Method for detecting residual quantity of dextran sulfate sodium in antibody

Abstract

The invention discloses a method for detecting the residual quantity of dextran sulfate sodium in an antibody. Specifically, the invention provides a method for detecting the residual quantity of dextran sulfate sodium in an antibody, which comprises the following steps: removing protein in the antibody, and reacting with a 2-aminopyridine solution; a high performance liquid chromatography method is adopted to detect a test sample and sodium dextran sulfate standard substances with different concentrations. The detection method disclosed by the invention is low in detection limit, high in accuracy and suitable for analyzing the residual quantity of dextran sulfate in the antibody.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a method for detecting the residual amount of sodium dextran sulfate. Background Art

[0002] Dextran sulfate is an anionic derivative of dextran, formed by the esterification reaction of dextran and chlorosulfonic acid, and exists in the form of sodium salt, also known as dextran sulfate sodium, dextran sulfate sodium or DSS (dextran sulfate sodium). The specific structure is shown in Formula I.

[0003]

[0004] Emicizumab is a modified humanized IgG4 monoclonal antibody produced in CHO cells by recombinant DNA technology for the treatment of hemophilia A. CHO cells (Chinese hamster ovary cells) are the main mammalian expression system for the production of recombinant proteins and antibodies. However, when CHO cells are cultured in vitro, cell agglomeration is prone to occur, which affects cell growth. Dextran sulfate sodium is a common anti-cell agglomeration agent that can promote single cell suspension by changing the charge on the cell surface, prevent cell agglomeration, support high-density cell culture, improve cell viability, and increase protein expression.

[0005] However, sodium dextran sulfate has certain toxicity and is one of the most common and effective drugs that induce ulcerative colitis. DSS can toxicly damage the colonic epithelial barrier and damage the integrity of the colonic epithelial barrier, thereby increasing the permeability of the intestinal mucosa, allowing large molecules to easily penetrate into the intestinal mucosal tissue, thereby triggering a series of inflammatory reactions. Therefore, dextran sulfate needs to be removed during the purification process and its residual level needs to be tested.

[0006] The document Yoshio Araki, Akira Andoh, et al. Application of 2-aminopyridine fluorescence labeling in the analysis of in vivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography. Journal of Chromatography B, 2001, 209-215 discloses a method of using 2-aminopyridine to react with dextran sulfate for labeling, and then using the physical property of dextran sulfate being insoluble in ethanol, adding ethanol to the reaction solution to form a precipitate containing dextran sulfate, adding water to dissolve it, and using gel chromatography to detect the dextran sulfate content. However, since dextran sulfate sodium will bind to proteins in antibodies, it is not feasible to separate and detect it using chromatography alone. In addition, the applicant found that the detection limit of the document method is relatively high. According to the document method, the product after the reaction of 2-aminopyridine and dextran sulfate is precipitated and then dissolved by ethanol, and the residual amount at the microgram level cannot be analyzed, which is not suitable for residual detection.

[0007] Therefore, it is necessary to establish a detection method with a low detection limit and suitable for the residual amount of sodium dextran sulfate in antibodies. Summary of the invention

[0008] The present invention aims to provide a method for detecting the residual amount of sodium dextran sulfate in an antibody which has the advantages of simple operation, low detection limit and high accuracy.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A method for detecting the residual amount of sodium dextran sulfate in an antibody comprises the following steps:

[0011] (1) Add trichloroacetic acid solution to the antibody to precipitate the antibody, and centrifuge to obtain the supernatant;

[0012] (2) taking the supernatant in step (1), adding 2-aminopyridine solution and incubating; then adding sodium cyanoborohydride solution to react, thereby obtaining a test solution;

[0013] (3) The test solution was tested by high performance liquid chromatography and the content was calculated according to the standard curve method;

[0014] The final concentration of 2-aminopyridine in the reaction system of step (2) is 50 μg / mL to 2000 μg / mL.

[0015] In some embodiments, the final concentration of 2-aminopyridine in the reaction system of step (2) is 50 μg / mL to 1000 μg / mL, preferably 50 μg / mL to 700 μg / mL, and more preferably 500 μg / mL.

[0016] In some embodiments, the standard curve of step (3) is obtained as follows: step (4) adding trichloroacetic acid solution and 2-aminopyridine solution to sodium dextran sulfate standard of gradient concentrations; then adding sodium cyanoborohydride solution to react to obtain sodium dextran sulfate standard solutions of different concentrations; using high performance liquid chromatography to detect the sodium dextran sulfate standard solution, and fitting by the least squares method to obtain the standard curve.

