Freeze drying method of instant fibrinogen

Abstract

The invention relates to a freeze drying method of instant fibrinogen, and belongs to the technical field of blood product preparation. The method comprises the steps of plasma pre-fusion, centrifugal separation, FI extraction, FI precipitation, dissolution and filtration, polyethylene glycol purification and separation, S / D inactivation, ethanol purification and separation, glycine purification and separation, ultrafiltration dialysis, preparation, degerming and subpackaging, freeze-drying and capping and the like. Wherein the freeze-drying process comprises four stages of pre-freezing, primary sublimation, desorption drying and plug pressing, and finally dry heat inactivation treatment is carried out. According to the method, efficient extraction and purification of fibrinogen are achieved by accurately controlling the temperature, the time, the pH value and the component proportion of various solutions in each step, and the obtained product has good instant dissolving performance and can be used in the medical fields of clinical hemostasis, tissue repair and the like.

Description

Technical Field

[0001] This invention belongs to the field of blood products technology, specifically relating to a freeze-drying method for rapidly soluble fibrinogen. Background Technology

[0002] Human fibrinogen (Fg) is a protein component in blood plasma, with a concentration as high as 2-4 g / L. It plays a crucial role in the coagulation system. The final stage of coagulation involves fibrinogen being converted into fibrin monomers by thrombin, which then cross-link with factor XIII to form an insoluble fibrin clot, achieving hemostasis. Patients with congenital fibrinogen deficiency or acquired fibrinogen deficiency due to disease are prone to coagulation dysfunction, and fibrinogen supplementation is currently the only reliable treatment.

[0003] Freeze-drying technology was first invented by the Englishman Wollaston in 1813. In 1909, Shsckell tested this method and achieved good results in the freeze-drying preservation of antitoxins, bacteria, rabies virus, and other biological products. The 2025 edition of the Chinese Pharmacopoeia lists all coagulation factor products as freeze-dried preparations. The entire freeze-drying process takes place in a low-temperature, low-pressure environment. After drying, the product volume remains unchanged, the material is porous and loose, has a long storage period, and is easily rehydrated to restore its original activity.

[0004] Fibrinogen has a molecular weight of 340 kDa and exhibits some hydrophobicity, resulting in a long reconstitution time after lyophilization. Human fibrinogen products are typically packaged in 25 ml vials with a liquid level of 25 mm. Significant differences in the temperature at which crystallization begins during pre-freezing between vials lead to substantial variations in the frozen crystal form and sublimation rate. Furthermore, some blood product companies have not conducted substantial comparative studies on their lyophilization processes, making it impossible to determine whether these are the optimal conditions. Therefore, the transition from optimal laboratory conditions to large-scale practical application in production facilities still faces numerous challenges. Summary of the Invention

[0005] This invention mainly designs a freeze-drying process that meets the requirements of large-scale industrialization, high efficiency, energy saving and emission reduction, and product quality that meets pharmacopoeia standards by screening freeze-drying formulations and optimizing freeze-drying process parameters.

[0006] The technical solution adopted by this invention to solve its technical problem is: a freeze-drying method for rapidly soluble fibrinogen, comprising the following steps:

[0007] 1. A method for freeze-drying rapidly soluble fibrinogen, characterized by comprising the following steps:

[0008] (1) Plasma pre-fusion and consolidation: Control the water bath temperature to less than 37°C, pre-fuse the plasma first, and then wash and consolidate it;

[0009] (2) Centrifugation: Take the combined plasma from step (1), centrifuge it, and collect the supernatant.

