A method for detecting the purity of a bispecific antibody protein

Abstract

The application provides a method for detecting the purity of a bispecific antibody protein, and a mobile phase is a mixed aqueous solution of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and arginine, 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 well separate the bispecific antibody from polymeric impurities, and has the advantages of rapidness, simplicity, low detection limit, high sensitivity, high stability 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 the purity of a bispecific antibody protein. BACKGROUND

[0002] Macrophages exert phagocytic effect requiring two signals to act simultaneously: one is the activation of "eat me" signal targeting the cell surface, and the other is the inactivation of "don't eat me" signal on the same cell surface. The lack of any one of the signals is not enough to trigger the occurrence of phagocytosis. CD24 is a "don't eat me" signal, tumor cells highly express CD24, which binds to Siglec-10 on the surface of macrophages to release "don't eat me" signal, thereby preventing tumor cells from being phagocytosed by macrophages. CD24 is highly expressed in many tumor cells and belongs to tumor-associated antigens (TAA). A bispecific antibody specifically recognizing CD24 and 4-1BB can specifically activate 4-1BB in the tumor microenvironment and reduce toxicity.

[0003] 4-1BB (CD137, TNFRSF9) is a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS). 4-1BB is expressed on the cell surface in monomer or dimer form, and after binding to its ligand (4-1BBL), it undergoes trimerization for signal transduction, and is a costimulatory molecule of CD8+ and CD4+ T cells, regulatory T cells (Tregs), NK cells and NKT cells, B cells and neutrophils, etc. On T cells, 4-1BB is not constitutively expressed, but is induced after T cell receptor (TCR) activation, and stimulates signal transduction through its natural ligand 4-1BBL or antibody agonist via TNFR-associated factor (TRAF)-2 and TRAF-1. Early signaling of 4-1BB involves K-63 polyubiquitination, activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways, and signaling leads to costimulation of T cells, cell proliferation, cytokine production, maturation and prolonged survival of CD8+ T cells.

[0004] Bispecific antibodies targeting CD24 and 4-1BB can specifically recognize CD24 and 4-1BB, activate the 4-1BB signaling pathway, activate CD8+ T cells, stimulate T cell proliferation and secretion of cytokines, and specifically kill tumor cells expressing CD24. By specifically binding to CD24-expressing tumor cells with anti-CD24 antibodies, the "don't eat me" signal is neutralized. Furthermore, bispecific antibodies can be enriched in the tumor microenvironment. Anti-4-1BB antibodies, acting as 4-1BB activating antibodies, can mimic the formation of a "trimer" from natural 4-1BBL, specifically activating CD8+ T cells and activating 4-1BB-expressing T and NK cells in PBMCs, secreting IL-2 and IFN-γ to achieve targeted killing of tumor cells. However, during cell culture, the protein molecules secreted by the cells may aggregate to varying degrees due to the liquid environment, mechanical environment, and the physicochemical properties of the molecules themselves. This is undesirable for protein production and will affect the yield and product quality. Furthermore, the large molecular weight of protein aggregates may affect their membrane-penetrating properties, leading to reduced bioavailability and potential immunogenicity, posing potential risks to patients after administration. Therefore, to further ensure the quality and safety of bispecific antibody drugs, it is necessary to establish a rapid, simple, sensitive, and stable detection method. Summary of the Invention

[0005] This invention provides a rapid, simple, low-detection-limit, high-sensitivity, and highly stable detection method for the purity detection of bispecific antibody proteins.

[0006] This invention provides a method for detecting the purity of bispecific antibody proteins. The method includes the following steps: Chromatographic conditions: the chromatographic column is packed with gel; the mobile phase is a mixed aqueous solution of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and arginine; wherein the concentration of disodium hydrogen phosphate dodecahydrate is 40-80 mM, and the concentration of sodium dihydrogen phosphate dihydrate is 20-60 mM; isocratic elution is used as the elution mode, and the chromatogram is recorded.

[0007] In some embodiments, the concentration of the 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 61 mM, and the concentration of sodium dihydrogen phosphate dihydrate is 39 mM.

[0010] In some embodiments, the arginine concentration is 100–300 mM; preferably, the arginine concentration is 150–250 mM; more preferably, the arginine concentration is 200 mM.

[0011] In some embodiments, the pH of the mobile phase is 6.5 to 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 3.5μm column.

[0013] In some embodiments, the method further includes the following steps: a mobile phase flow rate of 0.2–0.8 mL / min; a column temperature of 25–35 °C; an injection volume of 25–35 μL; a detection wavelength of 274–286 nm; and a sample chamber temperature of 5–15 °C.

