A method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum

Abstract

This invention discloses a method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum. The method includes the following steps: taking rabbit serum, filtering, adding a precipitant to the filtrate to precipitate, centrifuging, collecting the supernatant, and sequentially performing cation exchange chromatography, anion exchange chromatography, and cation exchange chromatography. This invention employs a strategy of alternating cation and anion exchange, gradually decreasing the particle size of the chromatographic medium, and gradually increasing the resolution. This allows for the separation of high-purity and high-activity rC1INH from rabbit serum with extremely low abundance, exhibiting high recovery rate and good batch-to-batch reproducibility. The purified rC1INH has a clearly defined composition and controllable quality, with a purity exceeding 95%. The purification method is simple to operate, fast in purification, and low in cost.

Description

Technical Field

[0001] This invention specifically relates to a method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum. Background Technology

[0002] Rabbit C1 esterase inhibitor, also known as rabbit complement 1 inhibitor (rC1INH), is a highly glycosylated protein found in rabbit serum, belonging to the serine protease inhibitor superfamily. Rabbit C1INH and human C1INH share high amino acid sequence homology (approximately 85%-90%), with highly conserved core domains, thus exhibiting identical core functions. C1INH primarily possesses the following two biological functions: 1) Maintaining vascular homeostasis: C1INH maintains vascular stability, preventing increased vascular permeability and angioedema. Under normal physiological conditions, it maintains the integrity of vascular endothelial cells and intravascular fluid balance by inhibiting the activity of related proteases; 2) Immune-inflammatory regulation: As a potent natural inhibitor of the classical complement and lectin pathways, as well as factor XII of the contact system and plasma kallikrein, C1INH can control the activation of the complement system, preventing excessive immune responses and inflammatory damage. It can bind to activated C1r or C1s to form a stable complex, inactivating C1 serine proteases and thus preventing the over-initiation of the complement cascade reaction, protecting body tissues from complement-mediated damage.

[0003] Rabbits are frequently used as laboratory animals to study the function and mechanism of action of C1INH, as well as for drug development. For example, transgenic rabbits carrying the human C1INH gene are currently commercially available, bred using genetic engineering technology to specifically express recombinant human C1INH (rhC1INH) in their mammary glands. This allows for the extraction of recombinant human C1INH from the transgenic rabbit's milk, providing an effective route for the production of recombinant protein drugs. The recombinant human C1INH produced by these transgenic rabbits has a completely identical amino acid sequence to the naturally occurring human C1INH, and shows no significant difference in molecular structure, physicochemical properties, and physiological function from C1INH derived from human serum. It can be used to treat or prevent human hereditary angioedema (HAE). Since this recombinant protein drug uses rabbits as a bioreactor, the stock solution of this recombinant therapeutic protein may contain rabbit endogenous C1INH (rC1INH) impurities. The presence of these impurities can easily cause abnormal immune responses in the body when rhC1INH is used in clinical applications. In order to ensure the safety of the drug, according to the relevant drug regulations, it is necessary to determine the residual amount of rhC1INH in the stock solution. In order to construct a corresponding detection method, it is necessary to prepare high-purity rC1INH as a standard for detection.

[0004] However, there are currently no reported methods for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum to obtain rC1INH standards that can be used for quality control of rhC1INH products. Although there are methods for isolating and purifying human C1 inhibitors from human blood, such as CN 106255532 A, these methods have not been found to be suitable for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum. Summary of the Invention

