Purification method of natural hemoglobin A2

Abstract

The invention discloses a purification method of natural hemoglobin A2, and relates to the technical field of protein purification. The method comprises the following steps: firstly removing glycosylated hemoglobin HbA1 through a boric acid column, then generating a polymer complex through specific binding of an antibody and a beta chain in hemoglobin A, further separating hemoglobin A from hemoglobin A2 in a centrifugal manner, and further preparing a purified HbA2 freeze-dried product. The purification method of the natural hemoglobin A2 provided by the invention is simple and efficient, and the purified natural hemoglobin A2 is high in purity, can be used for expanded production, and has a wide application value.

Description

Technical Field

[0001] This application relates to the technical field of protein purification, and particularly to a method for purifying natural hemoglobin A2. Background Art

[0002] Thalassemia (referred to as "thal") is a group of inherited hemolytic diseases caused by defects in globin genes (thal genes), which result in the disorder of globin peptide chain synthesis in hemoglobin. Thalassemia patients usually suffer from anemia because the body cannot produce enough hemoglobin. Hemoglobin is a protein that helps red blood cells carry oxygen. Without enough hemoglobin, red blood cells in the blood cannot function properly and cannot effectively transport oxygen to cells throughout the body, thereby causing tissue and organ hypoxia, which makes people feel tired.

[0003] Adult hemoglobin (Hb) usually consists of hemoglobin A (HbA, about 97%) and hemoglobin A2 (HbA2, about 3%). HbA can be further divided into non-glycated hemoglobin, namely natural hemoglobin HbA0, and glycated hemoglobin HbA1. Hemoglobin A2 is composed of 2 α-globins and 2 δ-globins. Its increase is a characteristic index of β-thalassemia patients. The HbA2 level in the normal population is in the range of 2.2%-3.3%, and the HbA2 level of typical β-thalassemia gene carriers is in the range of 4.0%-6.0%.

[0004] Traditional HbA2 detection methods are often interfered by heteroproteins in the sample, such as HbA1, and the accuracy is not good. In complex blood components, HbA2 coexists with other hemoglobins (HbA0, HbA1), and the separation and purification are difficult. Previous purification methods, such as chromatographic column separation and ion column chromatography, are difficult to completely remove interfering components with similar structures, resulting in large errors in subsequent quantitative analysis. Moreover, some separation methods are prone to cause denaturation and inactivation of the target protein, affecting the in-depth study of its natural characteristics and functions. In addition, the existing technology is cumbersome and time-consuming, and is not suitable for the rapid processing requirements of a large number of clinical samples. In view of this, it is urgent to develop a method for purifying HbA2 with high efficiency and capable of retaining natural activity. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, this application provides a method for purifying natural hemoglobin A2. First, glycated hemoglobin HbA1 is removed through a boric acid column, and then a multimeric complex is formed by the specific binding of an antibody to the β-chain in hemoglobin A, so that hemoglobin A and hemoglobin A2 can be separated by centrifugation, and then a purified HbA2 freeze-dried product is prepared. The method for purifying natural hemoglobin A2 provided by this application is simple and efficient, and the purified natural hemoglobin A2 has high purity, can be used for large-scale production, and has broad application value.

[0006] To achieve the above object, the present application adopts a technical solution including the following steps:

[0007] Fragmentation and extraction of red blood cells;

[0008] Pass through a perborate column to obtain a mixed solution enriched with HbA0 and HbA2;

[0009] The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex;

[0010] Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2;

[0011] Prepare a freeze-dried product of HbA2.

[0012] Beneficial technical effects:

[0013] Through the boric acid groups on the borate column, a covalent complex is formed with the cis-diol groups on glycated hemoglobin HbA1, so that glycated hemoglobin HbA1 in natural hemoglobin is targeted and captured by the borate column, achieving the effect of separating HbA1 and enriching HbA0 and HbA2, avoiding the interference of HbA1 in the subsequent purification process of HbA2, and significantly reducing the sample complexity during subsequent antibody treatment and further purification.

