阅读说明：本技术 用于纯化人纤维蛋白原的离子交换层析用缓冲液及填料 (Buffer solution and filler for ion exchange chromatography for purifying human fibrinogen ) 是由 鲁涛 牟蕾 李伟 余伟 王黔川 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种用于纯化人纤维蛋白原的离子交换层用缓冲液及填料,该离子交换层析用缓冲液包括平衡缓冲液,洗涤缓冲液和洗脱缓冲液。本发明属于血浆制品制备工艺领域。实验结果表明,利用本发明由平衡缓冲液,洗涤缓冲液和洗脱缓冲液组成的缓冲液,配合离子交换层析用填料对人纤维蛋白原进行纯化,能够明显提高所得产品中人纤维蛋白原含量和纯度,还能明显降低产品中的病毒灭活剂残留。本发明提供的离子交换层用缓冲液及填料在纯化人纤维蛋白原中具有良好的应用前景。(The invention discloses a buffer solution and a filler for an ion exchange layer for purifying human fibrinogen. The invention belongs to the field of plasma product preparation technology. Experimental results show that the buffer solution consisting of the equilibrium buffer solution, the washing buffer solution and the elution buffer solution is used in combination with the filler for ion exchange chromatography to purify the human fibrinogen, so that the content and the purity of the human fibrinogen in the obtained product can be obviously improved, and the virus inactivator residue in the product can be obviously reduced. The buffer solution and the filler for the ion exchange layer provided by the invention have good application prospects in purification of human fibrinogen.)

1. A buffer for ion exchange chromatography for purifying fibrinogen, comprising: the buffer solution for ion exchange chromatography comprises an equilibrium buffer solution, a washing buffer solution and an elution buffer solution; the balance buffer solution is 2-8 mM Tris buffer solution; the washing buffer solution consists of an aqueous solution of NaCl and Tris, wherein the concentration of NaCl is 20-60 mM, and the concentration of Tris is 2-8 mM; the elution buffer solution consists of NaCl and Tris aqueous solutions, wherein the concentration of NaCl is 70-130 mM, and the concentration of Tris is 2-8 mM.

2. The buffer for ion exchange chromatography according to claim 1, wherein: the pH value of the balance buffer solution is 8.0-9.0, the pH value of the washing buffer solution is 8.0-9.0, and the pH value of the elution buffer solution is 8.0-9.0.

3. The buffer for ion exchange chromatography according to claim 2, wherein: the balance buffer solution is a 5mM Tris buffer solution, and the pH value of the balance buffer solution is 8.5;

and/or, in the washing buffer, the concentration of NaCl is 30mM, the concentration of Tris is 5mM, and the pH value of the washing buffer is 8.5;

and/or, in the elution buffer, the concentration of NaCl is 100mM, the concentration of Tris is 5mM, and the pH value of the elution buffer is 8.5.

4. A composition for ion exchange chromatography for purifying fibrinogen, comprising: the composition comprises the following two components:

component 1: a buffer for ion exchange chromatography according to any one of claims 1 to 3;

and (2) component: the filler for ion exchange chromatography is polymethacrylate anion exchange resin.

5. The composition for ion exchange chromatography according to claim 4, characterized in that: the filler for ion exchange chromatography is selected from Capto Q, Fractogel EMD TMAE (M) or Fractogel EMD DEAE (M), preferably Fractogel EMD DEAE (M).

6. Use of the buffer solution for ion exchange chromatography according to any one of claims 1 to 3 and the composition for ion exchange chromatography according to any one of claims 4 to 5 for purifying fibrinogen.

7. Use according to claim 6, characterized in that: the purification of fibrinogen comprises the steps of: and (3) balancing chromatographic packing with a balance buffer solution, loading the fibrinogen subjected to virus inactivation onto a column, eluting with a washing buffer solution, eluting with an elution buffer solution, and collecting the eluate to obtain the purified fibrinogen.

8. Use according to claim 7, characterized in that: the fibrinogen after virus inactivation is prepared by the following method:

(a) taking the cryoprecipitate, adding a water phase solvent for dissolving to obtain a cryoprecipitate dissolved solution;

(b) adsorbing the cold precipitation solution with aluminum hydroxide, centrifuging, collecting supernatant, filtering, and retaining filtrate;

(c) adding polysorbate 80 and tributyl phosphate into the filtrate, and inactivating to obtain virus-inactivated fibrinogen.

