CN111849945A - Method for purifying human blood coagulation factor VIIa - Google Patents

Abstract

The invention provides a method for purifying human blood coagulation factor VIIa, which comprises the steps of purifying by adopting three steps of anion exchange chromatography, anion exchange chromatography and hydrophobic chromatography, inactivating viruses and removing viruses and filtering to prepare the human blood coagulation factor VIIa with purity, activity and safety meeting the medicinal requirements.

Description

The purification method of human blood coagulation factor VIIa

technical field

The invention relates to the technical field of biomedicine, in particular to a method for purifying human coagulation factor VIIa.

Background technique

Human factor VII (FVII) is a vitamin K-dependent serine protease that can initiate the extrinsic coagulation pathway or participate in the intrinsic coagulation pathway by activating human factor IX. FVII is synthesized in the liver and secreted into the blood, and circulates in the blood as a single-chain glycoprotein (zymogen) with a molecular weight of approximately 50,000 Da. The FVII zymogen is hydrolyzed by proteases at sites R152-I153, resulting in a double-chain molecule linked by a disulfide bond, which is converted to the active form FVIIa. Clinical studies have shown that FVIIa has a satisfactory therapeutic effect on thrombocytopenia and platelet dysfunction, severe trauma, and hemostasis in large-area surgical procedures.

The early preparation of human coagulation factor VII was prepared from fresh frozen plasma, which was adsorbed by barium sulfate and purified by multi-step chromatography. With the application of DEAE-Sephadex A-50 gel, it was gradually transformed into A-50 gel. It is mainly adsorbed and prepared by multi-step chromatographic purification. Because the content of human coagulation factor VII in plasma is extremely low, usually 200-400 ng/ml, the preparation of human coagulation factor VII is very difficult and the cost is extremely high; at the same time, the plasma extraction and preparation process is complicated, the batch difference is large, and the stability In addition, the use of plasma as a raw material greatly increases the risk of infection by foreign viruses. In recent years, the development of genetic engineering technology has provided the possibility for the recombinant production of FVII in vitro. NovoSeven of Denmark's Novo Nordisk introduced the cDNA gene of human coagulation factor VII into BHK cells (baby hamster kidney cells) for expression and production through genetic engineering technology, and obtained after activation and high purification. Bioactive pharmaceutical recombinant human coagulation factor VIIa.

Tomokiyo et al. reported a purification method for preparing human coagulation factor VIIa from plasma (Tomokiyo K, Yano H, Imamura M, et al. Large-scale production and properties of human plasma-derived activated Factor VII concentrate [J]. Vox Sanguinis , 2010, 84 (1): 54-64.), the specific steps include: using Q-Sepharose Fast Flow to capture FVII from plasma, then using immunoaffinity chromatography with anti-FVII antibody, and using DEAE-Sepharose after virus removal Purification was performed to obtain a FVII-FVIIa mixture, followed by in-solution activation to obtain FVIIa. Finally, the FVIIa preparation is obtained through the steps of dialysis, preparation filling, and freeze-drying.

Jurlander et al. disclosed the downstream purification process of Novo Nordisk's FVIIa (Jurlander B, ThimL, Klausen N K, et al. Recombinant Activated Factor VII (rFVIIa): Characterization, Manufacturing, and Clinical Development [J]. Seminars in Thrombosis and Hemostasis, 2001, 27(4): 373-384.): After harvesting the cell culture medium, a first step of anion exchange chromatography was performed to capture FVII, followed by virus inactivation and immunoaffinity chromatography, followed by a two-step anion exchange layer analysis to yield fully activated FVIIa.

CN1121723A discloses that Zn ²⁺ is added in the process of purification chromatography to perform controllable activation and degradation of FVII, and the chromatography steps are: anion exchange, immunoaffinity, anion exchange, anion exchange.

CN101268185A discloses adding hydrophobic chromatography process during purification to control product-related impurities in pharmaceuticals, such as varying levels of N-linked glycosylated sugar variants, oxidized forms, proteolytically degraded forms contained in post-elution peaks (heavy chain cleavage form) and aggregates, etc.

The existing downstream purification technology of human coagulation factor FVIIa usually adds an affinity chromatography step, and the FVIIa protein is specifically captured by a chromatographic medium connected with FVIIa antibody, but this technology has the following disadvantages: 1. High cost of industrialization, 2. The shedding of immunoaffinity chromatography ligands poses an associated safety risk.

SUMMARY OF THE INVENTION

The object of the present invention is at least to provide an improved purification process of human coagulation factor VIIa.

