A purification method and application of recombinant human coagulation factor VII

Abstract

The present invention discloses a purification method and application of recombinant human coagulation factor VII. The purification method comprises the following steps: adding a His tag to the coagulation factor VII gene sequence, then sequentially performing cell culture, clarification treatment, ultrafiltration concentration, nickel column affinity chromatography, anion chromatography, and finally self-activation to obtain recombinant human coagulation factor VII; wherein, during the nickel column affinity chromatography process, the chromatography loading concentration is 500-1000 μg / ml, and the equilibrium solution includes 0.5-1.0 mol / L NaCl. The purification method of the present invention improves the purity of recombinant coagulation factor VII and makes it more homogeneous. The recombinant coagulation factor VII of the present invention has a specific activity of 975 IU / mg, contains less other protein components, and can reduce the adverse reactions caused by other components; reduces the cost in the production process of recombinant coagulation factor VII, does not need to use specific monoclonal antibodies, and reduces the process cost; also reduces the production process steps of recombinant coagulation factor VII and improves the recovery rate.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a purification method and application of recombinant human coagulation factor VII. Background Art

[0002] Coagulation factor VII is a vitamin K-dependent serine protease that can initiate the extrinsic coagulation pathway or participate in the intrinsic coagulation pathway by activating human coagulation factor IX. Coagulation factor VII is synthesized in the liver and secreted into the blood, circulating in the blood in the form of a single-chain glycoprotein (zymogen) with a molecular weight of approximately 50 kDa. As an important coagulation factor in the coagulation pathway, activated coagulation factor VII can effectively overcome the defect of using factor VIII to treat hemophilia A bleeding, which causes patients to produce antibodies. It is currently the most commonly used drug for the clinical treatment of hemophilia A and B. Activated coagulation factor VII can also be used to treat clinical traumatic bleeding.

[0003] After consulting, there are currently two commonly used methods for preparing coagulation factor VII:

[0004] The first is to separate and extract from plasma. For example, Chinese invention patent CN111500563A discloses a method for purifying human coagulation factor VII from human plasma, which specifically includes the following steps: removing cold gel plasma, adsorbing the target protein containing coagulation factor VII on UniGel-80Q gel filler through the first anion chromatography, and loading the eluted sample on the second anion chromatography TMAE gel filler after treatment. The VII factor eluate can be obtained, and its specific activity is 12.43IU / mg. From a theoretical analysis, this scheme may not guarantee the purity of coagulation factor VII during the preparation of coagulation factor VII, and pathogenic substances are easily residual in blood products, requiring a strict inactivation procedure, and also having a certain impact on the titer of coagulation factor VII. Moreover, the separation and purification of coagulation factor VII from plasma requires a large amount of raw materials and high cost because the content of coagulation factor VII in plasma is extremely low. In the purification process, only two steps of ion chromatography are used, and it is difficult to ensure the purity of the final product, and other plasma protein components will inevitably appear, causing adverse reactions.

[0005] The second is to transfer human coagulation factor VII gene fragments into BHK and CHO cells for fermentation or to produce coagulation factor VII using rabbit mammary gland generators and then separate and purify. For example, Chinese invention patent CN102161701A discloses a method for separating and purifying high-purity activated coagulation recombinant human coagulation factor VII from cell culture fluid or plasma components, specifically comprising the following steps: first, the raw material is purified by affinity chromatography and the eluent containing coagulation recombinant human coagulation factor VII is collected, and then thrombin and magnesium ions are added to activate coagulation recombinant human coagulation factor VII, and finally the activated product is concentrated and the virus is inactivated to obtain the final product. In the process of preparing coagulation factor VII, this scheme needs to design and screen coagulation recombinant human coagulation factor VII monoclonal antibodies first and then couple them with affinity chromatography media. The whole process cost is high, and a set of additional monoclonal antibody expression and purification preparation technology is required, which is not conducive to the cost control of production preparation. In the production of coagulation factor VII using a rabbit mammary gland generator, due to the complex protein composition in rabbit milk, its purification difficulty is high. Moreover, in order to ensure the purity of coagulation factor VII during the purification and separation process, antigen-antibody affinity chromatography is often required to obtain coagulation factor VII through cell culture. It is necessary to design a monoclonal antibody that specifically adsorbs coagulation factor VII, and an additional set of monoclonal antibody expression and purification preparation technology is required, which is technically difficult and has high preparation costs.