[0010] (3) Extraction of FI: The supernatant from step (2) is separated by low-temperature ethanol method to obtain the precipitate, which is the FI precipitate;

[0011] (4) Dissolution and filtration of FI precipitate: Add the FI precipitate from step (3) to dissolution solution A, control the temperature of the dissolution solution to 20-30℃, dissolve for 0.5-2 hours, filter and collect filtrate A;

[0012] (5) Polyethylene glycol purification and separation: Polyethylene glycol (PEG) was added to the filtrate A in step (4) to a final concentration of 5%. After stirring for 0.5 to 1 h at 10 to 20 °C, the mixture was centrifuged and the PEG precipitate was collected. Solution B was added to the PEG precipitate and dissolved for 1 to 2 h at 20 to 30 °C. The mixture was then filtered and the filtrate B was collected.

[0013] (6) S / D inactivation: Add S / D reagent to filtrate B in step (5) until the final content of polysorbate 80 in filtrate B is 1% and the final content of tributyl phosphate is 0.3%. Inactivate at 24-26℃ for no less than 6 hours to obtain inactivation solution;

[0014] (7) Ethanol purification and separation: Slowly add 50% ethanol solution below -15℃ to the inactivation solution in step (6) until the ethanol content is 8%. After adding ethanol, cool the inactivation solution to -2.5~0℃, stir for 40~60 min and centrifuge to collect the ethanol precipitate. Dissolve the ethanol precipitate in solution B at 20~30℃ for 1~2 hours and filter to collect the filtrate C.

[0015] (8) Glycine purification and separation: Add glycine to the filtrate C from step (7) to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 2-10°C, stir for at least 60 min, centrifuge, and collect the precipitate. Dissolve the glycine precipitate in the collected precipitate with solution C. After dissolving at room temperature for 1-2 h, filter to obtain filtrate D.

[0016] (9) Ultrafiltration dialysis: Dialyze the filtrate D from step (8) with a constant weight of 3 to 4 times using the dissolving solution C, and then concentrate it by ultrafiltration to a protein concentration of 30 g / L or higher, which is the original solution;

[0017] (10) Preparation: Based on the protein content of the stock solution in step (9) and the target protein amount per unit dose, calculate the volume of the final preparation required. Dilute the stock solution to 25-28 g / L with dialysis buffer, stir evenly, and confirm that the pH value is 6.8-7.2.

[0018] (11) Sterilization and dispensing: The prepared stock solution is sterilized and filtered with a sterile filter, and then aseptically filled to obtain a semi-finished product;

[0019] (12) Freeze-drying and capping: The semi-finished product is transferred to a freeze dryer under aseptic conditions for freeze-drying. The freeze-drying process steps are as follows:

[0020] a Pre-freezing

[0021] After being packaged, the semi-finished products are placed in a freeze dryer. The plate temperature is kept at around -3°C for at least 120 minutes, and then cooled to around -45°C to begin pre-freezing and cooling, which is maintained for at least 180 minutes.

[0022] Then raise the temperature to around -15°C and maintain it for at least 120 minutes;

[0023] Continue cooling to around -45°C and maintain this temperature for at least 210 minutes;

[0024] When the condenser temperature drops below -45℃, start the vacuum pump and begin evacuation. Set the vacuum level to 10Pa.

[0025] b. A Sublimation

[0026] The plate is heated to about -20℃ and the temperature is controlled for at least 3000 minutes, while the vacuum degree is controlled at about 10Pa.

[0027] Sublimate until the semi-finished product water line disappears, raise the temperature to about -5℃, keep it at the temperature for at least 120 minutes, and control the vacuum degree at about 10Pa.

[0028] c. Analysis and drying

[0029] Raise the plate temperature to about 32°C and hold for at least 120 minutes, with the vacuum level controlled at about 10 Pa; then control the plate temperature at 33°C, set the vacuum level to 0 Pa, and hold for at least 600 minutes.

[0030] d. Plug

[0031] After the desorption and drying process is completed, the chamber is plugged and then vented to atmospheric pressure.

[0032] The temperature of the semi-finished product during the freeze-drying process must not exceed 35℃. The freeze-dried semi-finished product is then transferred to the capping room under aseptic conditions for capping and sealing.

