Purification method and preparation method of pegcetacoplan

Abstract

The present invention relates to the technical field of polypeptide drugs, and in particular to a purification method and preparation method of Pegcetacoplan. The purification method of Pegcetacoplan comprises: (a) performing reverse phase preparative liquid chromatography purification on the sample to be purified, and collecting the eluted fraction containing Pegcetacoplan; (b) after ultrafiltration treatment, performing cation exchange chromatography, and collecting the eluted fraction containing Pegcetacoplan; in the reverse phase preparative liquid chromatography purification, mobile phase A is an aqueous solution of reagent A; mobile phase B is acetonitrile; in the cation exchange chromatography, the equilibrium buffer is an aqueous solution of sodium acetate, sodium dihydrogen phosphate or potassium dihydrogen phosphate. The present invention is aimed at the structure and synthetic characteristics of Pegcetacoplan, combined with the reverse phase preparative liquid chromatography purification, ultrafiltration, and cation exchange chromatography steps, to obtain high-purity Pegcetacoplan while ensuring a high yield.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a purification method and a preparation method of Pegcetacoplan. Background Art

[0002] Peptides, as an important bioactive molecule, have a wide range of pharmacological activities, such as antibacterial, antiviral, and antitumor. In recent years, with the continuous development of biotechnology, peptide drugs have become a hot area in the research and development of new drugs. However, peptide molecules are often accompanied by many by-products and impurities during the synthesis process. The presence of these impurities will directly affect the activity and stability of peptide drugs. Therefore, the purification of peptides has become a key link in the preparation of high-activity and high-purity peptide drugs.

[0003] Pegcetacoplan is a PEGylated bicyclic peptide. Since the coupling reaction of PEG and polypeptide is a random nucleophilic reaction, the product of the reaction is a modified mixture. In addition, the substances in these modified mixtures have slight differences in molecular weight and surface charge, but other physical and chemical properties are very similar, which brings difficulties to the purification work.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One object of the present invention is to provide a method for purifying Pegcetacoplan, which can significantly improve the purity of Pegcetacoplan.

[0006] Another object of the present invention is to provide a method for preparing Pegcetacoplan, including the above-mentioned method for purifying Pegcetacoplan.

[0007] In order to achieve the above-mentioned object of the present invention, the present invention provides a method for purifying Pegcetacoplan, comprising the following steps:

[0008] (a) subjecting the sample to be purified to reverse phase preparative liquid chromatography, and collecting the eluted fractions containing Pegcetacoplan;

[0009] (b) subjecting the eluted fraction containing Pegcetacoplan to ultrafiltration, and then subjecting the eluted fraction containing Pegcetacoplan to cation exchange chromatography to collect the eluted fraction containing Pegcetacoplan;

[0010] In the reverse phase preparative liquid chromatography purification, mobile phase A is an aqueous solution of reagent A; mobile phase B is acetonitrile; the reagent A includes at least one of formic acid, trifluoroacetic acid, phosphoric acid and triethylamine;

[0011] In the cation exchange chromatography, the equilibrium buffer is an aqueous solution of reagent B, and the reagent B includes at least one of sodium acetate, sodium dihydrogen phosphate and potassium dihydrogen phosphate.

[0012] In a specific embodiment of the present invention, the mobile phase A is an aqueous solution of triethylamine and phosphoric acid. Further, in the mobile phase A, the volume fractions of triethylamine and phosphoric acid are 1.8% to 2.2% and 0.9% to 1.1%, respectively.

[0013] In a specific embodiment of the present invention, in the equilibration buffer, the concentration of the reagent B is 0.1 to 5 mM, preferably 3 to 5 mM. Furthermore, the pH of the equilibration buffer is 3 to 5.2.

[0014] In a specific embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, the chromatographic column is a reverse phase C18 chromatographic column. Further, the chromatographic column is a YMC reverse phase C18 chromatographic column.

[0015] In a specific embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, the mobile phase A and the mobile phase B are used for gradient elution; the gradient elution includes: within 0 to 60 minutes, the volume fraction of the mobile phase B changes from 20% to 28% to 46% to 60%.

[0016] In a specific embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, an ultraviolet detector with a detection wavelength of 220 nm is used for detection, and the eluted fractions containing Pegcetacoplan are collected.

[0017] In a specific embodiment of the present invention, the cation exchange chromatography comprises: loading the solution after the ultrafiltration treatment, then balancing with the equilibration buffer, and then eluting with an elution solvent. Further, the elution is performed with the equilibration buffer and mobile phase C, and the mobile phase C is an acetonitrile aqueous solution containing sodium dihydrogen phosphate and sodium chloride.

[0018] In a specific embodiment of the present invention, in the mobile phase C, the concentration of the sodium dihydrogen phosphate is 1-5 mM, the concentration of the sodium chloride is 0.4-0.6 M, and the volume fraction of acetonitrile in the acetonitrile aqueous solution is 15%-25%.

[0019] In a specific embodiment of the present invention, the elution comprises: within 0 to 15CV, the volume fraction of the mobile phase C changes from 0% to 40%.

[0020] In a specific embodiment of the present invention, in the cation exchange chromatography, the chromatography medium comprises at least one of NanoGel-50SP HP, Proteomix PRO30-S, Welch Precot SP Polymate, Monomix HC30-SP and BiolinkMaxgo 30S.

[0021] In a specific embodiment of the present invention, the ultrafiltration treatment comprises: ultrafiltration and concentrating the eluted fraction containing Pegcetacoplan by 8 to 12 times, ultrafiltration with water until the permeate conductivity is ≤ 0.1 μs / cm, and diluting the retentate to the original volume of the eluted fraction with a sodium dihydrogen phosphate aqueous solution. Furthermore, the concentration of the sodium dihydrogen phosphate aqueous solution is 0.1 to 5 mM, preferably 3 to 5 mM.

[0022] In a specific embodiment of the present invention, the purification method further comprises: subjecting the eluted fraction containing Pegcetacoplan in step (b) to salt conversion, concentration and lyophilization.