[0014] The mixed solution of enriched HbA0 and HbA2 can be treated with a slightly excessive polyclonal antibody against hemoglobin β or a monoclonal antibody against hemoglobin β, so that HbA0 specifically binds to the antibody to form a multimeric complex with a molecular weight significantly larger than that of HbA2; at the same time, the molecular weight of the antibody is also higher than that of HbA2, so that the multimeric complex and the excessive antibody can be efficiently separated from HbA2 by centrifugation. Moreover, the HbA2 prepared by this method has high purity, is easy to scale up production, and is suitable for large-scale preparation of HbA2 preparations. Description of the Drawings

[0015] Figure 1 It is a schematic flow chart of the purification method of natural hemoglobin A2.

[0016] Figure 2 It is a schematic diagram of separating HbA1 by a borate column and enriching HbA0 and HbA2. Detailed Embodiments

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer, the present application will be further described in detail below in conjunction with embodiments. However, this should not be construed as limiting the scope of the present application to the following examples. Without departing from the above method idea of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application.

[0018] In this application, the terms used are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0019] The singular forms "a", "the", and "any one" used in this application and the appended claims are intended to include the plural forms unless the context clearly indicates otherwise.

[0020] This application adopts a technical solution including the following steps:

[0021] Fragmentation and extraction of red blood cells;

[0022] Pass through a perborate column to obtain a mixed solution enriched with HbA0 and HbA2;

[0023] The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex;

[0024] Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2;

[0025] Prepare a freeze-dried product of HbA2.

[0026] In one possible implementation, the process of fragmenting and extracting the red blood cells includes:

[0027] Centrifuge the whole blood at 2500 - 3000 rpm for 5 - 10 min, remove the supernatant with a Pasteur pipette and retain the precipitate. Add 10 mM PBS buffer at pH 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2500 - 3000 rpm for 5 - 10 min again. Discard the supernatant and retain the precipitate, repeating two to three times;

[0028] Further add ultrapure water to the obtained precipitate, and oscillate and fragment at 37 °C and 200 - 300 rpm for 4 h; after the fragmentation is completed, centrifuge at 12000 - 13000 rpm for 10 - 15 min, discard the precipitate and retain the supernatant, which is the red blood cell extract.

[0029] In one possible implementation, the mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3.

[0030] In one possible implementation, the principle of passing through the perborate column to obtain a mixed solution enriched with HbA0 and HbA2 is as Figure 2 shown, and its process includes:

[0031] Equilibrate the borate column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2, and wait for sample loading after treatment;

[0032] Dialyze the red blood cell extract into the equilibration buffer of the boric acid column and change the solution three times.

[0033] After dialysis, centrifuge the equilibration buffer of the boric acid column at 12000 - 13000 rpm for 10 - 15 min, then filter it through a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2.

[0034] In a possible implementation, the pH value of the equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 is 8.5 - 8.9.

[0035] In a possible implementation, the process of the antibody specifically binding to the β-chain of HbA0 to form a multimeric complex includes:

[0036] Incubate the antibody with the mixed solution enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1 - 2 h.

[0037] Add 1 mM carbodiimide and react at room temperature for 30 - 60 min to obtain a mixed solution containing the multimeric complex.

[0038] In a possible implementation, the antibody includes any one of hemoglobin β polyclonal antibody and hemoglobin β monoclonal antibody.

[0039] In a possible implementation, the mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 is (1.2 - 1.5):1.

[0040] In a possible implementation, the process of centrifugally separating the multimeric complex and HbA2 to obtain enriched and purified HbA2 includes:

[0041] Centrifuge the mixed solution containing the multimeric complex at 4500 - 5000 rpm for 5 - 10 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain a supernatant enriched with HbA2.

[0042] Then centrifuge the supernatant enriched with HbA2 at 9000 - 10000 rpm for 10 - 20 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain purified HbA2.

[0043] In a possible implementation, the process of preparing the HbA2 freeze-dried product includes:

[0044] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times.

[0045] Then add mannitol with a concentration of 5% and PC300 with a concentration of 0.05%, and cool from room temperature to -50°C at a cooling rate of 1-5°C / min, and keep warm for 2-4 hours to obtain the freeze-dried product of HbA2.

[0046] In summary, the specific steps of a purification method of natural hemoglobin A2 provided by this application are as shown in the appendix Figure 1 as follows.

[0047] The following will specifically describe a purification method of natural hemoglobin A2 provided by this application in combination with different embodiments.