[0017] In some embodiments, the final concentration of the sodium dextran sulfate standard in the reaction system of step (4) is in the range of 0.5 μg / mL to 1000 μg / mL.

[0018] In some embodiments, the final concentration of 2-aminopyridine in the reaction system of step (4) is the same as the final concentration of 2-aminopyridine in the reaction system of step (2).

[0019] In some embodiments, the final concentration of the sodium dextran sulfate standard in step (4) ranges from 0.5 μg / mL to 750 μg / mL, preferably from 1 μg / mL to 750 μg / mL, and more preferably from 1 μg / mL to 100 μg / mL. In some specific embodiments, the final concentration of the sodium dextran sulfate standard in step (4) is 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 50 μg / mL, and 100 μg / mL, respectively.

[0020] In some embodiments, the final concentration of sodium cyanoborohydride in the reaction system of step (2) and step (4) is 10 μg / mL to 100 μg / mL, preferably 20 μg / mL to 40 μg / mL, and more preferably 30 μg / mL.

[0021] In some embodiments, the incubation temperature in step (2) and step (4) is 80°C to 120°C, preferably 100°C; the incubation time is 10 min to 20 min, preferably 13 min.

[0022] In some embodiments, the reaction temperature in step (2) and step (4) is 80°C to 120°C, preferably 90°C; the reaction time is 15h to 40h, preferably 18h to 22h, more preferably 20h.

[0023] In some embodiments, the mass concentration of the trichloroacetic acid solution in step (2) and step (4) is 20% to 50%, preferably 20% to 35%; more preferably 20%.

[0024] In some embodiments, the trichloroacetic acid solution is prepared by adding trichloroacetic acid to water.

[0025] Preferably, the trichloroacetic acid solution in step (4) is a trichloroacetic acid solution whose pH is adjusted to 6.0-6.7 by alkali; in some embodiments, the trichloroacetic acid solution with a pH of 6.0-6.7 is prepared by dissolving trichloroacetic acid in water and then adding NaOH to adjust the pH to 6.0-6.7.

[0026] In some embodiments, the solvents of the 2-aminopyridine solution, the sodium cyanoborohydride solution, and the sodium dextran sulfate standard solution are all water.

[0027] In some embodiments, the incubation temperature, incubation time, reaction temperature, reaction time and final concentration of sodium cyanoborohydride in the reaction system in step (2) and step (4) are the same.

[0028] In some specific embodiments, the steps (2) and (4) further comprise centrifuging and collecting the supernatant after the reaction.

[0029] In some specific embodiments, the step (2) and step (4) do not include the step of adding ethanol precipitation after the reaction with sodium cyanoborohydride.

[0030] In some embodiments, the chromatographic conditions of the high performance liquid chromatography are: the chromatographic column is a size exclusion chromatography column, and the pore size of the chromatographic column is Preferably

[0031] And / or, the mobile phase is a phosphate buffer with a pH of 3.0 to 7.0, preferably a pH of 5.0 to 7.0; more preferably 5.0; further preferably 4.4 g / L disodium hydrogen phosphate dodecahydrate and 13.7 g / L sodium dihydrogen phosphate dihydrate as the mobile phase, isocratic elution;

[0032] And / or, the column temperature in the high performance liquid chromatography method is 30°C to 60°C, preferably 40°C to 60°C, more preferably 50°C;

[0033] and / or, the flow rate is 0.8 mL / min to 1.5 mL / min, preferably 1.2 mL / min;

[0034] and / or, the injection volume is 30 μL to 50 μL, preferably 40 μL;

[0035] And / or, the detector is a fluorescence detector with an excitation wavelength of 320 nm and an emission wavelength of 400 nm.

[0036] In some specific embodiments, the chromatography column is Sepax Zenix SEC-150 (7.8x300 mm).

[0037] In some embodiments, the antibody is emicizumab.

[0038] In some embodiments, the antibody preparation is prepared by diluting the emicizumab injection solution with a PBS buffer having a pH value of 5.5 to 7.5.