[0033] (13) Dry heat: After capping, transfer to a water bath sterilizer for dry heat inactivation at 100±1℃ for 30 minutes to obtain the finished product.

[0034] Based on the above technical solution, the present invention can be further improved as follows.

[0035] As a preferred embodiment of the present invention, the formulation of the solution A includes: 29.4g sodium citrate, 9.1g lysine hydrochloride, and 6000IU sodium heparin per kilogram of water for injection, with a pH value of 6.8-7.0.

[0036] As a preferred embodiment of the present invention, the formulation of the solution B includes: 15g sodium citrate, 9g sodium chloride, 3.5g lysine hydrochloride, and 10g sucrose per kilogram of water for injection, with a pH value of 6.8 to 7.2.

[0037] As a preferred embodiment of the present invention, the formulation of the solution C includes: 10g sodium citrate, 2g sodium glutamate, and 20g arginine hydrochloride per kilogram of water for injection, with a pH value of 6.8 to 7.2.

[0038] As a preferred embodiment of the present invention, the S / D reagent formulation includes: 11% polysorbate 80 and 3.3% tributyl phosphate.

[0039] Human fibrinogen has a high viscosity, especially at low temperatures, and a large molecular weight, resulting in high protein concentration and large fill volume in the finished product. Therefore, its freeze-drying cycle is longer than that of other coagulation factor products. However, by designing and optimizing the freeze-drying process of this patent, the following effects can be achieved:

[0040] 1. During pre-freezing, there is a heat preservation process at -3℃. The purpose is to ensure that the temperature of the freeze-dried products remains consistent before entering the freezing stage, and to avoid large differences in the freezing process due to uneven temperature distribution of the freeze dryer's partition layer.

[0041] 2. In the pre-freezing process of freeze drying, the method of warming at -15℃ is adopted to avoid large differences between bottles, enhance crystallization, and increase the glass transition temperature.

[0042] 3. The first sublimation uses a sublimation temperature of -20℃, which slows down the heating rate of the partition and prevents the product from disintegrating or melting inside during the freeze-drying process. After the freeze-dried product is reconstituted, the foreign matter index is good. Attached Figure Description

[0043] Figure 1 The results of reconstitution time and visible foreign matter detection for samples from Examples 1-7 of this invention;

[0044] Figure 2The results of the detection of appearance, reconstitution time, and visible foreign matter of the samples in Examples 8-11 of this invention;

[0045] Figure 3 The results of reconstitution time and visible foreign matter detection are for the samples of Examples 12-18 of the present invention. Detailed Implementation

[0046] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0047] In the embodiments of the present invention, each embodiment adopts a complete technical solution. The formulations of the solution C in embodiments 1-7 are different. Compared with the technical solution, embodiments 8-11 are the same except for the pre-freezing and annealing parameters in the freeze-drying process. Compared with the technical solution, embodiments 12-18 are the same except for the primary sublimation parameters in the freeze-drying process.

[0048] The samples from Examples 1-7 were tested for reconstitution time and visible foreign matter; the samples from Examples 8-11 were tested for appearance, reconstitution time, and visible foreign matter; and the samples from Examples 12-18 were tested for reconstitution time and visible foreign matter.

[0049] The technical solution is as follows:

[0050] The formulation of solution A includes: 29.4g sodium citrate, 9.1g lysine hydrochloride, and 6000IU sodium heparin per kilogram of water for injection, with a pH of 6.9.

[0051] The formulation of solution B includes: 15g sodium citrate, 9g sodium chloride, 3.5g lysine hydrochloride, and 10g sucrose per kilogram of water for injection, with a pH of 7.0.

[0052] The formulation of solution C includes: 10g sodium citrate, 2g sodium glutamate, and 20g arginine hydrochloride per kilogram of water for injection, with a pH of 7.0.

[0053] The S / D reagent formulation includes: 11% polysorbate 80 and 3.3% tributyl phosphate.

