A method for detecting purity of a heterodimeric protein

Abstract

The application provides a detection method for the purity of a heterodimer protein, and a mobile phase is a mixed aqueous solution of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride, wherein the concentration of the disodium hydrogen phosphate dodecahydrate is 40-80 mM, and the concentration of the sodium dihydrogen phosphate dihydrate is 20-60 mM. The detection method can separate the dimer protein from impurities well, has the advantages of rapidness, simplicity, low detection limit, high sensitivity and the like, and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a method for detecting purity of a heterodimeric protein. BACKGROUND

[0002] Modern biological engineering often uses mammalian cells to produce protein drugs. The expression amount of protein in cell culture fluid changes with culture time, and frequent sampling is needed to monitor the expression amount in the late culture stage to find the best balance point between input and output. During the cell culture process, the protein molecules secreted by cells will be aggregated to different degrees due to the liquid environment, mechanical environment and physical and chemical properties of the molecules, which is undesirable for protein production and will affect the yield and product quality of the process. Therefore, monitoring the percentage of protein aggregates in the culture is also an important parameter for evaluating the culture process. In addition, protein aggregation has a great influence on the safety and effectiveness of protein drugs. Protein aggregates may affect their membrane penetration properties due to their large molecular weight, leading to reduced bioavailability, and may also produce immunogenicity, which will pose potential risks to patients after administration. Therefore, protein purity detection has become one of the important indicators for quality control of protein drugs.

[0003] Currently, there are three commonly used methods for protein content determination: affinity high performance liquid chromatography, non-reducing protein electrophoresis and size exclusion chromatography (SEC). Among them, affinity high performance liquid chromatography is used to determine the expression amount of protein. The expression amount of protein determined by this method is the sum of the aggregate form, monomer form and Fc-containing fragment of the protein, while only the monomer form of protein is required. Therefore, the result determined by this method is not the accurate expression amount of the target molecule (monomer form). Non-reducing protein electrophoresis needs to be operated under denaturing conditions, which may affect the determination of the content of aggregate components. Size exclusion chromatography (SEC) is a method that mainly separates proteins according to the difference in molecular size by using the unique characteristics of porous gel stationary phase. The detection conditions are relatively mild and do not greatly affect the native form of the protein. Therefore, size exclusion chromatography (SEC) is generally used for quality control of protein aggregate-related impurities in products in the industry. However, the currently disclosed size exclusion chromatography (SEC) often has problems such as poor separation degree or inability to separate aggregate impurities when used to detect aggregate impurities in proteins. In addition, some methods have poor stability and low detection sensitivity, and are not suitable for the separation and detection of low-concentration aggregate impurities.

[0004] Therefore, in order to further ensure the quality and safety of protein drugs, it is necessary to establish a rapid, simple, sensitive and stable size exclusion chromatography detection method. SUMMARY

[0005] In the present application, the inventors provide a rapid, simple, sensitive and stable detection method for detecting the purity of heterodimeric protein.

[0006] The present application provides a method for detecting the purity of heterodimeric protein, comprising the following steps: chromatographic conditions: the chromatographic column is filled with silica gel; the mobile phase is a mixed aqueous solution of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride; wherein the concentration of disodium hydrogen phosphate dodecahydrate is 40-80 mM, and the concentration of sodium dihydrogen phosphate dihydrate is 20-60 mM; the elution mode is isocratic elution; and the chromatogram is recorded.

[0007] In some embodiments, the concentration of disodium hydrogen phosphate dodecahydrate is 50-70 mM.

[0008] In some embodiments, the concentration of sodium dihydrogen phosphate dihydrate is 30-50 mM.

[0009] In some embodiments, the concentration of disodium hydrogen phosphate dodecahydrate is 60 mM, and the concentration of sodium dihydrogen phosphate dihydrate is 40 mM.

[0010] In some embodiments, the concentration of sodium chloride is 80-120 mM; preferably, the concentration of sodium chloride is 90-110 mM; more preferably, the concentration of sodium chloride is 100 mM.

