Method for detecting antibody purity in antibody expression cell fermentation broth

Abstract

The invention provides a method for detecting the purity of an antibody in an antibody expression cell fermentation broth. The detection method comprises the following steps: S1) pre-treating a sample to be detected to obtain a pre-treated product; s2) detecting the pretreatment product by using size exclusion chromatography high performance liquid chromatography to obtain the purity of the antibody in the fermentation liquor; wherein the pretreatment comprises the following steps: adjusting the pH value of a to-be-detected sample to 6.5-7.5, and adding sodium chloride into the to-be-detected sample until the concentration of sodium chloride in the to-be-detected sample is 100-500mM; the detection comprises the following steps: loading the pre-treated product to a chromatographic column of a size exclusion chromatography high performance liquid chromatography, and eluting; the elution comprises acid treatment, first equilibrium treatment, alkali treatment and second equilibrium treatment.

Description

Technical Field

[0001] The present invention relates to the field of antibody detection, and in particular to a method for detecting the purity of antibodies in a fermentation broth of antibody-expressing cells. Background Art

[0002] As a key therapeutic protein, monoclonal antibodies play an important role in the treatment of various diseases such as tumors, inflammation and autoimmune diseases. They are usually cultured in mammalian cells, especially Chinese hamster ovary cells (CHO). However, the production process of antibodies is affected by many conditions, including temperature, pH, ionic strength, shear force and oxygen supply, which may cause antibodies to form aggregates and fragments. Since the monomer purity of antibodies is crucial to drug efficacy and safety, the purity quality of antibodies must be considered in therapeutic applications.

[0003] In downstream process development, aggregates and fragments in antibodies can be removed through a variety of purification techniques. At present, the commonly used method is to detect samples pretreated with affinity chromatography by size exclusion high performance liquid chromatography (SEC-HPLC) to evaluate the initial purity of antibodies in the fermentation broth. However, this method has limitations because affinity chromatography specifically captures antibodies, resulting in a measured product purity that is higher than the actual initial purity, making it impossible to accurately assess the true quality of antibodies in the fermentation broth. This may lead to the erroneous selection of poor quality monoclonal antibodies in the monoclonal antibody screening stage, resulting in more yield losses in the subsequent purification process.

[0004] Therefore, the prior art lacks a method that can accurately determine the purity of antibodies in fermentation broth, which makes it difficult to screen out high-quality monoclonal antibodies. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for detecting the purity of antibodies in the fermentation broth of antibody-expressing cells, so as to solve the problem of low accuracy of the methods for detecting the antibody concentration in the fermentation broth in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a method for detecting the purity of an antibody in an antibody-expressing cell fermentation broth is provided, the detection method comprising: S1) pre-treating a sample to be tested to obtain a pre-treatment product; S2) detecting the pre-treatment product using size exclusion chromatography high performance liquid chromatography to obtain the purity of the antibody in the fermentation broth; wherein the pre-treatment comprises adjusting the pH of the sample to be tested to 6.5-7.5, and adding sodium chloride to the sample to be tested until the concentration of sodium chloride in the sample to be tested is 100-500 mM; the detection comprises: loading the pre-treatment product onto a chromatographic column of size exclusion chromatography high performance liquid chromatography, and eluting; the elution comprises: acid treatment, a first equilibrium treatment, an alkali treatment, and a second equilibrium treatment.

[0007] Further, S1) includes: obtaining a first pretreatment product after centrifuging and filtering the sample to be tested; adjusting the pH of the first pretreatment product to 6.5-7.5, and adding sodium chloride to the first pretreatment product until the concentration of sodium chloride in the first pretreatment product is 100-500mM, thereby obtaining a pretreatment product.

[0008] Furthermore, the mobile phase A used in the size exclusion chromatography HPLC was 100 mM PB and 250 mM NaCl, with a pH value of 6.5-7.2; and the mobile phase B was 100% isopropanol.

[0009] Furthermore, the volume ratio of mobile phase A to mobile phase B is 85-95:5-15; preferably, the loading amount of size exclusion chromatography HPLC is 50-100 μg.

[0010] Furthermore, the acid treatment includes: performing a first elution on the chromatographic column using an acid solution, the flow rate of the acid solution in the chromatographic column is 0.05 to 0.5 mL / min, and the time of the first elution is 0.5 to 2 hours.