[0023] In a specific embodiment of the present invention, the salt conversion treatment comprises: ultrafiltration and concentrating the eluted fraction containing Pegcetacoplan in step (b) by 4 to 6 times, and then adding a volume of 0.1% acetic acid aqueous solution with a volume of 5 times the volume of the concentrated material for ultrafiltration.

[0024] In a specific embodiment of the present invention, the membrane package used in the ultrafiltration is TeomaxTM Ⅱ CassetteRC 3K-D 0.1m 2 .

[0025] Another aspect of the present invention provides a method for preparing Pegcetacoplan, comprising the following steps:

[0026] (A) Pegcetacoplan intermediate cyclic peptide is coupled with PEG40K in a solvent to obtain a coupling solution;

[0027] (B) Purifying the coupling solution, wherein the purification is performed according to any one of the above-mentioned methods for purifying Pegcetacoplan.

[0028] In a specific embodiment of the present invention, in step (A), the mass ratio of the Pegcetacoplan intermediate cyclic peptide to the PEG40K is 1:(7-8).

[0029] In a specific embodiment of the present invention, the solvent comprises DMF.

[0030] In a specific embodiment of the present invention, the coupling reaction includes: dissolving the Pegcetacoplan intermediate cyclic peptide in a solvent to obtain a first solution; dissolving the PEG40K in a solvent to obtain a second solution; adding the second solution to the first solution at a flow rate of 100 to 140 mL / min, and then reacting at 30 to 35° C. for 1 to 2 hours.

[0031] In a specific embodiment of the present invention, step (B) further comprises: diluting the coupling solution by 8 to 12 times with water, filtering, and then performing the purification treatment.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention is based on the structure and synthetic characteristics of Pegcetacoplan, and combines reverse phase preparative liquid chromatography purification, ultrafiltration, and cation exchange chromatography steps to obtain high-purity Pegcetacoplan;

[0034] (2) The purification method of the present invention is simple to operate and easy to implement, and greatly reduces the use of organic reagents and reduces costs. DETAILED DESCRIPTION

[0035] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0036] Reversed-phase HPLC consists of a non-polar stationary phase and a polar mobile phase, and uses the difference in hydrophobicity of the solute for gradient elution separation and purification. Reversed-phase HPLC is efficient and rapid, suitable for relatively small-scale, high-value-added products, and is the main separation and purification method for peptides and the first choice for industrial production. However, studies have found that although reversed-phase HPLC can remove some small molecule impurities in peptides, it is difficult to separate them by membrane filtration because the physical and chemical properties of monosubstituted impurities and free PEG in the process are very similar to those of Pegcetacoplan, resulting in low product purity.

[0037] The inventors further discovered that due to the large difference in molecular weight between the PEG obtained in Pegcetacoplan and the polypeptide, after the PEG is modified on the intermediate cyclic peptide of Pegcetacoplan, there will be a shielding effect in the ion exchange chromatography, resulting in a weaker binding force between Pegcetacoplan and the chromatography medium, affecting the separation effect. Therefore, the existing purification methods of Pegcetacoplan cannot effectively remove the impurities formed in the coupling process of Pegcetacoplan. The present invention, based on the special structural characteristics of Pegcetacoplan, obtains high-purity Pegcetacoplan by combining a one-step reverse-phase preparative liquid chromatography purification and a one-step cation exchange chromatography.

[0038] Based on this, the present invention provides a method for purifying Pegcetacoplan, comprising the following steps:

[0039] (a) subjecting the sample to be purified to reverse phase preparative liquid chromatography, and collecting the eluted fractions containing Pegcetacoplan;

[0040] (b) subjecting the eluted fraction containing Pegcetacoplan to ultrafiltration, and then subjecting the eluted fraction containing Pegcetacoplan to cation exchange chromatography to collect the eluted fraction containing Pegcetacoplan;

[0041] In the reverse phase preparative liquid chromatography purification, mobile phase A is an aqueous solution of reagent A; mobile phase B is acetonitrile; reagent A includes at least one of formic acid, trifluoroacetic acid, phosphoric acid and triethylamine;

[0042] In the cation exchange chromatography, the equilibrium buffer is an aqueous solution of reagent B, and reagent B includes at least one of sodium acetate, sodium dihydrogen phosphate and potassium dihydrogen phosphate.

[0043] The present invention first uses reverse phase preparative liquid chromatography for purification, removes small molecule impurities and organic reagents under corresponding purification conditions, and prevents the generation of hydrolysis products of PEG modifiers; then uses ultrafiltration to remove organic reagents introduced in the reverse phase preparative liquid chromatography purification process, and then removes free PEG and monosubstituted impurities that are not conducive to separation by cation exchange chromatography. By using the purification method of the present invention, Pegcetacoplan with a purity of more than 99.5% can be obtained.

[0044] Among them, the mobile phase A and mobile phase B used in the reversed-phase preparative liquid chromatography purification, on the one hand, ensure the effective separation of various substances and the removal of small molecule impurities and organic reagents in the sample; on the other hand, prevent the generation of hydrolysis products of the PEG modifier to avoid affecting the stability of subsequent separation and purification steps.

[0045] Furthermore, further studies have found that the type of reagent B in the equilibration buffer used in the cation exchange chromatography process has a significant impact on the separation effect of free PEG and monosubstituted impurities and the yield of the target product. After reverse phase preparative liquid chromatography purification and ultrafiltration treatment, the present invention performs cation exchange chromatography loading and uses a suitable equilibration buffer to better separate the target product and impurities, while improving the purity and yield of the target product.

[0046] In a specific embodiment of the present invention, mobile phase A is an aqueous solution of triethylamine and phosphoric acid. Further, in mobile phase A, the volume fractions of triethylamine and phosphoric acid are 1.8% to 2.2% and 0.9% to 1.1%, respectively.

[0047] The present invention further optimizes the mobile phase A used in the reverse phase preparative liquid chromatography purification, so that the purity and yield of the target product are significantly improved. For example, in different embodiments, in the mobile phase A, the volume fraction of triethylamine can be 1.8%, 1.9%, 2%, 2.1%, 2.2% or a range consisting of any two thereof; the volume fraction of phosphoric acid can be 0.9%, 0.95%, 1%, 1.05%, 1.1% or a range consisting of any two thereof.