[0005] To address the above problems, this invention provides a method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum, comprising the following steps: 1) Take rabbit serum, filter, add precipitant to the filtrate to precipitate, centrifuge, and collect the supernatant; 2) Take the supernatant obtained in step 1), desalt and replace it in solution A, then load it onto a chromatography column packed with SP Big Beads, elute with solution B, and collect the eluent; 3) Take the eluent obtained in step 2), desalt and replace it in solution C, then load it onto a chromatography column packed with Q HP, and perform linear gradient elution with solutions C and D. Collect the eluent with enzyme activity ≥0.2 U / ml. 4) Take the eluent obtained in step 3), desalt and replace it in solution C, then load it onto a chromatography column packed with Mono S, and perform linear gradient elution with solutions C and D. Collect the eluent with enzyme activity ≥ 0.5 U / ml. The procedure for the linear gradient elution is as follows: using 20 column volumes, the mobile phase is linearly changed from 100% C solution to 100% D solution; Solution A is a buffer solution with a pH of 7.0 containing 20 mM NaCl. Solution B is a buffer solution with a pH of 7.0 containing 0.2-1 M NaCl; Solution C is a buffer solution containing 50 mM NaCl with a pH of 6.0~8.0; The D solution is a buffer solution containing 1 M NaCl with a pH of 6.0~8.0.

[0006] Further, the precipitant in step 1) includes ammonium sulfate and / or PEG-4000.

[0007] Furthermore, the ammonium sulfate is added until the saturation reaches 30-40%.

[0008] Further, the stirring temperature in step 1) is 2~25℃, and the stirring time is 6~12h.

[0009] Further, in step 1), the centrifugation speed is 10000 g, the temperature is 2-8℃, and the time is 25-35 min.

[0010] Further, the method for desalting and replacing the supernatant with solution A in step 2) is as follows: the supernatant is loaded onto a G25F desalting chromatography column equilibrated with solution A, and then eluted with solution A. Based on the UV signal response at 280 nm, the eluent is completely collected from the beginning to the end of the peak.

[0011] Furthermore, solution A is a 15-25mM sodium citrate buffer solution with pH 7.0 containing 20 mM NaCl, preferably a 20mM sodium citrate buffer solution with pH 7.0 containing 20 mM NaCl.

[0012] Further, the method for desalting and replacing the eluent with solution C in steps 3) and 4) is as follows: the eluent is loaded onto a G25F desalting chromatography column equilibrated with solution C, and then eluted with solution C. Based on the UV signal response at 280 nm, the eluent is completely collected from the beginning to the end of the peak.

[0013] Furthermore, the chromatography columns described in steps 3) and 4) are equilibrated with C solution before and after sample loading.

[0014] Furthermore, solution C is a 15-25 mM phosphate buffer containing 50 mM NaCl with a pH of 6.0-8.0, preferably a 20 mM phosphate buffer containing 50 mM NaCl with a pH of 6.0.

[0015] Further, solution B is a 15-25 mM sodium citrate buffer solution with pH 7.0 containing 0.2 M NaCl, preferably a 20 mM sodium citrate buffer solution with pH 7.0 containing 0.2 M NaCl. The D solution is a 15-25 mM phosphate buffer containing 1 M NaCl with a pH of 6.0-8.0, preferably a 20 mM phosphate buffer containing 1 M NaCl with a pH of 6.0.

[0016] This invention discloses a method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum. Based on the charge heterogeneity of the target protein rC1INH and impurities in rabbit serum, a strategy of alternating anions and cations, gradually decreasing the particle size of the chromatographic medium, and progressively increasing the resolution is employed. This method successfully isolates high-purity and highly active rC1INH from rabbit serum, which has extremely low abundance, with high recovery and good batch-to-batch reproducibility. The purified rC1INH has a clearly defined composition and controllable quality, with a purity exceeding 95%. The purification method is simple to operate, fast, and low-cost. The purified rC1INH standard can be used to prepare ELISA kits, making the kits highly sensitive, specific, and reproducible. These kits can detect the content of rC1INH in rabbit serum, plasma, or other biological samples, possessing extremely high biomedical research and application value.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 This is the capture and purification (SPBB) chromatography chromatogram of the present invention. Figure 2 This is the chromatographic pattern of the moderately purified (QHP) solution of the present invention. Figure 3 The fine purification (Mono S) chromatography pattern of this invention Figure 4 Electrophoresis diagrams for purity analysis of each step in this invention. Figure 5 SEC-HPLC chromatograms of purity analysis for each step of this invention. Figure 6 The LC-MS chromatogram of rC1INH isolated and purified in this invention. Figure 7 This invention provides a process route for purifying C1INH from rabbit serum. Detailed Implementation The raw materials, reagents and equipment used in the specific embodiments of the present invention are all purchased commercially. Among them, SPBig Beads is a strong cation exchange chromatography packing material with 6% highly cross-linked agarose as the matrix. Its functional group is sulfopropyl (SP), which is coupled to the matrix through chemically stable ether bonds. The particle size is usually between 100 and 300 µm. QHP is a high-resolution strong anion exchange chromatography packing material with 6% highly cross-linked agarose as the matrix. Its functional group is a quaternary ammonium group, which is coupled to the matrix through chemically stable ether bonds. The particle size is 34 μm. Mono S is a strong cation exchange chromatography packing material, which is a polystyrene / divinylbenzene resin with a matrix of monodisperse 10μm porous beads.