9. Use according to claim 8, characterized in that: in step (a), the cryoprecipitate is prepared from fresh frozen plasma; the aqueous phase solvent is Tris buffer solution;

and/or, in the step (b), the dosage of the aluminum hydroxide is 1 wt.% to 5 wt.% of the cryoprecipitate, the adsorption time is 10 to 20 minutes, and the adsorption temperature is 20 to 35 ℃;

and/or in the step (c), the use amount of polysorbate 80 is 0.5 wt.% to 2.0 wt.% of the filtrate, the use amount of tributyl phosphate is 0.1 wt.% to 0.5 wt.% of the filtrate, the inactivation time is 4 to 8 hours, and the inactivation temperature is 24 to 26 ℃.

10. Use according to claim 9, characterized in that: in the step (a), the aqueous phase solvent is 10-30 mM Tris buffer solution with pH of 6.0-7.5, preferably 20mM Tris buffer solution with pH of 6.8;

and/or, in step (b), the amount of aluminum hydroxide is 2 wt.% of the cryoprecipitate, the adsorption time is 15 minutes, and the adsorption temperature is 25 ℃;

in the step (c), the mass ratio of polysorbate 80 to tributyl phosphate is 10: 3, the polysorbate 80 is used in an amount of 1 wt.% of the filtrate, the inactivation time is 6 hours, and the inactivation temperature is 25 ℃.

Technical Field

The invention belongs to the field of plasma product preparation processes, and particularly relates to a buffer solution and a filler for an ion exchange layer for purifying human fibrinogen.

Background

Human Fibrinogen (Fg), also known as human coagulation factor i, is synthesized mainly by liver parenchymal cells, is one of the main components of plasma protein, and is rich in Fibrinogen content in plasma, about 2-4 g/L in normal human plasma, and is one of the "central" proteins in the blood coagulation system.

The human fibrinogen is a final substrate for the sequential activation of blood coagulation factors in the blood coagulation process, has a hemostatic function, can directly participate in the later stage of the blood coagulation process, and can also mediate platelet aggregation and influence blood viscosity. In the common pathway of blood coagulation, thrombin firstly cleaves amino ends Arg16-Gly17 of two A alpha chains of fibrinogen to release a pair of fibrinopeptide A to form a fibrin monomer I; releasing a pair of fibrinopeptides B at amino terminals Arg14-Gly15 of two B beta chains of the cracked fibrinogen to form a fibrin monomer II, exposing a polymerization part of the fibrin monomer, and forming an unstable soluble fibrin monomer through non-covalent combination. In activated blood coagulation factors XIII and Ca²⁺Under the action of the fibrin monomer, the fibrin monomer is mutually cross-linked to generate stable soluble fibrin, and the formed components of blood are wrapped in the stable soluble fibrin to form firm thrombus. Besides being involved in blood coagulation, human fibrinogen has other functions, such as binding to platelet membrane glycoprotein IIb/IIIa to mediate platelet aggregation reaction, and participating in atherosclerosis and tumor metastasis. Therefore, the preparation of high-purity human fibrinogen has important clinical value.

At present, the purification method of human fibrinogen is more adopted and is chromatographic purification. The Chinese patent application with the application number of 201711104891.5 discloses a preparation method of human fibrinogen, which comprises the following steps: (1) precipitating human plasma with low temperature ethanolDissolving the I in the extracting solution to obtain a component I solution; (2) performing S/D inactivation on the component I solution obtained in the step (1); (3) balancing a lysine affinity chromatography medium with a balance solution, then carrying out column chromatography on the solution obtained in the step (2), and collecting a penetration peak; (4) and (3) balancing the cation exchange chromatography medium with a balance solution, then carrying out column chromatography on the penetration peak obtained in the step (3), eluting the chromatographic column with an eluent after the chromatography is finished, finally eluting with an eluent, and collecting the elution peak to obtain the human fibrinogen solution. However, the purification steps of the method are complex, and lysine affinity column chromatography and cation exchange column chromatography are required to be carried out in sequence; in addition, the virus inactivation process of the method adopts an S/D method, but the human fibrinogen purified by the method also has a virus inactivator tributyl phosphate residue. It was found that tributyl phosphate half-Lethal (LD) of mice₅₀) 602mg/kg, which is harmful to The human body (The use of tri (n-butyl) phosphate reagents to inactive microorganisms viruses and human immunity viruses in plasma and plasma's subsequent reactivity. transfusion.1990 Sep; 30(7):591-8.). Therefore, there is a need to develop a fibrinogen purification method with simpler operation and less residual amount of tributyl phosphate as a virus inactivating agent.