[0054] (1) Plasma prefusion and consolidation

[0055] Control the water bath temperature at 37℃, pre-thaw the plasma, and then perform washing and merging.

[0056] (2) Centrifugal separation

[0057] Take the combined plasma from step (1), centrifuge it, and collect the supernatant.

[0058] (3) Extraction of FI

[0059] The supernatant from step (2) is separated by the low-temperature ethanol method to obtain the precipitate, which is FI precipitate;

[0060] (4) Dissolution and filtration of FI precipitate

[0061] Add the FI precipitate from step (3) to the dissolving solution A, control the temperature of the dissolving solution at 25°C, and filter after dissolving for 1.5 hours, and collect the filtrate A;

[0062] (5) Polyethylene glycol purification and separation

[0063] Polyethylene glycol (PEG) was added to the filtrate A in step (4) to a final concentration of 5%. After stirring for 0.75 h at 10–20 °C, the mixture was centrifuged and the PEG precipitate was collected. Solution B was added to the PEG precipitate and dissolved at 25 °C for 1.5 h. The mixture was then filtered and the filtrate B was collected.

[0064] (6) S / D inactivation

[0065] Add S / D reagent to filtrate B in step (5) until the final content of polysorbate 80 in filtrate B is 1% and the final content of tributyl phosphate is 0.3%. Inactivate at 25°C for no less than 6 hours to obtain inactivated solution.

[0066] (7) Ethanol purification and separation

[0067] Slowly add 50% ethanol solution below -15℃ to the inactivation solution in step (6) until the ethanol content is 8%. After adding ethanol, cool the inactivation solution to -1.5℃, stir for 50 min, centrifuge, and collect the ethanol precipitate. Dissolve the ethanol precipitate in solution B at 25℃ for 1.5 hours, filter, and collect the filtrate C.

[0068] (8) Glycine purification and separation

[0069] Add glycine to the filtrate C from step (7) to a final concentration of 1.8 mol / L. After it is fully dissolved, cool the solution to 6°C, stir for at least 60 min, centrifuge, and collect the precipitate. Dissolve the glycine precipitate in the collected precipitate with solution C, dissolve at room temperature for 1.5 h, and then filter to obtain filtrate D.

[0070] (9) Ultrafiltration dialysis

[0071] The filtrate D from step (8) was dialyzed 3.5 times by constant weight using dissolving solution C, and then concentrated by ultrafiltration to a protein concentration of 30 g / L or higher, which is the original solution.

[0072] (10) Preparation

[0073] Based on the protein content of the stock solution in step (9) and the target protein amount per unit dose, calculate the volume of the final formulation required. Dilute the stock solution to 27 g / L with dialysis buffer, stir evenly, and confirm that the pH value is 7.

[0074] (11) Sterilization and dispensing

[0075] The prepared stock solution is sterilized by filtering it with a sterile filter and then aseptically filled to obtain a semi-finished product.

[0076] (12) Freeze-drying and crimping

[0077] The semi-finished product is transferred to a freeze dryer under aseptic conditions for freeze drying. The freeze drying process steps are as follows:

[0078] a Pre-freezing

[0079] After being packaged, the semi-finished products are placed in a freeze dryer. The plate temperature is kept at around -3°C for at least 120 minutes, and then cooled to around -45°C to begin pre-freezing and cooling, which is maintained for at least 180 minutes.

[0080] Then raise the temperature to around -15°C and maintain it for at least 120 minutes;

[0081] Continue cooling to around -45°C and maintain this temperature for at least 210 minutes;

[0082] When the condenser temperature drops below -45℃, start the vacuum pump and begin evacuation. Set the vacuum level to 10Pa.

[0083] b. A Sublimation

[0084] The plate is heated to about -20℃ and the temperature is controlled for at least 3000 minutes, while the vacuum degree is controlled at about 10Pa.

[0085] Sublimate until the semi-finished product water line disappears, raise the temperature to about -5℃, keep it at the temperature for at least 120 minutes, and control the vacuum degree at about 10Pa.

