaCl, and pH 7.7, and then stored at room temperature in 20% ethanol.

[0075] 1) Flow-through condition screening

[0076] HCP, a difficult-to-remove impurity in the process, was first subjected to factor experiments (pH, conductivity, dynamic loading), and a response surface was established with HCP as the response. The results are detailed in Table 7 below. Figure 2-4 .

[0077] Table 7 Results of screening and testing of flow-through conditions for anion exchange chromatography

[0078]

[0079] Depend on Figure 2-4 It can be seen that HCP removal is favored under conditions of lower pH (≥7) and lower conductivity; HCP removal is favored under conditions of lower pH (≥7) and lower protein load; and HCP removal is favored under conditions of lower conductivity and lower protein load.

[0080] To further investigate the ability of anion exchange chromatography to simultaneously remove residual HCP and Protein A proteins, the Sweet Spot function in DoE was used for analysis, with acceptable values ​​for HCP and Protein A set at 90 ppm and 1.5 ppm, respectively. Figures 5A-5F The results showed that, under a loading of 200 mg / ml GEL, the optimal pH range was 7.3–7.9 as the conductivity increased from 2.3 ms / cm to 2.8 ms / cm.

[0081] To increase the robustness of the process, the operating margins for pH were set at 7.7±0.2, for conductivity at 2.6±0.2 ms / cm, and for loading at ≤200 mg / ml GEL for subsequent experiments.

[0082] Example 5. Cation exchange chromatography

[0083] Based on the screening and detection results in Example 5, the preferred specific process steps for the anion exchange chromatography study are as follows: Cation exchange chromatography is used as a purification step to remove trace process-related impurities. NanoGel 50SP chromatography packing material (Suzhou Nanomicro) is selected. 1M HAc is used as the pH adjustment solution, and 1M NaCl is used as the conductivity adjustment solution. The pH and conductivity of the anion exchange chromatography flow-through collection solution are adjusted to 4.9±0.2 and 4.5±0.4 ms / cm, respectively, and this is used as the loading solution for cation exchange chromatography. After equilibration of the chromatography column with 25mM NaAc-HAc, 28mM NaCl, pH 4.9, the sample is loaded using the pH- and conductivity-adjusted solution. After loading, the chromatography column is reequilibrated with 25mM NaAc-HAc, 28mM NaCl, pH 4.9. Next, elution fraction 1 was collected using 200 mM NaAc-HAc, pH 5.5 as elution buffer 1 for the first step. Then, elution fraction 2 was collected using 300 mM NaAc-HAc, pH 5.5 as elution buffer 2 for the second step. Fractions 1 and 2 were combined, sterilized, and filtered to prepare the cation exchange eluent. Finally, the column was regenerated using 20 mM Tris-HAc, 1 M NaCl, pH 7.7, and stored at room temperature in 20% ethanol.

[0084] 1) Screening of sample loading conditions

[0085] Since both the pH and conductivity of the sample solution can affect the dynamic loading of antibody proteins onto the column stock, a DoE (Domain of Effect) design was used to construct a response surface using a two-factor (pH, conductivity) two-level three-center assay. The high and low pH levels were pH 4.4, pH 5.4 and 4, 16 ms / cm, respectively, with dynamic loading as the response. The results are detailed in Table 8. Figure 6 .

[0086] Table 8 Experimental Design and Results of Sample Loading Conditions in Cation Exchange Chromatography

[0087] 1 4 4.4 91.62 2 16 4.4 90.7 3 4 5.4 102.24 4 16 5.4 17.66 5 4 4.9 106.79 6 16 4.9 58.08 7 10 4.4 103.25 8 10 5.4 62.61 9 10 4.9 88.51 10 10 4.9 88.46 11 10 4.9 88.17

[0088] Depend on Figure 6 It can be seen that when the conductivity is between 4.0 and 5.0 ms / cm and the pH fluctuates between 4.6 and 5.2, the dynamic loading of the packing material remains at its highest level (100 mg / mL Gel). Therefore, the process operating range for the pH of the sample solution and the equilibration solution was set to 4.9 ± 0.2, and the operating range for conductivity was set to 4.5 ± 0.4 ms / cm for subsequent experiments.

[0089] 2) Screening of elution conditions

[0090] Six NaAc-HAc fractions were linearly selected and eluted with pH 5.5 buffer (150 mM-330 mM). The yield and purity of SEC were observed, and the results are detailed in Table 9.

[0091] Table 9. Screening and detection results of cation exchange chromatography elution buffer 1

[0092] 150mM NaAc-HAc, pH 5.5 27.5 99.4 200mM NaAc-HAc, pH 5.5 62.8 98.9 250mM NaAc-HAc, pH 5.5 89.1 96.9 280mM NaAc-HAc, pH 5.5 90.1 95.7 300mM NaAc-HAc, pH 5.5 92.6 94.5 330mM NaAc-HAc, pH 5.5 113.0 91.9

[0093] As shown in Table 9, the yield of eluted products gradually increased with the increase of NaAc-HAc buffer concentration, but the purity of SEC decreased. Therefore, 200mM NaAc-HAc with a purity of over 98% and pH 5.5 was selected as elution buffer 1 for subsequent experiments.

[0094] To further improve the yield, after the first elution step, a second elution buffer was selected under full-load conditions.

[0095] Table 10 Screening and detection results of cation exchange chromatography elution buffer 2

[0096]

[0097] As shown in Table 10, the yield of the eluted product gradually increased with increasing NaAc-HAc buffer concentration, reaching a peak at 280 mM NaAc-HAc, pH 5.5. SEC purity showed a gradual decreasing trend, but remained at an acceptable level at 300 mM NaAc-HAc, pH 5.5. Taking all factors into consideration, 300 mM NaAc-HAc, pH 5.5 was selected as the elution buffer 2 for subsequent experiments.