[0048] Example 1:

[0049] As Figure 1 shown, a purification method of natural hemoglobin A2 includes the following steps:

[0050] 1. Crushing and extraction of red blood cells:

[0051] Centrifuge the whole blood at 2500 rpm for 5 minutes, remove the supernatant with a Pasteur pipette and retain the precipitate. Add PBS buffer with a concentration of 10 mM and a pH of 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2500 rpm for 5 minutes again. Discard the supernatant and retain the precipitate, and repeat two to three times;

[0052] Further add ultrapure water to the obtained precipitate, and oscillate and crush at 37°C and 200 rpm for 4 hours; after the crushing is completed, centrifuge at 12000 rpm for 10 minutes, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0053] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0054] 2. Pass through a boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0055] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH value is 8.5), and wait for sample loading after treatment;

[0056] Dialyze the red blood cell extract into the equilibration solution of the boric acid column and change the solution three times;

[0057] After dialysis, centrifuge the equilibration solution of the boric acid column at 12000 rpm for 10 minutes, then filter with a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2;

[0058] 3. The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex:

[0059] Incubate the polyclonal antibody against hemoglobin β with the mixture enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1 h; the mass ratio of the antibody to the mixture enriched with HbA0 and HbA2 is 1.2:1;

[0060] Add 1 mM carbodiimide and react at room temperature for 30 min to obtain a mixture containing a polymer complex;

[0061] 4. Centrifuge to separate the polymer complex and HbA2 to obtain purified HbA2:

[0062] Centrifuge the mixture containing the polymer complex at 4500 rpm for 5 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain the supernatant enriched with HbA2;

[0063] Then centrifuge the supernatant enriched with HbA2 at 9000 rpm for 10 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain purified HbA2;

[0064] 5. Prepare the freeze-dried product of HbA2:

[0065] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0066] Then add 5% mannitol and 0.05% PC300, and cool from room temperature to -50 °C at a cooling rate of 1 °C / min and keep warm for 2 h to obtain the freeze-dried product of HbA2.

[0067] Example 2:

[0068] As Figure 1 shown, a method for purifying natural hemoglobin A2 includes the following steps:

[0069] 1. Disruption and extraction of red blood cells:

[0070] Centrifuge the whole blood at 2600 rpm for 8 min, remove the supernatant with a Pasteur pipette and retain the precipitate, add PBS buffer at pH 7.4 of 10 mM to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2600 rpm for 8 min again, discard the supernatant and retain the precipitate, repeat two to three times;

[0071] Further add ultrapure water to the obtained precipitate, and disrupt it by shaking at 37 °C and 220 rpm for 4 h; after the disruption, centrifuge at 12500 rpm for 12 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0072] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0073] 2. Pass through the boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0074] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH 8.7), and wait for sample loading after treatment;

[0075] Dialyze the red blood cell extract into the equilibration solution of the boric acid column and change the solution three times;

[0076] After dialysis, centrifuge the equilibration solution of the boric acid column at 12500 rpm for 12 min, then filter it through a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2;

[0077] 3. The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex:

[0078] Incubate the hemoglobin β monoclonal antibody with the mixed solution enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1.5 h; the mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 is 1.3:1;

[0079] Add 1 mM carbodiimide and react at room temperature for 45 min to obtain a mixed solution containing the multimeric complex;

[0080] 4. Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2:

[0081] Centrifuge the mixed solution containing the multimeric complex at 4600 rpm for 7 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain a supernatant enriched with HbA2;

[0082] Then centrifuge the supernatant enriched with HbA2 at 9200 rpm for 15 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain purified HbA2;

[0083] 5. Prepare the freeze-dried product of HbA2:

[0084] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0085] Then add 5% mannitol and 0.05% PC300, and cool from room temperature to -50 °C at a cooling rate of 3 °C / min and keep warm for 3 h to obtain the freeze-dried product of HbA2.

