阅读说明：本技术 一种高浓度人纤维蛋白原的制备工艺 (Preparation process of high-concentration human fibrinogen ) 是由 刘敏亮 梁小明 罗二华 饶振 李雅文 廖凯 于 2020-06-05 设计创作，主要内容包括：本发明属于血液制品领域,具体涉及一种高浓度人纤维蛋白原的制备工艺,依次包括：组分I溶解、S/D灭活、低温乙醇沉淀、甘氨酸沉淀、加入氯化钙,阳离子交换层析、阴离子交换层析、超滤、浓缩、稀配、分装、冻干、干热灭活。本发明在人纤维蛋白原提取分离过程中采用了二步沉淀法和二步层析法,提高了人纤维蛋白的纯度,有效去除杂蛋白,可减少临床不良发应的发生,制备得到高浓度、高纯度、溶解性好的人纤维蛋白原,可减少临床用药次数,方便临床用药。(The invention belongs to the field of blood products, and particularly relates to a preparation process of high-concentration human fibrinogen, which sequentially comprises the following steps: dissolving component I, inactivating S/D, precipitating with low temperature ethanol, precipitating with glycine, adding calcium chloride, performing cation exchange chromatography, performing anion exchange chromatography, ultrafiltering, concentrating, diluting, packaging, lyophilizing, and inactivating with dry heat. The invention adopts a two-step precipitation method and a two-step chromatography method in the extraction and separation process of human fibrinogen, improves the purity of human fibrinogen, effectively removes foreign protein, can reduce the occurrence of clinical adverse reaction, prepares human fibrinogen with high concentration, high purity and good solubility, can reduce the frequency of clinical medication, and is convenient for clinical medication.)

1. A preparation process of high-concentration human fibrinogen is characterized by comprising the following steps:

(1) dissolving a component I: dissolving the component I by adopting a dissolving solution I with the weight being 10-15 times that of the component I;

(2) S/D inactivation: inactivating the dissolved component I in the step (1) by adopting S/D, and filtering and collecting to obtain filtrate I;

(3) low-temperature ethanol precipitation: treating and collecting the filtrate I obtained in the step (2) by adopting a low-temperature ethanol precipitation method to obtain a precipitate I, adding a dissolving solution II to dissolve the precipitate I, and filtering to obtain a filtrate II;

(4) and (3) glycine precipitation: obtaining a precipitate II by adopting a glycine precipitation method for the filtrate II obtained in the step (3), adding a dissolving solution to dissolve the precipitate II, and filtering to obtain a filtrate III;

(5) calcium chloride treatment: dialyzing filtrate III with solution III through 100KD ultrafiltration membrane; adding calcium chloride for reaction treatment, and filtering after the reaction to obtain filtrate IV;

(6) cation exchange chromatography: performing chromatography on the filtrate IV obtained in the step (5) by adopting cation exchange resin, balancing by using a dissolving solution (III) before filling the material on the column, collecting the flow-through liquid on the column, dialyzing by using a balanced liquid through an ultrafiltration membrane of 100KD, filtering and collecting a filtrate V;

(7) anion exchange chromatography: performing anion exchange resin chromatography on the filtrate V obtained in the step (6), balancing by using a balancing solution before filling the material on the column, and collecting the flow-through liquid on the column;

(8) concentration and diluted preparation: dialyzing the protein solution by using a 30KD ultrafiltration membrane to concentrate the protein solution, adding proline according to the volume of the protein solution until the final concentration is 1-3%, and adjusting the pH value to 6.5-7.5;

(9) subpackaging, freeze-drying and dry heat inactivation: and (4) filtering the product obtained in the step (8) by using a sterilizing filter element, subpackaging, freeze-drying, packaging and inactivating by using dry heat viruses.

2. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: in the step (1), adding 10-15 times of dissolving solution (I) by weight into the component I for dissolving, and stirring for 1-2 hours, wherein the water bath temperature is 30-37 ℃ during dissolving; wherein the dissolving solution (I) comprises sodium citrate, sodium chloride and sucrose.

3. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: in the step (3), cooling the filtrate I obtained in the step (2) to 0-5 ℃, adding 50%, -25 ℃ low-temperature ethanol until the final content of the ethanol is 6% -10%, controlling the temperature to be 0-5 ℃, stirring for 30 minutes, centrifuging under the centrifugation condition of 10000 revolutions per minute, controlling the temperature of the centrifuged liquid to be 0-5 ℃, and collecting precipitates to obtain a precipitate I; adding a dissolving solution II according to 10 times of the weight of the precipitate, stirring and dissolving for 1 hour at the dissolving temperature of 30-37 ℃, filtering by using a filter element with the diameter of 1 mu m, and collecting filtrate to obtain filtrate II; the dissolving solution (II) comprises sodium citrate, sodium chloride and arginine hydrochloride.

4. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: in the step (4), cooling the filtrate II obtained in the step (3) to 20-30 ℃, adding mixed powder of sodium chloride and glycine according to the volume of the filtrate until the final content of sodium chloride is 1.3-2.0 mol/L and the content of glycine is 1.3-2.0 mol/L, stirring for 30 minutes, controlling the temperature to be 20-30 ℃, centrifuging under the condition of 10000 r/min, controlling the temperature of the centrifuged liquid to be 20-30 ℃, collecting precipitate to obtain precipitate II, adding the precipitate II into a dissolving solution according to 10 times of the precipitate weight, stirring for dissolving for 1 hour, controlling the dissolving temperature to be 30-37 ℃, filtering by using a filter element with the diameter of 1 mu m, and collecting the filtrate to obtain filtrate III.

5. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: and (5) dialyzing the filtrate III obtained in the step (4) by using a dissolving solution III through a 100KD ultrafiltration membrane, adjusting the pH to 6.8-7.2, filtering by using a filter element with the diameter of 1 mu m, and collecting the filtrate to obtain a filtrate IV.

6. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: in the step (6), the cation exchange resin is CM-Sepharose-FF gel.

7. The process for producing high-concentration human fibrinogen according to any of claims 1 and 4 to 6, wherein: the dissolving solution (c) comprises sodium citrate, sodium chloride and arginine hydrochloride.

8. The process for producing high-concentration human fibrinogen according to claim 1 or 6, characterized in that: in the step (7), the anion exchange resin is Capto-Q XP gel.

9. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: the balance liquid comprises sodium citrate, sodium chloride and arginine hydrochloride; the dialysate comprises sodium citrate, sodium chloride and arginine hydrochloride.

10. The process for preparing high-concentration human fibrinogen according to claim 1, wherein: the specific parameters of the freeze drying in step (9) are set as follows:

firstly, putting the product into a cabinet at normal temperature;

reducing the temperature from normal temperature to-5 ℃ for 60 minutes, and keeping the temperature for 60 minutes;

③ reducing the temperature to-35 ℃ in 120 minutes, and keeping the temperature for 240 minutes;

fourthly, starting a vacuum pump until the vacuum reaches 0.3mbar and is stable;

fifthly, heating to-20 ℃ for 120 minutes, and keeping for 480 minutes;

sixthly, heating to 0 ℃ for 180 minutes and keeping for 1200 minutes;

seventhly, heating to 20 ℃ for 240 minutes, and keeping for 480 minutes;

eighthly, heating to 30 ℃ for 180 minutes, and keeping for 360 minutes;

ninthly, vacuumizing to a limit, plugging and discharging.

Technical Field

The invention belongs to the field of blood products, and particularly relates to a preparation process of high-concentration human fibrinogen.

Background

Fibrinogen (Fg), which is synthesized and secreted by hepatocytes, is a blood coagulation factor important in the body's hemostatic physiology. The storage stability is good, but the thermal stability is poor, irreversible precipitation can be formed at 56 ℃, and the Fg concentration in normal human plasma is 2.4-4.0 g/L.

Fg is an acute phase reaction protein, and abnormal blood content is a risk factor for diseases such as ischemic cardiovascular and cerebrovascular diseases. The increase of Fg is often a non-specific reaction of the body, and is commonly seen in toxemia, pneumonia, cholecystitis, pulmonary tuberculosis and other infections, nephrotic syndrome, rheumatic fever, malignant tumor, cerebral thrombosis, myocardial infarction and other aseptic inflammations; in addition, mild increase in Fg was observed during surgery, radiotherapy, and pregnancy. Fg is less reduced, but when it is less than 1.0g/L, bleeding can occur in the body. A congenital fibrinogen deficiency is a very rare genetic disease, inherited by autosomal recessive genes, in which the liver of the patient is unable to synthesize Fg; the secondary fibrinogen reduction is caused by fibrinolysis by fibrinolytic enzyme, such as early placenta stripping, thrombus formation caused by entry of amniotic fluid into blood vessel during delivery, disseminated intravascular coagulation, activation of plasminogen, increase of fibrinolytic enzyme activity in blood, fibrinolysis, consumption of original Fg in vivo, and reduction of the content of the fibrinogen; the reduction of Fg may also occur in severe liver parenchymal damage such as hepatic necrosis caused by various causes, advanced chronic liver disease, etc.