[0039] A method for detecting the residual amount of sodium dextran sulfate in emicizumab comprises the following steps:

[0040] 1) Preparation and determination of sodium dextran sulfate standard solution: adding trichloroacetic acid solution and 2-aminopyridine solution to sodium dextran sulfate standard solution with gradient concentrations; then adding sodium cyanoborohydride solution to react to obtain sodium dextran sulfate standard solutions of different concentrations; using high performance liquid chromatography to detect the sodium dextran sulfate standard solution, and fitting by the least square method to obtain a standard curve;

[0041] 2) Preparation of the test solution: Add PBS buffer with a pH value of 5.5 to 7.5 to dilute the emicizumab injection, add trichloroacetic acid solution to precipitate the antibody, centrifuge and take the supernatant; add 2-aminopyridine solution for incubation; then add sodium cyanoborohydride solution for reaction, centrifuge and take the supernatant to obtain the test solution;

[0042] 3) Determination of sodium dextran sulfate in emicizumab: The test solution was detected by high performance liquid chromatography, and the peak area in the test solution was substituted into the above standard curve to calculate the concentration of dextran sulfate in the test solution; the concentration of sodium dextran sulfate in emicizumab was obtained by multiplying the concentration of the test solution by the total dilution factor;

[0043] The final concentration of 2-aminopyridine in the reaction system of steps 1) and 2) is 50 μg / mL to 2000 μg / mL.

[0044] In some embodiments, the final concentration of 2-aminopyridine in the reaction system of steps 1) and 2) is 50 μg / mL to 1000 μg / mL, preferably 50 μg / mL to 700 μg / mL, and more preferably 500 μg / mL.

[0045] In some embodiments, in the method for detecting the residual amount of sodium dextran sulfate in emicizumab, the final concentration range of the sodium dextran sulfate standard in the reaction system of step 1) is 0.5 μg / mL to 1000 μg / mL, preferably 0.5 μg / mL to 750 μg / mL, more preferably 1 μg / mL to 750 μg / mL, and further preferably 1 μg / mL to 100 μg / mL.

[0046] In some embodiments, the final concentration of sodium cyanoborohydride in the reaction system of steps 1) and 2) is 10 μg / mL to 100 μg / mL, preferably 20 μg / mL to 40 μg / mL, and more preferably 30 μg / mL.

[0047] In some embodiments, steps 1) and 2) do not include the step of adding ethanol for precipitation after the reaction with sodium cyanoborohydride.

[0048] In some embodiments, the incubation temperature in steps 1) and 2) is 80°C to 120°C, preferably 100°C; the incubation time is 10min to 20min, preferably 13min; the reaction temperature in steps 1) and 2) is 80°C to 120°C, preferably 90°C; the reaction time is 15h to 40h, preferably 18h to 22h, more preferably 20h. The mass concentration of the trichloroacetic acid solution in steps 1) and 2) is 20% to 50%, preferably 20% to 35%; more preferably 20%; preferably, the trichloroacetic acid solution in step 1) is a trichloroacetic acid solution adjusted to pH 6.0 to 6.7 by alkali.

[0049] In some embodiments, the pH of the supernatant in step 2) is adjusted to 6.0-6.7 by alkali.

[0050] The technical effects of the present invention are as follows:

[0051] 1 The detection method of the present invention is suitable for detecting the residual amount of dextran sulfate at the microgram level, is easy to operate, saves detection time, and improves detection efficiency.

[0052] 2. By reducing the concentration of 2-aminopyridine, the peak area of ​​the labeled reagent can be reduced during the on-machine detection, thereby reducing damage to the instrument and reducing the operation time.

[0053] 3 The detection method of the present invention has good linear relationship, and the correlation coefficient can reach above 0.99; it has good repeatability, high accuracy, good solution stability and low detection limit.

[0054] the term

[0055] TCA is trichloroacetic acid.

[0056] 2-AP is 2-aminopyridine.

[0057] The term PBS buffer refers to phosphate buffered saline, the main components of which are Na2HPO4, KH2PO4, NaCl and KCl.

[0058] The term "standard curve method" is a quantitative method. When determining the content of a component in a sample, a chromatogram is made using the same chromatographic conditions as those used to draw the standard curve, the chromatographic peak area is measured, and then the component concentration in the sample injected into the chromatographic column is calculated on the standard curve based on the peak area. In this article, a standard curve between the concentration and peak area of ​​sodium dextran sulfate is constructed using the different sodium dextran sulfate standard concentrations as the horizontal axis and the peak area in the chromatogram corresponding to the sodium dextran sulfate standard solutions of different concentrations as the vertical axis.

[0059] The term "final concentration" refers to the concentration in the reaction system.