In some aspects, the present invention provides a method for purifying human coagulation factor VIIa, comprising the steps of: anion exchange chromatography, anion exchange chromatography, hydrophobic chromatography.

In some aspects, the present invention provides a method for purifying human coagulation factor VIIa, comprising the steps of: anion exchange chromatography, anion exchange chromatography, hydrophobic chromatography, and each step is performed in sequence.

In some aspects, the present invention provides a method for purifying human coagulation factor VIIa, comprising the following steps in sequence:

1) Anion exchange chromatography;

2) Inactivate the virus;

3) Anion exchange chromatography;

4) Hydrophobic chromatography;

5) In addition to virus filtering;

6) Ultrafiltration liquid change;

7) Sterilize and filter to obtain stock solution.

In some aspects, the present invention provides a method for purifying human coagulation factor VIIa, comprising the following steps in sequence:

1) Anion exchange chromatography;

2) virus inactivation is carried out to the product obtained in step 1);

3) using anion exchange chromatography to further purify the product obtained in step 2);

4) using hydrophobic chromatography to further purify the product obtained in step 3);

5) carry out virus-removing filtration to the product obtained in step 4);

6) carry out ultrafiltration and exchange liquid to the product obtained in step 5);

7) Sterilize and filter the product obtained in step 6) to obtain a stock solution.

In some aspects, the present invention first captures the protein of interest from a cell harvest fluid using anion exchange chromatography, wherein the anion exchange chromatography optionally includes (a) equilibrating the anion exchange material with an equilibration buffer, (b) eluting the anion exchange material with buffer rinsing the anion exchange material, and (c) eluting the anion exchange material with an elution buffer, wherein the equilibration buffer has a pH of 6-10, preferably a pH of about 8, and contains chlorinated chloride at a concentration of at least about 50 mM Sodium, preferably sodium chloride at a concentration of about 50 mM; elution buffer pH 6-10, preferably pH about 8, optionally containing sodium chloride at a concentration of at least about 50 mM; elution buffer pH 6-10 , preferably at a pH of about 8 and containing sodium chloride at a concentration of 50-800 mM, preferably at a concentration of about 500 mM.

In some aspects, the first step of anion exchange chromatography of the present invention comprises equilibrating the anion exchange material with an equilibration buffer and eluting the anion exchange material with an elution buffer, wherein the equilibration buffer has a pH of 6-10, preferably a pH of about 8, and contains sodium chloride at a concentration of at least about 50 mM, preferably at a concentration of about 50 mM; elution buffer pH 6-10, preferably pH about 8, and contains sodium chloride at a concentration of 50-800 mM , the preferred sodium chloride concentration is about 500 mM.

In some aspects, the first step of anion exchange chromatography of the present invention comprises equilibrating the anion exchange material with an equilibration buffer and eluting the anion exchange material with an elution buffer, wherein the equilibration buffer and the elution buffer are each independently selected From phosphate buffer, tris-hydrochloric acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, N-tris(hydroxymethyl)methylglycine buffer, N,N-dihydroxyl The ethyl glycine buffer, preferably the equilibration buffer and the elution buffer, respectively, are phosphate buffers, and most preferably the equilibration buffer and the elution buffer each comprise about 20 mmol/L of phosphate.

In some protocols, virus inactivation of the eluate from the first step of anion exchange chromatography is performed using the S/D (surfactant/detergent) method.

In some protocols, a second anion exchange chromatography is performed on the virus-inactivated product to remove Gla (γ-carboxyglutamic acid) deletion/partial deletion variants and other related impurities, wherein this step anion exchange layer The analysis optionally includes (a) equilibrating the anion exchange material with an equilibration buffer, (b) rinsing the anion exchange material with a wash buffer, and (c) eluting the anion exchange material with an elution buffer, wherein the equilibration buffer pH of 6-10, preferably about pH 8, and contains sodium chloride at a concentration of at least about 50 mM, preferably about 100 mM sodium chloride; wash buffer pH 6-10, preferably pH about 8, and contains sodium chloride at a concentration of at least about 50 mM and/or calcium chloride at a concentration of at least about 1 mM, preferably about 100 mM sodium chloride and about 2.25 mM calcium chloride; elution buffer pH 6-10, Preferably the pH is about 8 and contains sodium chloride at a concentration of at least about 50 mM and/or calcium chloride at a concentration of at least about 1 mM, preferably about 100 mM sodium chloride and about 5 mM calcium chloride.