[0086] Example 3:

[0087] As Figure 1 shown, a purification method of natural hemoglobin A2 includes the following steps:

[0088] 1. Red blood cell disruption and extraction:

[0089] Centrifuge the whole blood at 2750 rpm for 10 min, remove the supernatant with a Pasteur pipette and keep the precipitate, add 10 mM pH 7.4 PBS buffer to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2750 rpm for 10 min again, discard the supernatant and keep the precipitate, repeat two to three times;

[0090] Ultrapure water was further added to the obtained precipitate, and the precipitate was shaken and crushed at 37°C and 250 rpm for 4 hours; after the crushing, the precipitate was centrifuged at 12500 rpm for 15 minutes, and the precipitate was discarded to keep the supernatant, i.e., the red blood cell extract;

[0091] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to pure water is 1:3;

[0092] 2. Perboric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0093] The boric acid column was equilibrated with an equilibration buffer (pH 8.9) containing 50 mM taurine / NaOH and 20 mM MgCl2, and then treated before loading;

[0094] The red blood cell extract was dialyzed into the equilibrium solution of the boric acid column, and the solution was changed three times;

[0095] After dialysis, the equilibrium solution of the boric acid column was centrifuged at 12500 rpm for 15 min, filtered with a 0.22 μm filter membrane, and then loaded with samples to collect a mixed solution enriched with HbA0 and HbA2;

[0096] 3. The antibody specifically binds to the β chain of HbA0 to form a multimeric complex:

[0097] The hemoglobin β polyclonal antibody and the mixed solution enriched with HbA0 and HbA2 were incubated in PBS buffer at 37° C. for 2 hours; the mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 was 1.5:1;

[0098] 1 mM carbodiimide was added and reacted at room temperature for 60 min to obtain a mixed solution containing a polymer complex;

[0099] 4. Centrifuge to separate the polymer complex and HbA2 to obtain purified HbA2:

[0100] The mixed solution containing the polymer complex was centrifuged at 4750 rpm for 10 min, the precipitate was discarded and the supernatant was retained, and this process was repeated two to three times to obtain a supernatant enriched with HbA2;

[0101] Then, centrifuge the supernatant enriched with HbA2 at 9500 rpm for 20 min, discard the precipitate and retain the supernatant, and repeat this process two to three times to obtain purified HbA2;

[0102] 5. Preparation of freeze-dried HbA2 product:

[0103] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, and change the buffer every 4 h for three times;

[0104] Then, add mannitol at a concentration of 5% and PC300 at a concentration of 0.05%, and cool from room temperature to -50 °C at a cooling rate of 5 °C / min and keep it at this temperature for 4 h to obtain the freeze-dried HbA2 product.

[0105] Example 4:

[0106] As Figure 1 shown, a purification method of natural hemoglobin A2 includes the following steps:

[0107] 1. Disruption and extraction of red blood cells:

[0108] Centrifuge the whole blood at 2700 rpm for 7 min, remove the supernatant with a Pasteur pipette and retain the precipitate. Add PBS buffer at 10 mM pH 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, then centrifuge at 2700 rpm for 7 min again, discard the supernatant and retain the precipitate, and repeat this process two to three times;

[0109] Further add ultrapure water to the obtained precipitate, and disrupt it by shaking at 37 °C and 230 rpm for 4 h; after the disruption, centrifuge at 12400 rpm for 12 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0110] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0111] 2. Pass through a boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0112] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH value is 8.6), and wait for sample loading after treatment;

[0113] Dialyze the red blood cell extract into the equilibration solution of the boric acid column, and change the solution three times;

[0114] After dialysis, centrifuge the equilibration solution of the boric acid column at 12600 rpm for 12 min, then filter it with a 0.22 μm filter membrane, and then load the sample to collect the mixed solution enriched with HbA0 and HbA2;

[0115] 3. Specific binding of the antibody to the β-chain of HbA0 to form a multimeric complex:

[0116] Incubate the hemoglobin β monoclonal antibody with the mixture enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1.2 h; the mass ratio of the antibody to the mixture enriched with HbA0 and HbA2 is 1.4:1;

[0117] Add 1 mM carbodiimide and react at room temperature for 40 min to obtain a mixture containing a polymer complex;

[0118] 4. Centrifuge to separate the polymer complex and HbA2 to obtain purified HbA2:

[0119] Centrifuge the mixture containing the polymer complex at 4700 rpm for 6 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain the supernatant enriched with HbA2;

[0120] Then centrifuge the supernatant enriched with HbA2 at 9600 rpm for 15 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain purified HbA2;

[0121] 5. Prepare the freeze-dried product of HbA2:

[0122] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0123] Then add 5% mannitol and 0.05% PC300, cool from room temperature to -50 °C at a cooling rate of 2 °C / min, and keep warm for 2.5 h to obtain the freeze-dried product of HbA2.