[0020] G25F: It is a common gel filtration chromatography packing material composed of dextran and cross-linking agent (such as epichlorohydrin) through ether bonding. It is mainly used for buffer replacement, desalting and removal of small molecules in biological samples such as proteins, polysaccharides and nucleic acids.

[0021] Example 1: Isolation and purification of rClINH from rabbit serum 1) Take rabbit serum, filter it through a 0.45μm filter, add ammonium sulfate to the filtrate until its saturation reaches 35%, stir at 2~8℃ for 8~12h, then centrifuge at 10000 g speed and 2~8℃ for 30 min, and collect the supernatant; 2) Take the supernatant obtained in step 1) and load it onto a G25F desalting chromatography column equilibrated with solution A. Elute with solution A again. Based on the UV signal response at 280 nm, collect the eluent completely from the peak start to the peak end. Load it onto an SP BigBeads chromatography column equilibrated with solution A. After reequilibration with solution A, elute with solution B and collect the eluent. 3) Take the eluent obtained in step 2) and load it onto a G25F desalting column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the start to the end of the peak. Load it onto a Q HP column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.4 U / ml. 4) Take the eluent obtained in step 3) and load it onto a G25F desalting chromatography column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the start to the end of the peak. Load the eluent onto a Mono S chromatography column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.87 U / ml. The procedure for linear gradient elution is as follows: using 20 column volumes (CV), the mobile phase is linearly changed from 100% C solution to 100% D solution; Solution A is a 20mM sodium citrate buffer solution with a pH of 7.0 containing 20mM NaCl; Solution B is a 20 mM sodium citrate buffer solution with a pH of 7.0 containing 0.2 M NaCl; Solution C is a 20 mM phosphate buffer solution containing 50 mM NaCl with a pH of 6.0; Solution D is a 20 mM phosphate buffer containing 1 M NaCl with a pH of 6.0.

[0022] Example 2: Isolation and purification of rClINH from rabbit serum 1) Take rabbit serum, filter it through a 0.45μm filter, add ammonium sulfate to the filtrate until its saturation reaches 40%, stir at room temperature for 6-10 h, then centrifuge at 10000 g speed and 2-8℃ for 30 min, and collect the supernatant. 2) Take the supernatant obtained in step 1) and load it onto a G25F desalting chromatography column equilibrated with solution A. Elute with solution A again. Based on the UV signal response at 280 nm, collect the eluent completely from the peak start to the peak end. Load it onto an SP BigBeads chromatography column equilibrated with solution A. After reequilibration with solution A, elute with solution B and collect the eluent. 3) Take the eluent obtained in step 2) and load it onto a G25F desalting chromatography column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the beginning to the end of the peak. Load it onto a Q HP chromatography column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.24 U / ml. 4) Take the eluent obtained in step 3) and load it onto a G25F desalting chromatography column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the beginning to the end of the peak. Load the eluent onto a Mono S chromatography column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.52 U / ml. The procedure for linear gradient elution is as follows: using 20 column volumes (CV), the mobile phase is linearly changed from 100% C solution to 100% D solution; Solution A is a 15 mM sodium citrate buffer containing 20 mM NaCl with a pH of 7.0; Solution B is a 15 mM sodium citrate buffer solution with a pH of 7.0 containing 0.2 M NaCl; Solution C is a 15 mM phosphate buffer solution containing 50 mM NaCl with a pH of 7.0. Solution D is a 15 mM phosphate buffer containing 1 M NaCl with a pH of 7.0.