In some aspects, the second step of anion exchange chromatography of the invention comprises (a) equilibrating the anion exchange material with an equilibration buffer, (b) rinsing the anion exchange material with an elution buffer, and (c) using an elution buffer Elute the anion exchange material, wherein the equilibration buffer has a pH of 6-10, preferably a pH of about 8, and contains sodium chloride at a concentration of at least about 50 mM, preferably about 100 mM sodium chloride; the wash buffer pH is 6-10, preferably at a pH of about 8, and comprising sodium chloride at a concentration of at least about 50 mM and/or calcium chloride at a concentration of at least about 1 mM, preferably about 100 mM sodium chloride and about 2.25 mM calcium chloride; The elution buffer has a pH of 6-10, preferably a pH of about 8, and contains sodium chloride at a concentration of at least about 50 mM and/or calcium chloride at a concentration of at least about 1 mM, preferably about 100 mM sodium chloride and about 5 mM of calcium chloride.

In some aspects, the second step of anion exchange chromatography of the present invention comprises equilibrating the anion exchange material with an equilibration buffer, rinsing the anion exchange material with an elution buffer, and eluting the anion exchange material with an elution buffer, wherein the equilibration buffer The buffer solution, elution buffer and elution buffer can be independently selected from phosphate buffer, tris-hydrochloric acid buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, N-tri( Hydroxymethyl)methylglycine buffer, N,N-dihydroxyethylglycine buffer, preferably equilibration buffer, elution buffer and elution buffer are tris buffer respectively, most preferably equilibration buffer The buffer, elution buffer and elution buffer respectively include about 10 mmol/L of tris-hydrochloric acid.

In some schemes, the eluate obtained by the second anion exchange chromatography is further subjected to hydrophobic chromatography to remove product-related impurities such as polymers, oxides and degradation products, and the hydrophobic chromatography step optionally includes ( a) equilibrating the HIC material with an equilibration buffer, (b) eluting the HIC material with an elution buffer, and (c) eluting the HIC material with an elution buffer, wherein the step of chromatography The sample solution contains salts and/or zwitterions, and the salts can be selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride, sodium acetate, sodium sulfate, potassium acetate, potassium chloride and potassium sulfate, so The zwitterion may be selected from: glycine, alanine, leucine and isoleucine, preferably ammonium acetate.

In some aspects, the hydrophobic chromatography of the present invention comprises equilibrating the HIC material with an equilibration buffer and eluting the HIC material with an elution buffer, wherein the equilibration buffer comprises 10-30 mmol/L glycylglycine , preferably about 10 mmol/L glycylglycine, and 1-20 mol/L ammonium acetate, preferably about 1.8 mol/L ammonium acetate, and the pH of the equilibration buffer is 6-10, preferably about 6; elution buffer The liquid contains 10-30mmol/L glycylglycine, preferably about 10mmol/L glycylglycine, and 10-100mmol/L trisodium citrate dihydrate, preferably about 30mmol/L trisodium citrate dihydrate, And the pH of the elution buffer is 6-10, preferably about pH 6.

In some protocols, the hydrophobic chromatography eluate product is subjected to virus-removing filtration, followed by ultrafiltration exchange and sterile filtration to obtain a stock solution.

In some aspects, the methods for purifying human factor VIIa provided herein do not comprise an affinity chromatography step.

In some aspects, the methods for purifying human factor VIIa provided herein do not include an immunoaffinity chromatography step coupled to an antibody to factor VIIa on an affinity chromatography medium.

In some aspects, the present invention provides methods for the purification of human factor VIIa, which is recombinant human factor VIIa produced under cell culture conditions. In some embodiments, the human factor VIIa of the invention is not plasma-derived human factor VIIa.

Another object of the present invention is to provide a method for reducing Gla variants in human coagulation factor VIIa, the method for reducing Gla variants comprising sequentially performing the following steps: anion exchange chromatography, anion exchange chromatography, hydrophobic chromatography, in In some embodiments, the human factor VIIa is recombinant human factor VIIa, and in other embodiments, the method does not comprise affinity chromatography.

It is also an object of the present invention to provide a method for reducing the content of polymers, degradants and oxides in a recombinant protein, in some specific embodiments, the recombinant protein is recombinant human coagulation factor VIIa.