[0011] Furthermore, the acid in the acid solution includes one or more of acetic acid and / or citric acid; the concentration of the acid in the acid solution is 100-200 mM.

[0012] Furthermore, the first equilibrium treatment includes performing a second elution on the chromatographic column using mobile phase A; the flow rate of mobile phase A in the chromatographic column is 0.2 to 0.5 mL / min, and the time of the second elution is 0.5 to 2 hours.

[0013] Furthermore, the alkaline treatment includes performing a third elution on the chromatographic column using an alkaline solution, and then performing a second equilibrium treatment after the chromatographic column is allowed to stand for 0.5 to 2 hours; the flow rate of the alkaline solution in the chromatographic column is 0.05 to 0.5 mL / min, and the time for the third elution is 0.5 to 2 hours.

[0014] Furthermore, the alkali in the alkali solution includes sodium hydroxide; and the concentration of the alkali in the alkali solution is 0.01-0.1M.

[0015] Furthermore, the second equilibrium treatment includes performing a fourth elution on the chromatographic column using mobile phase A, the flow rate of mobile phase A in the chromatographic column is 0.2 to 0.5 mL / min, and the time of the fourth elution is 0.5 to 2 hours.

[0016] By applying the technical solution of the present invention, the sample to be tested (fermentation broth) is subjected to the above-mentioned pretreatment to obtain a pretreatment product with a pH of 6.5-7.5 and a sodium chloride concentration of 100-500 mM. The pretreatment product is loaded into a size exclusion chromatography high performance liquid chromatography, and then acid treatment, a first equilibrium treatment, an alkali treatment and a second equilibrium treatment are sequentially performed to detect the concentration of the monoclonal antibody. Compared with the prior art, the detection method of the present application can more accurately reflect the concentration of the monoclonal antibody in the fermentation broth, improve the accuracy of the screening, and avoid the consequences of reduced yield due to screening errors. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 The SEC-HPLC mass spectrum of the detection result according to Example 1 of the specification of the present invention is shown.

[0019] Figure 2 The SEC-HPLC mass spectrum of the detection result of Comparative Example 3 according to the specification of the present invention is shown. DETAILED DESCRIPTION

[0020] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0021] Terminology explanation:

[0022] Chromatographic resolution: It is the main indicator for quantitatively describing the separation of two adjacent components in a mixture in a chromatographic column. Chromatographic resolution is equal to the ratio of the difference between the chromatographic retention values ​​of two adjacent components to half of the sum of the baseline widths of the chromatographic peaks of the two components.

[0023] As mentioned in the background technology, the fermentation broth treated with affinity chromatography will be specifically captured. At this time, the quality of the product will be higher than its actual initial quality. There is no way to objectively judge the quality of the antibodies in the fermentation broth. In clone screening, clones with poor quality will be mistakenly selected, which will result in a greater loss of yield in the subsequent purification process. Therefore, in this application, the inventors attempt to develop a new method for detecting the purity of antibodies in fermentation broth, which can accurately detect the concentration of monoclonal antibodies in fermentation broth compared to the prior art. Based on this, a series of protection schemes of this application are proposed.

[0024] In a first typical embodiment of the present application, a method for detecting the purity of an antibody in an antibody-expressing cell fermentation broth is provided, the method comprising:

[0025] S1) pre-treating the sample to be tested to obtain a pre-treatment product; S2) detecting the pre-treatment product using size exclusion chromatography high performance liquid chromatography to obtain the purity of the antibody in the fermentation broth; wherein the pre-treatment includes adjusting the pH of the sample to be tested to 6.5-7.5, and adding sodium chloride to the sample to be tested until the concentration of sodium chloride in the sample to be tested is 100-500mM; the detection includes: loading the pre-treatment product onto a chromatographic column of size exclusion chromatography high performance liquid chromatography, and eluting; the elution includes: acid treatment, a first equilibrium treatment, an alkali treatment, and a second equilibrium treatment.

[0026] The fermentation broth of monoclonal antibodies contains a variety of components, including soluble and insoluble substances. When the fermentation broth sample is directly used for SEC-HPLC detection, the chromatographic column may be blocked. In addition, the insoluble components will affect the effect and life of subsequent chromatographic separation, while complex components such as cell debris, proteins and cell metabolites will interfere with chromatographic analysis and affect accuracy. Therefore, in the prior art, the fermentation broth is not directly passed through SEC-HPLC for monoclonal antibody detection.