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

[0015] In some implementations, the column temperature is 30°C.

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

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

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

[0019] In some embodiments, the bispecific antibody comprises: (a) a first antibody or an antigen-binding fragment thereof that specifically binds to CD24, and (b) a second antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB.

[0020] In some embodiments, the first antibody or its antigen-binding fragment comprises a heavy chain and a light chain, and the second antibody or its antigen-binding fragment comprises an scFv; wherein the scFv is attached to the C-terminus of the heavy chain of the first antibody or its antigen-binding fragment.

[0021] In some embodiments, the first antibody or its antigen-binding fragment comprises HCDR1, HCDR2 and HCDR3 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 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0022] In some embodiments, the first antibody includes a heavy chain variable region VH as shown in SEQ ID NO:13 and a light chain variable region VL as shown in SEQ ID NO:14.

[0023] In some embodiments, the scFv comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0024] In some embodiments, the scFv includes a heavy chain variable region VH as shown in SEQ ID NO:15, and a light chain variable region VL as shown in SEQ ID NO:16.

[0025] In some embodiments, the concentration of the heterodimeric protein is 3 mg / mL.

[0026] In some implementations, the method further includes the following steps: (1) preparation of mobile phase: weigh an appropriate amount of disodium hydrogen phosphate dodecahydrate, an appropriate amount of sodium dihydrogen phosphate dihydrate, and an appropriate amount of arginine, dissolve them in water, filter, and obtain the solution; (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. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of a bispecific antibody.

[0028] Figure 2 The image shows the protein chromatographic detection pattern in Comparative Example 1.

[0029] Figure 3 The protein chromatogram is shown in Comparative Example 2.

[0030] Figure 4 The image shows the protein chromatographic detection pattern in Comparative Example 3.

[0031] Figure 5 The protein chromatogram is shown in Comparative Example 4.

[0032] Figure 6 This is the protein chromatographic detection chromatogram from Example 1. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. It should also be understood that the terminology used in the embodiments of the present invention is for describing specific implementations and not for limiting the scope of protection of the present invention. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0034] Comparative Example 1

[0035] 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 bring the volume to 1 L. Filter through a 0.45 μm filter membrane to obtain the mobile phase. Prepare the test protein (i.e., the bispecific antibody solution) according to Table 1. The structure of the bispecific antibody that specifically recognizes CD24 and 4-1BB is shown below. Figure 1 As shown in the figures. The HCDR1, HCDR2, and HCDR3 sequences of the heavy chain of the anti-CD24 antibody are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. The HCDR1, HCDR2, and HCDR3 sequences of the variable region (VH) of the heavy chain of the anti-4-1BB antibody scFv are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. The LCDR1, LCDR2, and LCDR3 sequences of the variable region (VL) of the light chain of the anti-4-1BB antibody scFv are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. The LCDR1, LCDR2, and LCDR3 sequences of the light chain of the anti-CD24 antibody are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The anti-CD24 antibody heavy chain and the anti-4-1BB antibody scFv are linked using the (GGGGS)3 linker peptide, and the VH and VL in the scFv are also linked using the (GGGGS)3 linker peptide. The heavy chain sequence of the bispecific antibody is shown in SEQ ID NO:17, and the light chain sequence is shown in SEQ ID NO:18.

[0036] Table 1. Protein components and their contents to be tested

[0037]

[0038]

[0039] Take the protein to be tested, dilute it with the mobile phase to a protein concentration of 3 mg / mL, centrifuge at 12000 rpm for 5 min, and collect the supernatant to obtain the test solution. A high-performance liquid chromatograph (HPLC) was used, Waters E2695. A TSK gel G3000SWXL (300 mm × 7.8 mm) gel size exclusion column was used, with isocratic elution using the mobile phase at a flow rate of 1.0 mL / min for 16 min. The injection volume was 30 μL, the column temperature was 25℃, the sample pan temperature was 10℃, and a UV detector was used at a detection wavelength of 280 nm.

[0040] The content of immunoglobulin monomers and macromolecular polymers (HMWs) was calculated using the area normalization method.

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

[0042] Macromolecular polymer content = (Macromolecular polymer peak area / Total peak area) × 100%

[0043] The results are as follows Figure 2 As shown, the content of macromolecular polymer impurities was 2.34%, and the separation degree between immunoglobulin monomers and macromolecular polymer impurities was 1.25, which failed to achieve baseline separation. This method is not suitable for product quality control.

