CN117426158B

CN117426158B - Freeze-drying production process of biological product DBT

Info

Publication number: CN117426158B
Application number: CN201410233598.9A
Authority: CN
Inventors: 万华印; 黄志霞; 李若冰; 万德夫; 付岩霖; 朱晋辉; 李敏
Original assignee: Guangzhou Zhongwei Biotechnology Co ltd
Current assignee: Guangzhou Zhongwei Biotechnology Co ltd
Priority date: 2014-05-28
Filing date: 2014-05-28
Publication date: 2017-12-29
Anticipated expiration: 2034-05-28

Abstract

The invention relates to the technical fields of biological materials, biological pharmacy, biological products, medical appliances and the like, in particular to a freeze-drying production process of biological products DBT of which main components are derived from mammals and can be absorbed by biological organisms. The freeze-drying production process is a layered vacuum freeze-drying method, wherein the main components are frozen in a layered and sequential manner, so that interaction between the main components is avoided, and the main components are combined and then are freeze-dried into a three-layer spongy structural protein patch in a vacuum manner, and the spongy structural protein patch can be used for stopping bleeding, sealing defective tissues, preventing tissue adhesion and promoting wound healing after being sheared and packaged. In the preparation of the freeze-dried preparation, the components such as albumin and emulsifying agent, such as polysorbate 80, are added, so that the appearance of the freeze-dried finished product can be obviously improved, the qualified finished product yield can be increased, and the thermal stability of biological product DBT can be improved.

Description

Freeze-drying production process of biological product DBT

Technical Field

The invention relates to the technical fields of biopharmaceuticals, biological products, biological materials, medical appliances and the like, in particular to a freeze-drying production process of biological products DBT of which main components are derived from animals and can be absorbed by biological organisms.

Background

The rapid and thorough hemostasis in the operation reduces the bleeding amount, keeps the operation field clear, and is one of the cores of the basic operation of the surgical operation. In various first aid such as frequent traffic and accident, the problems of rapid and effective hemostasis, pain relief of patients, life saving and the like are also commonly faced. Currently, common topical hemostatics are fibrin glue, gelatin sponge, oxidized cellulose, microfibril collagen, thrombin, alginic acid fiber and the like. The local hemostatic material is widely used for surgical hemostasis, and the hemostatic effect of the local hemostatic material is fully confirmed in clinical and animal experiments. The use of the local hemostatic can not only reduce bleeding amount, simplify operation, shorten operation time, but also promote wound healing.

However, various hemostatic materials currently used have many disadvantages in terms of hemostatic speed, adhesive effect, tissue compatibility, ease of operation, and the like. Common collagen sponges, gelatin sponges and the like have poor viscosity when used for hemostasis, are easy to fall off, and have tissue compatibility which is inferior to that of fibrinogen and other coagulation factors. Oxidized cellulose, alginic acid fiber and the like are not as histocompatible as biological materials composed of protein components. The fibrin glue is very tedious in use and operation, needs to be prepared in advance for a certain time, has poor adhesive strength, and has poor use effect on wound surfaces with large bleeding amount.

In the human coagulation mechanism, thrombin acts on fibrinogen, cleaving 4 peptide bonds at the ends of its 2 a alpha and 2B beta chains, releasing 2 fibrinopeptides a and two fibrinopeptides B, resulting in fibrin monomers. The fibrin monomer is spontaneously connected into an unstable fibrin polymer, and amide transfer occurs in the soluble fibrin polymer under the action of XIIIa and calcium ions to form stable covalent bonds, so that stable fibrin clots which are transversely, longitudinally and cross-connected are generated, and finally, the coagulation process is completed.

In this patent study, the following patents were searched and compared:

suspension of cn02804097.X containing fibrinogen, thrombin and alcohol and method for coating carrier

Method for preparing CN200810167199.1 instant fibrinogen and thrombin combined preparation

CN03126852.8 quick biological hemostatic sealing device and manufacturing process thereof