[0060] Instruction Manual

[0061] Figure 1 This is a graph showing the corresponding peak areas for different labeling reaction times in Example 1;

[0062] Figure 2 This is a graph showing the correspondence between different DSS concentrations and peak areas in Example 3;

[0063] Figure 3 This is a linear relationship diagram for determination of DSS content in Example 4;

[0064] Figure 4 The chromatogram of the sample detection of Sample-3 in Example 4;

[0065] Figure 5 This is the HPLC chromatogram of DSS detection after adding 5 mg / mL 2-AP in Example 6-1;

[0066] Figure 6 For Example 6-1 Figure 5 Subsequent HPLC instrument post-equilibrium diagram;

[0067] Figure 7 This is the HPLC chromatogram detected by DSS after adding 500 μg / mL 2-AP in Example 6-2;

[0068] Figure 8 For Example 6-2 Figure 7 Magnified image of . DETAILED DESCRIPTION

[0069] The present invention is described in more detail below by way of examples, but these specific descriptions are only used to illustrate the technical solution of the present invention and do not constitute any limitation to the present invention.

[0070] The reagents and raw materials used in the present invention are all commercially available. Experimental methods without specific conditions are usually carried out under conventional conditions.

[0071] The method for detecting the content of sodium dextran sulfate in the embodiments adopts HPLC detection, the chromatograph is Agilent 1260 Infinity II, the mobile phase is composed of 4.4 g / L disodium hydrogen phosphate dodecahydrate and 13.7 g / L sodium dihydrogen phosphate dihydrate, isocratic elution, column temperature: 50°C; flow rate 1.2 mL / min; injection volume 40 μL; chromatographic column is Sepax Zenix SEC-150 (7.8x300 mm); Agilent 1260 FLD detector, excitation wavelength 320 nm, emission wavelength 400 nm.

[0072] In the experiment, DSS is dextran sulfate sodium with an average molecular weight of about 5kD.

[0073] Example 1 Effect of different labeling reaction times on dextran sulfate detection results

[0074] The solution was prepared as follows:

[0075] DSS solution (0.2 mg / mL): weigh 20 mg of dextran sulfate sodium, add water to make up to 100 mL, and mix well.

[0076] 2-AP solution (20 mg / mL): weigh 20 mg of 2-aminopyridine, add 1 mL of water, and mix well.

[0077] Sodium cyanoborohydride solution (1 mg / mL): weigh 5 mg of sodium cyanoborohydride, add 5 mL of water, and mix well.

[0078] 20% TCA (pH 6.0-6.7): Weigh 2 g of trichloroacetic acid, dissolve it in water, adjust the pH to 6.0-6.7 with 1 M NaOH, and then make up to 10 mL and mix well.

[0079] Take 25μL of DSS solution, add 5μL of 2-AP solution, add 20μL of 20% TCA (pH6.0~6.7), add water to make up the volume to 194μL, incubate at 100℃ for 13min; add 6μL of sodium cyanoborohydride solution, react at 90℃ for different time. After the reaction is completed, take out the sample, centrifuge at 8000rpm for 5min, take 2 / 3 of the supernatant, and detect by HPLC. The results are shown in Table 1 and Figure 1 shown.

[0080] Table 1 Reaction time of different labels

[0081] Reaction time (h) DSS peak area (LU*s) 1 153.009 3 285.938 5 400.485 18 893.456 20 914.551

[0082] The results showed that the measured peak area increased linearly when heated for 1 h, 3 h, and 5 h, and the peak areas were close to each other at 18 h and 20 h after heating, indicating that the reaction was close to equilibrium.

[0083] Example 2 Effect of Dextran Sulfate Labeling on Dextran Sulfate Detection Results

[0084] 0.2 mg / mL DSS solution, 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0085] Take 1 μL of DSS solution, add different volumes of 2-AP solution, then add 20 μL of 20% TCA solution with a concentration of 200 mg / mL and pH 6.0, add water to make the final volume of the solution 194 μL, incubate at 100°C for 13 min; add 6 μL of 1 mg / mL sodium cyanoborohydride, heat at 90°C for 20 h, centrifuge to take the supernatant, and detect by HPLC. The results are shown in Table 2.

[0086] Table 2 The influence of different ratios of DSS and 2-AP on the detection results

[0087]

[0088]

[0089] The results showed that when the molar ratio of 2-AP:DSS was higher than 2500:1, the peak area with the corresponding peak was detected, and DSS at a concentration of 1 μg / mL could be detected.