[0048] In a specific embodiment of the present invention, in the balancing buffer, the concentration of reagent B is 0.1 to 5 mM, preferably 3 to 5 mM. Further, the pH of the balancing buffer is 3 to 5.2. In actual operation, the pH of the balancing buffer is adjusted by using the corresponding acid in reagent B in the balancing buffer. For example, when reagent B is sodium acetate, acetic acid is used to adjust the pH; when reagent B is sodium dihydrogen phosphate or potassium dihydrogen phosphate, phosphoric acid is used to adjust the pH.

[0049] In a specific embodiment of the present invention, in the equilibration buffer, reagent B is sodium dihydrogen phosphate.

[0050] The present invention further optimizes the type, concentration and pH of the reagent B of the equilibrium buffer in the cation exchange chromatography, and cooperates with the reverse phase preparative liquid chromatography purification and ultrafiltration steps in the previous order, which can significantly improve the purity and yield of the target product. For example, in different embodiments, the concentration of reagent B can be 0.1mM, 1mM, 2mM, 3mM, 4mM, 5mM or a range consisting of any two thereof; the pH of the equilibrium buffer can be 3, 3.5, 4, 4.5, 5, 5.2 or a range consisting of any two thereof.

[0051] In a specific embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, the chromatographic column is a reverse phase C18 chromatographic column. Further, the chromatographic column is a YMC reverse phase C18 chromatographic column.

[0052] Among them, the specifications of the YMC reverse phase C18 chromatographic column are 20×250 mm, 10 μm, 120 Å. The use of the YMC reverse phase C18 chromatographic column is more conducive to the separation of Pegcetacoplan.

[0053] In a specific embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, mobile phase A and mobile phase B are used for gradient elution; the gradient elution includes: within 0 to 60 minutes, the volume fraction of mobile phase B changes from 20% to 28% to 46% to 60%.

[0054] In the reverse phase preparative liquid chromatography purification of the present invention, on the basis of using specific mobile phase A and mobile phase B, the elution procedure is further adjusted for the types of impurities pre-separated and removed in the purification stage, so as to effectively remove the pre-separated substances in this stage in a shorter time, and ensure high purity and yield. For example, in different embodiments, in gradient elution, within 0 to 60 minutes, the volume fraction of mobile phase B can be changed from 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% or any two thereof, to 46%, 48%, 50%, 52%, 54%, 55%, 56%, 58%, 60% any two thereof.

[0055] In a preferred embodiment of the present invention, in the reverse phase preparative liquid chromatography purification, the gradient elution comprises: within 0 to 60 min, the volume fraction of the mobile phase B changes from 25% to 26% to 46% to 50%.

[0056] In a specific embodiment of the present invention, during the reverse phase preparative liquid chromatography purification, an ultraviolet detector with a detection wavelength of 220 nm is used for detection, and the eluted fractions containing Pegcetacoplan are collected.

[0057] In a specific embodiment of the present invention, the cation exchange chromatography comprises: loading the solution after ultrafiltration treatment, then balancing with an equilibrium buffer, and then eluting with an elution solvent. Further, eluting with an equilibrium buffer and a mobile phase C, the mobile phase C is an acetonitrile aqueous solution containing sodium dihydrogen phosphate and sodium chloride.

[0058] In actual operation, equilibrium buffer is used for equilibrium until the absorption of the eluent at a detection wavelength of 220 nm is 0, and then subsequent elution is performed.

[0059] In a specific embodiment of the present invention, in mobile phase C, the concentration of sodium dihydrogen phosphate is 1-5 mM, the concentration of sodium chloride is 0.4-0.6 M, and the volume fraction of acetonitrile in the acetonitrile aqueous solution is 15%-25%.

[0060] In cation exchange chromatography, after loading, a specific equilibration buffer is used for equilibration to fully remove impurities and avoid elution of the target product to ensure the yield; after equilibration, a specific elution solvent is used for elution. For example, in different embodiments, in mobile phase C, the concentration of sodium dihydrogen phosphate can be 1mM, 2mM, 3mM, 4mM, 5mM, any two of which are in the range; the concentration of sodium chloride can be 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, or any two of which are in the range; in acetonitrile aqueous solution, the volume fraction of acetonitrile can be 15%, 18%, 20%, 22%, 25%, or any two of which are in the range. In a preferred embodiment, in mobile phase C, the concentration of sodium dihydrogen phosphate is 3mM, the concentration of sodium chloride is 0.5M, and the volume fraction of acetonitrile in acetonitrile aqueous solution is 20%.

[0061] In a specific embodiment of the present invention, the elution comprises: within 0 to 15 CV, the volume fraction of the mobile phase C changes from 0% to 40%.

[0062] In a specific embodiment of the present invention, in the cation exchange chromatography, the chromatography medium comprises at least one of NanoGel-50SPHP, Proteomix PRO30-S, Welch Precot SP Polymate, Monomix HC30-SP and Biolink Maxgo30S, preferably NanoGel-50SP HP.

[0063] Cation exchange chromatography mainly uses the difference in electrostatic interaction between the chromatographic medium and the substance to be separated to achieve separation, and there are many factors affecting the electrostatic interaction, including but not limited to the type of chromatographic medium, the type of equilibrium buffer and elution solvent, and the type of substance to be separated. The present invention is directed to the substance to be separated after reverse phase preparative liquid chromatography purification and ultrafiltration treatment, and is combined with the corresponding equilibrium buffer, elution solvent, etc. When NanoGel-50SP HP is used as the chromatographic medium, compared with other chromatographic media, while significantly improving the purity of the target product, it can also increase the sample loading and improve efficiency.

[0064] In a specific embodiment of the present invention, the ultrafiltration treatment comprises: ultrafiltration and concentrating the eluted fraction containing Pegcetacoplan by 8 to 12 times, ultrafiltration with water until the permeate conductivity is ≤ 0.1 μs / cm, and diluting the retentate with a sodium dihydrogen phosphate aqueous solution to the original volume of the eluted fraction. Furthermore, the concentration of the sodium dihydrogen phosphate aqueous solution is 0.1 to 5 mM, preferably 3 to 5 mM.