[0124] Example 5:

[0125] As Figure 1 shown, a method for purifying natural hemoglobin A2 includes the following steps:

[0126] 1. Disruption and extraction of red blood cells:

[0127] Centrifuge the whole blood at 2800 rpm for 6 min, remove the supernatant with a Pasteur pipette and retain the precipitate, add PBS buffer at pH 7.4 of 10 mM to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2800 rpm for 6 min again, discard the supernatant and retain the precipitate, repeat two to three times;

[0128] Further add ultrapure water to the obtained precipitate, oscillate and disrupt at 37 °C and 260 rpm for 4 h; after disruption, centrifuge at 12700 rpm for 13 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0129] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0130] 2. Pass through a boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0131] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH 8.8), and wait for sample loading after treatment;

[0132] Dialyze the red blood cell extract into the equilibration solution of the boric acid column and change the solution three times;

[0133] After dialysis, centrifuge the equilibration solution of the boric acid column at 12,800 rpm for 13 min, then filter it through a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2;

[0134] 3. The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex:

[0135] Incubate the polyclonal antibody against hemoglobin β with the mixed solution enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1.8 h; the mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 is 1.4:1;

[0136] Add 1 mM carbodiimide and react at room temperature for 50 min to obtain a mixed solution containing a multimeric complex;

[0137] 4. Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2:

[0138] Centrifuge the mixed solution containing the multimeric complex at 4800 rpm for 8 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain a supernatant enriched with HbA2;

[0139] Then centrifuge the supernatant enriched with HbA2 at 9700 rpm for 18 min, discard the precipitate and retain the supernatant, repeat two to three times to obtain purified HbA2;

[0140] 5. Prepare a freeze-dried product of HbA2:

[0141] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4 and change the solution once every 4 h for three times;

[0142] Then add 5% mannitol and 0.05% PC300, cool from room temperature to -50 °C at a cooling rate of 4 °C / min, and keep warm for 3.5 h to obtain a freeze-dried product of HbA2.

[0143] Example 6:

[0144] As Figure 1 shown, a method for purifying natural hemoglobin A2 includes the following steps:

[0145] 1. Fragmentation and extraction of red blood cells:

[0146] Centrifuge the whole blood at 3000 rpm for 10 min, remove the supernatant with a Pasteur pipette and retain the precipitate. Add PBS buffer with a concentration of 10 mM and a pH of 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, then centrifuge at 3000 rpm for 5 min, discard the supernatant and retain the precipitate, repeating two to three times;

[0147] Further add ultrapure water to the obtained precipitate, and break it by shaking at 37 °C and 300 rpm for 4 h; after the fragmentation is completed, centrifuge at 13000 rpm for 15 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0148] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0149] 2. Pass through a boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0150] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH value is 8.5), and wait for sample loading after treatment;

[0151] Dialyze the red blood cell extract into the equilibration solution of the boric acid column and change the solution three times;

[0152] After dialysis, centrifuge the equilibration solution of the boric acid column at 13000 rpm for 10 min, then filter it with a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2;

[0153] 3. The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex:

[0154] Incubate the hemoglobin β monoclonal antibody with the mixed solution enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 2 h; the mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 is 1.5:1;

[0155] Add 1 mM carbodiimide and react at room temperature for 60 min to obtain a mixed solution containing a multimeric complex;

[0156] 4. Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2:

[0157] Centrifuge the mixed solution containing the multimeric complex at 5000 rpm for 10 min, discard the precipitate and retain the supernatant, repeating two to three times to obtain a supernatant enriched with HbA2;

[0158] Centrifuge the supernatant enriched with HbA2 at 10,000 rpm for 20 min, discard the precipitate and retain the supernatant, and repeat two to three times to obtain purified HbA2;

[0159] 5. Preparation of freeze-dried HbA2 product:

[0160] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0161] Then add mannitol at a concentration of 5% and PC300 at a concentration of 0.05%, cool from room temperature to -50 °C at a cooling rate of 1 °C / min, and keep warm for 4 h to obtain the freeze-dried HbA2 product.