In some embodiments of the invention, the purification of recombinant human factor VIIa excludes the step of affinity chromatography. Antibody proteins conjugated to affinity media may contaminate or interfere with the purified product during the immunoaffinity chromatography step. The present invention excludes affinity chromatography, can avoid the risk of anti-FVIIa antibody shedding, thereby obtaining a recombinant protein product with higher purity and less immunogenicity.

The method for purifying human coagulation factor VIIa provided by the present invention has the following beneficial effects: adopting two steps of anion exchange and one step of hydrophobic chromatography, a total of three steps of chromatography can be used to prepare and obtain a human blood coagulation factor whose purity, activity and safety meet the requirements for medicinal use VIIa, an additional two-step virus inactivation and virus removal filtration process ensures that the risk of virus contamination of the final product is excluded. The method of the present invention does not contain an immunoaffinity chromatography step, has low industrialization cost and avoids the risk of anti-FVIIa antibody falling off; in addition, the three-step chromatography process is simple and easy to scale up for production.

Explanation and Definition

Unless otherwise indicated, for the purposes of this application, the following terms used in this specification and claims shall have the following meanings.

"Coagulation factor": refers to the various protein components involved in the blood coagulation process, including coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII.

"Recombinant human coagulation factor": a human coagulation factor produced by recombinant DNA technology or recombinant RNA technology. Recombinant protein is the application of gene recombination technology to obtain a recombinant vector with a gene fragment that can be translated into a target protein, and then transfer it into a host cell that can express the target protein to express a specific recombinant protein molecule. The production of recombinant proteins mainly includes four systems: prokaryotic expression system, mammalian cell expression system, eukaryotic expression system and insect cell expression system.

"Antibody": used in its broadest sense, specifically covering whole monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two whole antibodies, and antibody fragments so long as they have desired biological activity.

"Gla (gamma-carboxyglutamic acid) deletion/partial deletion variant": native human factor VII undergoes post-translational modifications, including microbial K-dependent carboxylation, resulting in 10 Glas at the N-terminus of FVII or FVIIa ( γ-carboxyglutamic acid) residues. FVIIa produced by gene recombination technology is prone to form variants with varying degrees of Gla during the expression process, which are called Gla (γ-carboxyglutamic acid) deletion/partial deletion variants, in which Gla (γ-carboxyglutamic acid) modification The higher the ratio, the higher the specific activity of the protein.

"Affinity chromatography" refers to a purification and extraction method that combines chromatography technology with the specific reaction of antigen and antibody. The usual practice is to chemically bind the antibody (or antigen) to the column material with molecular sieve function such as agarose and dextran. )Combine.

The word "comprise" or "comprise" and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, ie, "including but not limited to".

Unless otherwise stated, "about" in the present invention means within ±5% of the specified numerical range given, preferably within ±2%, more preferably within ±1%. For example a pH of about 5.5 means a pH of 5.5±5%, preferably a pH of 5.5±2%, more preferably a pH of 5.5±1%.

Herein, "polymer" refers to a polymer produced by the aggregation of recombinant proteins due to the interaction of amino acid residues on the peptide chain or the aggregation of molecules due to hydrophobic interaction or electrostatic interaction.

Herein, "degradant" refers to a product with a smaller molecular weight than that of the recombinant protein produced by the deamidation or peptide chain cleavage of the recombinant protein in an aqueous solution.

Description of drawings

Figure 1 is a graph showing the effect of the second step anion exchange chromatography to remove GLa (γ-carboxyglutamic acid) deletion/partial deletion variants (measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)). Lane 1 is the loading solution, lane 2 is the flow-through solution, lane 3 is the elution peak 1, lane 4 is the elution peak 2, lane 5 is the elution peak, and M is the molecular weight marker.

Detailed ways

Example 1 Purification of FVIIa

Downstream purification of FVIIa proceeds through the following seven steps:

The first step is anion exchange chromatography.

The clarified cell culture broth containing FVIIa was loaded onto a TMAE anion chromatography column pre-equilibrated with buffer A1 (20 mmol/L hydrated disodium hydrogen phosphate, 50 mmol/L sodium chloride, pH 8.0-8.2). Equilibration after loading was performed with the same buffer, and then FVIIa was eluted with buffer B1 (20 mmol/L hydrated disodium hydrogen phosphate, 500 mmol/L sodium chloride, pH 8.0-8.2).

The second step, S/D virus inactivation.

Triton X-100 (polyethylene glycol octyl phenyl ether) and TNBP (tributyl phosphate) were added to the TMAE eluted product to make the final concentrations of 2% and 0.45%, respectively, and maintained at 25°C for 45min for inactivation Lipid-enveloped virus.