[0086] c. Analysis and drying

[0087] Raise the plate temperature to about 32°C and hold for at least 120 minutes, with the vacuum level controlled at about 10 Pa; then control the plate temperature at 33°C, set the vacuum level to 0 Pa, and hold for at least 600 minutes.

[0088] d. Plug

[0089] After the desorption and drying process is completed, the chamber is plugged and then vented to atmospheric pressure.

[0090] The temperature of the semi-finished product during the freeze-drying process must not exceed 35℃. The freeze-dried semi-finished product is then transferred to the capping room under aseptic conditions for capping and sealing.

[0091] (13) Dry heat

[0092] After capping, the product is transferred to a water bath sterilizer for dry heat inactivation at 100°C for 30 minutes to obtain the finished product.

[0093] The formulations of the lyophilization solution C in Examples 1-7 differ, mainly in the selection of the lyophilization protection formula. The specific lyophilization formulas are as follows:

[0094] The specific parameter designs for Examples 8-11 are as follows:

[0095] The specific parameter designs for Examples 12-18 are as follows:

[0096] The reconstitution time and visible foreign matter were measured in samples from Examples 1-7, and the results are as follows:

[0097] The results showed that the freeze-dried formulation of Example 4 had a significant advantage in reconstitution time, and the reconstituted solution was clear with no visible foreign matter.

[0098] The samples from Examples 8-11 were tested for appearance, reconstitution time, and visible foreign matter. The results are as follows:

[0099] The results showed that setting the annealing condition to -15℃ resulted in a freeze-dried product with a delicate appearance, full shape, shortest reconstitution time, clear consistency after reconstitution, no visible foreign matter, and compliance with regulations.

[0100] The reconstitution time and visible foreign matter were measured in samples from Examples 12-18, and the results are as follows:

[0101] The results showed that the reconstitution time did not exceed 10 minutes at different primary sublimation temperatures. However, to improve the freeze-drying rate and prevent melting during mass production due to excessively high sublimation temperatures, a primary sublimation temperature of -20℃ was ultimately selected. The data indicated that the reconstitution time was better at a vacuum level of 0.1 mbar than at 0.07 mbar, therefore, a vacuum level of 0.1 mbar was chosen. Sublimation under these conditions ensured that the product would not melt, and that its appearance and reconstitution time met the requirements.