[0162] Comparative Example 1:

[0163] A purification method for natural hemoglobin A2, comprising the following steps:

[0164] 1. Disruption and extraction of red blood cells:

[0165] Centrifuge the whole blood at 2,500 rpm for 5 min, remove the supernatant with a Pasteur pipette and retain the precipitate, add PBS buffer at 10 mM pH 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2,500 rpm for 5 min again, discard the supernatant and retain the precipitate, and repeat two to three times;

[0166] Further add ultrapure water to the obtained precipitate, and shake and disrupt at 37 °C and 200 - 300 rpm for 4 h; after the disruption, centrifuge at 12,000 rpm for 10 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0167] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0168] 2. Specific binding of the antibody to the β-chain of HbA0 to form a multimeric complex:

[0169] Incubate the hemoglobin β polyclonal antibody with the red blood cell extract in PBS buffer at 37 °C for 1 h; the mass ratio of the antibody to the red blood cell extract is 1.2:1;

[0170] Add 1 mM carbodiimide and react at room temperature for 30 min to obtain a mixed solution containing the multimeric complex;

[0171] 3. Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2:

[0172] Centrifuge the mixed solution containing the multimeric complex at 4,500 rpm for 5 min, discard the precipitate and retain the supernatant, and repeat two to three times to obtain the supernatant enriched with HbA2;

[0173] Then, centrifuge the supernatant enriched with HbA2 at 9000 rpm for 10 min, discard the precipitate and retain the supernatant, and repeat this two to three times to obtain purified HbA2;

[0174] 4. Preparation of freeze-dried HbA2 product:

[0175] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0176] Then, add mannitol with a concentration of 5% and PC300 with a concentration of 0.05%, and cool from room temperature to -50 °C at a cooling rate of 1 °C / min, and keep it warm for 2 h to obtain the freeze-dried HbA2 product.

[0177] Comparative Example 2:

[0178] A method for purifying natural hemoglobin A2, comprising the following steps:

[0179] 1. Disruption and extraction of red blood cells:

[0180] Centrifuge the whole blood at 2750 rpm for 10 min, use a Pasteur pipette to remove the supernatant and retain the precipitate, add PBS buffer at pH 7.4 to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2750 rpm for 10 min again, discard the supernatant and retain the precipitate, and repeat this two to three times;

[0181] Further add ultrapure water to the obtained precipitate, and oscillate and disrupt it at 37 °C and 250 rpm for 4 h; after the disruption, centrifuge at 12500 rpm for 15 min, discard the precipitate and retain the supernatant, which is the red blood cell extract;

[0182] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to the pure water is 1:3;

[0183] 2. Pass through a boric acid column to obtain a mixed solution enriched with HbA0 and HbA2:

[0184] Equilibrate the boric acid column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 (pH value is 8.9), and wait for sample loading after treatment;

[0185] Dialyze the red blood cell extract into the equilibration solution of the boric acid column, and change the solution three times;

[0186] After dialysis, centrifuge the equilibration solution of the boric acid column at 12500 rpm for 15 min, then filter it with a 0.22 μm filter membrane, and then load the sample to collect the mixed solution enriched with HbA0 and HbA2;

[0187] 3. Centrifuge and separate the mixture of HbA0 and HbA2 to obtain purified HbA2:

[0188] The mixed solution containing the polymer complex was centrifuged at 4750 rpm for 10 min, the precipitate was discarded and the supernatant was retained, and this process was repeated two to three times to obtain a supernatant enriched with HbA2;

[0189] The HbA2-enriched supernatant was then centrifuged at 9500 rpm for 20 min, the precipitate was discarded and the supernatant was retained, and this was repeated two to three times to obtain purified HbA2;

[0190] 4. Preparation of HbA2 lyophilized product:

[0191] The purified HbA2 was dialyzed into 10 mM pH 7.4 PBS buffer, and the solution was changed every 4 h for three times;

[0192] Then, 5% mannitol and 0.05% PC300 were added, and the temperature was lowered from room temperature to -50°C at a cooling rate of 5°C / min and kept warm for 4 hours to obtain a lyophilized HbA2 product.