Disclosure of Invention

The invention aims to provide a buffer solution and a filler for an ion exchange layer for purifying human fibrinogen, and application of the buffer solution and the filler in purifying the fibrinogen, so as to simplify the purification steps of the fibrinogen, improve the purity of the fibrinogen and reduce the residual amount of a virus inactivating agent.

The invention provides a buffer solution for ion exchange chromatography for purifying fibrinogen, which comprises an equilibrium buffer solution, a washing buffer solution and an elution buffer solution; the balance buffer solution is 2-8 mM Tris buffer solution; the washing buffer solution consists of an aqueous solution of NaCl and Tris, wherein the concentration of NaCl is 20-60 mM, and the concentration of Tris is 2-8 mM; the elution buffer solution consists of an aqueous solution of NaCl and Tris, wherein the concentration of NaCl is 70-130 mM, and the concentration of Tris is 2-8 mM.

Further, the pH value of the balance buffer solution is 8.0-9.0, the pH value of the washing buffer solution is 8.0-9.0, and the pH value of the elution buffer solution is 8.0-9.0.

Further, the equilibration buffer is 5mM Tris buffer, and the pH of the equilibration buffer is 8.5;

and/or, in the washing buffer, the concentration of NaCl is 30mM, the concentration of Tris is 5mM, and the pH value of the washing buffer is 8.5;

and/or, in the elution buffer, the concentration of NaCl is 100mM, the concentration of Tris is 5mM, and the pH value of the elution buffer is 8.5.

The present invention also provides a composition for ion exchange chromatography for purifying fibrinogen, the composition comprising the following two components:

component 1: the above-mentioned buffer solution for ion exchange chromatography;

and (2) component: the filler for ion exchange chromatography is polymethacrylate anion exchange resin.

Further, the filler for ion exchange chromatography is selected from Capto Q, Fractogel EMD TMAE (M) or Fractogel EMD DEAE (M), preferably Fractogel EMD DEAE (M).

The invention also provides the buffer solution for ion exchange chromatography and the application of the composition for ion exchange chromatography in purifying fibrinogen.

Further, the purification of fibrinogen comprises the steps of: and (3) balancing chromatographic packing with a balance buffer solution, loading the fibrinogen subjected to virus inactivation onto a column, eluting with a washing buffer solution, eluting with an elution buffer solution, and collecting the eluate to obtain the purified fibrinogen.

Further, the virus inactivated fibrinogen is prepared according to the following method:

(a) taking the cryoprecipitate, adding a water phase solvent for dissolving to obtain a cryoprecipitate dissolved solution;

(b) adsorbing the cold precipitation solution with aluminum hydroxide, centrifuging, collecting supernatant, filtering, and retaining filtrate;

(c) adding polysorbate 80 and tributyl phosphate into the filtrate, and inactivating to obtain virus-inactivated fibrinogen.

Further, in step (a), the cryoprecipitate is a cryoprecipitate prepared from fresh frozen plasma; the aqueous phase solvent is Tris buffer solution;

and/or, in the step (b), the dosage of the aluminum hydroxide is 1 wt.% to 5 wt.% of the cryoprecipitate, the adsorption time is 10 to 20 minutes, and the adsorption temperature is 20 to 35 ℃;

and/or in the step (c), the use amount of polysorbate 80 is 0.5 wt.% to 2.0 wt.% of the filtrate, the use amount of tributyl phosphate is 0.1 wt.% to 0.5 wt.% of the filtrate, the inactivation time is 4 to 8 hours, and the inactivation temperature is 24 to 26 ℃.