[0011] In some embodiments, the pH of the mobile phase is 6.5-7.5; preferably, the pH of the mobile phase is 7.0.

[0012] In some embodiments, the chromatographic column is a Waters XBridge BEH SEC 3.5 μm chromatographic column.

[0013] In some embodiments, the method further comprises the following steps: the flow rate of the mobile phase is 0.2-0.8 mL / min; the column temperature is 25-35℃; the injection volume is 25-35 μL; the detection wavelength is 274-286 nm; and / or the sample chamber temperature is 5-15℃.

[0014] In some embodiments, the flow rate of the mobile phase is 0.5 mL / min.

[0015] In some embodiments, the column temperature is 30℃.

[0016] In some embodiments, the injection volume is 30 μL.

[0017] In some embodiments, the detection wavelength is 280 nm.

[0018] In some embodiments, the sample chamber temperature is 10°C.

[0019] In some embodiments, the heterodimeric protein comprises HCDR1, HCDR2 and HCDR3 of heavy chain 1 as shown in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 of light chain as shown in the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; preferably, the heterodimeric protein comprises heavy chain 1 as shown in the amino acid sequence of SEQ ID NO: 7, heavy chain 2 as shown in the amino acid sequence of SEQ ID NO: 8, and light chain as shown in the amino acid sequence of SEQ ID NO: 9.

[0020] In some embodiments, the heterodimeric protein concentration is 3 mg / mL.

[0021] In some embodiments, the method further comprises the following steps: (1) mobile phase preparation: weigh an appropriate amount of disodium hydrogen phosphate dodecahydrate, an appropriate amount of sodium dihydrogen phosphate dihydrate, and an appropriate amount of sodium chloride, dissolve in water, filter, and obtain; (2) preparation of test sample solution: take an appropriate amount of the protein to be tested, dilute it with the mobile phase, centrifuge, and take the supernatant as the test sample solution. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Protein chromatogram detection graph for Comparative Example 1.

[0023] Figure 2 Protein chromatogram detection graph for Comparative Example 2.

[0024] Figure 3 Protein chromatogram detection graph for Comparative Example 3.

[0025] Figure 4 Protein chromatogram detection graph for Comparative Example 4.

[0026] Figure 5 Protein chromatogram detection graph for Comparative Example 5.

[0027] Figure 6 Protein chromatogram detection graph for Example 1. DETAILED DESCRIPTION

[0028] The present application is further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the present application is not limited to the embodiments described below, which are presented as specific examples only. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present application. Various modifications and improvements throughout the scope of the inventive concept are intended to be included within the scope of the present application and the appended claims and any equivalents thereof.

[0029] Comparative Example 1

[0030] Weigh 21.84 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 6.08 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 5.84 g of sodium chloride (NaCl), dissolve in water, and dilute to 1 L to obtain the mobile phase. Prepare the test protein (i.e., the test heterodimeric protein solution) according to Table 1, wherein the heterodimeric protein comprises a heavy chain 1 as shown in the amino acid sequence of SEQ ID NO: 7, a heavy chain 2 as shown in the amino acid sequence of SEQ ID NO: 8, and a light chain as shown in the amino acid sequence of SEQ ID NO: 9. The light chain and the heavy chain 1 are complexed to form a targeting moiety with binding specificity for a tumor antigen or immune checkpoint, and the heavy chain 2 comprises an Fc region and an immunomodulator fused to the Fc region, and the light chain, the heavy chain 1, and the heavy chain 2 are complexed to form the heterodimeric protein.