[0065] Research has found that if the material after the coupling reaction is directly subjected to ultrafiltration treatment, due to the interference of a large amount of organic solvents in the system, it is easy to cause the penetration of the target product and it is difficult to separate the impurities. The present invention performs the above-mentioned ultrafiltration treatment after reversed-phase preparative liquid chromatography purification, on the one hand, removes the organic solvent introduced during the reversed-phase preparative liquid chromatography purification process, and then uses sodium dihydrogen phosphate aqueous solution to replace the liquid, while avoiding the degradation of the target product, reducing the ionic strength of the sample, so as to achieve further separation and purification in the subsequent cation exchange chromatography.

[0066] For example, in different embodiments, the concentration of sodium dihydrogen phosphate in the sodium dihydrogen phosphate aqueous solution used to dilute the retentate may be 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or a range consisting of any two of them.

[0067] In a specific embodiment of the present invention, the purification method further comprises: subjecting the eluted fraction containing Pegcetacoplan in step (b) to salt conversion, concentration and lyophilization.

[0068] In a specific embodiment of the present invention, the salt conversion treatment comprises: ultrafiltration and concentrating the eluted fraction containing Pegcetacoplan in step (b) by 4 to 6 times, and then adding 0.1% acetic acid aqueous solution with a volume of 5 times the volume of the concentrated material for ultrafiltration.

[0069] In a specific embodiment of the present invention, the membrane package used for ultrafiltration is TeomaxTM Ⅱ Cassette RC3K-D 0.1m 2 The membrane package used for ultrafiltration is suitable for ultrafiltration after reverse phase preparative liquid chromatography purification, and is also suitable for ultrafiltration in salt conversion treatment.

[0070] Another aspect of the present invention provides a method for preparing Pegcetacoplan, comprising the following steps:

[0071] (A) Pegcetacoplan intermediate cyclic peptide is coupled with PEG40K in a solvent to obtain a coupling solution;

[0072] (B) Purifying the coupling solution according to any of the above-mentioned purification methods for Pegcetacoplan.

[0073] Among them, the intermediate cyclic peptide of Pegcetacoplan can be self-made or purchased. Specifically, the structural formula of the intermediate cyclic peptide of Pegcetacoplan is as follows:

[0074] .

[0075] PEG40K has the following general structural formula, where n is an integer between 800 and 900;

[0076] In a specific embodiment of the present invention, in step (A), the mass ratio of the intermediate cyclic peptide of Pegcetacoplan to PEG40K is 1:(7-8).

[0077] For example, in different embodiments, the mass ratio of the intermediate cyclic peptide of Pegcetacoplan to PEG40K can be 1:7, 1:7.2, 1:7.5, 1:7.8, 1:8 or a range consisting of any two of them.

[0078] In a particular embodiment of the invention, the solvent comprises DMF.

[0079] In a specific embodiment of the present invention, the coupling reaction includes: dissolving the Pegcetacoplan intermediate cyclic peptide in a solvent to obtain a first solution; dissolving PEG40K in a solvent to obtain a second solution; adding the second solution to the first solution at a flow rate of 100 to 140 mL / min, and then reacting at 30 to 35° C. for 1 to 2 hours.

[0080] In actual operation, in the first solution, the concentration of the Pegcetacoplan intermediate cyclic peptide can be 6-8 mg / mL; in the second solution, the concentration of PEG40K can be 20-30 mg / mL, and the flow rate of the second solution added to the first solution can be 100 mL / min, 110 mL / min, 120 mL / min, 130 mL / min, 140 mL / min or a range composed of any two thereof. The above process parameters are adjusted to improve the yield of the target product.

[0081] In a specific embodiment of the present invention, step (B) further comprises: diluting the coupling solution by 8 to 12 times with water, filtering, and then performing purification treatment.

[0082] Example 1

[0083] This embodiment provides a method for purifying Pegcetacoplan, comprising the following steps:

[0084] (1) The coupling solution was diluted 10 times with purified water and then loaded onto a chromatographic column for reverse phase preparative liquid chromatography purification, and the eluted fractions containing Pegcetacoplan were collected; the chromatographic column was a YMC reverse phase C18 chromatographic column (20×250 mm, 10 μm, 120 Å), and the detection wavelength was 220 nm; mobile phase A and mobile phase B were used for gradient elution, mobile phase A was an aqueous solution containing 2% (volume fraction) triethylamine and 1% (volume fraction) phosphoric acid, and mobile phase B was acetonitrile; the gradient elution was: within 0 to 60 min, the volume fraction of mobile phase B changed from 25% to 50%.

[0085] The preparation of the coupling liquid includes: taking 137.5 mg of the finished product of the intermediate cyclic peptide of Pegcetacoplan, adding it to 20 mL of DMF, and ultrasonically dissolving it to obtain a first solution; taking 1 g of PEG40K and dissolving it in 40 mL of DMF, and ultrasonically dissolving it to obtain a second solution; under stirring conditions, adding the second solution to the first solution at a flow rate of 120 mL / min, after the dropwise addition is completed, stirring and reacting at 35° C. for 1 hour to obtain a coupling liquid; in the coupling liquid, the purity of the target product is 38.51%.

[0086] (2) The eluted fraction containing Pegcetacoplan collected in step (1) was concentrated 10 times by ultrafiltration, and then purified water was added to ultrafilter until the permeate conductivity was 0.1 μs / cm, and then the remaining portion was supplemented with 3 mM sodium dihydrogen phosphate aqueous solution to the original volume of the eluted fraction. The membrane package used for ultrafiltration was TeomaxTM Ⅱ Cassette RC 3K-D 0.1m 2 .

[0087] (3) Loading the solution obtained in step (2) onto a NanoGel-50SP HP cation exchange chromatography column. After loading, equilibrate the cation exchange chromatography column with an equilibration buffer until the absorbance of the eluent at 220 nm is 0; then, eluting with the equilibration buffer and mobile phase C (the sum of the volume percentages of the equilibration buffer and mobile phase C is 100%), and collecting the eluted fractions containing Pegcetacoplan;

[0088] The equilibrium buffer was an aqueous solution of 3 mM sodium dihydrogen phosphate (pH 5.2); the mobile phase C was an acetonitrile aqueous solution containing 3 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 20%; during elution, the volume fraction of mobile phase C changed from 0% to 40% within 0 to 15 CV.