Further, in the step (a), the aqueous phase solvent is 10-30 mM of Tris buffer solution with pH of 6.0-7.5, preferably 20mM of Tris buffer solution with pH of 6.8;

and/or, in step (b), the amount of aluminum hydroxide is 2 wt.% of the cryoprecipitate, the adsorption time is 15 minutes, and the adsorption temperature is 25 ℃;

in the step (c), the mass ratio of polysorbate 80 to tributyl phosphate is 10: 3, the polysorbate 80 is used in an amount of 1 wt.% of the filtrate, the inactivation time is 6 hours, and the inactivation temperature is 25 ℃.

Further, the fibrinogen is human fibrinogen.

Definitions of terms used in connection with the present invention: the initial definitions provided for by terms herein apply to that term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

The S/D method is a virus inactivation method, and refers to a method of disrupting the lipid membrane of enveloped viruses with an organic solvent/detergent mixture (S/D). Once the lipid membrane is disrupted, the virus is no longer able to bind to the infected cells.

Cryoprecipitate is a plasma product containing factor VIII and fibrinogen, and can be used for treating patients who lack factor VIII and fibrinogen and have bleeding or hemophilia.

Tris is an abbreviation for Tris hydroxymethyl aminomethane.

Aqueous phase solvent refers to water or an aqueous solution of a non-organic phase.

The room temperature means 25. + -. 2 ℃.

The purification method provided by the invention is simple and convenient to operate, low in cost and good in industrial application prospect.

The buffer solution consisting of the equilibrium buffer solution, the washing buffer solution and the elution buffer solution is used for purifying the human fibrinogen by matching with the filler for ion exchange chromatography, so that the content and the purity of the human fibrinogen in the obtained product can be obviously improved, and the virus inactivating agent residue in the product can be obviously reduced. The buffer solution and the filler for the ion exchange layer provided by the invention have good application prospects in purification of human fibrinogen.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a linear elution chromatogram. Wherein the abscissa is the chromatography volume; the ordinate is the percentage of liquid in pump B in the elution buffer; the blue curve is the UV280 absorption value; a green line parallel to the ordinate is the indicator line indicating that the percentage of liquid in pump B in the elution buffer here is 10%; the green curve is the percentage of the B pump; the brown curve is the conductance value.

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 purification of human fibrinogen

The first step is as follows: preliminary purification

1. Taking a cryoprecipitate prepared from fresh frozen plasma, and adding the cryoprecipitate into a Tris buffer solution with the weight ratio of 1:3 and the pH value of 6.8 at room temperature for dissolving to obtain a cryoprecipitate dissolving solution;

2. adding aluminum hydroxide (the amount of the aluminum hydroxide is 2 wt.% of the cryoprecipitate) into the cryoprecipitate dissolving solution, and stirring for 15 minutes at room temperature to perform adsorption treatment;

3. centrifuging the system obtained after adsorption at 4000g for 20 minutes, taking the supernatant, filtering the supernatant through a 0.45-micrometer filter membrane, and keeping the filtrate;

the second step is that: inactivation of viruses

Performing virus inactivation on the obtained filtrate according to an S/D method: and (3) adding polysorbate 80 (the dosage of the polysorbate 80 is 1 wt.% of the filtrate) and tributyl phosphate (the dosage of the tributyl phosphate is 0.3 wt.% of the filtrate) into the filtrate obtained in the first step, and inactivating the mixture at 25 ℃ for 6 hours to obtain a sample to be loaded on the column.

The third step: anion exchange chromatography purification

Fractogel EMD DEAE (M) is used as anion exchange chromatography packing to purify a sample to be loaded on a column, and the method comprises the following specific steps:

and (2) balancing the anion exchange chromatography packing Fractogel EMD DEAE (M) with 10 times of column volume of an equilibrium buffer solution, loading the to-be-loaded column sample obtained in the second step onto the column, eluting the chromatography column with the washing buffer solution, eluting with the elution buffer solution, and collecting the eluent to obtain the human fibrinogen solution.

Wherein the balance buffer solution is a Tris buffer solution with the concentration of 5mM and the pH value of 8.5; the pH value of the washing buffer solution is 8.5, the washing buffer solution consists of NaCl and Tris aqueous solutions, and the concentration of NaCl in the washing buffer solution is 30mM, and the concentration of Tris in the washing buffer solution is 5 mM; the elution buffer has a pH of 8.5 and consists of an aqueous solution of NaCl and Tris, wherein the concentration of NaCl in the elution buffer is 100mM and the concentration of Tris in the elution buffer is 5 mM.