[0031] Table 1: Components and their contents of the test protein

[0032] Component Amount Heterodimeric protein 10.00 g / L Polysorbate 80 0.20 g / L Histidine 1.72 g / L Histidine hydrochloride 1.87 g / L Trehalose 51.35 g / L Water for injection q.s. to 1 L

[0033] Take the test protein, dilute it with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, take the supernatant, and obtain the test sample solution. Use a high-performance liquid chromatograph, source: Waters, model: E2695. Use a TSKgel G3000SWXL (300 mm x 7.8 mm) gel exclusion chromatography column, isocratic elution with the mobile phase, flow rate: 1.0 mL / min, elution time: 16 min, injection volume: 30 μL, column temperature: 30 °C, sample tray temperature: 10 °C, use a UV detector, and detection wavelength: 280 nm.

[0034] According to the area normalization method, calculate the contents of the heterodimeric protein monomer, large molecular weight (HMW) aggregate, and small molecular weight (LMW) fragment.

[0035] Heterodimeric protein monomer content = (main peak area / total peak area) x 100%

[0036] Macromolecular aggregate content = (macromolecular aggregate peak area / total peak area) × 100%

[0037] The results are as follows Figure 1 As shown, the macromolecular aggregate impurity content is 1.09%, and the resolution between the heterodimeric protein monomer and the macromolecular aggregate impurity is 1.63, which meets the resolution requirements. However, there is no resolution between the heterodimeric protein monomer and the small molecule fragment impurities, making it impossible to accurately calculate the content of the heterodimeric protein monomer. This method is not suitable for product quality control.

[0038] Comparative Example 2

[0039] Weigh 21.84 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 6.08 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 5.84 g of sodium chloride (NaCl), dissolve them in water and dilute to 1 L, filter through a 0.45 μm filter membrane to obtain the mobile phase. Take the protein to be tested (the preparation method of the protein to be tested is the same as that of Comparative Example 1), dilute it with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 minutes, and take the supernatant to obtain the test solution. Use a high performance liquid chromatograph from Waters, model E2695. Use an Agilent AdvanceBio SEC ( A 2.7 μm, 300 mm × 7.8 mm) chromatographic column was used, and isocratic elution was performed with the mobile phase at a flow rate of 1.0 mL per minute, an elution time of 16 minutes, an injection volume of 30 μL, a column temperature of 30°C, a sample tray temperature of 10°C, and a UV detector with a detection wavelength of 280 nm.

[0040] The contents of heterodimeric protein monomers, large molecular weight aggregates (HMW) and small molecular weight fragments (LMW) were calculated by area normalization method.

[0041] Heterodimeric protein monomer content = (main peak area / total peak area) × 100%

[0042] Macromolecular aggregate content = (macromolecular aggregate peak area / total peak area) × 100%

[0043] The results are as follows Figure 2 As shown, the content of macromolecular aggregate impurities is 0.88%, and the separation degree between heterodimeric protein monomers and macromolecular aggregate impurities is 1.65. However, there is no separation degree between heterodimeric protein monomers and small molecule fragment impurities, and the content of heterodimeric protein monomers cannot be accurately calculated. This method is not suitable for product quality control.

[0044] Comparative Example 3

[0045] Take twelve hydrated disodium hydrogen phosphate (Na2HPO4·12H2O) 4.37 g, sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) 1.22 g, arginine 6.97 g, dissolve in water and constant volume to 0.2 L, filter through 0.45 μm filter membrane, the mobile phase is obtained. Take the test protein (the test protein preparation method is the same as that of comparative example 1), dilute with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant, the test sample solution is obtained. Use high performance liquid chromatograph, source from Waters, model E2695. Use Waters XBridge BEH SEC( 3.5 μm, 300 mm x 7.8 mm) chromatographic column, isocratic elution with mobile phase, flow rate is 0.5 mL per minute, elution time is 30 minutes, injection volume is 30 μL, column temperature is 30℃, sample tray temperature is 10℃, use ultraviolet detector, detection wavelength is 280 nm.

[0046] According to the area normalization method, the content of the heterodimer protein monomer, the large molecular weight polymer (HMW) and the small molecular weight fragment (LMW) is calculated.