[0006] Another example is the Chinese invention patent CN114438061A, which discloses a method for purifying coagulation factor VII, which specifically includes the following steps: defatting the rabbit milk of transgenic rabbits for coagulation factor VII to obtain defatted rabbit milk; subjecting the defatted rabbit milk to SPFast Flow cationic chromatography to obtain a first purified solution containing coagulation factor VII; subjecting the first purified solution to HeparinBestarose HP chromatography column chromatography to obtain a second purified solution containing coagulation factor VII; subjecting the second purified solution to Butyl-650M chromatography to obtain a third purified solution containing coagulation factor VII. This solution requires a multi-step process in the preparation of coagulation factor VII, which has a high production cost for coagulation factor VII, and the multi-step process requires more complex detection methods for the stability of the final product to ensure the final quality of the product. Moreover, coagulation factor VII is obtained by a rabbit mammary gland generator, because the components in rabbit milk are complex, a multi-step chromatography process is required to remove the effects of casein on affinity chromatography loading and purity, and adding a multi-step process before affinity chromatography will reduce the recovery rate of coagulation factor VII. Summary of the invention

[0007] In view of the above problems existing in the prior art, the present invention provides a purification method and application of recombinant human coagulation factor VII.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In a first aspect, a method for purifying recombinant human coagulation factor VII comprises the following steps:

[0010] A His tag is added to the coagulation factor VII gene sequence, and then cell culture, clarification, ultrafiltration concentration, nickel column affinity chromatography, anion chromatography, and finally self-activation are performed to obtain recombinant human coagulation factor VII; wherein, during the nickel column affinity chromatography process, the chromatography loading concentration is 500~1000μg / ml, and the equilibrium solution includes 0.5~1.0mol / L NaCl.

[0011] Furthermore, in the nickel column affinity chromatography process, a chromatographic filler model GP-NTA chromatography column is used.

[0012] Furthermore, the final conductivity of the nickel column affinity chromatography is 2-4 ms / cm.

[0013] Furthermore, the base sequence obtained by adding a His tag to the coagulation factor VII gene sequence is shown in SEQ ID NO.1.

[0014] Furthermore, the clarification treatment specifically includes: subjecting the cell collection liquid to centrifugal treatment at 6000 rpm for 20 minutes, collecting the centrifugal supernatant and then treating it through a 0.65 μm filter to obtain a clarified treatment liquid.

[0015] Furthermore, the ultrafiltration concentration specifically includes: ultrafiltration and concentration of the clarified treated liquid through a 10Kd membrane package, concentrating the recombinant human coagulation factor VII to 500-1000 μg / ml, and washing and filtering with 20 m mol / L Tris-Hcl, 0.5 mol / L NaCl, pH 8.0 solution for about 5-10 times the volume to obtain an ultrafiltration concentrate.

[0016] Furthermore, the nickel column affinity chromatography specifically includes: loading the ultrafiltration concentrate onto a GP-NTA chromatography column, balancing 5 to 10 column bed volumes with a balance solution containing 0.5 to 1.0 mol / L NaCl, and a loading linear flow rate of 1 to 2 cm / min; balancing 5 to 10 column bed volumes with a balance solution containing 0.5 to 1.0 mol / L NaCl; washing 5 to 10 column bed volumes with a 20 m mol / L Tris-Hcl, pH 8.0 solution; and finally eluting with a 20 m mol / L Tris-Hcl, 100 m mol / L imidazole, pH 8.0 solution to obtain a nickel column collection liquid.