[0044] Comparative Example 2

[0045] 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 bring the volume to 1 L. Filter through a 0.45 μm filter membrane to obtain the mobile phase. Take the test protein (prepared in the same way as Comparative Example 1), dilute with the mobile phase to a protein concentration of 3 mg / mL, centrifuge at 12000 rpm for 5 min, and collect the supernatant to obtain the test solution. A high-performance liquid chromatograph (HPLC) system (Waters, model E2695) was used. The HPLC system was an Agilent AdvanceBio SEC. A 2.7 μm (300 mm × 7.8 mm) column was used, with isocratic elution of the mobile phase at a flow rate of 0.5 mL per minute for 30 minutes. The injection volume was 30 μL, the column temperature was 30 °C, the sample pan temperature was 10 °C, and an ultraviolet detector was used with a detection wavelength of 280 nm.

[0046] The content of immunoglobulin monomers and macromolecular polymers (HMWs) was calculated using the area normalization method.

[0047] Immunoglobulin monomer content = (main peak area / total peak area) × 100%

[0048] Macromolecular polymer content = (Macromolecular polymer peak area / Total peak area) × 100%

[0049] The results are as follows Figure 3As shown, the content of macromolecular polymer impurities is 1.67%, and the separation degree between immunoglobulin monomers and macromolecular polymer impurities is 2.54. However, there is a tailing phenomenon after the peak of immunoglobulin monomers, with a peak tailing factor reaching 1.652. The protein interacts with the charged stationary phase on the surface, and these interactions cause the protein sample to be adsorbed, resulting in peak tailing. This method is not suitable for product quality control.

[0050] Comparative Example 3

[0051] 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 bring to a final volume of 1 L. Filter through a 0.45 μm filter membrane to obtain the mobile phase. Take the test protein (prepared in the same way as Comparative Example 1), dilute with the mobile phase to a protein concentration of 3 mg / mL, centrifuge at 12000 rpm for 5 min, and collect the supernatant to obtain the test solution. A high-performance liquid chromatograph (HPLC) was used, Waters E2695. A Waters XBridge BEH SEC (…) was employed. A 3.5 μm (300 mm × 7.8 mm) chromatographic column was used, with isocratic elution of the mobile phase at a flow rate of 0.5 mL per minute for 30 minutes. The injection volume was 30 μL, the column temperature was 30 °C, the sample pan temperature was 10 °C, and an ultraviolet detector was used with a detection wavelength of 280 nm.

[0052] The content of immunoglobulin monomers and macromolecular polymers (HMWs) was calculated using the area normalization method.

[0053] Immunoglobulin monomer content = (main peak area / total peak area) × 100%

[0054] Macromolecular polymer content = (Macromolecular polymer peak area / Total peak area) × 100%

[0055] The results are as follows Figure 4 As shown, the content of macromolecular polymer impurities is 2.20%, and the separation degree between immunoglobulin monomers and macromolecular polymer impurities is 2.66. However, there is a tailing phenomenon after the peak of immunoglobulin monomers, with a peak tailing factor reaching 1.522. The protein interacts with the charged stationary phase on the surface, and these interactions cause the protein sample to be adsorbed, resulting in peak tailing. This method is not suitable for product quality control.

[0056] Comparative Example 4

[0057] 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 bring to a final volume of 1 L. Filter through a 0.45 μm filter membrane to obtain mobile phase A. Mobile phase B is acetonitrile. Take the test protein (prepared in the same way as Comparative Example 1), dilute with mobile phase A to a protein concentration of 3 mg / mL, centrifuge at 12000 rpm for 5 min, and collect the supernatant to obtain the test solution. A high-performance liquid chromatograph (HPLC) was used, Waters E2695. A Waters XBridge BEH SEC (…) was employed. A 3.5 μm (300 mm × 7.8 mm) column was used for isocratic elution with mobile phase A:mobile phase B = 90:10 (v / v), the flow rate was 0.5 mL / min, the elution time was 30 min, the injection volume was 30 μL, the column temperature was 30 °C, the sample pan temperature was 10 °C, and an ultraviolet detector was used with a detection wavelength of 280 nm.

[0058] The content of immunoglobulin monomers and macromolecular polymers (HMWs) was calculated using the area normalization method.