[0047] The content of the heterodimer protein monomer is (main peak area / total peak area) x 100%

[0048] The content of the large molecular weight polymer is (large molecular weight polymer peak area / total peak area) x 100%

[0049] The results are shown in Figure 3 The content of the large molecular weight polymer impurity is 1.23%, the separation degree of the heterodimer protein monomer and the large molecular weight polymer impurity is 1.99, but the separation degree of the heterodimer protein monomer and the small molecular weight polymer impurity does not meet the requirements, and the content of the heterodimer protein monomer cannot be accurately calculated. This method is not suitable for quality control of the product.

[0050] Comparative example 4

[0051] Take twelve hydrated disodium hydrogen phosphate (Na2HPO4·12H2O) 21.84 g, sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) 6.08 g, sodium chloride (NaCl) 5.84 g, dissolve in water and constant volume to 1 L, filter through 0.45 μm filter membrane, the mobile phase A is obtained. The mobile phase B is acetonitrile. Take the test protein (the test protein preparation method is the same as that of comparative example 1), dilute with the mobile phase (mobile phase A: mobile phase B = 80:20 (v / v)) to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant, the test sample solution is obtained. Use high performance liquid chromatograph, source from Waters, model E2695. Use Agilent AdvanceBio SEC( 2.7 pm, 300 mm x 7.8 mm) column with mobile phase A: mobile phase B = 80:20 (v / v) for isocratic elution at a flow rate of 0.5 mL per minute, elution time of 30 minutes, injection volume of 30 pL, column temperature of 30 °C, sample tray temperature of 10 °C, using a UV detector at a detection wavelength of 280 nm.

[0052] The content of the heterodimer protein monomer, large molecular weight aggregate (HMW) and small molecular weight fragment (LMW) was calculated by area normalization method.

[0053] The content of the heterodimer protein monomer = (main peak area / total peak area) x 100%

[0054] The content of the large molecular weight aggregate = (large molecular weight aggregate peak area / total peak area) x 100%

[0055] The results are shown in Table 1. Figure 4 As shown in Table 1, the large molecular weight aggregate impurity in the test protein could not be effectively separated, and the use of acetonitrile destroyed the natural state of the analyte, resulting in a small molecular weight aggregate impurity content as high as 3.63%, indicating that this method is not suitable for product quality control.

[0056] Comparative Example 5

[0057] Weigh 21.84 g of disodium hydrogen phosphate dodecahydrate (Na2HP04-12H20), 6.08 g of sodium dihydrogen phosphate dihydrate (NaH2P04-2H20), and 29.20 g of sodium chloride (NaCl), dissolve them in water, and dilute to 1 L. Filter through a 0.45 pm filter membrane to obtain the mobile phase. Take the test protein (the preparation method of the test protein is the same as that of Comparative Example 1), dilute it with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12,000 rpm for 5 min, and take the supernatant to obtain the test sample solution. A high performance liquid chromatograph (Waters, E2695) was used. A Waters XBridge BEH SEC (3.5 pm, 300 mm x 7.8 mm) column was used for isocratic elution with the mobile phase at a flow rate of 0.5 mL per minute, an elution time of 30 minutes, an injection volume of 30 pL, a column temperature of 30 °C, a sample tray temperature of 10 °C, and a UV detector at a detection wavelength of 280 nm. 3.5 pm, 300 mm x 7.8 mm) column with mobile phase A: mobile phase B = 80:20 (v / v) for isocratic elution at a flow rate of 0.5 mL per minute, elution time of 30 minutes, injection volume of 30 pL, column temperature of 30 °C, sample tray temperature of 10 °C, using a UV detector at a detection wavelength of 280 nm.

[0058] The content of the heterodimer protein monomer, large molecular weight aggregate (HMW) and small molecular weight fragment (LMW) was calculated by area normalization method.