[0017] Furthermore, the anion chromatography specifically includes: loading the nickel column collection liquid onto Q Sepharose FF anion chromatography filler, washing 5 to 10 column bed volumes with 20m mol / L Tris-Hcl, pH 8.0 solution, removing imidazole in the nickel column collection liquid, and eluting with 20m mol / L Tris-Hcl, 0.5mol / L NaCl, pH 7.2 solution to obtain anion chromatography collection liquid.

[0018] Furthermore, the self-activation specifically includes: activating the anion chromatography collection liquid by solution incubation self-activation method, ultrafiltration dialysis through a 10Kd membrane package, replacing the buffer with 20m mol / L Tris-Hcl, 0.5mol / L NaCl, 10m mol / L CaCl2, pH7.2, and concentrating it to a protein concentration of 3000~4000μg / ml through a 10kD ultrafiltration membrane package, placing it at 4°C for activation, and the activation time is 24 to 48h.

[0019] In a second aspect, a method for purifying recombinant human coagulation factor VII according to the first aspect is used in the preparation of a drug for preventing or treating hemorrhagic diseases.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] ① Improving the purity of recombinant coagulation factor VII and making it more homogeneous. The specific activity of the recombinant coagulation factor VII of the present invention is 975 IU / mg, and it contains less other protein components, which can reduce the adverse reactions caused by other components;

[0022] ② Reduce the cost of recombinant coagulation factor VII production process, no need to use specific monoclonal antibodies, and reduce process costs;

[0023] ③ Reduce the production process steps of recombinant coagulation factor VII and improve the recovery rate. The recovery rate of clarification treatment is 85%~90%, the recovery rate of ultrafiltration concentration is 90%~100%, the recovery rate of nickel column is 65%~75%, and the recovery rate of anion chromatography is 70%~80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 The electrophoresis test results of samples at each stage of GP-NTA nickel column; in the figure, the order of spotting is:

[0026] 1. Collect cell supernatant

[0027] 2. Ultrafiltration concentration flow through

[0028] 3. Ultrafiltration concentrate

[0029] 4. GP-NTA chromatography flow-through

[0030] 5.GP-NTA chromatography washing

[0031] 6.GP-NTA chromatography collection liquid;

[0032] Figure 2 The electrophoresis test results of samples from each step of the purification process are shown in the figure. The order of spotting is:

[0033] 1. Empty CHO supernatant

[0034] 2. Collect cell supernatant

[0035] 3. Ultrafiltration concentrate

[0036] 4.Q Sepharose FF chromatography collection fluid

[0037] 5. Self-activating samples;

[0038] Figure 3 The electrophoresis test results of samples at each stage of Cytiva nickel column packing are shown in the figure. In the figure, the electrophoresis order is:

[0039] 1. Ultrafiltration concentrate

[0040] 2. Ultrafiltration concentration flow through

[0041] 3. Ni Sepharose 6FF flow-through

[0042] 4. Ni Sepharose Excel Flow Through

[0043] 5. Ni Sepharose 6FF collection solution

[0044] 6.Ni Sepharose Excel collection solution. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0046] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0047] The term "comprises" or "comprising" as used in the present invention means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0048] The terms "optionally", "any", "arbitrarily" or "any" used in the present invention mean that the event or environment described later may but need not occur, and the description includes occasions where the event or environment occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.

[0049] The term "about" used in the present invention means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0050] The term "and / or" used in the present invention should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0051] Example 1 Method for Purifying Recombinant Human Coagulation Factor VII

[0052] A method for purifying recombinant human coagulation factor VII, comprising the following steps:

[0053] Preparation of cell harvest fluid: Add a His tag to the coagulation factor VII gene sequence, and the obtained base sequence is shown in SEQ ID NO.1. The constructed base sequence is transferred into CHO cells. CHO cells are suspended and cultured in a bioreactor with a cell inoculation density of 1×10 6 / ml, cultured at 37℃ for 5 days; then transferred to 34℃ for expression culture for 12-16 days, and the supernatant samples were harvested for electrophoresis (see Figure 1 , Lane 1 and Figure 2 , Lane 2). The results of ELISA kit detection at different culture days are as follows (Table 1):

[0054] Table 1: Expression levels at different culture days

[0055]

[0056] The base sequence SEQ ID NO.1 is as follows:

[0057]

[0058] The amino acid sequence of the recombinant human coagulation factor VII after expression is shown in SEQ ID NO.2, specifically: MVSQALRLLCLLLGLQGCLAAGGVAKASGGETRDMPWKPGPHRVFVTQEEAHGVLHRRRRANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLL VNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGA TALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFPHHHHHHH-.