Clinically, Fg is mainly used for treating congenital and acquired fibrinogen reduction or deficiency, severe liver injury, liver cirrhosis, disseminated intravascular coagulation, postpartum hemorrhage and blood coagulation disorder caused by major surgery, trauma or internal hemorrhage and the like.

Currently commercially available human fibrinogen is 0.5 g/vial, with a volume of 25ml after reconstitution, a protein concentration of about 2.5%, and a purity of about 80%. Because the Fg redissolution process takes long time, the whole process needs 15-30 minutes, the product and the sterilized injection water are preheated to 30-37 ℃, then the preheated sterilized injection water is injected according to the label amount of the bottle label, and the product is placed in a water bath at 30-37 ℃ and slightly shaken to be dissolved. For clinical emergency hemostasis, it is necessary to develop high concentration, high purity, fast dissolving Fg (1 g/vial, protein concentration 5%). Not only can save clinical time, but also can reduce the infusion times.

At present, only a few manufacturers of human fibrinogen in China, such as Shanghai rice and Hualan organisms, exist. Most manufacturers adopt the component I as the raw material for precipitation, and adopt the cold precipitation as the raw material individually, the preparation process is a low-temperature ethanol method, the concentration of the product is about 2.5%, the purity is about 80%, and the redissolution time is longer. Problems with solubility and reconstitution time should be noted during the manufacturing process. The difficulty of dissolution and even the precipitation of a large amount of denatured protein after dissolution are caused by the activation of fibrinogen. Fibrinogen is easily activated, especially in the presence of thrombin, thrombin activates originally water-soluble fibrinogen into fibrin monomers, which are polymerized to finally form water-insoluble fibrin, and the fibrin is then twisted into lumps to form fibrin glue, so that difficulty in filtration and long redissolution time of products are easily caused in the process preparation process. Because the product contains a small amount of the fibronectin, the fibronectin has poor heat stability, and is easy to denature particularly during dry heat inactivation, so that the protein is easy to separate out during redissolution.

As can be seen, there are still some problems with the existing human fibrinogen: (1) the purity is not high, the impurity content of the product is excessive, and adverse reactions such as disseminated intravascular coagulation, rash, tachycardia, fever and the like are easy to occur in clinical use; (2) the product has low solubility, most products need to be dissolved in water bath at 30-37 ℃, the dissolving time is about 20 minutes, and even more than 30 minutes, the dissolving time is too long when the product is used, the clinical use is inconvenient, and particularly under the emergency condition; (3) the product contains a small amount of thrombin and fibronectin, and a large amount of denatured protein can be separated even after some products are activated after being dissolved; (4) the yield is not high, and especially human fibrinogen is extracted from the cryoprecipitate; (5) the existing product has the protein concentration of 2.5 percent, contains about 4 percent of arginine hydrochloride and 1.3 percent of sodium citrate as a protective agent, and needs to be dissolved for many times during clinical use.

The invention patent of human fibrinogen is compared with the prior domestic invention patent of human fibrinogen by inquiring the invention patent of human fibrinogen, such as:

ZL200810046747.5 discloses a preparation method of human fibrinogen preparation, which comprises the steps of dissolving plasma, preparing component I, and performing two-step low-temperature ethanol precipitation to prepare human fibrinogen. The content of human fibrinogen protein prepared by the method is 2.5-3.5%, and arginine hydrochloride and sodium citrate are used as prescription protective agents.

ZL201110078557.3 discloses a method for extracting human fibrinogen from component I by column chromatography, which comprises FI precipitation dissolution and filtration, S/D inactivation, first glycine precipitation, DEAE-650M column chromatography, second glycine precipitation, preparation, split charging and freeze drying, and dry heat inactivation. The method adopts two-step glycine precipitation combined with DEAE-650M column chromatography, the protein content of the prepared product is 2.5%, arginine hydrochloride and sodium citrate are used as prescription protective agents, and the purity is about 85%.