[0059] The content of the heterodimer protein monomer = (main peak area / total peak area) x 100%

[0060] The content of the large molecular weight aggregate = (large molecular weight aggregate peak area / total peak area) x 100%

[0061] The results are as follows Figure 5 As shown, the macromolecular aggregate impurity content is 1.95%, the resolution between heterodimeric protein monomers and macromolecular aggregate impurities is 2.59, and the small molecular aggregate impurity content is 1.33%. This indicates that while high salt concentrations can weaken ionic interactions between the protein and the stationary phase, they can also increase hydrophobic interactions, leading to an increase in the aggregate impurity content of the analyte. This method is not suitable for product quality control.

[0062] Example 1

[0063] Weigh 21.84 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 6.08 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 5.84 g of sodium chloride (NaCl), dissolve them in water and dilute to 1 L, filter through a 0.45 μm filter membrane to obtain a mobile phase. Take the protein to be tested (the preparation method of the protein to be tested is the same as that of Comparative Example 1), dilute it with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 minutes, and take the supernatant to obtain the test solution. Use a high performance liquid chromatograph from Waters, model E2695. Use Waters XBridge BEH SEC ( A 3.5 μm, 300 mm × 7.8 mm) chromatographic column was used, and isocratic elution was performed with the mobile phase at a flow rate of 0.5 mL per minute, an elution time of 30 minutes, an injection volume of 30 μL, a column temperature of 30°C, a sample tray temperature of 10°C, and a UV detector with a detection wavelength of 280 nm.

[0064] The contents of heterodimeric protein monomers, large molecular weight aggregates (HMW) and small molecular weight fragments (LMW) were calculated by area normalization method.

[0065] Heterodimeric protein monomer content = (main peak area / total peak area) × 100%

[0066] Macromolecular aggregate content = (macromolecular aggregate peak area / total peak area) × 100%

[0067] The results are as follows Figure 6 As shown, the content of large molecular aggregate impurities is 1.01%, the separation degree of heterodimeric protein monomers and large molecular aggregate impurities is 2.08, the content of small molecular aggregate impurities is 1.87%, and the separation degree of heterodimeric protein monomers and small molecular aggregate impurities is 1.69. The content of heterodimeric protein monomers is 97.12%.

[0068] Example 2 System suitability test

[0069] Take the protein to be tested (the preparation method of the protein to be tested is the same as that of Comparative Example 1), dilute it with the mobile phase (the preparation method of the mobile phase is the same as that of Example 1) to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12,000 rpm for 5 min, and take the supernatant to obtain a system suitability solution.

[0070] Inject 1 needle of the blank solution (mobile phase) and 6 needles of the system suitability solution. The results are shown in Table 2.

[0071] Table 2 System suitability test results

[0072]

[0073] It can be seen that the RSD of the peak area percentage of the main peak is ≤2.0%, the RSD of the retention time of the main peak is ≤2.0%, the theoretical plate number of the main peak is ≥2500, and the separation degree of the main peak from the macromolecular aggregate peak is ≥1.5, and the system suitability is good.

[0074] Example 3 Specificity test

[0075] Preparation of the mobile phase: weigh 21.84 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 6.08 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), and 5.84 g of sodium chloride (NaCl), dissolve and dilute to 1 L with water, and filter through a 0.45 μm filter membrane to obtain the mobile phase.

[0076] Preparation of the protein to be tested: the preparation method is the same as that of Comparative Example 1.

[0077] 1. Specificity destruction solution

[0078] Specificity oxygen destruction solution: take 100 μL of the protein to be tested, add 10 μL of 30% H2O2 solution, and stand at room temperature for 2 h, then dilute with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12,000 rpm for 5 min, and take the supernatant as the specificity oxygen destruction solution.

[0079] Specificity heat destruction solution: take 100 μL of the protein to be tested, place it in a 50°C water bath for 1 h, dilute with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12,000 rpm for 5 min, and take the supernatant as the specificity heat destruction solution.