[0193] Comparative Example 3:

[0194] A method for purifying natural hemoglobin A2 comprises the following steps:

[0195] 1. Red blood cell disruption and extraction:

[0196] The whole blood was centrifuged at 3000 rpm for 10 min, the supernatant was removed with a Pasteur pipette and the precipitate was retained, 10 mM PBS buffer (pH 7.4) was added to the precipitate, the precipitate was blown up with a Pasteur pipette, and then centrifuged at 3000 rpm for 5 min, the supernatant was discarded and the precipitate was retained, and this was repeated two to three times;

[0197] Ultrapure water was further added to the obtained precipitate, and the precipitate was shaken and crushed at 37°C and 200-300 rpm for 4 hours; after the crushing, the precipitate was centrifuged at 13000 rpm for 15 minutes, and the precipitate was discarded to keep the supernatant, i.e., the red blood cell extract;

[0198] The mass ratio of the whole blood to the PBS buffer is 1:3; the mass ratio of the whole blood to pure water is 1:3;

[0199] 2. Centrifuge the red blood cell extract to obtain purified HbA2:

[0200] The red blood cell extract was centrifuged at 5000 rpm for 10 min, the precipitate was discarded and the supernatant was retained, and this process was repeated two to three times to obtain a supernatant enriched with HbA2;

[0201] Centrifuge the supernatant enriched with HbA2 at 10,000 rpm for 20 min, discard the precipitate and retain the supernatant, and repeat two to three times to obtain purified HbA2;

[0202] 3. Preparation of freeze-dried HbA2 product:

[0203] Dialyze the purified HbA2 into 10 mM PBS buffer at pH 7.4, change the solution every 4 h, and change the solution three times;

[0204] Then add mannitol at a concentration of 5% and PC300 at a concentration of 0.05%, cool from room temperature to -50 °C at a cooling rate of 1 °C / min, and keep warm for 4 h to obtain a freeze-dried HbA2 product.

[0205] Use a hemoglobin analyzer to detect the content of each protein in the prepared purified HbA2, and the results are shown in Table 1.

[0206] Table 1 Test results of the content of each protein in the purified HbA2 prepared in Examples 1-6 and Comparative Examples 1-3

[0207]

[0208] As can be seen from Table 1, the concentration of HbA2 in Examples 1-6 is higher than that in Comparative Examples 1-3, and at the same time, the concentrations of HbA0 and HbA1 in Examples 1-6 are significantly lower than those in Comparative Examples 1-3.

[0209] This is because in this application, the boric acid groups on the boric acid column form a covalent complex with the cis-diol groups on the glycated hemoglobin HbA1, so that the glycated hemoglobin HbA1 in natural hemoglobin is targeted and captured by the boric acid column, achieving the effect of separating HbA1 and enriching HbA0 and HbA2, avoiding the interference of HbA1 in the subsequent purification process of HbA2, and significantly reducing the sample complexity in subsequent antibody treatment and further purification.

[0210] The mixed solution of enriched HbA0 and HbA2 can be treated with a slightly excessive amount of hemoglobin β polyclonal antibody or hemoglobin β monoclonal antibody, so that HbA0 specifically binds to the antibody to form a multimeric complex with a molecular weight significantly larger than that of HbA2; at the same time, the molecular weight of the antibody is also higher than that of HbA2, so that the multimeric complex and the excessive antibody can be efficiently separated from HbA2 by centrifugation to obtain high-purity HbA2.

[0211] The red blood cell extract prepared in Comparative Example 1 does not have a perboric acid column, so it is unable to effectively and specifically form a covalent complex with the cis-diol group on the glycated hemoglobin HbA1 to remove the glycated hemoglobin HbA1; however, due to the specific binding of the antibody to HbA0, the HbA0 content in the final product is very low, the HbA2 content is relatively high, and there are more HbA1 impurities therein.

[0212] The red blood cell extract prepared in Comparative Example 2 was not subjected to the specific binding of the antibody and HbA0 but was passed through a boric acid column. For the same reason, the final product had a high HbA0 content, a low HbA2 content, and very little HbA1 impurity.

[0213] The red blood cell extract prepared in Comparative Example 3 was neither subjected to the specific binding of the antibody and HbA0 nor subjected to the perboric acid column, so the final product had a high HbA0 content and a low HbA2 content, and contained a large amount of HbA1 impurities.

[0214] The above results show and describe the basic principles and main features of the present application as well as the advantages of the present application.

[0215] Those skilled in the art should understand that the present application is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present application. Without departing from the spirit and scope of the present application, the present application may have various changes and improvements, and these changes and improvements fall within the scope of the present application for which protection is sought. The scope of protection sought in the present application is defined by the equivalents of the attached claims.