Example 2 buffer for ion exchange chromatography for purification of fibrinogen

The buffer solution for ion exchange chromatography for purifying fibrinogen in this example consists of an equilibration buffer solution, a washing buffer solution and an elution buffer solution, wherein the equilibration buffer solution is a Tris buffer solution with 5mM and pH of 8.5; the pH value of the washing buffer solution is 8.5, the washing buffer solution consists of a NaCl aqueous solution and a Tris buffer solution, wherein the concentration of NaCl in the washing buffer solution is 30mM, and the concentration of Tris in the washing buffer solution is 5 mM; the pH value of the elution buffer solution is 8.5, and the elution buffer solution consists of a NaCl aqueous solution and a Tris buffer solution, wherein the concentration of NaCl in the elution buffer solution is 100mM, and the concentration of Tris in the elution buffer solution is 5 mM.

Example 3 ion exchange chromatography composition for purifying fibrinogen

This example ion exchange chromatography composition for purifying fibrinogen consisted of the following two components:

component 1: buffer for ion exchange chromatography for purifying fibrinogen described in example 2;

and (2) component: packing for ion exchange chromatography: fractogel EMD DEAE (M).

The advantageous effects of the present invention are described below by way of experimental examples.

Experimental example 1 evaluation of adsorption Effect

1. Experimental methods

The filtrates after the first step of the cold precipitation and adsorption treatment in example 1 were used as test samples to test the purity of human fibrinogen and the adsorption effects of blood coagulation factors II, VII, IX and X in the samples before and after the initial purification.

2. Results of the experiment

Table 1 human fibrinogen and adsorbed coagulation factors II, VII, IX, X (n ═ 3) in samples before and after primary purification

Test index	Before preliminary purification	After preliminary purification
			Protein content mg/ml (n ═ 8)	34.65	33.01
Coagulable protein content mg/ml (n ═ 8)	21.88	21.49
			Purity% (n ═ 8)	63.15	65.10
FII IU/ml(n＝2)	0.32	0.11
			FVII IU/ml(n＝2)	0.54	0.08
FIX IU/ml(n＝2)	0.85	0.23
			FX IU/ml(n＝2)	0.13	0.04

In table 1, protein content refers to the total protein content in the sample; the content of the coagulable protein, namely the content of human fibrinogen, and the test method comprises the following steps: incubating the sample with a thrombin solution, separating the precipitate, and determining the protein content, namely the content of the coagulable protein; purity is the percentage of coagulable protein content in the sample.

As can be seen from the results in Table 1, the method of the invention can primarily improve the purity of human fibrinogen after the adsorption treatment by aluminum hydroxide, and the process has excellent removal effect on blood coagulation factors FII, VII, IX and X which affect the stability of human fibrinogen.

Experimental example 2 evaluation of Virus inactivation Effect

1. Experimental methods

The sample to be subjected to virus inactivation obtained in the second step of example 1 was used as a test sample to evaluate the virus inactivation effect.

2. Results of the experiment

TABLE 2 SD Virus inactivation Effect (n ═ 3)

Indicative of virus	PRV virus	Sindbis virus	HIV virus
				Inactivating effect	≥4.75logTCID50/ml	≥4.02logPFU/ml	≥5.5logTCID50/m

As can be seen from the results in Table 2, the method of the present invention for inactivating SD virus can effectively inactivate lipid-enveloped virus, and can reach the national standard.

EXAMPLE 3 chromatography Medium screening experiment

1. Experimental methods

The procedure of example 1 was followed except that the anion exchange chromatography packing in the third step was replaced with Capto Q, Fractogel EMD TMAE (M) or Fractogel EMD DEAE (M) in table 3. The gel loading and yield were compared when the three anion exchange chromatography packing materials were used separately.

The gel loading refers to the amount of target protein that can be adsorbed by the gel, and the higher the loading, the better the adsorption effect, and the less gel is needed to separate a sample of the same volume. The yield is the ratio of the target protein obtained by removing impurities such as foreign proteins in the chromatography step to the target protein before chromatography.