[0027] Due to the above problems, in the prior art, the fermentation broth is usually subjected to affinity chromatography to remove insoluble substances and other impurities that affect the detection in the fermentation broth, and to specifically capture the antibodies to reduce the interference of other components, so as to improve the accuracy of subsequent purity detection and the reliability of evaluation. Affinity chromatography, as the first purification step, can significantly improve the efficiency and effect of subsequent SEC-HPLC purification and reduce unnecessary sample loss.

[0028] The pH conditions during affinity chromatography elution are generally low (usually 3.0-3.7), and low pH conditions can promote the formation of more antibody aggregates. However, due to the different binding forces between aggregates and monomers and the filler, monomers and aggregates will separate. After affinity purification and specific capture, the antibody sample purity is higher, more monomers are polymerized, the aggregate content increases, and the quality of the product is better than the original quality. Therefore, the test results of the samples after affinity chromatography cannot truly represent the quality of the antibody in the fermentation broth, resulting in the measured product purity being higher than the actual initial purity, and thus unable to accurately evaluate the true quality of the antibody in the fermentation broth, resulting in erroneous screening, and in the subsequent antibody production and purification process, and then in the subsequent purification process, causing more yield losses.

[0029] In the present application, the sample to be tested (fermentation broth) is pre-treated and the sample elution step is added to achieve the purpose of directly detecting the purity of the antibody in the fermentation broth by SEC-HPLC. Moreover, the detection method of the present application is used to detect the above-mentioned sample to be tested. Compared with the prior art, the detection result is more accurate and can objectively reflect the purity of the antibody in the fermentation broth, further improving the accuracy of the monoclonal antibody screening stage, and avoiding the adverse effects of low-quality monoclonal antibodies mistakenly screened out in the subsequent production and purification process on the product yield.

[0030] During the pretreatment process of the present application, the pH of the sample to be tested is adjusted to 6.5-7.5 (6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5) to make the sample to be tested more stable in this pH range, reduce secondary interactions, and prevent separation, thereby affecting the subsequent column process; and eliminate the interference and influence of pH on the subsequent SEC-HPLC experimental results, thereby improving the accuracy of the detection results.

[0031] The purpose of adjusting the salt concentration of the sample to be tested in the present application is to reduce the interaction and aggregation of the antibody and the filler, maintain the stable state of the antibody, which is beneficial to improve the separation effect of the subsequent SEC-HPLC, increase the resolution of the peaks between the monomer and the dimer, and help improve the detection accuracy of the subsequent SEC-HPLC.

[0032] Furthermore, by adding a post-processing step to SEC-HPLC, the present application can enable the normal operation of SEC-HPLC, avoiding the phenomenon of overpressure, reduced resolution, and reduced detectability after an increase in the number of test samples. This is helpful for the stability, repeatability, and accuracy of the detection system of the present application, and is conducive to screening out truly high-quality monoclonal antibodies, and promoting the research and development process of drugs for the treatment of tumors, inflammation, and autoimmune diseases.

[0033] In a preferred embodiment, the antibody-expressing cells in the above-mentioned antibody-expressing cell fermentation broth include cells of a mammalian cell expression system; preferably, the cells of the mammalian cell expression system include but are not limited to CHO cells and 293 cells.

[0034] In a preferred embodiment, S1) includes: centrifuging and filtering the sample to be tested to obtain a first pretreatment product; adjusting the pH of the first pretreatment product to 6.5-7.5, and adding sodium chloride to the first pretreatment product until the concentration of sodium chloride in the first pretreatment product is 100-500mM to obtain a pretreatment product.

[0035] In a preferred embodiment, the mobile phase A used in size exclusion chromatography HPLC is 100mM PB and 250mM NaCl, with a pH value of 6.5-7.2; the mobile phase B is 100% isopropanol; preferably, the volume ratio of mobile phase A to mobile phase B is 85-95:5-15.

[0036] In a preferred embodiment, the acid treatment comprises: performing a first elution on the chromatographic column using an acid solution, the flow rate of the acid solution in the chromatographic column is 0.05 to 0.5 mL / min, more preferably 0.1 to 0.2 mL / min, and the time of the first elution is 0.5 to 2 hours.

[0037] In a preferred embodiment, the acid in the acid solution includes acetic acid and / or citric acid; the concentration of the acid in the acid solution is 100-200 mM.