[0023] Example 3: Isolation and purification of rClINH from rabbit serum 1) Take rabbit serum, filter it through a 0.45μm filter, add ammonium sulfate to the filtrate until its saturation reaches 40%, stir at room temperature for 6-10 h, then centrifuge at 10000 g speed and 2-8℃ for 30 min, and collect the supernatant. 2) Take the supernatant obtained in step 1) and load it onto a G25F desalting chromatography column equilibrated with solution A. Elute with solution A again. Based on the UV signal response at 280 nm, collect the eluent completely from the peak start to the peak end. Load it onto an SP BigBeads chromatography column equilibrated with solution A. After reequilibration with solution A, elute with solution B and collect the eluent. 3) Take the eluent obtained in step 2) and load it onto a G25F desalting chromatography column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the beginning to the end of the peak. Load it onto a Q HP chromatography column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.33 U / ml. 4) Take the eluent obtained in step 3) and load it onto a G25F desalting chromatography column equilibrated with solution C. Elute with solution C again. Based on the UV signal response at 280 nm, collect the eluent completely from the start to the end of the peak. Load it onto a Mono S chromatography column equilibrated with solution C. After reequilibration with solution C, perform linear gradient elution and collect the eluent with enzyme activity ≥0.71 U / ml. The procedure for linear gradient elution is as follows: using 20 column volumes (CV), the mobile phase is linearly changed from 100% C solution to 100% D solution; Solution A is a 25mM sodium citrate buffer containing 20 mM NaCl with a pH of 7.0; Solution B is a 25 mM sodium citrate buffer solution with a pH of 7.0 containing 0.2 M NaCl; Solution C is a 25 mM phosphate buffer solution containing 50 mM NaCl with a pH of 8.0; Solution D is a 25 mM phosphate buffer containing 1 M NaCl with a pH of 8.0.

[0024] The following experimental examples illustrate the beneficial effects of the present invention.

[0025] Experiment 1: Verification of the effectiveness of isolating and purifying rClINH from rabbit serum 1. Ammonium sulfate precipitation 1.1 The thawed rabbit serum was filtered through a 0.45 μm filter, and ammonium sulfate was added to the filtered serum to a 35% saturation. The mixture was stirred overnight at 2-8°C (or stirred at room temperature for 6 h). 1.2 The ammonium sulfate-treated rabbit serum was centrifuged under the following conditions: 10,000 g speed, 2-8℃ for 30 min. The supernatant was collected after centrifugation. 1.3 Desalination and solution replacement: After centrifugation, the supernatant was loaded in a certain volume onto a G25F desalting chromatography column equilibrated with solution A (pH 7.0, 20 mM sodium citrate buffer, 20 mM NaCl). Elution was then continued using solution A. The protein elution peak was detected by a UV detector (280 nm), and the conductivity (reflecting the salt concentration) was monitored. When the UV signal reached a peak, the eluent was collected until the peak ended, and the target protein fraction was replaced with fresh solution A.

[0026] 2. SP BB Capture Chromatography: After desalting and buffer replacement, the sample is loaded onto an SP Big Beads column equilibrated with solution A. After reequilibration with solution A, it is eluted with solution B (pH 7.0, 20 mM sodium citrate buffer, 0.2 M NaCl). The eluted sample is collected, which is the crude pure rClINH. The capture chromatography chromatogram is shown below. Figure 1 As shown.

[0027] from Figure 1 It is evident that a large number of impurity proteins did not bind to the chromatography packing material and were removed by flow-through. The target protein rC1INH bound to the chromatography packing material and was eluted by a certain concentration of sodium chloride, appearing in the eluent.