[0059] Immunoglobulin monomer content = (main peak area / total peak area) × 100%

[0060] Macromolecular polymer content = (Macromolecular polymer peak area / Total peak area) × 100%

[0061] The results are as follows Figure 5 As shown, the content of macromolecular polymer impurities was 1.78%, and the separation degree between immunoglobulin monomers and macromolecular polymer impurities was 2.76. However, a significant small molecule fragment peak appeared after the immunoglobulin monomer peak, with a small molecule fragment impurity content of 0.81%. The use of acetonitrile did not improve the adsorption between the sample and the stationary phase and destroyed the native state 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 34.84 g of L-Arginine. Dissolve in water and bring the volume to 1 L. Filter through a 0.45 μm filter membrane to obtain the mobile phase. Take the test protein (prepared in the same way as Comparative Example 1), dilute with the stock buffer (preparation method shown in Table 3) to a protein concentration of 3 mg / mL, centrifuge at 12000 rpm for 5 min, and collect the supernatant to obtain the test solution. A high-performance liquid chromatograph (HPLC) was used, Waters E2695. A Waters XBridge BEH SEC (…) was employed. A 3.5 μm (300 mm × 7.8 mm) chromatographic column was used, with isocratic elution of the mobile phase at a flow rate of 0.5 mL per minute for 30 minutes. The injection volume was 30 μL, the column temperature was 30 °C, the sample pan temperature was 10 °C, and an ultraviolet detector was used with a detection wavelength of 280 nm.

[0064] The content of immunoglobulin monomers and macromolecular polymers (HMWs) was calculated using the area normalization method.

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

[0066] Macromolecular polymer content = (Macromolecular polymer peak area / Total peak area) × 100%

[0067] The results are as follows Figure 6 As shown, the content of macromolecular polymer impurities was 2.02%, the content of immunoglobulin monomers was 97.97%, the separation degree between immunoglobulin monomers and macromolecular polymer impurities was 2.37, and the peak tailing factor of immunoglobulin monomers was 1.363. This is because arginine was added to the mobile phase. Arginine can bind to the analyte, thereby hindering the interaction between the analyte and the stationary phase.

[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 stock buffer (preparation is shown in Table 3) to a protein concentration of about 3 mg / mL, mix well to obtain the system suitability solution.

[0070] One syringe of blank solution (i.e., stock solution buffer) and six syringes of system suitability solution were administered. The results are shown in Table 2.

[0071] Table 2 System Suitability Test Results

[0072] Injection Main peak retention time (min) Main peak peak area Main peak to aggregate peak resolution Blank / / / 1 13.217 18799465 2.68 2 13.213 18770855 2.68 3 13.218 18782598 2.68 4 13.216 18791368 2.67 5 13.219 18785958 2.69 6 13.225 18794112 2.68 RSD (%) 0.030 0.054 /

[0073] It can be seen that the percentage of main peak area RSD is ≤2.0%, the retention time of main peak RSD is ≤2.0%, and the separation degree between main peak and aggregate peak is ≥1.5, indicating good system applicability.

[0074] Example 3 Specificity Test

[0075] Preparation of specific solution: Take the test protein (the test protein is prepared in the same way as Comparative Example 1), dilute it with the stock buffer (as above) to a protein concentration of 3 mg / mL, and mix well to obtain the specific solution.

[0076] Preparation of blank solutions for each buffer: Take each buffer (stock buffer, affinity chromatography (PA) buffer, virus inactivation (VI) buffer, anion exchange chromatography (AEX) buffer, cation exchange chromatography (CEX) buffer, nanofiltration exchange buffer (VF) buffer, ultrafiltration exchange buffer (UF / DF) buffer) as blank solutions for each buffer.

[0077] Table 3 Buffer Preparation

[0078]

[0079] One injection was administered with the specific solution and blank solutions of each buffer solution. The results are shown in Table 4.

[0080] Table 4 Results of specificity test

[0081] Condition Main peak peak area High molecular weight aggregate content (%) Immunoglobulin monomer content (%) Stock buffer / / / Affinity chromatography (PA) buffer / / / Virus inactivation (VI) buffer / / / Anion exchange chromatography (AEX) buffer / / / Cation exchange chromatography (CEX) buffer / / / Virus filtration (VF) buffer / / / Ultrafiltration / diafiltration (UF / DF) buffer / / / Specificity solution 18729831 0.7 99.3

[0082] No peaks appeared at the specific solution elution positions for each buffer blank solution, thus not interfering with detection. Specificity is good.