[0038] The effect of acid treatment is to elute the alkaline impurities in the articles to be tested. If the flow rate of acid in the process of acid treatment is too fast, it will cause the chromatogram to appear overpressure phenomenon (referring to the situation that the pressure suddenly rises and exceeds the normal working pressure in the chromatographic system), and the effect of removing impurities is poor, affecting the accuracy of the final result. But the flow rate is too slow to cause the process flow to be lengthened, and the filler is in the acidic condition for a long time, which will affect the life of the filler in the chromatogram. The flow rate, time and concentration of the acid solution of acid treatment are controlled in the above range by the present application, the overall effect of acid treatment is improved, and the effect of subsequent alkali treatment is not affected, when the number of sample treatment is many, the stable operation of SEC-HPLC instrument can still be guaranteed, the consequence of the detection result accuracy decline caused by the decrease in chromatographic resolution and the increase in column pressure alarm is avoided, which is conducive to improving the accuracy of the final detection result.

[0039] In a preferred embodiment, the first equilibration treatment includes performing a second elution on the chromatographic column using mobile phase A; the flow rate of mobile phase A in the chromatographic column is 0.2 to 0.5 mL / min, and the time of the second elution is 0.5 to 2 hours.

[0040] In a preferred embodiment, the alkaline treatment includes performing a third elution on the chromatographic column using an alkaline solution, and then performing a second equilibrium treatment after the chromatographic column is allowed to stand for 0.5 to 2 hours; the flow rate of the alkaline solution in the chromatographic column is 0.05 to 0.5 mL / min, more preferably 0.1 to 0.2 mL / min, and the time for the third elution is 0.5 to 2 hours.

[0041] In a preferred embodiment, the alkali in the alkaline solution includes one or more of sodium hydroxide; the concentration of the alkali in the alkaline solution is 0.01-0.1M.

[0042] The role of alkali treatment is to remove other impurities such as acidic impurities, cells and acidic substances remaining from acid treatment attached to the filler. The flow rate of the alkaline solution in the present application during the alkali treatment is to extend the contact time between the alkali and the chromatographic column, and further achieve the purpose of removing impurities. The flow rate, elution time and concentration of the alkaline solution in the alkali treatment are controlled within the above range to avoid the chromatographic column being in alkaline conditions for a long time, which affects the life of the chromatographic column filler, and the consequences of increased column pressure and reduced chromatographic resolution. The effect of alkali treatment can also be further improved, which is conducive to improving the accuracy of the final test results. In a preferred embodiment, the second equilibrium treatment includes using mobile phase A to perform a fourth elution on the chromatographic column, and the flow rate of mobile phase A in the chromatographic column is 0.2-0.5 mL / min, and the time of the fourth elution is 0.5-2 hours.

[0043] In a preferred embodiment, the detection comprises detecting using a high performance liquid chromatography or an ultra high performance liquid chromatography.

[0044] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0045] Example 1

[0046] The purity of the antibody in the fermentation broth was detected by SEC-HPLC, and the steps were as follows:

[0047] 1. Sample pretreatment:

[0048] 1.1 Centrifugal filtration: The fermentation broth expressed by CHO cells was subjected to secondary centrifugation, first at 1000g for ten minutes at 4°C, then at 10000g for half an hour, the supernatant was taken, and then filtered through a 0.2um filter.

[0049] 1.2 Adjusting pH and NaCl concentration: Take the above supernatant, adjust its pH to 7.0, and add 500 mM NaCl to obtain a pre-treated product.

[0050] 2. Sample testing:

[0051] 2.1 Column activation: For a new column used for the first time, 1) flush it with mobile phase (100 mM PB-250 mM NaCl pH 6.8) at a flow rate of 0.5 mL / min for 2 hours;

[0052] 2) Connect the detector and inject 50-100 μg of standard solution more than 4 times continuously;

[0053] 2.2 Column balancing: 1) All modules of the Purge system; 2) Mobile phase pipelines used by Prime; 3) Adjust the mobility ratio to the initial gradient, 95% mobile phase A (100mM PB-250mM NaCl pH 6.8), 5% mobile phase B (100% IPA isopropanol); 4) Control the flow rate of mobile phase A and mobile phase B to 0.8mL / min and balance the system until the baseline is stable.