[0080] Specificity alkali destruction solution: take 100 μL of the protein to be tested, add 20 μL of 0.1 mol / L sodium hydroxide solution, stand at room temperature for 1 h, then neutralize with 20 μL of 0.1 mol / L hydrochloric acid solution, dilute with the mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12,000 rpm for 5 min, and take the supernatant as the specificity alkali destruction solution.

[0081] Specificity acid destruction solution: take 100 μL of the protein to be tested, add 5 μL of 0.1 mol / L hydrochloric acid solution, and place at room temperature for 1 h. Then, neutralize with 5 μL of 0.1 mol / L sodium hydroxide solution, dilute with mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant as the specificity acid destruction solution.

[0082] Specificity light destruction solution: take 100 μL of the protein to be tested, place in a light box, and irradiate with sunlight intensity of 4500 Lux and ultraviolet light intensity of 255 uw / cm 2 for 2 h. Then, dilute with mobile phase to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant as the specificity light destruction solution.

[0083] 2. Specificity excipient destruction solution

[0084] Specificity oxygen excipient destruction solution: take 100 μL of the protein to be tested, and prepare in the same way as the oxygen destruction solution as the specificity oxygen excipient destruction solution.

[0085] Specificity heat excipient destruction solution: take 100 μL of the protein to be tested, and prepare in the same way as the heat destruction solution as the specificity heat excipient destruction solution.

[0086] Specificity base excipient destruction solution: take 100 μL of the protein to be tested, and prepare in the same way as the base destruction solution as the specificity base excipient destruction solution.

[0087] Specificity light excipient destruction solution: take 100 μL of the protein to be tested, and prepare in the same way as the light destruction solution as the specificity light excipient destruction solution.

[0088] Specificity acid excipient destruction solution: take 100 μL of the protein to be tested, and prepare in the same way as the acid destruction solution as the specificity acid excipient destruction solution.

[0089] 3. Specificity blank solution

[0090] Specificity base blank solution: take 100 μL of the mobile phase, and prepare in the same way as the base destruction solution as the specificity base blank solution.

[0091] Specificity acid blank solution: take 100 μL of the mobile phase, and prepare in the same way as the acid destruction solution as the specificity acid blank solution.

[0092] Specificity oxygen blank solution: take 100 μL of the mobile phase, and prepare in the same way as the oxygen destruction solution as the specificity oxygen blank solution.

[0093] 4. Specificity non-destruction solution

[0094] Take 100 μL of the protein to be tested, dilute to a solution with a protein concentration of about 3 mg / mL with the mobile phase, centrifuge at 12000 rpm for 5 min, and take the supernatant as the specificity non-destructive solution.

[0095] Each buffer blank solution: take each buffer of the protein to be tested (see Table 3 for details) as the buffer blank solution.

[0096] Table 3 Buffer composition

[0097]

[0098]

[0099] Take each specificity destructive solution, specificity auxiliary destructive solution, specificity blank solution, and specificity non-destructive solution each into a needle. The results are shown in Table 4.

[0100] Table 4 Specificity test results

[0101]

[0102] It can be seen that each auxiliary destructive solution and the original buffer solution do not have peaks at the peak position of the specificity solution, and do not interfere with sample detection; the separation degree between each destructive solution macromolecular impurity and heterologous dimer protein monomer is greater than 1.5, and the method can sensitively detect the peak type change of each specificity destructive solution, and has good specificity.

[0103] Example 4 Quantitative limit test

[0104] Quantitative limit solution preparation: take the protein to be tested (preparation method same as Comparative Example 1), dilute to a solution with a protein concentration of 0.006 mg / mL with the mobile phase (mobile phase preparation method same as Example 3), centrifuge at 12000 rpm for 5 min, take the supernatant as the quantitative limit solution, and prepare 6 parallel samples.

[0105] Take 6 quantitative limit solutions each into a needle, determine the quantitative limit with a main peak signal-to-noise ratio of about 10:1, calculate the amount that can be reliably detected, and the test results are shown in Table 5.