[0083] Example 4 Limit of Quantitation Test

[0084] Preparation of limit of quantitation solution: Take the protein to be tested (prepared in the same way as comparative example 1), dilute it with the stock buffer (as above) to a protein concentration of 0.004 mg / mL, centrifuge at 12000 rpm for 5 min, take the supernatant as the limit of quantitation solution, and prepare 6 parallel solutions.

[0085] Six solutions with the limit of quantitation were injected once each. The limit of quantitation was determined with a signal-to-noise ratio of approximately 10:1 for the main peak. The amount that could be reliably detected was calculated. The experimental results are shown in Table 5.

[0086] Table 5 Results of Limit of Quantitation Test

[0087]

[0088] The signal-to-noise ratio of the limit-of-quantitation (LOQ) solutions was greater than 10. The RSD of the retention time of the main peak in the six LOQ solutions was less than 10.0%, the RSD of the peak area of ​​the main peak was less than 15.0%, and the LOQ concentration was 0.004 mg / mL, accounting for 0.13% of the concentration of the test sample. The sensitivity of this method is sufficient to detect the content of polymers and immunoglobulin monomers in this product.

[0089] Example 5: Linearity and Range Test

[0090] Preparation of linear solutions: Take the protein to be tested (prepared in the same way as Comparative Example 1), dilute it with the stock buffer (as above) to a protein concentration of 0.004 mg / mL, 0.3 mg / mL, 1.2 mg / mL, 2.4 mg / mL, 3.0 mg / mL and 4.5 mg / mL, mix well to prepare linear solutions, and prepare two copies of each concentration.

[0091] Take each linear solution, inject one injection into each linear solution, detect and record the chromatogram, and perform linear regression on the sample concentration using the peak area of ​​the main peak, obtain the regression equation and correlation coefficient (R2), and examine the linearity of this method. The experimental results are shown in Table 6.

[0092] Table 6. Results of Linearity and Range Tests

[0093]

[0094] The linear regression equation was Y = 6226415X + 88705.33, with a correlation coefficient R² > 0.99. The linear relationship was good in the protein concentration range of 0.004–4.5 mg / mL (LOQ-150%).

[0095] Example 6 Accuracy Test

[0096] Accuracy solution preparation: Take the protein to be tested (preparation method is the same as Comparative Example 1), dilute the sample with stock buffer (as above), and prepare solutions with concentrations of 80% (2.4 mg / mL), 100% (3.0 mg / mL) and 120% (3.6 mg / mL) respectively. Mix well as the accuracy solutions, and prepare 3 copies of each concentration.

[0097] Inject one sample of each accuracy solution, detect and record the chromatogram. Inject one sample of each accuracy solution, record the percentage of polymer and main peak area, and calculate the relative standard deviation of the main peak area percentage to examine the accuracy of the method. Specific experimental results are shown in Table 7.

[0098] Table 7 Accuracy Test Results

[0099]

[0100]

[0101] The peak area percentage RSD of the main peak is less than 2.0%, indicating good accuracy within the range of 80% to 120%.

[0102] Example 7 Repeatability Test

[0103] Preparation of repeatability solutions: Take the protein to be tested (prepared in the same way as comparative example 1), dilute it with the stock buffer (as above) to a protein concentration of about 3 mg / mL, mix well to prepare a repeatability solution, and prepare 6 parallel solutions.

[0104] Inject one sample of each reproducible solution, detect and record the chromatogram, record the percentage of polymer and main peak area, and calculate the relative standard deviation of the main peak area percentage to examine the repeatability of the method. Specific results are shown in Table 8.

[0105] Table 8 Results of Repeatability Tests

[0106] Number Aggregate peak peak area percent (%) Main peak peak area percent (%) 1 0.7 99.3 2 0.7 99.3 3 0.7 99.3 4 0.7 99.3 5 0.7 99.3 6 0.7 99.3 RSD (%) / 0.0

[0107] The percentage of peak area of ​​the 6-needle main peak is less than 2.0%, indicating good repeatability.

[0108] Example 8 Intermediate Precision Test

[0109] Preparation of intermediate precision solution: Prepare intermediate precision solution according to the method for preparing repeatable solution.

[0110] The experimenters were changed to experimenters A and B, and the intermediate precision of the method was examined according to the repeatability test procedure. The percentage of polymer and main peak area was recorded, and the relative standard deviation (RSD%) of the main peak area percentage was calculated to examine the intermediate precision of the method. The specific results are shown in Table 9.

[0111] Table 9 Summary of Precision Test Results

[0112]

[0113]

[0114] The RSD of the main peak area percentage in the 12-needle intermediate precision solution was <2.0%, indicating good intermediate precision.