[0089] (4) The eluted fraction containing Pegcetacoplan collected in step (3) is concentrated by ultrafiltration 5 times, and then a 0.1% acetic acid aqueous solution with a volume 5 times the volume of the concentrated material is added for ultrafiltration, followed by lyophilization to obtain pure Pegcetacoplan.

[0090] Embodiments 2 to 5

[0091] Examples 2 to 5 refer to the purification method of Pegcetacoplan in Example 1, except that the purification parameters of the reverse phase preparative liquid chromatography in step (1) are different. The parts not mentioned are the same as those in Example 1. The specific differences are as follows:

[0092] The mobile phase A in Example 2 is different from that in Example 1. The mobile phase A in Example 2 is an aqueous solution of TFA with a volume fraction of 1%;

[0093] The gradient elution in Example 3 is different from that in Example 1. The gradient elution in Example 3 is as follows: within 0 to 60 min, the volume fraction of mobile phase B changes from 26% to 46%;

[0094] The gradient elution in Example 4 is different from that in Example 1. The gradient elution in Example 4 is as follows: within 0 to 60 min, the volume fraction of mobile phase B changes from 28% to 48%;

[0095] The gradient elution in Example 5 is different from that in Example 1. The gradient elution in Example 5 is as follows: within 0 to 60 minutes, the volume fraction of mobile phase B changes from 20% to 60%.

[0096] The purity of the eluted fractions containing Pegcetacoplan collected in step (1) in the purification methods of Examples 1 to 5 was tested and the yield was calculated. The results are shown in Table 1.

[0097] Table 1 Purity and yield test results of different examples

[0098]

[0099] From the above test results, it can be seen that the reverse phase preparative liquid chromatography purification process of Example 3 has higher purity and yield than other processes. It should be noted that the RP-HPLC purity in Table 1 is detected by HPLC in a reverse phase system, mainly detecting polypeptide-related impurities, which can reflect relative purity.

[0100] Embodiments 6 to 9

[0101] Examples 6 to 9 refer to the purification method of Pegcetacoplan in Example 3, except that the cation exchange chromatography parameters in step (3) are different. The unmentioned parts are the same as those in Example 3. The specific differences are as follows:

[0102] The equilibration buffer in Example 6 is different from that in Example 3. The equilibration buffer in Example 6 is an aqueous solution of sodium acetate with a concentration of 3 mM;

[0103] The equilibration buffer in Example 7 is different from that in Example 3. The equilibration buffer in Example 7 is an aqueous solution of potassium dihydrogen phosphate with a concentration of 3 mM;

[0104] The equilibration buffer in Example 8 is different from that in Example 3. The equilibration buffer in Example 8 is an aqueous solution of sodium dihydrogen phosphate with a concentration of 0.5 mM;

[0105] The equilibration buffer in Example 9 is different from that in Example 3. The equilibration buffer in Example 9 is an aqueous solution of sodium dihydrogen phosphate with a concentration of 5 mM.

[0106] The purity of the eluted fractions containing Pegcetacoplan collected in step (3) in the purification methods of Examples 3 and Examples 6 to 9 was tested, and the yield was calculated. The results are shown in Table 2. In the subsequent examples, the purity of the eluted fractions collected in step (3) was tested by ion chromatography HPLC, which can detect both polypeptide impurities and PEG impurities, and can reflect the actual total purity.

[0107] Table 2 Purity and yield test results of different examples

[0108]

[0109] From the above test results, it can be seen that the eluted fractions obtained in Examples 6 to 8 contain some free PEG and some monosubstituted PEG, and although the eluted fraction obtained in Example 9 can separate impurities well, the yield of the target product is low. In the purification method of Example 3, not only can the impurities be separated at a high level and the purity be significantly improved, but the yield of the product can also be guaranteed, which better meets the production requirements.

[0110] Embodiments 10 to 13

[0111] Examples 10 to 13 refer to the purification method of Pegcetacoplan in Example 3, except that the cation exchange chromatography parameters in step (3) are different. The unmentioned parts are the same as those in Example 3. The specific differences are as follows:

[0112] The mobile phase C in Example 10 is different from that in Example 3. The mobile phase C in Example 10 is an acetonitrile aqueous solution containing 0.5M sodium chloride with a volume fraction of 20%;

[0113] The mobile phase C in Example 11 is different from that in Example 3. The mobile phase C in Example 11 is an acetonitrile aqueous solution containing 3 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 40%;

[0114] The mobile phase C in Example 12 is different from that in Example 3. The mobile phase C in Example 12 is an acetonitrile aqueous solution containing 3 mM sodium acetate and 0.5 M sodium chloride with a volume fraction of 20%;

[0115] The mobile phase C in Example 13 is different from that in Example 3. The mobile phase C in Example 13 is an acetonitrile aqueous solution containing 5 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 20%.

[0116] The purity of the eluted fraction containing Pegcetacoplan collected in step (3) in the purification methods of Example 3 and Examples 10 to 13 was tested and the yield was calculated. The results are shown in Table 3.

[0117] Table 3 Purity and yield test results of different examples

[0118]

[0119] Embodiments 14 to 17

[0120] Examples 14 to 17 refer to the purification method of Pegcetacoplan in Example 3, except that the cation exchange chromatography parameters in step (3) are different. The unmentioned parts are the same as those in Example 3. The specific differences are as follows:

[0121] In Example 14, the cation exchange chromatography column is a Proteomix PRO30-S cation exchange chromatography column;

[0122] In Example 15, the cation exchange chromatography column is a Biolink Maxgo 30S cation exchange chromatography column;

[0123] In Example 16, the cation exchange chromatography column is a Welch Precot SP Polymate cation exchange chromatography column;

[0124] In Example 17, the cation exchange chromatography column is a Monomix HC30-SP cation exchange chromatography column.

[0125] The purity of the sample loading amount corresponding to step (3) and the collected elution fractions containing Pegcetacoplan in the purification methods of Example 3 and Examples 14 to 17 were tested, and the yield was calculated. The results are shown in Table 4.