Claims

1. A purification method of natural hemoglobin A2, characterized in that, It includes the following steps: Fragmentation and extraction of red blood cells; Pass through a perborate column to obtain a mixed solution enriched with HbA0 and HbA2; The antibody specifically binds to the β-chain of HbA0 to form a multimeric complex; Centrifuge to separate the multimeric complex and HbA2 to obtain purified HbA2; Prepare a freeze-dried product of HbA2.

2. The purification method of a natural hemoglobin A2 according to claim 1, characterized in that, The process of fragmentation and extraction of the red blood cells includes: Centrifuge the whole blood at 2500 - 3000 rpm for 5 - 10 min, remove the supernatant with a Pasteur pipette and retain the precipitate. Add PBS buffer solution with a pH of 7.4 and a concentration of 10 mM to the precipitate, blow up the precipitate with a Pasteur pipette, and then centrifuge at 2500 - 3000 rpm for 5 - 10 min again. Discard the supernatant and retain the precipitate, repeating two to three times; Further add ultrapure water to the obtained precipitate, and oscillate and fragment at 37 °C and 200 - 300 rpm for 4 h; after the fragmentation is completed, centrifuge at 12000 - 13000 rpm for 10 - 15 min, discard the precipitate and retain the supernatant, which is the red blood cell extract.

3. The purification method of a natural hemoglobin A2 according to claim 2, wherein The mass ratio of the whole blood to the PBS buffer solution is 1:3; the mass ratio of the whole blood to the pure water is 1:

3.

4. The purification method of a natural hemoglobin A2 according to claim 1, characterized in that, The process of passing through the perborate column to obtain a mixed solution enriched with HbA0 and HbA2 includes: Equilibrate the perborate column with an equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2, and wait for sample loading after treatment; Dialyze the red blood cell extract into the equilibration solution of the perborate column, changing the solution three times; After the dialysis is completed, centrifuge the equilibration solution of the perborate column at 12000 - 13000 rpm for 10 - 15 min, then filter with a 0.22 μm filter membrane, and then load the sample to collect a mixed solution enriched with HbA0 and HbA2.

5. A purification method of natural hemoglobin A2 according to claim 4, characterized in that, The pH value of the equilibration buffer containing 50 mM taurine / NaOH and 20 mM MgCl2 is 8.5 - 8.

9.

6. A purification method of natural hemoglobin A2 according to claim 1, characterized in that, The process of the antibody specifically binding to the β-chain of HbA0 to form a multimeric complex includes: Incubate the antibody with the mixed solution enriched with HbA0 and HbA2 in PBS buffer at 37 °C for 1 - 2 h; Add 1 mM carbodiimide and react at room temperature for 30 - 60 min to obtain a mixed solution containing a multimeric complex.

7. The purification method of a natural hemoglobin A2 according to claim 6, characterized in that, The antibody includes any one of hemoglobin β polyclonal antibody and hemoglobin β monoclonal antibody.

8. The purification method of a natural hemoglobin A2 according to claim 6, characterized in that, The mass ratio of the antibody to the mixed solution enriched with HbA0 and HbA2 is (1.2 - 1.5):

1.

9. A purification method of natural hemoglobin A2 according to claim 1, characterized in that, The process of centrifuging to separate the multimeric complex and HbA2 to obtain enriched and purified HbA2 includes: Centrifuge the mixed solution containing the multimeric complex at 4500 - 5000 rpm for 5 - 10 min, discard the precipitate and retain the supernatant, repeating two to three times to obtain a supernatant enriched with HbA2; Then centrifuge the supernatant enriched with HbA2 at 9000 - 10000 rpm for 10 - 20 min, discard the precipitate and retain the supernatant, repeating two to three times to obtain purified HbA2.

10. A purification method of natural hemoglobin A2 according to claim 1, characterized in that, The process of preparing the freeze-dried product of HbA2 includes: Dialyze the purified HbA2 into 10 mM PBS buffer with a pH of 7.4, change the solution once every 4 h, and change the solution three times; Then add mannitol with a concentration of 5% and PC300 with a concentration of 0.05%, and cool from room temperature to -50°C at a cooling rate of 1 - 5°C / min, and keep warm for 2 - 4 h to obtain the freeze-dried product of HbA2.

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