CN201710941269.3 discloses a method for preparing human fibrinogen by a double-layer chromatography, which comprises the steps of precipitating, dissolving and filtering a component I; S/D virus inactivation; q Sepharose Fast Flow anion exchange chromatography; primary ultrafiltration concentration; heparin affinity chromatography; performing secondary ultrafiltration concentration; detecting and preparing; subpackaging and freeze-drying; capping and dry heat inactivation. The protein content of the prepared product is 2.2-2.4%, and glycine and sodium citrate are used as protective agents.

CN201810054783.X discloses a preparation method of human fibrinogen, which comprises the steps of preparation of a component I, DEAE-650M column chromatography, first-step glycine precipitation, second-step glycine precipitation and detection preparation; subpackaging and freeze-drying; capping and dry heat inactivation. The protein content of the prepared product is 2.4%, and glycine, arginine hydrochloride, mannitol and sodium citrate are used as protective agents.

In view of the preparation process and clinical application conditions of human fibrinogen in the current market, it is particularly necessary to develop a new extraction process of human fibrinogen with high purity, high concentration and good solubility.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation process of high-concentration human fibrinogen.

The technical scheme adopted by the invention is as follows: a preparation process of high-concentration human fibrinogen comprises the following steps:

(1) dissolving a component I: dissolving the component I by adopting a dissolving solution I with the weight being 10-15 times that of the component I;

(2) S/D inactivation: inactivating the dissolved component I in the step (1) by adopting S/D, and filtering and collecting to obtain filtrate I;

(3) low-temperature ethanol precipitation: treating and collecting the filtrate I obtained in the step (2) by adopting a low-temperature ethanol precipitation method to obtain a precipitate I, adding a dissolving solution II to dissolve the precipitate I, and filtering to obtain a filtrate II;

(4) and (3) glycine precipitation: obtaining a precipitate II by adopting a glycine precipitation method for the filtrate II obtained in the step (3), adding a dissolving solution to dissolve the precipitate II, and filtering to obtain a filtrate III;

(5) calcium chloride treatment: dialyzing filtrate III with solution III through 100KD ultrafiltration membrane; adding calcium chloride for reaction treatment, and filtering after the reaction to obtain filtrate IV;

(6) cation exchange chromatography: performing chromatography on the filtrate IV obtained in the step (5) by adopting cation exchange resin, balancing by using a dissolving solution (III) before filling the material on the column, collecting the flow-through liquid on the column, dialyzing by using a balanced liquid through an ultrafiltration membrane of 100KD, filtering and collecting a filtrate V;

(7) anion exchange chromatography: performing anion exchange resin chromatography on the filtrate V obtained in the step (6), balancing with a balancing solution before filling the material on the column, collecting the flow-through liquid on the column,

(8) concentration and diluted preparation: dialyzing the protein solution by using a 30KD ultrafiltration membrane to concentrate the protein solution, adding proline according to the volume of the protein solution until the final concentration is 1-3%, and adjusting the pH value to 6.5-7.5;

(9) subpackaging, freeze-drying and dry heat inactivation: and (4) filtering the product obtained in the step (8) by using a sterilizing filter element, subpackaging, freeze-drying, packaging and inactivating by using dry heat viruses.

Dissolving the component I, inactivating by S/D, performing 1-step low-temperature ethanol precipitation, and removing part of foreign proteins; then glycine precipitation is carried out for 1 step, and part of impure protein is further removed. According to the invention, two precipitation methods of low-temperature ethanol precipitation and glycine precipitation which are different in principle are combined in the preparation process, so that the foreign protein can be removed by different principles, and the purity of the human fibrinogen is improved.

Calcium chloride is added into the protein solution to activate the existing prothrombin into thrombin, and the thrombin is removed by cation exchange resin, so that the product is prevented from being activated in the storage and use processes, and the clinical medication risk is reduced; the protein solution is subjected to anion exchange chromatography to remove foreign proteins such as fibronectin, so that the purity of human fibrinogen is further improved; proline is added into the prescription of the product to increase the solubility and stability of the product; the advantages of the process are benefited, the product concentration is improved, and meanwhile, the auxiliary material dosage of the product is not obviously improved, namely, the risk of the auxiliary material is reduced; can be dissolved at room temperature when being dissolved, and is convenient for clinical use.

Preferably, in the step (1), the component I is dissolved by adding 10-15 times of dissolving solution (I) by weight, and stirring for 1-2 hours, wherein the water bath temperature is 30-37 ℃ during dissolving; wherein the dissolving solution (I) comprises sodium citrate, sodium chloride and sucrose.