[0126] Table 4 Test results of sample loading, purity and yield of different examples

[0127]

[0128] From the above test results, it can be seen that compared with the other embodiments, the chromatographic column packings of Examples 15 to 17 are not suitable for the purification of Pegcetacoplan, and the chromatographic column packing of Example 14 has certain limitations for the separation of impurities. By comparison, it can be seen that the cation exchange chromatography medium used in Example 3 is more helpful in improving the purity of the target product, ensuring the yield of the product, and better meeting the production needs in the system of the present invention.

[0129] Embodiments 18 to 20

[0130] Examples 18 to 20 refer to the purification method of Pegcetacoplan in Example 3, except that the cation exchange chromatography parameters in step (3) are different. The unmentioned parts are the same as those in Example 3. The specific differences are as follows:

[0131] The equilibration buffer in Example 18 is different from that in Example 3. The equilibration buffer in Example 18 is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM, and phosphoric acid is used to adjust the pH to 3;

[0132] The equilibration buffer in Example 19 is different from that in Example 3. The equilibration buffer in Example 19 is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM, and phosphoric acid is used to adjust the pH to 4;

[0133] The equilibration buffer in Example 20 is different from that in Example 3. The equilibration buffer in Example 20 is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM, and phosphoric acid is used to adjust the pH to 5.

[0134] The purity of the sample loading amount corresponding to step (3) and the collected elution fractions containing Pegcetacoplan in the purification methods of Example 3 and Examples 18 to 20 were tested, and the yield was calculated. The results are shown in Table 5.

[0135] Table 5 Test results of sample loading, purity and yield of different examples

[0136]

[0137] Examples 21 to 24

[0138] Examples 21 to 24 refer to the purification method of Pegcetacoplan in Example 3, except that the ultrafiltration treatment in step (2) is different. The unmentioned parts are the same as those in Example 3. The specific differences are as follows:

[0139] Step (2) of Example 21 comprises: ultrafiltration and concentration of the eluted fraction containing Pegcetacoplan collected in step (1) by 10 times, then adding purified water to ultrafiltration until the conductivity of the permeate is 0.1 μs / cm, and then making up the remaining portion with 0.1 mM sodium dihydrogen phosphate aqueous solution to the original volume of the eluted fraction;

[0140] Step (2) of Example 22 comprises: ultrafiltration and concentration of the eluted fraction containing Pegcetacoplan collected in step (1) by 10 times, then adding purified water for ultrafiltration until the permeate conductivity is 0.1 μs / cm, and then making up the remaining portion with 5 mM sodium dihydrogen phosphate aqueous solution to the original volume of the eluted fraction;

[0141] Step (2) of Example 23 comprises: ultrafiltration and concentration of the eluted fraction containing Pegcetacoplan collected in step (1) by 10 times, then adding purified water to ultrafiltration until the permeate conductivity is 0.1 μs / cm, and then making up the remaining portion with 3 mM sodium citrate aqueous solution to the original volume of the eluted fraction;

[0142] Step (2) of Example 24 comprises: ultrafiltration and concentration of the elution fraction containing Pegcetacoplan collected in step (1) by 10 times, then adding purified water and ultrafiltration until the conductivity of the permeate is 0.1 μs / cm, and then making up the remaining portion with water to the volume of the original elution fraction.

[0143] The purity of the sample loading amount corresponding to step (3) and the collected elution fractions containing Pegcetacoplan in the purification methods of Example 3 and Examples 21 to 24 were tested, and the yield was calculated. The results are shown in Table 6.

[0144] Table 6 Test results of sample loading, purity and yield of different examples

[0145]

[0146] Comparative Example 1

[0147] Comparative Example 1 provides a method for purifying Pegcetacoplan, comprising the following steps:

[0148] (1) After diluting the coupling solution 10 times with purified water, add 5 times the volume of purified water for ultrafiltration, and collect the ultrafiltration liquid. The membrane package used for ultrafiltration is TeomaxTM Ⅱ Cassette RC 3K-D 0.1m 2 ;

[0149] The preparation of the coupling liquid includes: taking 137.5 mg of the finished product of the intermediate cyclic peptide of Pegcetacoplan, adding it to 20 mL of DMF, and ultrasonically dissolving it to obtain a first solution; taking 1 g of PEG40K and dissolving it in 40 mL of DMF, and ultrasonically dissolving it to obtain a second solution; under stirring conditions, adding the second solution to the first solution at a flow rate of 120 mL / min, after the dropwise addition is completed, stirring the reaction at 35° C. for 1 hour to obtain a coupling liquid.

[0150] (2) The liquid containing Pegcetacoplan collected in step (1) is loaded onto a chromatographic column for purification by reverse phase preparative liquid chromatography, and the eluted fraction containing Pegcetacoplan is collected; wherein the chromatographic column is a YMC reverse phase C18 chromatographic column (20×250 mm, 10 μm, 120 Å), and the detection wavelength is 220 nm; mobile phase A and mobile phase B are used for gradient elution, wherein mobile phase A is an aqueous solution containing 2% (volume fraction) triethylamine and 1% (volume fraction) phosphoric acid, and mobile phase B is acetonitrile; the gradient elution is: within 0 to 60 minutes, the volume fraction of mobile phase B changes from 26% to 46%;

[0151] (3) Loading the solution obtained in step (2) onto a NanoGel-50SP HP cation exchange chromatography column. After loading, equilibrate the cation exchange chromatography column with an equilibration buffer until the absorbance of the eluent at 220 nm is 0; then, eluting with the equilibration buffer and mobile phase C (the sum of the volume percentages of the equilibration buffer and mobile phase C is 100%), and collecting the eluted fractions containing Pegcetacoplan;

[0152] The equilibrium buffer is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM; the mobile phase C is an acetonitrile aqueous solution containing 3 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 20%; during elution, the volume fraction of mobile phase C changes from 0% to 40% within 0 to 15 CV.

[0153] (4) The eluted fraction containing Pegcetacoplan collected in step (3) was concentrated by ultrafiltration 5 times, and then a 0.1% acetic acid aqueous solution with a volume of 5 times the volume of the concentrated material was added for ultrafiltration, and then freeze-dried to obtain pure Pegcetacoplan.