[0115] Example 9 Durability Test

[0116] Preparation of test solution: Take the protein to be tested (preparation method is the same as Comparative Example 1), dilute it with stock buffer (as above) to a protein concentration of about 3 mg / mL, and mix well to prepare a robustness solution.

[0117] Fine-tuning the wavelength and column temperature, the test sample solutions were analyzed to examine the robustness of the method. The standard conditions were a wavelength of 280 nm and a column temperature of 30 °C. Based on these standard conditions, the test wavelengths were changed to 278 nm and 282 nm, and the column temperatures were changed to 25 °C and 35 °C. Chromatograms were detected and recorded, including the percentage of polymer and main peak area. The relative standard deviation (RSD) of the main peak area percentage was calculated to examine the impact of different conditions on the sample detection results, thereby determining the robustness of the method. The experimental results are shown in Table 10.

[0118] Table 10 Durability Test Results

[0119]

[0120] Under all conditions, the percentage of the main peak area (RSD) is less than 2.0%, indicating good durability.

[0121] Example 10 Solution Stability

[0122] Preparation of stability solution: Take the protein to be tested (prepared in the same way as comparative example 1), dilute it with the stock buffer (as above) to a protein concentration of about 3 mg / mL, and mix well to obtain the stability solution.

[0123] The stability solution was taken and placed at 10℃. Samples were injected at 8h, 16h, 24h, and 30h after placement, with one injection at each time point, to investigate the stability of the solution at 10℃. The experimental results are shown in Table 11.

[0124] Table 11 Results of solution stability test

[0125] Stability condition Aggregate peak peak area percent (%) Main peak peak area percent (%) 8h 0.7 99.3 16h 0.7 99.3 24h 0.7 99.3 30h 0.7 99.3 RSD % / 0.0

[0126] At each time point, the percentage of the main peak area (SD) was less than 2.0%, indicating that the solution had good stability at 10℃ for 30 hours.

[0127] It is evident that this method exhibits strong specificity, high accuracy, good precision, good linearity within an appropriate range, good robustness, and good solution stability, making it suitable for the detection of protein purity.

[0128] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for detecting the purity of a bispecific antibody protein, characterized in that, The method comprises the following steps: The chromatographic conditions are as follows: the chromatographic column is filled with gel, and the chromatographic column is a Waters XBridge BEH SEC, 200Å, 3.5 µm, 300 mm*7.8 mm; the mobile phase is a mixed aqueous solution of twelve sodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate and arginine, wherein the concentration of twelve sodium hydrogen phosphate dihydrate is 40-80 mM, the concentration of sodium dihydrogen phosphate dihydrate is 20-60 mM, isocratic elution is used as the elution mode, the detection wavelength is 274-286 nm, and a chromatogram is recorded; the bispecific antibody comprises: (a) a first antibody or an antigen binding fragment thereof specifically binding to CD24, and (b) a second antibody or an antigen binding fragment thereof specifically binding to 4-1BB; the first antibody or the antigen binding fragment thereof comprises a heavy chain and a light chain, and the second antibody or the antigen binding fragment thereof comprises an scFv; wherein the scFv is connected to the C-terminal of the heavy chain of the first antibody or the antigen binding fragment thereof, the first antibody or the antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and comprises LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; and the scFv comprises HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, and comprises LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:

12.

2. The detection method according to claim 1, characterized in that, The concentration of the twelve sodium hydrogen phosphate dihydrate is 50-70 mM.

3. The method of claim 1, wherein, The concentration of the sodium dihydrogen phosphate dihydrate is 30-50 mM.

4. The assay of any one of claims 1-3, wherein, The concentration of the arginine is 100-300 mM.

5. The detection method according to claim 4, characterized in that, The concentration of the arginine is 150-250 mM.

6. The method of claim 1, wherein, The pH of the mobile phase is 6.5-7.

5.

7. The method of claim 1, wherein, 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 °C; the injection volume is 25-35 µL; and the sample chamber temperature is 5-15 °C.

8. The method of claim 1, wherein, The method further comprises the following steps: (1) Preparation of the mobile phase: an appropriate amount of twelve sodium hydrogen phosphate dihydrate, an appropriate amount of sodium dihydrogen phosphate dihydrate and an appropriate amount of arginine are weighed, dissolved in water, and filtered to obtain the mobile phase; (2) Preparation of the test sample solution: an appropriate amount of the protein to be tested is diluted with the mobile phase, centrifuged, and the supernatant is taken as the test sample solution.

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