CN2621681Y quick hemostatic dressing material

Preparation method of CN1234425Y absorbable fibrin hemostatic patch

CN03142013.3 instant fibrinogen preparation resistant to dry heat treatment

CN201110103540.9 fibrin hemostatic plaster and its preparing process

Preparation method of CN200810046747.5 human fibrinogen preparation

In the above patent, similar to the main component of biological product DBT of this study, there are "suspension of fibrinogen, thrombin and alcohol and method for coating carrier thereof" of CN02804097.X, CN03126852.8 rapid biological hemostatic sealing device and manufacturing process thereof, CN201110103540.9 fibrin hemostatic plaster and manufacturing method thereof, preparation method of absorbable fibrin hemostatic plaster of CN1234425Y, etc. Hemostatic materials are disclosed in these patents as containing fibrinogen, thrombin, dressing, and the like. The fibrinogen contained in the cn02804097.x suspension containing fibrinogen, thrombin and alcohol and method of coating the carrier is definitely human fibrinogen, while other patents include human and other mammalian fibrinogen. The preparation method adopted in the suspension containing fibrinogen, thrombin and alcohol and the method for coating the carrier thereof of the CN02804097.X and the preparation method of the absorbable fibrin hemostatic plaster of the CN1234425Y is a method for uniformly mixing the fibrinogen and the thrombin by ethanol and coating the mixture on the carrier. The CN03126852.8 quick biological hemostatic sealing device and its making process, CN201110103540.9 fibrin hemostatic plaster and its making process, and the low temperature organic solvent mixing and coating process, and also the layering freeze drying process.

The biological product DBT of the research is a very effective local hemostatic material, and can be used for the purposes of auxiliary hemostasis, wound closure and the like in surgery and emergency occasions. Has the characteristics of easy use, easy carrying, rapid hemostasis, complete absorption by human body, good compatibility with human body, small side effect and the like.

The major components of the biological product DBT of this study contained fibrinogen, thrombin and collagen from human and other mammalian sources. The main difference between this study and the above invention is the formulation of the lyophilized formulation. Specifically, the biological product DBT biological material of the research contains auxiliary components such as albumin, emulsifying agent and the like in the preparation of the freeze-dried solution of the preparation.

Because fibrinogen and thrombin in biological product DBT can produce catalytic reaction in liquid state, so that the fibrinogen can be converted into fibrin, and the fibrinogen can lose coagulability and viscosity, and can not play the role of stopping bleeding and sealing. It is therefore necessary to avoid premature reaction in the preparation process of the biological DBT. Some similar products are prepared by respectively freeze-drying fibrinogen and thrombin, grinding into powder, mixing the fibrinogen and thrombin in a low-temperature organic solvent, coating the mixture on a collagen sponge, and volatilizing the organic solvent. The method needs to freeze-dry three components such as fibrinogen, thrombin, collagen and the like respectively, so that the freeze-drying workload is increased; when the coating is carried out again, the main components are easy to be unevenly distributed, and the coating operation has high requirements on equipment and environment; in addition, the product obtained by the process is not beautiful.

Albumin is a major plasma protein that can be extracted from human plasma or other mammalian plasma, and is itself an important blood product. Albumin has a higher inactivation temperature relative to fibrinogen and thrombin. The albumin is added, so that a certain auxiliary effect of heat stabilization can be achieved on fibrinogen and thrombin, and the quality guarantee period of the product is prolonged. In addition, the addition of an appropriate amount of albumin to the thrombin solution is helpful to the preparation process, and can bond the fibrinogen layer, the thrombin layer and the collagen layer without delamination. If albumin is not added, the fibrinogen layer is easily peeled off and a complete product cannot be formed.

The preparation process of biological product DBT adopts a layering freeze vacuum drying method, but the fibrinogen layer and the collagen layer have different relative molecular weights, viscosities, thicknesses, water contents and the like, so that how to ensure the appearance and the yield of the product after drying becomes a difficult point. Through multiple experiments, the fibrinogen layer is easy to crack after freeze-drying, fragile during shearing and split charging, and easy to adhere to a freeze-drying mould box and not easy to peel after freeze-drying, and the yield of the freeze-dried preparation is low due to the reasons. Valuable raw materials are wasted, and energy and equipment loss required by freeze-drying are also wasted.

After a proper amount of emulsifying agent is added into the biological product DBT component, the freeze-drying problem can be effectively solved, and the qualified finished product yield is improved by one to two times. Common emulsifying agents recorded in Chinese pharmacopoeia include polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, polyoxyl (40) stearate, sucrose stearate, sodium lauryl sulfate, triethanolamine, soybean lecithin, egg yolk lecithin, hydrogenated castor oil, poloxamer, span 20, span 40, span 60, span 80, span 85 and the like. Polysorbate 80, polysorbate 60, egg yolk lecithin, sucrose stearate and the like also have the function of a solubilizer, allowing a proper amount of the same to be applied to injections.