[0106] Table 5 Quantitative limit test results

[0107]

[0108]

[0109] The signal-to-noise ratio of the quantitative limit solution is greater than 10, the RSD of the main peak retention time of 6 quantitative limit solutions is less than or equal to 10.0%, the RSD of the main peak area is less than or equal to 15.0%, the quantitative limit concentration is 0.006 mg / mL, which is 0.2% of the concentration of the test product. The sensitivity of the method is sufficient to detect the content of the product polymer impurities and the content of the heterologous dimer protein monomer.

[0110] Example 5 Linearity and Range Test

[0111] Linear solution preparation: Take the protein to be tested (preparation method same as Comparative Example 1), dilute with mobile phase (mobile phase preparation method same as Example 3) to protein concentrations of 0.006 mg / mL, 0.3 mg / mL, 1.2 mg / mL, 2.4 mg / mL, 3.0 mg / mL, 4.5 mg / mL, centrifuge at 12000 rpm for 5 min, take the supernatant as the linear solution, and prepare two parallel solutions for each concentration.

[0112] Take each linear solution, inject 1 needle of each linear solution, detect and record the chromatogram, respectively, and linearly regress the average value of the main peak area to the sample concentration to obtain the regression equation and the correlation coefficient (R 2 ), and investigate the linearity of the method. The test results are shown in Table 6.

[0113] Table 6 Linearity and Range Test Results

[0114]

[0115] The linear regression equation is Y = 4266746X - 84730.33, the correlation coefficient R 2 ≥ 0.99, and the protein concentration is in the range of 0.006-4.5 mg / mL, with good linear relationship.

[0116] Example 6 Accuracy Test

[0117] Accuracy solution preparation: Take the protein to be tested (preparation method same as Comparative Example 1), respectively, prepare solutions with concentrations of 80% (2.4 mg / mL), 100% (3.0 mg / mL) and 120% (3.6 mg / mL), centrifuge at 12000 rpm for 5 min, take the supernatant as the accuracy solution, and prepare 3 parallel solutions for each concentration.

[0118] Take each accuracy solution and inject 1 needle, detect and record the chromatogram, take each accuracy solution and inject 1 needle, record the percentage of the area of the polymer, main peak and fragment peak, and calculate the relative standard deviation to investigate the accuracy of the method. The specific results are shown in Table 7.

[0119] Table 7 Accuracy Test Results

[0120]

[0121] The peak area percentage RSD of the main peak was less than or equal to 2.0%, and the accuracy was good within the range of 80% to 120%.

[0122] Repeatability test of Example 7

[0123] Preparation of the repeatability solution: The test protein (prepared according to the method of Comparative Example 1) was diluted with the mobile phase (prepared according to the method of Example 3) to a solution with a protein concentration of about 3 mg / mL. The solution was centrifuged at 12,000 rpm for 5 min, and the supernatant was taken as the repeatability solution. Six solutions were prepared in parallel.

[0124] One injection was taken from each of the repeatability solutions, and the chromatogram was detected and recorded. The peak area percentages of the aggregate, main peak, and fragment peak were recorded, and the relative standard deviation was calculated to investigate the repeatability of the method. The specific results are shown in Table 8.

[0125] Table 8: Results of the repeatability test

[0126]

[0127]

[0128] The peak area percentage RSD of the main peak was less than or equal to 2.0% for 6 injections, and the repeatability was good.

[0129] Intermediate precision test of Example 8

[0130] Preparation of the intermediate precision solution: The intermediate precision solution was prepared according to the method of preparing the repeatability solution.

[0131] The intermediate precision of the method was investigated by changing the experimental personnel and following the operation of the repeatability test. The peak area percentages of the aggregate, main peak, and fragment peak were recorded, and the relative standard deviation (RSD%) was calculated to investigate the intermediate precision of the method. The specific results are shown in Table 9.

[0132] Table 9: Summary of the precision test results

[0133]

[0134] The peak area percentage RSD of the main peak was less than or equal to 2.0% for 12 injections of the intermediate precision solution, and the intermediate precision was good.