[0054] 2.3 Injection: 1) First run 2 times with 20 μL of water each time and a 10 μL buffer (50 mM Tris-Hac pH 7.4) as a blank control.

[0055] 2) Start sampling. The first sample is the pure protein of the monoclonal antibody, which is used as a marker to determine the peak position of the antibody.

[0056] 3) The third sample is the pre-treatment product;

[0057] 4) The last sample is 20 μL of water.

[0058] 3. Post-processing:

[0059] 3.1 Acid treatment: 1) Regenerate the column with 120 mM HAc at a flow rate of 0.1 mL / min for 1 hour;

[0060] 3.2 First equilibration treatment: 1) Use mobile phase A for equilibration at a flow rate of 0.5 mL / min for 1 hour;

[0061] 3.3 Alkali treatment: 1) Use 0.05M NaOH for flushing at a flow rate of 0.1mL / min. After flushing for 1 hour, pause for 30 minutes.

[0062] 3.4 Second equilibration treatment: 1) Use mobile phase A for equilibration and adjust the flow rate to 0.5 mL / min for 1 hour;

[0063] 4. Column preservation: 1) Use mobile phase B at 0.5 mL / min for 1 hour and then store.

[0064] The test results of this embodiment are as follows Figure 1 And as shown in Table 1.

[0065] Table 1

[0066] Peak time (min) type Width(min) Peak area Peak height Peak area % 6.389 BB 1.60 195.49 5.97 2.44 7.666 BB 0.96 43.48 1.88 0.54 8.490 BM 1.42 3697.93 248.65 45.90 10.919 MM 1.77 411.98 22.82 5.14 11.977 MM 1.93 711.39 12.41 8.87 13.544 MM 0.49 633.13 29.65 7.90 13.789 MM 0.63 484.75 20.29 6.05 14.564 MM 0.51 328.16 13.29 4.09 15.238 MM 0.69 483.96 18.66 6.04 15.697 MM 0.38 78.23 4.49 0.98 16.105 MM 0.69 91.90 4.62 1.15 16.973 BV 0.91 724.67 41.99 9.04 17.756 VV 0.77 150.84 5.43 1.88

[0067] The SEC column pressure range of the embodiment of the present application is set at <500 bar. When the column pressure exceeds this range, the instrument will report an error and cannot continue to operate.

[0068] When the method of Example 1 is used to detect antibodies in the fermentation broth, even if more than 100 samples are detected, the chromatographic resolution does not decrease, and there is no phenomenon of column pressure overpressure.

[0069] Example 2

[0070] Sample processing: The fermentation broth expressed by CHO cells was subjected to secondary centrifugation, first at 1000g for ten minutes at 4°C, then at 10000g for half an hour, the supernatant was taken, and then filtered through a 0.2um filter.

[0071] The pH of the filtered supernatant was adjusted to 3.5, 4.5, 5.5, 6.5 and 7.5, and allowed to stand for half an hour. If the supernatant became turbid, it was filtered again. The state of the supernatant and the column flow were observed and recorded. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074] Due to the small particle size of the SEC column, if the sample is turbid and does not meet the loading standard, it will cause column blockage; when the pH is 6.5-7.5, the sample meets the loading standard, and after more than 100 samples, there is no significant difference in column pressure and resolution.

[0075] Example 3

[0076] Processing of samples to be tested: The fermentation broth expressed by CHO cells was subjected to secondary centrifugation, first at 1000g for ten minutes at 4°C, then at 10000g for half an hour, the supernatant was taken, and then filtered through a 0.2um filter.

[0077] The pH of the filtered supernatant was adjusted to 7.0, and different concentrations of NaCl were added, namely 500 mM NaCl, 1 M NaCl and 1.5 M NaCl. The mixture was allowed to stand for 1 hour, and the condition of the supernatant was observed. The results are shown in Table 3.

[0078] Table 3

[0079]

[0080] Example 4

[0081] The only difference from the detection method in Example 1 is that during the acid treatment, the flow rate is set to 0.5 mL / min, and the resolution of the chromatographic column decreases after detecting more than 50 samples (serious tailing peaks appear during chromatographic operation).

[0082] Example 5

[0083] The only difference from the detection method in Example 1 is that during the alkali treatment, the flow rate is set to 0.5 mL / min. The chromatographic column is over-pressurized during the alkali washing process. The pressure exceeds the set range of <500 bar, and the instrument alarms and cannot operate normally.