The formula composition and the proportion of components contained in the biological product DBT, including main components of fibrinogen, thrombin, albumin, XIII coagulation factor, collagen and the like and auxiliary components of saccharides, sugar alcohols, amino acids, salts, vitamins, emulsifying agents and the like, have very important influences on the preparation, appearance, stability, efficacy and the like of the product. The biological product DBT with good stability, attractive and complete appearance and obvious efficacy is obtained by optimizing the formula composition and proportion of the product.

By adopting the freeze-drying production process, only one freeze-drying operation is needed, special equipment and environment are not needed, and the appearance of the product is more attractive. Through experiments, the biological product DBT product prepared by the freeze-drying production process has good stability, attractive appearance and obvious clinical effect.

Disclosure of Invention

The invention aims to provide a freeze-drying process method for biological product DBT, and the biological product DBT produced by the freeze-drying process has attractive and complete appearance and high yield.

The freeze-drying production process of the biological product DBT is a method of carrying out vacuum freeze-drying on 3 solutions by adopting layering and gradual freezing and combining, wherein the solution (1) is a fibrinogen mixture solution, the solution (2) is a thrombin mixture solution and the solution (3) is a collagen solution.

The 1 st solution of the freeze-drying production process of biological product DBT is fibrinogen mixture solution (1):

the 2 nd process for freeze-drying biological product DBT is thrombin mixture solution (2):

the 3 rd kind of freeze-drying production process of the biological product DBT is collagen solution (3), and collagen is contained in 1-40 mg/ml.

The freeze-drying process of the biological product DBT comprises the steps of layering and freezing the solution (1), the solution (2), the solution (3) or the solution (3), the solution (1), the solution (2) or the solution (3), the solution (2), the solution (1) or the solution (2), the solution (1) and the solution (3).

The above-mentioned biological DBT layering freezing sequence, its freeze-drying production steps are preferably:

1) Preparing fibrinogen mixture solution, thrombin mixture solution and collagen solution respectively;

2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.2-8 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

3) The thrombin mixture solution is mixed according to 1 to 50 mu l/cm ² Uniformly spraying the (area) amount on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 0.5-15 mm, and then freezing for 5-60 minutes at-70-5 ℃;

5) Cooling the cold trap of the freeze dryer water catcher to-70 to-40 ℃ and then preserving heat for 30-60 minutes;

6) Starting a vacuum pump, and after the material pressure is reduced to 5-60 Pa, raising the temperature of the shelf to-30 to-15 ℃;

7) After the temperature of the material reaches the set shelf temperature for 0.1-3 hours, the shelf temperature is raised to-20 to-10 ℃;

8) After the temperature of the material reaches the set shelf temperature for 0.1-2 hours, the shelf temperature is raised to-5 ℃;

9) After the temperature of the material reaches the set shelf temperature for 0.1-2 hours, the shelf temperature is raised to 30-35 ℃;

10 After the material temperature reaches the set shelf temperature for 0.1-2 hours, the freeze-drying is finished.

2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.25-5 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

3) The thrombin mixture solution is mixed according to the volume of 2 to 40 mu l/cm ² Uniformly spraying the (area) amount on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 1-12 mm, and then freezing for 5-60 minutes at-70-5 ℃;

7) After the temperature of the material reaches the set shelf temperature for 0.25-3 hours, the shelf temperature is raised to-20 to-10 ℃;

8) After the temperature of the material reaches the set shelf temperature for 0.25-2 hours, the shelf temperature is raised to-5 ℃;

9) After the temperature of the material reaches the set shelf temperature for 0.25-2 hours, the shelf temperature is raised to 30-35 ℃;

10 After the temperature of the material reaches the set shelf temperature for 0.25-2 hours, the freeze-drying is finished.