[0135] Durability test of Example 9

[0136] Preparation of the test sample solution: The test protein (prepared according to the method of Comparative Example 1) was diluted with the mobile phase (prepared according to the method of Example 3) to a solution with a protein concentration of about 3 mg / mL. The solution was centrifuged at 12,000 rpm for 5 min, and the supernatant was taken as the durability solution.

[0137] Fine-tune the wavelength, flow rate, column temperature, respectively, to detect the test solution, and investigate the durability of the method. The test wavelength is 278 nm, 280 nm, 282 nm, the test flow rate is 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, and the test column temperature is 25℃, 30℃, 35℃, respectively. Detect and record the chromatogram, record the percentage of peak area of the main peak, the fragment peak, and calculate the relative standard deviation (RSD), investigate the influence of different conditions on the test results of the sample to determine the durability of the method. The test results are shown in Table 10.

[0138] Table 10 Durability test results

[0139]

[0140] Under each condition, the RSD of the main peak area percentage is ≤2.0%, and the durability is good.

[0141] Example 10 Solution stability

[0142] Stability solution preparation: Take the protein to be tested (preparation method same as Comparative Example 1), dilute with mobile phase (mobile phase preparation method same as Example 3) to a solution with a protein concentration of about 3 mg / mL, centrifuge at 12000 rpm for 5 min, and take the supernatant as the stability solution.

[0143] Take the stability solution, place it at 10℃, and detect it at 0h and 24h after placement, respectively, with 1 injection at each time point, to investigate the solution stability at 10℃. The test results are shown in Table 11.

[0144] Table 11 Solution stability test results

[0145]

[0146] Under each condition, the SD of the main peak area percentage is ≤2.0, and the solution is stable at 10℃ for 0h and 24h.

[0147] It can be seen that the method has strong specificity, high accuracy, good precision, good linearity in the appropriate range, good durability, and good solution stability, and can be used for the determination of protein purity.

[0148] The protection content of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and the appended claims are protected.

Claims

1. A method for detecting the purity of a heterodimeric protein, characterized in that: The method comprises the following steps: Chromatographic conditions: the chromatographic column is Waters XBridge BEH SEC The method comprises the following steps: a 3.5 μm chromatographic column; a mobile phase comprising a mixed aqueous solution of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride, wherein the concentration of disodium hydrogen phosphate dodecahydrate is 60 mM, the concentration of sodium dihydrogen phosphate dihydrate is 40 mM, and the concentration of sodium chloride is 100 mM; the elution mode is isocratic elution; the detection wavelength is 274-286 nm; and a chromatogram is recorded; the heterodimeric protein comprises HCDR1, HCDR2, and HCDR3 of the heavy chain 1 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and LCDR1, LCDR2, and LCDR3 of the light chain as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:

6.

2. The detection method according to claim 1, characterized in that The pH of the mobile phase is 6.5-7.

5.

3. The detection method according to claim 2, characterized in that The method further comprises the following steps: the mobile phase flow rate is 0.2-0.8 mL / min; the column temperature is 25-35° C.; the injection volume is 25-35 μL; and the sample chamber temperature is 5-15° C.

4. The detection method according to claim 3, characterized in that The heterodimeric protein comprises a heavy chain 1 as shown in the amino acid sequence of SEQ ID NO: 7, a heavy chain 2 as shown in the amino acid sequence of SEQ ID NO: 8, and a light chain as shown in the amino acid sequence of SEQ ID NO:

9.

5. The detection method according to any one of claims 1 to 4, characterized in that The method further comprises the following steps: (1) Preparation of mobile phase: Weigh appropriate amounts of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and sodium chloride, dissolve in water, and filter to obtain the mobile phase. (2) Preparation of test solution: Take an appropriate amount of the protein to be tested, dilute it with the mobile phase, centrifuge, and take the supernatant as the test solution.

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