[0090] Example 3 Dextran sulfate detection linear range

[0091] 0.2 mg / mL DSS solution, 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0092] Take different volumes of DSS solution, add 5 μL of 2-AP solution, then add 20 μL of 20% TCA solution at pH 6.0, add water to make the final volume of the solution 194 μL, incubate at 100°C for 13 min; add 6 μL of sodium cyanoborohydride, heat at 90°C for 15 h, centrifuge and take the supernatant, and detect the content of dextran sulfate by HPLC. The results are shown in Table 3 and Figure 2 .

[0093] Table 3 Peak area results at different DSS concentrations

[0094]

[0095] The results show that the linear correlation coefficient R 2Greater than 0.99, the linear relationship is good. Too high DSS concentration will cause the sample response to exceed the upper limit of the peak height under the current gain, resulting in inaccurate peak area.

[0096] Example 4 Detection of protein samples

[0097] 1. Preparation and determination of DSS standard solution

[0098] 0.2 mg / mL DSS solution, 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0099] Take different volumes of DSS solution, add 5 μL of 2-AP solution, then add 20 μL of 20% TCA solution with pH 6.0, add water to make the final volume of the solution 194 μL, incubate at 100°C for 13 min; add 6 μL of sodium cyanoborohydride, heat at 90°C for 20 h, centrifuge and take the supernatant to obtain the standard solution, and use HPLC to detect the content of dextran sulfate. The specific sample addition amount is shown in Table 4. Using the different concentrations of sodium dextran sulfate standards as the horizontal axis and the peak area in the chromatogram corresponding to the different concentrations of sodium dextran sulfate standard solutions as the vertical axis, a linear fit was performed to obtain the linear relationship formula of the standard curve y=38.673x+6.4943. The specific results are shown in Tables 5 and Figure 3 As shown in Figure 6.8-7.0min is the DSS peak.

[0100] Table 4: Sample volume of each solution

[0101]

[0102] Table 5 Peak area values ​​at different DSS concentrations

[0103]

[0104] 2. Determination of DSS in samples

[0105] 20 mg / mL 2-AP solution and 1 mg / mL sodium cyanoborohydride solution were prepared as in Example 1.

[0106] 20% TCA (pH not adjusted): weigh 2 g of trichloroacetic acid, add water to make up to 10 mL, and mix well.

[0107] Protein product solution: The sample is 30 mg (1 ml) of emicizumab injection. Take the sample and add PBS buffer with a pH value of 7.4 to dilute it to 1.5 mg / mL to obtain the protein product solution.

[0108] Determination of the test sample: Take 0.5mL of the protein product solution, add 0.25mL of water, vortex mix, add 0.25mL of 20% TCA (pH not adjusted) at a concentration of 200mg / mL to precipitate the protein, centrifuge and take 0.5mL of the supernatant, adjust the pH to between 6.0 and 6.7, and then make up to 1mL. Take 100μL of the above pH-adjusted solution, add 5μL of 2-AP solution and 89μL of water. Incubate at 100℃ for 13min; add 6μL of sodium cyanoborohydride, heat at 90℃ for 20h, centrifuge and take the supernatant to obtain the test solution, and detect by HPLC. Substitute the peak area in the test sample into the linear relationship formula obtained in the above "Preparation of standard solution" to calculate the concentration of dextran sulfate in the test solution. Subtract the concentration of the test solution * the total dilution factor (160 times) to obtain the DSS concentration in the sample. The results are shown in Table 6. The sample detection chromatogram of Sample-3 is shown in Figure 4 .

[0109] Table 6 DSS concentration in different protein samples

[0110]

[0111] 3. Accuracy

[0112] Take the above sample sample-3 for spike recovery test. The specific operation is that the sample is 30mg (1ml) of emicizumab injection, take the sample, add PBS buffer with a pH value of 7.4 to dilute to 1.5mg / mL, and obtain the protein product solution. Take 2 portions of 0.5mL of the protein product, add 3.2mg / mL and 6.4mg / mL DSS solution (prepared in water) respectively, and add 20% TCA, 2-AP and sodium cyanoborohydride to the rest, and operate as in Example 4 under "Determination of DSS in the sample". The results are shown in Table 7.

[0113] Table 7 Accuracy results

[0114]

[0115] The results showed that the recovery rates were all in the range of 80% to 120%, indicating that the method had good accuracy.

[0116] 4 Detection limit

[0117] According to the method of “standard solution preparation” in Example 4, DSS of different concentrations was prepared and tested by HPLC. The results are shown in Table 8.