The layering freezing sequence of the biological product DBT is characterized in that the freeze-drying production steps are preferably as follows:

2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.5-3 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

3) The thrombin mixture solution is mixed according to 3 to 30 mu l/cm ² Uniformly spraying the (area) amount on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 2-10 mm, and then freezing for 5-60 minutes at-70-5 ℃;

7) After the temperature of the material reaches the set shelf temperature for 0.5-2 hours, the shelf temperature is raised to-20 to-10 ℃;

8) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to-5 ℃;

9) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to 30-35 ℃;

10 After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the freeze-drying is finished.

2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.75-2 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

3) The thrombin mixture solution is mixed according to 5 to 20 mu l/cm ² Uniformly spraying the (area) amount on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 3-9 mm, and then freezing for 5-60 minutes at-70-5 ℃;

7) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to-20 to-10 ℃;

8) After the temperature of the material reaches the set shelf temperature for 0.5-1 hour, the shelf temperature is raised to-5 ℃;

9) After the temperature of the material reaches the set shelf temperature for 0.5-1 hour, the shelf temperature is raised to 30-35 ℃;

10 After the temperature of the material reaches the set shelf temperature for 0.5-1 hour, the freeze-drying is finished.

2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to make the thickness of the fibrinogen mixture solution be 1mm, and freezing the fibrinogen mixture solution at the temperature of-45 ℃ for 35 minutes;

3) The thrombin mixture solution was prepared at a concentration of 10. Mu.l/cm ² Uniformly spraying the amount of (area) on the solidified fibrinogen layer solid, and then freezing at-45 ℃ for 25 minutes;

4) Uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen layer and thrombin layer solids to enable the thickness of the collagen solution to be 5mm, and then freezing the collagen solution at-45 ℃ for 40 minutes;

5) Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 30 minutes;

6) Starting a vacuum pump, and after the material pressure is reduced to 30Pa, raising the temperature of the shelf to-26 ℃;

7) After the temperature of the material reaches the set shelf temperature for 1 hour, the shelf temperature is raised to-15 ℃;

8) After the temperature of the material reaches the set shelf temperature for 1 hour, the shelf temperature is raised to 0 ℃;

9) After the temperature of the material reaches the set shelf temperature for 0.5 hour, the shelf temperature is raised to 35 ℃;

10 After the material temperature reaches the set shelf temperature for 0.5 hour, the freeze-drying is finished.

The freeze-drying production process of biological product DBT has the finished product shearing package environment temperature of 4-35 ℃ and relative humidity of 10-90%o after freeze-drying

The freeze-drying production process of the biological product DBT comprises the steps of adopting a medicinal plastic hard box for the inner package of the freeze-dried biological product DBT, filling the medicinal plastic hard box into a composite aluminum foil bag for packaging the medicine, sealing the medicinal plastic hard box, and filling a medicinal drying agent into the bag.

The biological DBT can be made into various shapes such as sheet, tube, cone, cylinder, sphere, etc.

The biological DBT can be used for auxiliary hemostasis in surgery and emergency treatment: includes hemostasis of various wounds and substantial organ sections, hemostasis of surgical bleeding during operation, sealing of defective tissues, preventing tissue adhesion and promoting wound healing.

Drawings

FIG. 1 is a schematic representation of the lyophilization profile of biological DBT

Detailed Description

In order to describe the technical content, the achieved objects and effects of the present invention in detail, the following detailed description is made with reference to the embodiments.

Example 1

Fibrinogen, thrombin, albumin and the like are extracted from pig blood plasma according to a conventional mature blood product preparation method, and collagen is extracted from pig tendons.

Respectively dissolving fibrinogen, thrombin, collagen and the like, and adding auxiliary components to ensure that the contents of the components respectively reach:

(1) fibrinogen mixed solution:

(2) thrombin mixed solution:

(3) collagen solution:

collagen 16mg/ml 8mg/cm ² 8mg/cm ²

Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to make the thickness of the fibrinogen mixture solution be 1mm, and freezing the fibrinogen mixture solution at the temperature of-45 ℃ for 25 minutes; at a rate of 1ml/100cm ² Uniformly spraying the thrombin mixture solution on the solidified fibrinogen solids, and then freezing at-45 ℃ for 15 minutes; uniformly pouring the collagen solution which is cooled to 4 ℃ in advance on the solidified fibrinogen and thrombin solid to make the thickness of the collagen solution be 5mm, and freezing the collagen solution at the temperature of-45 ℃ for 60 minutes. Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 60 minutes; starting a vacuum pump, and after the material pressure is reduced to 15-30 Pa, raising the temperature of the shelf to-25 ℃; after the temperature of the material reaches-25 ℃ and is maintained for 1 hour, the temperature of the shelf is raised to-15 ℃; after the temperature of the material reaches-15 ℃ and is maintained for 1 hour, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ and is maintained for 1 hour, the temperature of the shelf is raised to 35 ℃; after the temperature of the material reaches 35 ℃ and is maintained for 1 hour, the freeze-drying is finished, and the freeze-drying curve is shown in the following figure 1. Taking out biological product DBT, shearing, sealing and packaging according to the specification at 20deg.C and 80% humidity.