The third step is anion exchange chromatography.

The conductance of the S/D virus inactivation product was adjusted to below 12 mS/cm with buffer D (10 mmol/L tris, pH 8.0-8.2). The sample was loaded onto a Q Sepharose High Performance anion chromatography column pre-equilibrated with buffer A2 (10 mmol/L tris, 100 mmol/L sodium chloride, pH 8.0-8.2). Equilibrate after loading with the same buffer as pre-equilibration, and then use buffer B2 (10mmol/L Tris+100mmol/L NaCl+2.25mM calcium chloride solution, pH 8.0-8.2) Rinse was performed, followed by elution with eluent C1 (10 mmol/L tris(hydroxymethylaminomethane)+100 mmol/L sodium chloride+5 mM calcium chloride solution, pH 8.0-8.2), and the eluate was collected.

The fourth step is hydrophobic chromatography.

The conductivity of the anion exchange chromatography eluate from the previous step was adjusted to 100 mS/cm with saturated ammonium acetate. The sample was loaded onto a PhenylSepharose High Performance hydrophobic chromatography column pre-equilibrated with buffer A3 (10 mmol/L glycylglycine, 1.8 mol/L ammonium acetate, pH 6.0). Equilibrate with the same buffer as pre-equilibration, and use buffer 35% B3 (B3: 10 mmol/L glycylglycine + 30 mmol/L trisodium citrate dihydrate, pH 6.0) for elution after Elution was carried out with 10 column volume gradients of 35%-100% B3, and the eluate was collected in sections, and the qualified fractions were combined after RP-HPLC and SEC-HPLC purity detection.

The fifth step, in addition to virus filtering.

With the hydrophobic eluent (10mmol/L glycylglycine+30mmol/L trisodium citrate dihydrate, pH 6.0) rinsing the 20nm virus-removing filter membrane that has been washed with water, after the hydrophobic chromatography gained product is carried out virus-removing filtration, Control filter pressure 3bar.

The sixth step is to change the fluid by ultrafiltration.

The ultrafiltration membrane package (10kDa) was washed with water and equilibrated with replacement buffer (10mmol/L glycylglycine, 40mmol/L sodium chloride, 10mmol/L calcium chloride, 0.5g/L methionine, pH 6.0) . Then, the product after virus removal and filtration is concentrated and exchanged, and then replaced with the exchange buffer, and then the mother solution of polysorbate 80, sucrose and mannitol is added to protect the target protein.

The seventh step is sterile filtration.

Sterile filtration was performed using a 0.22 μm filter.

Using the above purification process, the purity of the final solution obtained is shown in Table 1 below.

Table 1 Final stock solution purity table

Example 2 Determination of polymer content

The molecular weight distribution of recombinant human coagulation factor VIIa was detected by a molecular weight exclusion chromatography column, and the phosphate-sodium chloride mobile phase system was used for elution. Purity was determined by peak area normalization using a UV detector. Use a ThermoUltimate 3000 high performance liquid chromatograph with a UV detector, equilibrate the column to a stable baseline, mobile phase: 50 mmol/L disodium hydrogen phosphate, 300 mmol/L sodium chloride, pH 7.5. The parameters are shown in Table 2 below.

Table 2 Gel chromatography liquid analysis parameters

The results show that the method of the present invention can obviously remove the polymer in rFVIIa by using the size exclusion chromatography-high performance liquid chromatography method.

Example 3 Determination of content of oxides and degradation products

The purity of recombinant human coagulation factor VIIa for injection was detected by reversed-phase chromatography. With the increase of organic phase, the components with different retention abilities were eluted to achieve separation. The recombinant human coagulation factor VIIa was determined by UV detector and peak area normalization method. Coagulation factor VIIa purity. Using a Thermo Ultimate 3000 high performance liquid chromatograph with UV detector, equilibrate the column to a stable baseline, mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile. The parameters are shown in Table 3 below.

Table 3 Reversed-phase chromatography liquid analysis parameters

The results show that the reversed-phase chromatography-high performance liquid chromatography method can detect that the method of the present invention can obviously remove oxides and degradation products in rFVIIa.

Example 4 Determination of Gla (γ-carboxyglutamic acid) variants

Gla (gamma-carboxyglutamic acid) deletion/partial deletion variants of rFVIIa were determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is a denaturing polyacrylamide gel electrophoresis method. The principle is based on the fact that most proteins can be combined with the anionic surfactant sodium dodecyl sulfate (SDS) to form a complex by weight, so that the protein molecules can be combined. The negative charge is far greater than the net charge of the natural protein molecule, eliminating the charge effect of different protein molecules and allowing proteins to be separated by molecular size.