[0059] Clarification treatment, ultrafiltration concentration, i.e. nickel column chromatography pre-treatment: the cell collection solution is centrifuged at 6000rpm for 20min, the centrifugal supernatant is collected and then processed through a 0.65μm filter to obtain a clarified treatment solution; ultrafiltration concentration to 500μg / ml: the clarified treatment solution is ultrafiltered and concentrated through a 10Kd membrane package to concentrate recombinant human coagulation factor VII to 500μg / ml, and washed with 20m mol / L Tris-HCl, 0.5 mol / L NaCl, pH8.0 solution for about 5 to 10 times the volume to obtain an ultrafiltration concentrate; the electrophoresis results of the samples during the ultrafiltration process (see Figure 1 Lanes 1, 2, and 3: before ultrafiltration, diafiltration, and ultrafiltration collection, respectively);

[0060] Nickel column affinity chromatography: The ultrafiltration concentrate was loaded onto a GP-NTA chromatography column provided by Suzhou Bojin Biotechnology Co., Ltd., and 5-10 column bed volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl, with a loading linear flow rate of 1-2cm / min; 5-10 column bed volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl; 5-10 column bed volumes were rinsed with 20m mol / L Tris-Hcl, pH 8.0 solution until the conductivity dropped to 2ms / cm; finally, 20m mol / L Tris-Hcl, 100m mol / L imidazole, pH 8.0 solution was used for elution to obtain a nickel column collection solution, and the conductivity of the nickel column collection solution was controlled at 2-4ms / cm. Too high a conductivity would result in flow-through during anion chromatography, resulting in a reduced recovery rate. Electrophoresis of samples at each stage of nickel column chromatography (see Figure 1 , Lanes 3, 4, 5, and 6 are GP-NTA chromatography loading sample, GP-NTA chromatography flow-through sample, GP-NTA chromatography washing sample, and GP-NTA chromatography collection sample, respectively);

[0061] (4) Anion chromatography: The nickel column collected solution was loaded onto Q Sepharose FF anion chromatography filler and washed with 20mmol / L Tris-HCl, pH8.0 solution for 5-10 column volumes to remove imidazole from the nickel column collected solution. The solution was eluted with 20mmol / L Tris-HCl, 0.5mol / L NaCl, pH7.2 solution to obtain the electrophoretic pattern of the anion chromatography collected solution (see Figure 2 , lane 4);

[0062] (5) Self-activation includes: activating the coagulation factor VII by the solution incubation self-activation method using the anion chromatography collection solution, ultrafiltration dialysis of the coagulation factor VII through a 10Kd membrane package, replacing the buffer with 20m mol / L Tris-Hcl, 0.5mol / L NaCl, 10m mol / L CaCl2, pH7.2, and concentrating it through a 10kD ultrafiltration membrane package to a protein concentration of 3000~4000μg / ml, placing it at 4℃ for activation, and the activation time is 24~48h. The electrophoresis of the sample after activation (see Figure 2 , lane 5).

[0063] Example 2 Effect of sample target protein concentration on nickel column chromatography

[0064] The ultrafiltration concentrates of different sample protein concentrations were loaded onto the GP-NTA chromatography column provided by Suzhou Bojin Biotechnology Co., Ltd., and 5 to 10 column volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl, and the loading linear flow rate was 1 to 2cm / min; 5 to 10 column volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl; 5 to 10 column volumes were washed with 20m mol / L Tris-Hcl, pH8.0 solution; and finally eluted with 20m mol / L Tris-Hcl, 100m mol / L imidazole, pH8.0 solution to obtain nickel column collection liquid. The effects of different sample concentrations on nickel column chromatography are shown in Table 2:

[0065] Table 2: Effect of sample concentration on nickel column chromatography

[0066]

[0067] According to Table 2, when the loading concentration of recombinant human coagulation factor VII is lower than 500μg / ml during the loading process, sample flow-through is likely to occur, resulting in a decrease in the chromatographic recovery rate and sample loss. As the concentration of the loaded recombinant human coagulation factor VII continues to increase, the recovery rate does not increase. At the same time, because the flux of the cell collection fluid decreases as the concentration multiple increases, the process time will be extended as the concentration multiple increases. Therefore, it is more appropriate to load the sample concentration between 500 and 1000μg / ml. If it exceeds 1000μg / ml, there will be difficulties in ultrafiltration concentration, and the culture medium contained in the cell supernatant has a certain viscosity. After being concentrated more than 10 times, the use of a 10 kd millipore membrane package will result in high pressure and slow flow rate at the permeation end.

[0068] The target protein in the cell supernatant is 80-100 μg / ml. The inventors have tried to directly load the cell supernatant onto GP-NTA without concentration, but weak adsorption occurred. The target protein was present in the loading flow through electrophoresis, and non-specifically adsorbed proteins were mostly collected during elution.

[0069] Example 3 Effects of different nickel column models on nickel column chromatography

[0070] The ultrafiltration concentrate was loaded onto different nickel column chromatography fillers. The present embodiment used NiSepharose 6FF, Ni Sepharose Excel produced by Cytiva and GP-NTA chromatography columns produced by Bojin. The same volume of ultrafiltration concentrate was loaded onto different nickel column chromatography columns, and 5 to 10 column bed volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl, and the loading linear flow rate was 1 to 2cm / min; 5 to 10 column bed volumes were balanced with a balance solution containing 20m mol / L Tris-Hcl and 0.5 mol / L NaCl; 5 to 10 column bed volumes were rinsed with 20m mol / L Tris-Hcl, pH 8.0 solution; and finally eluted with 20m mol / L Tris-Hcl, 100m mol / L imidazole, pH 8.0 solution to obtain nickel column collection liquid. The effects of different types of nickel columns on chromatography are shown in Table 3:

[0071] Table 3: Effects of different nickel columns on chromatography

[0072]

[0073] according to Figure 1 , Figure 3 , Table 3 shows that:

[0074] Figure 1 The middle lanes 3, 4, and 6 correspond to the GP-NTA loading sample, loading flow-through, and elution collection, respectively;

[0075] Figure 3 The middle lanes 1, 3, and 5 correspond to Ni Sepharose 6FF loading sample, loading flow-through, and elution collection, respectively;

[0076] Figure 3 The middle lanes 1, 4, and 6 correspond to Ni Sepharose Excel loading sample, loading flow-through, and elution collection, respectively;

[0077] According to the electrophoresis pattern, it can be seen that GP-NTA has better adsorption for the recombinant human coagulation factor VII designed by the present invention. Ni Sepharose 6FF and Ni Sepharose 6FF show obvious recombinant human coagulation factor VII flow-through bands, that is, the adsorption effect of the target protein on Ni Sepharose 6FF and Ni Sepharose 6FF is poor; no recombinant human coagulation factor VII bands are seen in the GP-NTA flow-through, and they are concentrated in the elution collection stage. For the recombinant human coagulation factor VII of the present invention, GP-NTA has better adsorption.