[0098] Example 6. Three consecutive batches of pilot production

[0099] Using the preferred scheme determined in the above embodiments, three consecutive 200L pilot-scale production batches were conducted. The product yield, process impurities, and product impurities were tested, and the results are detailed in Table 11. As shown in Table 11, the yields of the three batches were consistently between 75.6% and 81.7%. Process impurities HCP, DNA, and Protein A all met the requirements. Product-related impurities, such as reduced CE-SDS and SEC, had purities above 99%, and non-reduced CE-SDS had purities above 95%. The charge purity CEX peak was between 67.3% and 70.8%, and the acidic peak was between 24.9% and 28.2%, demonstrating good batch-to-batch consistency and comparable processes. Therefore, the downstream purification process of this invention can produce antibody proteins that meet regulatory requirements, exhibiting excellent performance in terms of purity, yield, and impurity removal.

[0100] Table 11 Results of Three Consecutive Batches of Pilot Production

[0101]

Claims

1. A downstream purification process for monoclonal antibodies, characterized in that, Includes the following steps: a) Deep filtration: Deep filtration of the culture medium of cells expressing monoclonal antibodies is performed using a series of deep capsule filters and sterile filters. b) Affinity chromatography: The affinity chromatography column is equilibrated with a neutral salt-containing equilibration buffer. The product obtained in step a) is loaded onto the affinity chromatography column, reequilibrated with the equilibration buffer, and then eluted sequentially with elution buffer 1 and elution buffer 2. Finally, elution is performed with elution buffer. Elution buffer 1 is 500 mM Tris-HAc, pH 7.4; elution buffer 2 is 50 mM NaAc-HAc, pH 5.0; and elution buffer is 50 mM NaAc-HAc, pH 3.

7. The packing material of the affinity chromatography column is Protein A affinity packing material. c) Low acid inactivation of virus: Adjust the pH of the product obtained in step b) to 3.4 ± 0.4 and place at room temperature for 1-4 hours; d) Anion exchange chromatography: Neutralize the product obtained in step c) to pH 7.3-7.9 and adjust the conductivity to 2.3-2.8 ms / cm. Load the sample onto anion exchange chromatography column and collect the flow-through peak; and e) Cation exchange chromatography: Equilibrate the cation exchange chromatography column using a low-salt equilibration buffer. Adjust the pH of the product obtained in step d) to 4.6-5.2 and the conductivity to 4.0-5.0 ms / cm. Load the product onto the cation exchange chromatography column and reequilibrate using the low-salt equilibration buffer. Elute in steps using two high-salt elution buffers and combine the elution products to obtain the purified monoclonal antibody. The low-salt equilibration buffer is 25 mM NaAc-HAc, 28 mM NaCl, pH 4.

9. The two high-salt elution buffers are elution buffer 1 and elution buffer 2, respectively. Elution buffer 1 is 200 mM NaAc-HAc, pH 5.5, and elution buffer 2 is 300 mM NaAc-HAc, pH 5.

5.

2. The method according to claim 1, characterized in that, The pore size of the deep capsule filter used in step a) is 0.2-0.8 µm, and / or the pore size of the sterilizing filter is 0.22 µm.

3. The method according to claim 1 or 2, characterized in that, The equilibration buffer used in step b) is 50 mM Tris-HAc, 150 mM NaCl, pH 7.

2.

4. The method according to claim 1 or 2, characterized in that, In step c), the product obtained in step b) is adjusted to pH 3.7 ± 0.1 and left to stand at room temperature for 1 hour.

5. The method according to claim 4, characterized in that, The room temperature is 18-26°C.

6. The method according to claim 4, characterized in that, In step c), the pH of the product obtained in step b) is adjusted using 1M HAc.

7. The method according to claim 1 or 2, characterized in that, After inactivating the virus with low acid in step c), the pH is neutralized to pH 6.5 ± 0.

5.

8. The method according to claim 7, characterized in that, After inactivating the virus with low acid in step c), the pH was neutralized to pH 6.5 ± 0.5 using 1M Tris-HAc at pH 9.

0.

9. The method according to claim 1 or 2, characterized in that, In step d), the product obtained in step c) is neutralized to pH 7.7 ± 0.2 and the conductivity is adjusted to 2.6 ± 0.2 ms / cm.

10. The method according to claim 9, characterized in that, In step d), the pH is adjusted using 1M Tris-HAc, pH 9.0, and / or the conductivity is adjusted using water for injection.

11. The method according to claim 9, characterized in that, In step d), the loading amount is ≤200mg / ml GEL.

12. The method according to claim 9, characterized in that, In step d), before loading the sample, the anion exchange chromatography column is equilibrated with 20 mM Tris-HAc, 15.7 mM NaCl, and pH 7.7 equilibration buffer.

13. The method according to claim 1 or 2, characterized in that, In step e), the product obtained in step d) is adjusted to pH 4.9 ± 0.2 and the conductivity is adjusted to 4.5 ± 0.4 ms / cm.

14. The method according to claim 13, characterized in that, In step e), the pH is adjusted using 1M HAc, and / or the conductivity is adjusted using 1M NaCl.

15. The method according to claim 1 or 2, characterized in that, Before loading the sample in step d), and / or before loading the sample in step e), and / or after combining the elution products in step e), a sterile filtration step is performed.

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