Using Bio-Rad/PowerPac Basic equipment, the specific method is as follows: placing the gel in the electrophoresis tank and adding electrode buffer. Add 5 μl of molecular weight standard to the sample addition hole of the gel, add the reference solution and the test solution to the sample addition hole adjacent to the sample addition hole, and cover the electrophoresis tank. In voltage regulation mode, adjust the power supply voltage to 140V, and stop electrophoresis when bromophenol blue migrates to the bottom of the gel. Pour off the electrode buffer and remove the gel for staining and destaining.

The results measured by this method are shown in Figure 1. The results show that the third step of anion exchange chromatography in Example 1 can elute the Gla deletion/partial deletion variants.

In light of the present disclosure, although the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that, without departing from the concept, spirit and scope of the present invention, the methods described herein can be The method and the steps or order of steps of the method can be changed.

The disclosures of all documents cited herein are hereby incorporated by reference to the extent that they provide exemplary, procedural and other details supplementing those described herein.

Claims (10)

1. a purification method of human blood coagulation factor VIIa, comprises the following steps that carry out successively: 1) Anion exchange chromatography; 2) Inactivate the virus; 3) Anion exchange chromatography; 4) Hydrophobic chromatography; 5) In addition to virus filtering; 6) Ultrafiltration liquid change; 7) Sterilize and filter to obtain stock solution. 2. method according to claim 1 is characterized in that, described method comprises the following steps that are carried out in sequence: 1) Anion exchange chromatography; 2) virus inactivation is carried out to the product obtained in step 1); 3) using anion exchange chromatography to further purify the product obtained in step 2); 4) using hydrophobic chromatography to further purify the product obtained in step 3); 5) carry out virus-removing filtration to the product obtained in step 4); 6) carry out ultrafiltration and exchange liquid to the product obtained in step 5); 7) Sterilize and filter the product obtained in step 6) to obtain a stock solution. 3. The method according to any one of claims 1-2, wherein in the step 1), anion exchange chromatography is used to capture the target protein from the cell harvest liquid, and this step anion exchange chromatography optionally includes (a) equilibrates the anion exchange material, (b) rinses the anion exchange material, and (c) elutes the anion exchange material. 4. The method according to any one of claims 1-3, wherein in the step 2), S/D method is used for virus inactivation. 5. The method of any one of claims 1-4, wherein step 3) optionally comprises (a) balancing the anion exchange material, (b) rinsing the anion exchange material, and (c) Elution of anion exchange material, wherein the buffer used for equilibration has a pH of 6-10, preferably a pH of about 8, and contains sodium chloride at a concentration of at least about 50 mM, preferably about 100 mM; the buffer used for rinsing The pH is 6-10, preferably about 8, and contains sodium chloride at a concentration of at least about 50 mM and/or calcium chloride at a concentration of at least about 1 mM, preferably about 100 mM sodium chloride and about 2.25 mM chlorine calcium chloride; the elution buffer is pH 6-10, preferably pH about 8, and contains sodium chloride at a concentration of at least 50 mM and/or calcium chloride at a concentration of at least 1 mM, preferably the elution buffer contains about 100 mM sodium chloride and about 5 mM calcium chloride. 6. The method according to any one of claims 1-5, wherein in the step 4), optionally comprising (a) equilibrium hydrophobic interaction chromatography material, (b) eluting the hydrophobic interaction chromatography material, and (c) elution of the hydrophobic interaction chromatography material, wherein the sample solution of this step of chromatography contains salts and/or zwitterions, and the salts can be selected from: ammonium acetate, ammonium sulfate, ammonium chloride, sodium chloride , sodium acetate, sodium sulfate, potassium acetate, potassium chloride and potassium sulfate, the zwitterions may be selected from: glycine, alanine, leucine and isoleucine. 7. The method according to claim 6, wherein the salt and/or zwitterion is preferably ammonium acetate. 8. The method of any one of claims 1-7, wherein the method does not comprise an affinity chromatography step. 9 . The method according to claim 8 , wherein the affinity chromatography is immunoaffinity chromatography coupled with a coagulation factor VIIa antibody on an affinity chromatography medium. 10 . 10. The method according to any one of claims 1-9, wherein the human coagulation factor VIIa is recombinant human coagulation factor VIIa produced under cell culture conditions.

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