Gel loading ═ volume on column sample x protein concentration)/volume of chromatography column;

note: the sample volume on the column refers to the volume from the beginning of loading to the obvious rise of the ultraviolet absorption peak and the appearance of a large amount of protein flowing through.

The yield is the total amount of fibrinogen in the sample after chromatography/the total amount of fibrinogen in the sample before chromatography.

2. Results of the experiment

TABLE 3 results of chromatography Medium screening experiments

As can be seen from Table 3, the gel loading of the three chromatographic packings was, in order from large to small, Fractogel EMD DEAE (M) > Fractogel EMD TMAE (M) > Capto Q, and the yield was, in order from large to small, Fractogel EMD TMAE (M) > Fractogel EMD DEAE (M) > Capto Q. In terms of balanced consideration of both loading capacity and yield, Fractogel EMD TMAE (M) requires a large chromatography gel volume, is high in cost, inconvenient to operate and not suitable for large-scale production, so Fractogel EMD DEAE (M) is selected as a purification chromatography medium.

EXAMPLE 4 elution buffer screening experiment

1. Experimental methods

The procedure of example 1 was followed, except that the NaCl concentration in the elution buffer of the third step was changed, and a linear gradient elution was performed, which was specifically performed by:

the liquid (pH 8.5) in pump A was 5mM Tris buffer, and the liquid (pH 8.5) in pump B was composed of Tris buffer and aqueous NaCl, with a Tris concentration of 5mM and a NaCl concentration of 1M. And taking the liquid obtained by mixing the pump A and the pump B as an elution buffer solution, gradually increasing the proportion of the liquid in the pump B in the elution buffer solution, and performing linear gradient elution.

2. Results of the experiment

The chromatogram is shown in figure 1, and the detection shows that a hetero-protein peak appears when the proportion of the liquid in the B pump is 3% -4%, and the elution peak obtained when the proportion of the liquid in the B pump reaches 10% is rich in human fibrinogen. Therefore, when the proportion of the liquid in the B pump in the elution buffer solution reaches 10%, the elution solution rich in human fibrinogen can be obtained. Here, the concentration of Tris was 5mM and the concentration of NaCl was 100mM, respectively, in the elution buffer.

Experimental example 5 verification experiment for purification Effect of human fibrinogen

1. Experimental methods

Test samples: purified human fibrinogen (i.e., post-chromatography sample) prepared in the third step of inventive example 1; the sample to be loaded on the column (i.e., the sample before chromatography) obtained in the second step of example 1 of the present invention.

The following criteria were tested:

coagulable protein (i.e., human fibrinogen) content: incubating the sample with a thrombin solution, separating the precipitate, and determining the protein content, namely the content of the coagulable protein;

purity: percent coagulable protein content in the sample;

yield of coagulable protein: percentage of total amount of coagulable protein of the sample after chromatography and before chromatography;

polysorbate 80 residue: measuring by a colorimetric method;

tributyl phosphate residue: and (4) measuring by gas chromatography.

2. Results of the experiment

Table 4 human fibrinogen purification effect (n ═ 8)

Test index	Pre-chromatography samples	Post-chromatography samples
			Content of coagulable protein (mg/ml)	3.81±0.27	4.35±0.51
Purity (%)	63.29±0.05	72.99±0.06
			Yield of coagulable protein	/	～70％
Polysorbate 80 residual u g/ml	1000	28.13±2.47
			Residual mu g/ml of tributyl phosphate	300	Not detected out

In table 4, the coagulable protein refers to human fibrinogen in the product.

As can be seen from Table 4, the content and purity of human fibrinogen in the product purified by the method of the present invention are significantly improved, and the yield is also higher; meanwhile, the purification method of the invention can obviously reduce the virus inactivator residue, and tributyl phosphate is not detected in the obtained product.

In summary, the present invention provides a buffer and a filler for an ion exchange layer for purifying human fibrinogen. Experimental results show that the buffer solution consisting of the equilibrium buffer solution, the washing buffer solution and the elution buffer solution is used in combination with the filler for ion exchange chromatography to purify the human fibrinogen, so that the content and the purity of the human fibrinogen in the obtained product can be obviously improved, and the virus inactivator residue in the product can be obviously reduced. The buffer solution and the filler for the ion exchange layer provided by the invention have good application prospects in purification of human fibrinogen.