Claims

1. A method for freeze-drying of lyophilized fibrinogen, characterized in that, Comprising the following steps: (1) Plasma pre-fusion, merging Control the water bath temperature less than 37℃, first pre-fusion of plasma, after completion, then washing and merging; (2) Centrifugal separation Take the merged plasma of step (1), centrifugal separation, collect the supernatant; (3) FI extraction The supernatant of step (2) is separated by low temperature ethanol method to obtain the precipitate, which is FI precipitate; (4) FI precipitate dissolution and filtration Add the FI precipitate of step (3) to the dissolution liquid A, control the temperature of the dissolution liquid to 20-30℃, dissolve for 0.5-2 hours and then filter, collect the filtrate A; (5) PEG purification and separation Add PEG to the filtrate A of step (4) to a final concentration of 5%, stir for 0.5-1 hour at 10-20℃, then centrifugal separation, collect the PEG precipitate; Add dissolution liquid B to the PEG precipitate, dissolve for 1-2 hours at 20-30℃, then filter, collect the filtrate B; (6) S / D inactivation Add S / D reagent to the filtrate B of step (5) to a final concentration of 1% polysorbate 80 and 0.3% tributyl phosphate, inactivate at 24-26℃ for not less than 6 hours to obtain the inactivated liquid; (7) Ethanol purification and separation Slowly add 50% ethanol solution below -15℃ to the inactivated liquid of step (6) to a concentration of 8%, cool the inactivated liquid to -2.5-0℃ after adding ethanol, stir for 40-60 min, then centrifugal separation, collect the ethanol precipitate; dissolve the ethanol precipitate with dissolution liquid B at 20-30℃ for 1-2 hours, then filter, collect the filtrate C; (8) Glycine purification and separation Add glycine to the filtrate C of step (7) to a final concentration of 1.8 mol / L, cool the solution to 2-10℃ after fully dissolving, stir for at least 60 min, then centrifugal separation, collect the precipitate; dissolve the collected precipitate with dissolution liquid C, filter after dissolving at room temperature for 1-2 hours to obtain the filtrate D; (9) Ultrafiltration dialysis Dialyze the filtrate D of step (8) with dissolution liquid C for 3-4 times of constant weight, concentrate by ultrafiltration to a protein concentration of more than 30 g / L, which is the stock solution; (10) Preparation Based on the protein content of the stock solution of step (9) and the target protein mass per unit dose, calculate the volume of the final preparation required, dilute the stock solution to 25-28 g / L with dialysis buffer, stir uniformly, and confirm that the pH value is 6.8-7.2; (11) Sterilization and dispensing Sterilize the prepared stock solution with a sterilization filter, then perform sterile filling, which is the semi-finished product; (12) Freeze-drying and capping Transfer the semi-finished product to a freeze dryer under sterile conditions for freeze-drying treatment, and the freeze-drying process steps are as follows: a Pre-freezing Put the dispensed semi-finished product into the freeze dryer, and when the plate temperature reaches about -3℃, keep it for at least 120 min, then cool it to about -45℃ to start pre-freezing, maintain for at least 180 min; Then increase the temperature to about -15℃ and maintain for at least 120 min; Continue to cool to about -45℃ and maintain for at least 210 min; When the condenser temperature drops to -45℃ or below, start the vacuum pump and begin vacuuming, with a vacuum setting of 10 Pa; b Primary sublimation Raise the plate layer temperature to about -20℃ and control the temperature for at least 3000 min, with a vacuum degree of about 10 Pa; Sublimate until the semi-finished product water line disappears, raise the temperature to about -5℃, and maintain the temperature for at least 120 min, with a vacuum degree of about 10 Pa; c Analytical drying Raise the plate layer temperature to about 32℃ and maintain the temperature for at least 120 min, with a vacuum degree of about 10 Pa; then control the plate layer temperature at 33℃ and set the vacuum degree to 0 Pa, and maintain the temperature for at least 600 min. d Pressing After analytical drying, press, and then release the box to atmospheric pressure; The temperature of the semi-finished product during the freeze-drying process should not exceed 35℃, and the freeze-dried semi-finished product is transferred to the capping room under sterile conditions for capping and sealing. (13) Dry heat After capping, transfer to a water bath sterilization cabinet for 100±1℃ dry heat inactivation for 30 min, and the finished product is obtained.

2. A method of freeze-drying fibrinogen according to claim 1, characterized in that: The formula of the dissolving solution A includes: 29.4 g of sodium citrate, 9.1 g of lysine hydrochloride, 6000 IU of heparin sodium per kilogram of injection water, and the pH value is 6.8-7.

0.

3. A method of freeze-drying fibrinogen according to claim 1, characterized in that: The formula of the dissolving solution B includes: 15 g of sodium citrate, 9 g of sodium chloride, 3.5 g of lysine hydrochloride, 10 g of sucrose per kilogram of injection water, and the pH value is 6.8-7.

2.

4. A method of freeze-drying fibrinogen according to claim 1, characterized in that: The formula of the dissolving solution C includes: 10 g of sodium citrate, 2 g of glutamic acid sodium, 20 g of arginine hydrochloride per kilogram of injection water, and the pH value is 6.8-7.

2.

5. A method of freeze-drying fibrinogen according to claim 1, characterized in that: The formula of the S / D reagent includes: polysorbate 80 content 11%, tributyl phosphate content 3.3%.

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