[0154] Comparative Example 2

[0155] Comparative Example 2 provides a method for purifying Pegcetacoplan, comprising the following steps: (1) diluting the coupling solution 10 times with purified water, adding 5 times the volume of purified water for ultrafiltration, and collecting the ultrafiltration liquid. The membrane package used for ultrafiltration is TeomaxTM Ⅱ Cassette RC 3K-D 0.1m 2 ;

[0156] The preparation of the coupling liquid includes: taking 137.5 mg of the finished product of the intermediate cyclic peptide of Pegcetacoplan, adding it to 20 mL of DMF and ultrasonically dissolving it to obtain a first solution; taking 1 g of PEG40K and dissolving it in 40 mL of DMF, ultrasonically dissolving it to obtain a second solution; under stirring conditions, adding the second solution to the first solution at a flow rate of 120 mL / min, after the dropwise addition is completed, stirring and reacting at 35° C. for 1 hour to obtain a coupling liquid. (2) Loading the solution obtained in step (1) onto a NanoGel-50SP HP cation exchange chromatography column, after the loading is completed, using an equilibrium buffer to equilibrate the cation exchange chromatography column until the absorption of the eluent at 220 nm is 0; then using an equilibrium buffer and mobile phase C (the sum of the volume percentages of the equilibrium buffer and mobile phase C is 100%) for elution, and collecting the eluted fraction containing Pegcetacoplan;

[0157] The equilibrium buffer is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM; the mobile phase C is an acetonitrile aqueous solution containing 3 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 20%; during elution, the volume fraction of mobile phase C changes from 0% to 40% within 0 to 15 CV. (3) The liquid containing Pegcetacoplan collected in step (1) is loaded onto a chromatographic column for reverse phase preparative liquid chromatography purification, and the eluted fraction containing Pegcetacoplan is collected; wherein the chromatographic column is a YMC reverse phase C18 chromatographic column (20×250 mm, 10 μm, 120 Å), and the detection wavelength is 220 nm; mobile phase A and mobile phase B are used for gradient elution, mobile phase A is an aqueous solution containing 2% (volume fraction) triethylamine and 1% (volume fraction) phosphoric acid, and mobile phase B is acetonitrile; the gradient elution is: within 0 to 60 minutes, the volume fraction of mobile phase B changes from 26% to 46%; (4) The eluted fraction containing Pegcetacoplan collected in step (3) is ultrafiltered and concentrated 5 times, and then a volume of 0.1% acetic acid aqueous solution with a volume fraction of 5 times the volume of the concentrated material is added for ultrafiltration, and then freeze-dried to obtain pure Pegcetacoplan.

[0158] Comparative Example 3

[0159] Comparative Example 3 provides a method for purifying Pegcetacoplan, comprising the following steps:

[0160] (1) Dilute the coupling solution 10 times with the equilibration solution and load it onto the NanoGel-50SP HP cation exchange chromatography column. After loading, use the equilibration buffer to equilibrate the cation exchange chromatography column until the absorption of the eluent at 220 nm is 0; then use the equilibration buffer and mobile phase C (the sum of the volume percentages of the equilibration buffer and mobile phase C is 100%) for elution, and collect the eluted fractions containing Pegcetacoplan;

[0161] The equilibrium buffer is an aqueous solution of sodium dihydrogen phosphate with a concentration of 3 mM; the mobile phase C is an acetonitrile aqueous solution containing 3 mM sodium dihydrogen phosphate and 0.5 M sodium chloride with a volume fraction of 20%; during elution, the volume fraction of mobile phase C changes from 0% to 40% within 0 to 15 CV.

[0162] The preparation of the coupling liquid includes: taking 137.5 mg of the finished product of the intermediate cyclic peptide of Pegcetacoplan, adding it to 20 mL of DMF, and ultrasonically dissolving it to obtain a first solution; taking 1 g of PEG40K and dissolving it in 40 mL of DMF, and ultrasonically dissolving it to obtain a second solution; under stirring conditions, adding the second solution to the first solution at a flow rate of 120 mL / min, after the dropwise addition is completed, stirring the reaction at 35° C. for 1 hour to obtain a coupling liquid.

[0163] (2) The Pegcetacoplan-containing fraction collected in step (1) is loaded onto a chromatographic column for purification by reverse phase preparative liquid chromatography, and the eluted fraction containing Pegcetacoplan is collected; wherein the chromatographic column is a YMC reverse phase C18 chromatographic column (20×250 mm, 10 μm, 120 Å), and the detection wavelength is 220 nm; mobile phase A and mobile phase B are used for gradient elution, wherein mobile phase A is an aqueous solution containing 2% (volume fraction) triethylamine and 1% (volume fraction) phosphoric acid, and mobile phase B is acetonitrile; the gradient elution is: within 0 to 60 minutes, the volume fraction of mobile phase B changes from 26% to 46%;

[0164] (3) The eluted fraction containing Pegcetacoplan collected in step (2) is concentrated by ultrafiltration 5 times, and then a 0.1% acetic acid aqueous solution with a volume 5 times the volume of the concentrated material is added for ultrafiltration, and then freeze-dried to obtain pure Pegcetacoplan.

[0165] In the purification methods of Comparative Examples 1 to 3, part of the sample flowed through during the first ultrafiltration process in Comparative Examples 1 and 2, and the yield requirement could not be met. In Comparative Example 3, the sample completely flowed through during the ion purification process, and sample purification could not be achieved.

[0166] Comparative Examples 4-5

[0167] Comparative Examples 4-5 refer to the purification method of Pegcetacoplan in Example 1, except that: the purification parameters of the reverse phase preparative liquid chromatography in step (1) are different, and the parts not mentioned are the same as those in Example 1. The specific differences are as follows:

[0168] The mobile phase A of Comparative Example 4 is different: the mobile phase A of Comparative Example 4 is an aqueous solution containing 50 mM sodium bicarbonate;

[0169] The mobile phase B of Comparative Example 5 is different: the mobile phase B of Comparative Example 5 is methanol.