Preferably, in the step (3), the filtrate I obtained in the step (2) is cooled to 0-5 ℃, 50% of low-temperature ethanol with the temperature of-25 ℃ is added until the final content of the ethanol is 6% -10%, the temperature is controlled to be 0-5 ℃, the mixture is stirred for 30 minutes, centrifugation is carried out, the centrifugation condition is 10000 revolutions per minute, the temperature of the centrifuged liquid is controlled to be 0-5 ℃, and the precipitate is collected to obtain a precipitate I; adding a dissolving solution II according to 10 times of the weight of the precipitate, stirring and dissolving for 1 hour at the dissolving temperature of 30-37 ℃, filtering by using a filter element with the diameter of 1 mu m, and collecting filtrate to obtain filtrate II; the dissolving solution (II) comprises sodium citrate, sodium chloride and arginine hydrochloride.

Preferably, in the step (4), the filtrate II obtained in the step (3) is cooled to 20-30 ℃, mixed powder of sodium chloride and glycine is added according to the volume of the filtrate until the final content of sodium chloride is 1.3-2.0 mol/L and the content of glycine is 1.3-2.0 mol/L, the mixture is stirred for 30 minutes, the temperature is controlled to be 20-30 ℃, centrifugation is carried out, the centrifugation condition is 10000 r/min, the temperature of the centrifuged liquid is controlled to be 20-30 ℃, precipitate II is obtained by collecting the precipitate, the precipitate II is added into a dissolving solution according to 10 times of the weight of the precipitate, the dissolving solution is stirred and dissolved for 1 hour, the dissolving temperature is 30-37 ℃, a filter element with the diameter of 1 mu m is used for filtering, and the filtrate III is obtained by collecting the filtrate.

Preferably, in the step (5), the filtrate III obtained in the step (4) is dialyzed by a 100KD ultrafiltration membrane through a dissolving solution III, the pH value is adjusted to 6.8-7.2, the filtrate is filtered by a filter element with the diameter of 1 mu m, and the filtrate IV is collected.

Preferably, in step (6), the cation exchange resin is CM-Sepharose-FF gel.

Preferably, the dissolving solution (c) comprises sodium citrate, sodium chloride and arginine hydrochloride.

Preferably, in step (7), the anion exchange resin is Capto-Q XP gel.

Preferably, the balance liquid comprises sodium citrate, sodium chloride and arginine hydrochloride; the dialysate comprises sodium citrate, sodium chloride and arginine hydrochloride.

The invention can prepare 5% high concentration human fibrinogen, and proline, arginine hydrochloride and sodium citrate are used as auxiliary materials, which can improve the stability and dissolution time of human fibrinogen.

Preferably, the specific parameters for freeze-drying in step (9) are set as follows:

firstly, putting the product into a cabinet at normal temperature;

reducing the temperature from normal temperature to-5 ℃ for 60 minutes, and keeping the temperature for 60 minutes;

③ reducing the temperature to-35 ℃ in 120 minutes, and keeping the temperature for 240 minutes;

fourthly, starting a vacuum pump until the vacuum reaches 0.3mbar and is stable;

fifthly, heating to-20 ℃ for 120 minutes, and keeping for 480 minutes;

sixthly, heating to 0 ℃ for 180 minutes and keeping for 1200 minutes;

seventhly, heating to 20 ℃ for 240 minutes, and keeping for 480 minutes;

eighthly, heating to 30 ℃ for 180 minutes, and keeping for 360 minutes;

ninthly, vacuumizing to a limit, plugging and discharging.

The freeze-drying process which is researched by self can reduce the water content in the product, improve the stability of the product and improve the appearance of the product.

The invention has the following beneficial effects:

1. at present, the plasma raw material is in the shortage day by day, the purity and the yield of human fibrinogen are improved by extracting the fibrinogen from FI, and the comprehensive utilization rate of the plasma can be improved, so that the comprehensive competitiveness of enterprises is improved.

2. The two-step precipitation method and the two-step chromatography method are adopted in the extraction and separation process of the human fibrinogen, so that the purity of the human fibrinogen is improved, the foreign protein is effectively removed, and the occurrence of clinical adverse reactions can be reduced.

3. The preparation method has the advantages of preparing human fibrinogen with high concentration, high purity and good solubility, reducing the frequency of clinical medication and facilitating clinical medication.

The comparison of the key quality indexes of the product prepared by the method of the invention and human fibrinogen described in Chinese pharmacopoeia (2015 edition, three parts) is shown in the following table.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 is a schematic flow chart of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.