[0118] Table 8 Detection Limits

[0119]

[0120] 5. Repeatability

[0121] Referring to the method of "standard solution preparation" in Example 4, a sample with a DSS concentration of 8.0 μg / mL was prepared in parallel in triplicate and tested by HPLC. The results are shown in Table 9. The results show good repeatability.

[0122] Table 9 Repeatability

[0123]

[0124] Example 5 Sample stability

[0125] According to the experimental method of Example 4, the test solution was placed at 6°C and room temperature (25°C-30°C) for 3 hours and 6 hours, and then the content of dextran sulfate in each sample was detected. The results are shown in Table 10.

[0126] Table 10 Sample stability

[0127]

[0128] The results showed that there was basically no difference in the test results at 6°C and room temperature, and the sample solution was stable.

[0129] Example 6 Effect of different 2-AP concentrations on test results

[0130] Example 6-1

[0131] 0.2 mg / mL DSS solution, 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0132] Take 15 μL of DSS solution, add 50 μL of 2-AP solution, then add 20 μL of 20% TCA solution with a concentration of 200 mg / mL and pH 6.0, add water to make the final volume of the solution 194 μL, incubate at 100°C for 13 min; add 6 μL of 1 mg / mL sodium cyanoborohydride, heat at 90°C for 20 h, centrifuge to take the supernatant, and perform HPLC detection. The results are shown in Figure 5 and Figure 6 The chromatographic conditions in the experimental method were flow rate 1.0 mL / min, column temperature 30°C, PMT value 10, and the rest were the same as above.

[0133] like Figure 5 As shown in the figure, no obvious DSS peak was observed, and a flat-top peak appeared for a long time after 9 minutes of peak elution time. This was because the response value of 5 mg / mL 2-AP was too high, exceeding the conventional detection value of the fluorescence detector; a flat-top peak appeared for a long time after 9 minutes of peak elution time, and flat-top peaks continued to appear during the baseline equilibration process, indicating that an excess of 2-AP remained in the system and could not be eluted. After the empty needle was used to equilibrate the system, it was found that the residue still existed (see for details). Figure 6), it took 80 minutes to basically rinse out the residue, which required a long time for flushing and balancing.

[0134] Example 6-2

[0135] Refer to Example 6-1, except that 5 μL of 2-AP solution was added to make the final concentration of 2-AP in the detection solution 500 μg / mL. Figure 7 and Figure 8 .

[0136] The results showed that the DSS peak was at 6.5-7.5 min, the system peak was at 8-12 min, and the 2-AP peak was at 13 min. After reducing the 2-AP concentration, the gain value (PMT value) of the fluorescence detector was increased to 15. The higher the gain value, the higher the sample peak area at the same concentration, and the lower the detection limit. At the same time, equilibrium can be reached in 60 minutes, reducing the equilibrium time.

[0137] Comparative Example 1

[0138] Take the protein sample in the test solution preparation in Example 4, use -20°C frozen ethanol, or add acetonitrile, or heat denaturation to precipitate the protein, and the remaining operations refer to the test solution preparation in Example 4. No DSS peak is detected by HPLC.

[0139] Comparative Example 2

[0140] Comparative Example 2-1

[0141] Take dextran sulfate, add 2-aminopyridine (prepared by 2-aminopyridine + hydrochloric acid + water, pH 6.7) and adjust the volume to 25mL. After incubation at 100℃ for 13min, add 1.5mL reducing agent (600mg sodium cyanoborohydride + 1.5mL water), vortex mix and take 200μL solution and place it in a dry thermostat, react at 90℃ for 20h. After heating, add four times the volume of saturated sodium chloride ethanol, mix and place in a centrifuge, centrifuge at 500g for 5min, discard the supernatant and retain the precipitate, rinse the precipitate with ethanol several times, place it in a vacuum concentrator to remove the remaining ethanol, dissolve it in water, and detect it by HPLC. The results are shown in Table 11.

[0142] Table 11

[0143]

[0144] This method uses the physical property of dextran sulfate being insoluble in ethanol, and precipitates the product by adding ethanol to remove the labeling agent 2-aminopyridine. The results show that when the DSS content in the test sample is low, below 0.5 mg / mL, it may be because the content of dextran sulfate in the product is low and no precipitation can be formed, so the product will be discarded with the solution and the supernatant, resulting in the inability to detect dextran sulfate when the content is low.