[0170] Comparative Examples 6-7

[0171] Comparative Examples 6-7 refer to the purification method of Pegcetacoplan in Example 1, except that the cation exchange chromatography parameters in step (3) are different, and the unmentioned parts are the same as those in Example 1. The specific differences are as follows:

[0172] The equilibration buffer in Comparative Example 6 was different: the equilibration buffer in Comparative Example 7 was an aqueous solution of 3 mM sodium citrate at pH 5;

[0173] The equilibration buffer in Comparative Example 7 is different: the equilibration buffer in Comparative Example 8 is an aqueous solution of ammonium acetate at pH 5.

[0174] The purity of the eluted fractions containing Pegcetacoplan collected in step (3) in the purification methods of Comparative Examples 4 to 7 was tested and their yields were calculated. The results are shown in Table 7.

[0175] Table 7 Purity and yield test results of different comparative examples

[0176]

[0177] Note: The yields in Comparative Examples 4 and 5 are the yields of the reverse phase step; the yields in Comparative Examples 6 and 7 are the yields of the cation exchange chromatography step.

[0178] According to the above test results, the present invention targets the structure and synthesis characteristics of Pegcetacoplan, combines reverse phase preparative liquid chromatography purification, ultrafiltration, and cation exchange chromatography steps, and can obtain high-purity Pegcetacoplan. The purity of the obtained Pegcetacoplan product can reach 99.98%, and the overall yield can reach 77.34%.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for purifying pegcetacoplan, characterized in that: The steps include: (a) subjecting the sample to be purified to reverse phase preparative liquid chromatography, and collecting the eluted fractions containing Pegcetacoplan; (b) subjecting the eluted fraction containing Pegcetacoplan to ultrafiltration, and then subjecting the eluted fraction containing Pegcetacoplan to cation exchange chromatography to collect the eluted fraction containing Pegcetacoplan; In the reverse phase preparative liquid chromatography purification, mobile phase A is an aqueous solution of triethylamine and phosphoric acid; mobile phase B is acetonitrile; In the reverse phase preparative liquid chromatography purification, the chromatographic column is a reverse phase C18 chromatographic column; The ultrafiltration treatment comprises: ultrafiltration and concentration of the eluted fraction containing Pegcetacoplan by 8 to 12 times, ultrafiltration with water until the permeate conductivity is ≤ 0.1 μs / cm, and diluting the retentate with sodium dihydrogen phosphate aqueous solution to the original volume of the eluted fraction; the membrane package used in the ultrafiltration is TeomaxTM Ⅱ Cassette RC 3K-D 0.1m 2 ; In the cation exchange chromatography, the equilibration buffer is an aqueous solution of reagent B, and the reagent B includes at least one of sodium acetate, sodium dihydrogen phosphate and potassium dihydrogen phosphate; in the cation exchange chromatography, the chromatographic medium is NanoGel-50SP HP; the cation exchange chromatography includes: loading the solution after the ultrafiltration treatment, then equilibrate with the equilibration buffer, and then elute with an elution solvent; eluting with the equilibration buffer and mobile phase C, and the mobile phase C is an acetonitrile aqueous solution containing sodium dihydrogen phosphate and sodium chloride; The preparation of the sample to be purified includes: coupling the intermediate cyclic peptide of Pegcetacoplan and PEG40K in a solvent to obtain a coupling solution; the mass ratio of the intermediate cyclic peptide of Pegcetacoplan to the PEG40K is 1: (7-8); the coupling reaction includes: dissolving the intermediate cyclic peptide of Pegcetacoplan in a solvent to obtain a first solution; dissolving the PEG40K in a solvent to obtain a second solution; adding the second solution to the first solution, and then reacting at 30-35° C. for 1-2 hours; The structural formula of the Pegcetacoplan intermediate cyclic peptide is as follows: 。 2. The purification method according to claim 1, characterized in that In the mobile phase A, the volume fractions of triethylamine and phosphoric acid are 1.8% to 2.2% and 0.9% to 1.1% respectively.

3. The purification method according to claim 1, characterized in that In the equilibration buffer, the concentration of the reagent B is 0.1-5 mM.

4. The purification method according to claim 1, characterized in that The pH of the equilibrium buffer is 3-5.

2.

5. The purification method according to claim 1, characterized in that In the reverse phase preparative liquid chromatography purification, the mobile phase A and the mobile phase B are used for gradient elution; the gradient elution includes: within 0 to 60 minutes, the volume fraction of the mobile phase B changes from 20% to 28% to 46% to 60%.

6. The purification method according to claim 1, characterized in that In the reverse phase preparative liquid chromatography purification, an ultraviolet detector with a detection wavelength of 220 nm is used for detection, and the eluted fractions containing Pegcetacoplan are collected.

7. The purification method according to claim 1, characterized in that In the mobile phase C, the concentration of the sodium dihydrogen phosphate is 1-5 mM, the concentration of the sodium chloride is 0.4-0.6 M, and the volume fraction of acetonitrile in the acetonitrile aqueous solution is 15%-25%.

8. The purification method according to claim 7, characterized in that The elution comprises: within 0 to 15 CV, the volume fraction of the mobile phase C changes from 0% to 40%.

9. The purification method according to claim 1, characterized in that The concentration of the sodium dihydrogen phosphate aqueous solution is 0.1-5 mM.

10. The purification method according to claim 1, characterized in that: The purification method further comprises: subjecting the eluted fraction containing Pegcetacoplan in step (b) to salt conversion, concentration and freeze-drying.

11. The purification method according to claim 10, characterized in that: The salt conversion treatment comprises: ultrafiltration and concentrating the eluted fraction containing Pegcetacoplan in step (b) by 4 to 6 times, and then adding a volume of 0.1% acetic acid aqueous solution with a volume of 5 times the volume of the concentrated material for ultrafiltration.

12. The purification method according to claim 1, characterized in that The second solution is added to the first solution at a flow rate of 100-140 mL / min.

13. The purification method according to claim 1, characterized in that The preparation of the sample to be purified further comprises: diluting the coupling solution by 8 to 12 times with water and filtering.

14. A method for preparing Pegcetacoplan, characterized in that it comprises the purification method according to any one of claims 1 to 11.

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