[0084] Example 6

[0085] The only difference from the detection method in Example 1 is that during the acid treatment, the acid concentration in the acid solution is 1 MHAc, the resolution of the chromatographic column is reduced, and the peak tailing phenomenon occurs.

[0086] Example 7

[0087] The only difference from the detection method in Example 1 is that during the acid treatment process, the acid concentration in the acid solution is 50mMHAc. After detecting more than 100 samples, the chromatographic column is over-pressurized, and the pressure exceeds the set range of <500bar. The instrument alarms and cannot operate normally.

[0088] Example 8

[0089] The only difference from the detection method in Example 1 is that during the alkali treatment, the concentration of the alkali in the alkali solution is 1 M NaOH, the resolution of the chromatographic column decreases, and a tailing phenomenon occurs.

[0090] Example 9

[0091] The only difference from the detection method in Example 1 is that during the alkali treatment process, the concentration of alkali in the alkali solution is 0.01MNaOH. After more than 100 samples are detected, the column pressure gradually increases, and the pressure exceeds the set range of <500bar. The instrument alarms and cannot operate normally.

[0092] Comparative Example 1

[0093] The only difference from the detection method in Example 1 is that no alkaline treatment and second equilibrium treatment are performed during the elution process, and the remaining steps are exactly the same as in Example 1. After more than 50 samples are detected, the column pressure increases, and the pressure exceeds the set range of <500 bar, the instrument alarms, and cannot operate normally.

[0094] Comparative Example 2

[0095] 1. Take 5 mL of equilibrium solution (50 mM Tris-HAc, 150 mM NaCl, pH 7.4) to treat the affinity filler;

[0096] 2. Take 5 mL of alkali (0.1 M NaOH) to treat the affinity filler;

[0097] 3. Take 5 mL of equilibrium solution (50 mM Tris-HAc, 150 mM NaCl, pH 7.4) to treat the affinity filler;

[0098] 4. Take the fermentation broth containing 20-40 mg of protein sample for sample loading;

[0099] 5. Take 5 mL of equilibrium solution (50 mM Tris-HAc, 150 mM NaCl, pH 7.4) to treat the affinity filler;

[0100] 6. Take 10 mL of eluent (50 mM NaAc-HAc, pH 3.0-3.7) to elute the sample and collect the sample;

[0101] 7. Filter the eluted sample using a 0.22um syringe filter;

[0102] 8. Take 50-100 μg sample for SEC-HPLC experiment;

[0103] Column balancing: 1) All modules of the Purge system; 2) Mobile phase lines used by Prime; 3) Adjust the flow ratio to the initial gradient, 95% mobile phase A (100mM PB-250mM NaCl pH 6.8), 5% mobile phase B (100% IPA isopropanol); 4) Control the flow rate of mobile phase A and mobile phase B to 0.8mL / min to balance the system until the baseline is stable.

[0104] Injection: 1) First load 20 μL of water twice and 20 μL of eluent once as blank control.

[0105] 2) Start sampling. The first sample is the pure protein of the monoclonal antibody, which is used as a marker to determine the peak position of the antibody.

[0106] 3) The third sample is the pre-treatment product;

[0107] 4) The last sample is 20 μL of water.

[0108] Cleaning residue: Use the mobile phase as the sample, run the normal sample analysis gradient, and load 20 μL each time to clean the column;

[0109] The test results of this comparative example are as follows Figure 2 And as shown in Table 4.

[0110] Table 4

[0111] Peak time (min) type Width(min) Peak area Peak height Peak area % 7.566 MM 1.52 41.68 1.58 0.90 8.485 BB 2.05 4590.18 310.04 99.10

[0112] Compare Figure 1 ,Table 1, Figure 2The test results of Table 4 (Example 1 and Comparative Example 2) show that the peak position of the antibody in the fermentation broth is consistent with the peak position of the pure protein. The sample after affinity treatment basically does not contain fragments, but a lot of fragments can be observed in the test results of Example 1 of the present application, and these fragments are basically HCP (Host cell protein). It can be seen that most of the HCP molecular weight is lower than that of the antibody, and can be effectively separated by SEC-HPLC without affecting the accuracy of the test.