[0028] 3. Q HP intermediate purification 3.1 Desalination and solution replacement: The crude purified rC1INH was loaded at a certain volume onto a G25F desalting chromatography column equilibrated with C solution (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl). Elution was then continued using C solution. The protein elution peak was detected by a UV detector (280 nm), and the conductivity (reflecting salt concentration) was monitored. When the UV signal peaked, the eluent was collected until the peak ended. The target protein fraction was then replaced with fresh C solution as the loading solution for this chromatography step. 3.2 Linear Elution: The desalted sample was loaded onto a QHP chromatography column equilibrated with solution C (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl). After reequilibration, linear elution with sodium chloride concentration was performed. The low-salt solution was solution C (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl), and the high-salt solution was solution D (pH 6.0, 20 mM phosphate buffer, 1 M NaCl). Elution was performed for 20 column volumes (CV), i.e., 20 CV to complete the linear gradient change of salt concentration from low to high. This step of linear elution further separated and purified the target product. The location of the target protein was located by enzyme kinetic substrate colorimetric method. The enzyme activity of the target fraction was 0.4 U / ml (other fractions had no activity or very low activity). This fraction was collected as the moderately purified eluent containing rC1INH. The moderately purified eluent containing rC1INH was then used for further purification. The chromatogram of the moderately purified eluent is shown below. Figure 2 As shown.

[0029] from Figure 2 It is evident that by utilizing QHP to address the isoelectric point differences between different proteins, and combining this with linear elution, the purity of rC1INH protein in SP BB eluent can be further improved.

[0030] 4. Mono S fine purification 4.1 Desalting and Buffer Replacement: Load a certain volume of Q HP elution buffer (i.e., moderately purified elution buffer containing rC1INH) onto a G25F desalting chromatography column equilibrated with C solution (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl). Then continue elution with C solution. Detect the protein elution peak using a UV detector (280 nm) and monitor the conductivity (reflecting salt concentration). When the UV signal reaches a peak, start collecting the elution buffer until the peak ends. Replace the target protein component with fresh C solution as the sample for this chromatography step.

[0031] 4.2 Linear Elution: The desalted sample was loaded onto a Mono S chromatography column equilibrated with solution C (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl). After reequilibration with solution C, linear elution with increasing sodium chloride concentration was performed. The low-salt solution was solution C (pH 6.0, 20 mM phosphate buffer, 50 mM NaCl), and the high-salt solution was solution D (pH 6.0, 20 mM phosphate buffer, 1 M NaCl). Elution was performed for 20 column volumes (CV), i.e., 20 CV to complete the linear gradient change in salt concentration from low to high. The target protein was located using enzyme kinetics substrate colorimetric method. The enzyme activity of the target fraction was 0.87 U / ml. This fraction was the rC1INH purified sample. The finely purified chromatographic pattern is shown below. Figure 3 As shown.

[0032] from Figure 3 It is evident that using Mono S column chromatography can further improve the purity of the target protein rC1INH.

[0033] 5. Purity Analysis (SDS-PAGE Silver Staining Method): After three-step purification, the purity of the target protein increased progressively, and the purity of the final finely purified sample was above 95%. (See attached image) Figure 4 .

[0034] 6. Purity Analysis (SEC-HPLC): After three-step purification, the purity of the final finely purified sample is above 95%, see [reference needed]. Figure 5 .

[0035] 7. Sequence Identification (LC-MS): Three proteases—trypsin, chymotrypsin, and Glu-C—as well as trypsin + chymotrypsin and trypsin + Glu-C combination enzymes, were used to digest the purified rC1INH sample and generate liquid chromatography-mass spectra. The amino acid sequence coverage reached over 83.5%. (See attached image). Figure 6 .

[0036] Following the same separation and purification process described above ( Figure 7 Multiple batches of rabbit serum were isolated and purified. The purity of the finely purified samples obtained was above 95%, and the amino acid sequence coverage was above 83.5%. The yield was up to 31 mg / L serum, indicating that the separation and purification process of this invention has good batch-to-batch reproducibility and can stably obtain high yield, high purity and high activity rClINH.