[0078] Example 4 Effects of different equilibrium washing conditions on nickel column chromatography

[0079] The same ultrafiltration concentrate was loaded onto the GP-NTA chromatography column, and 5 to 10 column volumes were balanced with different salt concentrations and imidazole concentrations, and the loading linear flow rate was 1 to 2 cm / min; 5 to 10 column volumes were balanced with different salt concentrations and imidazole concentrations; 5 to 10 column volumes were washed with 20 mmol / L Tris-Hcl, pH 8.0 solution; and finally eluted with 20 mmol / L Tris-Hcl, 100 mmol / L imidazole, pH 8.0 solution to obtain nickel column collection liquid. The effects of different loading sample concentrations on nickel column chromatography are shown in Table 4:

[0080] Table 4: Effects of different equilibrium washing conditions on nickel column chromatography

[0081]

[0082] According to Table 4, adding low-concentration imidazole to the equilibrium solution will cause desorption of some samples during the re-equilibrium process, which will reduce the recovery rate. The purpose of adding low imidazole is to reduce the nonspecific adsorption of impurities on the nickel column. During the experiment, it was found that high-concentration sodium chloride has the same effect. When the imidazole concentration exceeds 10m mol / L, the recovery rate will be further reduced, and the sample may not even bind to the nickel column; when the sodium chloride concentration exceeds 1 mol / L, the sample will salt out and the sample turbidity will increase. Experiments 1, 4, and 5 in Table 4 are comparative experiments in which the sodium chloride concentration in the equilibrium solution gradually increases. It can be seen that as the sodium chloride concentration increases, the sample purity also increases, but the recovery rate also decreases. Balancing the recovery rate and purity, it is a better solution to add sodium chloride with a concentration of 0.5~1 mol / L in the equilibrium solution.

[0083] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for purifying recombinant human coagulation factor VII, characterized in that: It includes the following steps: A His tag is added to the coagulation factor VII gene sequence, and the obtained base sequence is shown in SEQ ID NO.

1. The constructed base sequence is transferred into cells, and then cell culture, clarification treatment, ultrafiltration concentration, nickel column affinity chromatography, anion chromatography, and finally self-activation are performed to obtain recombinant human coagulation factor VII; wherein, The ultrafiltration concentration specifically includes: ultrafiltration and concentration of the clarified treatment liquid through a membrane package to concentrate the recombinant human coagulation factor VII to 500-1000 μg / ml, and diafiltration of 5-10 times the volume to obtain an ultrafiltration concentrate; In the nickel column affinity chromatography process, the chromatography filler model used is GP-NTA chromatography column; The nickel column affinity chromatography specifically comprises: loading the ultrafiltration concentrate onto the chromatography column, balancing 5 to 10 column bed volumes with a balance solution containing 0.5 to 1.0 mol / L NaCl and 20 m mol / L Tris-Hcl, and the loading linear flow rate is 1 to 2 cm / min; balancing 5 to 10 column bed volumes with a balance solution containing 0.5 to 1.0 mol / L NaCl and 20 m mol / L Tris-Hcl; washing 5 to 10 column bed volumes with a 20 mmol / L Tris-Hcl, pH 8.0 solution; and finally eluting with a 20 m mol / L Tris-Hcl, 100 mmol / L imidazole, pH 8.0 solution to obtain a nickel column collection solution; the loading sample concentration of the ultrafiltration concentrate is 500 to 1000 μg / ml; and the balance solution does not contain imidazole.

2. The method for purifying recombinant human coagulation factor VII according to claim 1, characterized in that: The final conductivity of the nickel column affinity chromatography is 2-4 ms / cm.

3. The method for purifying recombinant human coagulation factor VII according to claim 1, characterized in that: The clarification treatment specifically includes: subjecting the cell collection fluid to centrifugal treatment, collecting the centrifugal supernatant and then treating it through a filter to obtain a clarified treatment fluid.

4. The method for purifying recombinant human coagulation factor VII according to claim 1, characterized in that: The anion chromatography specifically comprises: loading the nickel column collected liquid onto Q Sepharose FF anion chromatography filler, washing 5 to 10 column bed volumes, removing imidazole in the nickel column collected liquid, eluting, and obtaining anion chromatography collected liquid.

5. The method for purifying recombinant human coagulation factor VII according to claim 1, characterized in that: The self-activation specifically includes: activating the anion chromatography collection liquid by solution incubation self-activation method, ultrafiltration dialysis through a membrane package, replacing the buffer and concentrating it through an ultrafiltration membrane package to a protein concentration of 3000-4000 μg / ml, and activating it for 24-48 hours.

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