[0113] In addition, the sample purity after affinity treatment reached 99.1%, and the aggregate accounted for only 0.9%. The results of direct loading of the fermentation broth showed that the actual aggregate proportion of the product reached 2.98%, and the aggregate distribution was also different from that of pure protein. Without considering cell debris, the mass purity of the antibody was 97.02%. Therefore, there is a large error in using the purity mass detected by SEC-HPLC after affinity chromatography to represent the purity mass of the antibody in the fermentation broth. It is more accurate to use the detection method of this application to detect antibodies in the fermentation broth.

[0114] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: using the detection method of the present application, it is avoided that the quality of the monoclonal antibody caused by affinity chromatography is forced to improve, thereby causing wrong screening, resulting in subsequent purification and processing difficulties, resulting in a large loss of yield. The detection method of the present application is realized by adding the steps of pre-treatment and post-treatment in the SEC-HPLC detection process, and accurately regulating the parameter conditions in the treatment process, so as to achieve the purpose of being able to directly use SEC-HPLC to detect the antibody purity in the fermentation broth, and accurately detect the antibody purity in the fermentation broth, and after detecting 100 samples, the system can still remain stable, and there is no peak tailing and column pressure increase. The phenomenon. Therefore, the inspection method of the present application is used to help screen out truly high-quality monoclonal antibodies, improve the yield of the final product, and has high repeatability and high system stability, which is more suitable for promotion relative to the detection method of the prior art.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting the purity of antibodies in antibody-expressing cell fermentation broth, characterized in that: The detection method comprises: S1) performing pretreatment on the sample to be tested to obtain a pretreatment product; S2) detecting the pre-treatment product by size exclusion chromatography-high performance liquid chromatography to obtain the purity of the antibody in the fermentation broth; The pretreatment includes adjusting the pH of the sample to be tested to 6.5-7.5, and adding sodium chloride to the sample to be tested until the concentration of sodium chloride in the sample to be tested is 100-500 mM; The detection comprises: loading the pre-treatment product onto a chromatographic column of size exclusion chromatography high performance liquid chromatography and then eluting; The elution comprises: acid treatment, a first equilibrium treatment, an alkali treatment and a second equilibrium treatment.

2. The detection method according to claim 1, characterized in that: The S1) comprises: performing centrifugal treatment and filtering treatment on the sample to be tested to obtain a first pre-treatment product; The pH of the first pre-treatment product is adjusted to 6.5-7.5, and sodium chloride is added to the first pre-treatment product until the concentration of sodium chloride in the first pre-treatment product is 100-500 mM, to obtain the pre-treatment product.

3. The detection method according to claim 1, characterized in that: The mobile phase A used in the size exclusion chromatography HPLC is 100 mM PB and 250 mM NaCl, with a pH value of 6.5 to 7.2; Mobile phase B was 100% isopropanol.

4. The detection method according to claim 3, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 85-95:5-15; Preferably, the sample loading amount of the size exclusion chromatography HPLC is 50-100 μg.

5. The detection method according to claim 1, characterized in that: The acid treatment comprises: performing a first elution on the chromatographic column using an acid solution, the flow rate of the acid solution in the chromatographic column is 0.05 to 0.5 mL / min, and the time of the first elution is 0.5 to 2 hours.

6. The detection method according to claim 5, characterized in that: The acid in the acid solution includes one or more of acetic acid and / or citric acid; The acid concentration of the acid solution is 100-200 mM.

7. The detection method according to claim 3, characterized in that: The first equilibrium treatment includes performing a second elution on the chromatographic column using the mobile phase A; the flow rate of the mobile phase A in the chromatographic column is 0.2 to 0.5 mL / min, and the time of the second elution is 0.5 to 2 hours.

8. The detection method according to claim 1, characterized in that: The alkaline treatment comprises performing a third elution on the chromatographic column using an alkaline solution, and then performing the second equilibration treatment after the chromatographic column is left to stand for 0.5 to 2 hours; The flow rate of the alkaline solution in the chromatographic column is 0.05 to 0.5 mL / min, and the time of the third elution is 0.5 to 2 hours.

9. The detection method according to claim 8, characterized in that: The alkali in the alkali solution includes sodium hydroxide; The concentration of the alkali in the alkali solution is 0.01-0.1M.

10. The detection method according to claim 3, characterized in that: The second equilibrium treatment includes performing a fourth elution on the chromatographic column using the mobile phase A, the flow rate of the mobile phase A in the chromatographic column is 0.2 to 0.5 mL / min, and the time of the fourth elution is 0.5 to 2 hours.

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