[0145] Comparative Example 2-2

[0146] Protein product solution: The sample is 30 mg (1 ml) of emicizumab injection. Take the sample and add PBS buffer with a pH value of 7.4 to dilute it to 1.5 mg / mL to obtain the protein product solution.

[0147] Take 0.5 mL of the protein product solution, add 0.25 mL of water, vortex mix, add 0.25 mL of 20% TCA (without pH adjustment) with a concentration of 200 mg / mL to precipitate the protein, centrifuge and take 0.5 mL of the supernatant, adjust the pH to between 6.0 and 6.7, and then make up to 1 mL. Take 100 μL of the solution after pH adjustment, refer to the method of Comparative Example 2-1, HPLC detection, no DSS peak in the chromatogram, no detection.

[0148] Comparative Example 3

[0149] Refer to Comparative Example 2, but without the ethanol precipitation step.

[0150] Take dextran sulfate, add 2-aminopyridine (prepared by 2-aminopyridine + hydrochloric acid + water, pH 6.7) and adjust the volume to 25mL. After incubation at 100℃ for 13min, add 1.5mL reducing agent (600mg sodium cyanoborohydride + 1.5mL water), vortex mix and take 200μL and place it in a dry thermostat, and heat at 90℃ for 20h. After the reaction is completed, take out the sample, centrifuge at 8000rpm for 5min, take 2 / 3 of the supernatant, and detect it by HPLC. The results are shown in Table 12. The results show that in the target linear range of 0.5μg / mL to 500μg / mL, there is no peak without ethanol precipitation step, and the DSS content is not detected.

[0151] Table 12

[0152]

[0153] Comparative Example 4

[0154] Comparative Example 4-1

[0155] 0.2 mg / mL DSS solution, 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0156] Take different volumes of DSS mother solution, add 5μL of 20mg / mL 2-AP mother solution, then add 20μL of 20% TCA solution at pH 6.0 and add water to make the final volume of the solution 194μL, incubate at 100℃ for 13min; add 6μL of sodium cyanoborohydride and heat at 90℃ for 15h. After heating, add four times the volume of saturated sodium chloride in ethanol, mix well and place in a centrifuge, centrifuge at 500g for 5min, discard the supernatant and retain the precipitate, rinse the precipitate with ethanol several times, place it in a vacuum concentrator to remove the remaining ethanol, add water to dissolve, and use HPLC to detect the content of dextran sulfate. The results show that when the final concentration of DSS in the reaction system is 10μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL or 10μg / mL, there is no obvious precipitation in the sample after concentration. HPLC detection results show that there is no DSS peak in the chromatogram and it is not detected.

[0157] Comparative Example 4-2

[0158] 20 mg / mL 2-AP solution, 1 mg / mL sodium cyanoborohydride solution and 20% TCA were prepared as in Example 1.

[0159] Protein product solution: The sample is 30 mg (1 ml) of emicizumab injection. Take the sample and add PBS buffer with a pH value of 7.4 to dilute it to 1.5 mg / mL to obtain the protein product solution.

[0160] Take 0.5mL of protein product solution, add 0.25mL of water, vortex mix, add 0.25mL of 20% TCA (without pH adjustment) with a concentration of 200mg / mL to precipitate protein, centrifuge and take 0.5mL of supernatant, adjust the pH to between 6.0 and 6.7, and then make up to 1mL. Take 100μL of the solution after pH adjustment, and refer to Comparative Example 4-1 for the rest of the operation. After concentration, the sample has no obvious precipitation and the chromatogram has no DSS peak.

Claims

1. A method for detecting the residual amount of sodium dextran sulfate in an antibody, comprising the following steps: (1) Add trichloroacetic acid solution to the antibody to precipitate the antibody, and centrifuge to obtain the supernatant; (2) adding 2-aminopyridine solution to the supernatant in step (1) and incubating; then adding sodium cyanoborohydride solution to react to obtain a test solution; (3) The test solution was tested by high performance liquid chromatography and the content was calculated according to the standard curve method; The final concentration of 2-aminopyridine in the reaction system of step (2) is 50 μg / mL to 2000 μg / mL; preferably 50 μg / mL to 1000 μg / mL; more preferably 50 μg / mL to 700 μg / mL, and further preferably 500 μg / mL.

2. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1, characterized in that: The standard curve of step (3) is obtained by the following method: step (4) adding trichloroacetic acid solution and 2-aminopyridine solution to the sodium dextran sulfate standard of gradient concentrations; then adding sodium cyanoborohydride solution to react to obtain sodium dextran sulfate standard solutions of different concentrations; using high performance liquid chromatography to detect the sodium dextran sulfate standard solution, and fitting by the least squares method to obtain a standard curve; Preferably, the final concentration of the sodium dextran sulfate standard in the reaction system of step (4) is in the range of 0.5 μg / mL to 1000 μg / mL; And / or, the final concentration of 2-aminopyridine in the reaction system of step (4) is the same as the final concentration of 2-aminopyridine in the reaction system of step (2).

3. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 2, characterized in that: The final concentration of the sodium dextran sulfate standard in step (4) is in the range of 0.5 μg / mL to 750 μg / mL, preferably 1 μg / mL to 750 μg / mL, and more preferably 1 μg / mL to 100 μg / mL; And / or, the final concentration of sodium cyanoborohydride in the reaction system of step (2) and step (4) is 10 μg / mL to 100 μg / mL, preferably 20 μg / mL to 40 μg / mL, and more preferably 30 μg / mL. And / or, in step (2) and step (4), the incubation temperature is 80° C. to 120° C., preferably 100° C.; the incubation time is 10 min to 20 min, preferably 13 min; And / or, in the step (2) and the step (4), the reaction temperature is 80°C to 120°C, preferably 90°C; the reaction time is 15h to 40h, preferably 18h to 22h, more preferably 20h.

4. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1, characterized in that: The mass concentration of the trichloroacetic acid solution in step (2) and step (4) is 20% to 50%, preferably 20% to 35%; More preferably 20%; Preferably, the trichloroacetic acid solution in step (4) is a trichloroacetic acid solution adjusted to a pH of 6.0 to 6.7 by alkali.

5. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1 or 2, characterized in that: The solvents of the 2-aminopyridine solution, the sodium cyanoborohydride solution and the sodium dextran sulfate standard solution are all water.

6. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1, characterized in that: The steps (2) and (4) do not include the step of adding ethanol for precipitation after the reaction with sodium cyanoborohydride.

7. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a size exclusion chromatographic column, and the pore size of the chromatographic column is Preferably and / or, the mobile phase is a phosphate buffer with a pH of 3.0 to 7.0, preferably a pH of 5.0 to 7.0; more preferably 5.0; further preferably 4.4 g / L disodium hydrogen phosphate dodecahydrate and 13.7 g / L sodium dihydrogen phosphate dihydrate as the mobile phase, Isocratic elution; And / or, the column temperature in the high performance liquid chromatography method is 30°C to 60°C, preferably 40°C to 60°C, more preferably 50°C; and / or, the flow rate is 0.8 mL / min to 1.5 mL / min, preferably 1.2 mL / min; and / or, the injection volume is 30 μL to 50 μL, preferably 40 μL; And / or, the detector is a fluorescence detector with an excitation wavelength of 320 nm and an emission wavelength of 400 nm.

8. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 1, characterized in that: The antibody is emicizumab.

9. The method for detecting the residual amount of sodium dextran sulfate in an antibody according to claim 8, characterized in that: The antibody is prepared by adding PBS buffer having a pH value of 5.5 to 7.5 to dilute the emicizumab injection.

10. A method for detecting the residual amount of sodium dextran sulfate in emicizumab, comprising the following steps: 1) Preparation and determination of sodium dextran sulfate standard solution: adding trichloroacetic acid solution and 2-aminopyridine solution to sodium dextran sulfate standard solution with gradient concentrations; then adding sodium cyanoborohydride solution to react to obtain sodium dextran sulfate standard solutions of different concentrations; using high performance liquid chromatography to detect the sodium dextran sulfate standard solution, and fitting by the least square method to obtain a standard curve; 2) Preparation of the test solution: Add PBS buffer with a pH value of 5.5 to 7.5 to dilute the emicizumab injection, add trichloroacetic acid solution to precipitate the antibody, centrifuge and take the supernatant; add 2-aminopyridine solution for incubation; then add sodium cyanoborohydride solution for reaction, centrifuge and take the supernatant to obtain the test solution; 3) Determination of sodium dextran sulfate in emicizumab: The test solution was detected by high performance liquid chromatography, and the peak area in the test solution was substituted into the above standard curve to calculate the concentration of dextran sulfate in the test solution; the concentration of the test solution multiplied by the total dilution factor to obtain the concentration of sodium dextran sulfate in emicizumab; the final concentration of 2-aminopyridine in the reaction system of steps 1) and 2) was 50 μg / mL to 2000 μg / mL.

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