[0037] In summary, this invention, based on the charge heterogeneity of the target protein rC1INH and impurities in rabbit serum, employs a strategy of alternating cations and anions, gradually decreasing the particle size of the chromatographic medium, and progressively increasing the resolution. This allows for the separation of high-purity and highly active rC1INH from rabbit serum, which has extremely low abundance, with high recovery and good batch-to-batch reproducibility. The purified rC1INH has a clearly defined composition and controllable quality, with a purity exceeding 95%. The purification method is simple to operate, fast, and low-cost. The purified rC1INH standard can be used to prepare ELISA kits, making the kits highly sensitive, specific, and reproducible. These kits can detect the content of rC1INH in rabbit serum, plasma, or other biological samples, possessing extremely high biomedical research and application value.

Claims

1. A method for isolating and purifying rabbit C1 esterase inhibitors from rabbit serum, characterized in that: It includes the following steps: 1) Take rabbit serum, filter, add precipitant to the filtrate to precipitate, centrifuge, and collect the supernatant; 2) Take the supernatant obtained in step 1), desalt and replace it in solution A, then load it onto a chromatography column packed with SP Big Beads, elute with solution B, and collect the eluent; 3) Take the eluent obtained in step 2), desalt and replace it in solution C, then load it onto a chromatography column packed with Q HP, and perform linear gradient elution with solutions C and D. Collect the eluent with enzyme activity ≥0.2 U / ml. 4) Take the eluent obtained in step 3), desalt and replace it in solution C, then load it onto a chromatography column packed with Mono S, and perform linear gradient elution with solutions C and D. Collect the eluent with enzyme activity ≥ 0.5 U / ml. The procedure for the linear gradient elution is as follows: using 20 column volumes, the mobile phase is linearly changed from 100% C solution to 100% D solution; Solution A is a buffer solution with a pH of 7.0 containing 20 mM NaCl. Solution B is a buffer solution with a pH of 7.0 containing 0.2-1 M NaCl; Solution C is a buffer solution containing 50 mM NaCl with a pH of 6.0~8.0; The D solution is a buffer solution containing 1 M NaCl with a pH of 6.0~8.

0.

2. The method according to claim 1, characterized in that: Step 1) The precipitant includes ammonium sulfate and / or PEG-4000.

3. The method according to claim 2, characterized in that: The ammonium sulfate is added until the saturation reaches 30-40%.

4. The method according to claim 1, characterized in that: Step 1) The stirring temperature is 2~25℃, and the time is 6~12h.

5. The method according to claim 1, characterized in that: Step 1) The centrifugation speed is 10000 g, the temperature is 2-8℃, and the time is 25-35 min.

6. The method according to claim 1, characterized in that: Step 2) The method for desalting and replacing the supernatant with solution A is as follows: the supernatant is loaded onto a G25F desalting chromatography column equilibrated with solution A, and then eluted with solution A. Based on the UV signal response at 280 nm, the eluent is completely collected from the beginning to the end of the peak.

7. The method according to claim 1 or 6, characterized in that: Solution A is a 15-25mM sodium citrate buffer solution with pH 7.0 containing 20 mM NaCl, preferably a 20mM sodium citrate buffer solution with pH 7.0 containing 20 mM NaCl.

8. The method according to claim 1, characterized in that: The method for desalting and replacing the eluent with solution C in steps 3) and 4) is as follows: the eluent is loaded onto a G25F desalting chromatography column equilibrated with solution C, and then eluted with solution C. Based on the UV signal response at 280 nm, the eluent is completely collected from the beginning to the end of the peak.

9. The method according to claim 1, characterized in that: The chromatography column described in steps 3) and 4) is equilibrated with C solution before and after sample loading; The C solution is a 15-25 mM phosphate buffer containing 50 mM NaCl with a pH of 6.0-8.0, preferably a 20 mM phosphate buffer containing 50 mM NaCl with a pH of 6.

0.

10. The method according to claim 1, characterized in that: Solution B is a 15-25 mM sodium citrate buffer solution with pH 7.0 containing 0.2 M NaCl, preferably a 20 mM sodium citrate buffer solution with pH 7.0 containing 0.2 M NaCl. The D solution is a 15-25 mM phosphate buffer containing 1 M NaCl with a pH of 6.0-8.0, preferably a 20 mM phosphate buffer containing 1 M NaCl with a pH of 6.0.

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