The DBT of the biological product after freeze-drying has uniform appearance thickness, no phenomena of cracking, delamination, sticking, and the like, the yield is 98.4%, and the finished product inspection qualification rate is 100%.

After the biological product DBT is stored for 3 months under the condition of 37 ℃ and 90% relative humidity, the color appearance is not obviously different from that of the initial reference substance, and the viscosity is normal; after 6 months of storage, the color appearance slightly changed from the initial control, and the setting time and adhesive strength were not greatly different when used (Table 1).

TABLE 1 Property Change of biological products DBT after 37 ℃ preservation

Example 2

The product produced in example 1 above was packaged at a temperature/humidity of 4 ℃/30%, 15 ℃/70%, 25 ℃/50%, 35 ℃/90%, and stored at a relative humidity of 8% for 12 months at 2 ℃. The test results are shown in the following table.

TABLE 2 stability of biological DBT products under different packaging conditions

Example 3

Fibrinogen, thrombin, albumin and the like are extracted from human plasma according to a conventional mature blood product preparation method, and collagen is extracted from pig tendons.

(1) fibrinogen mixed solution:

(2) thrombin mixed solution:

(3) collagen solution:

collagen 12.4mg/ml 6.2mg/cm ²

Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to enable the thickness of the fibrinogen mixture solution to be 1.5mm, and freezing the fibrinogen mixture solution at the temperature of-40 ℃ for 30 minutes; the thrombin mixture solution was prepared at 15. Mu.l/cm ² Uniformly spraying the amount of (area) on the solidified fibrinogen solid, and then freezing at-40 ℃ for 20 minutes; uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen and thrombin solid to make the thickness of the collagen solution be 5mm, and then freezing the collagen solution at the temperature of minus 40 ℃ for 60 minutes; cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 60 minutes; starting a vacuum pump, and after the material pressure is reduced to 20Pa, raising the temperature of the shelf to-25 ℃; after the temperature of the material reaches-25 ℃ for 3 hours, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ for 2 hours, the temperature of the shelf is raised to 35 ℃; and after the temperature of the material reaches 35 ℃ for 2 hours, the freeze-drying is finished. And taking out the biological product DBT, shearing, sealing and packaging according to the specification.

The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 96.3 percent.

Packaging at 20deg.C/75% temperature/humidity, and storing at 20deg.C and 50% relative humidity for 12 months.

Example 4

A fibrinogen mixture solution, a thrombin mixture solution, and a collagen solution were prepared as in example 1. Pouring the fibrinogen mixture solution into a freeze-drying mould box to enable the thickness of the fibrinogen mixture solution to be 1.5mm, and freezing the fibrinogen mixture solution at the temperature of-50 ℃ for 20 minutes; the thrombin mixture solution was prepared at 8. Mu.l/cm ² Uniformly spraying the amount of (area) on the solidified fibrinogen solid, and then freezing at-50 ℃ for 10 minutes; uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen and thrombin solid to make the thickness of the collagen solution be 4mm, and then freezing the collagen solution at-50 ℃ for 45 minutes.

Cooling the cold trap of the freeze dryer water catcher to-65 ℃ and then preserving heat for 60 minutes; starting a vacuum pump, and after the material pressure is reduced to 15Pa, raising the temperature of the shelf to-28 ℃; after the temperature of the material reaches-28 ℃ for 2.5 hours, the temperature of the shelf is raised to-5 ℃; after the temperature of the material reaches-5 ℃ for 2 hours, the temperature of the shelf is raised to 33 ℃; and after the temperature of the material reaches 33 ℃ for 1.5 hours, the freeze-drying is finished. The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 88.5 percent.

Packaging at 15 deg.C/40% temperature/humidity, and storing at 25deg.C and 60% relative humidity for 12 months.

Example 5

A fibrinogen mixture solution, a thrombin mixture solution, and a collagen solution were prepared as in example 3.

Pouring the fibrinogen mixture solution into a freeze-drying mould box to make the thickness of the fibrinogen mixture solution be 1.0mm, and freezing the fibrinogen mixture solution at the temperature of-50 ℃ for 20 minutes; the thrombin mixture solution was prepared at a concentration of 10. Mu.l/cm ² Uniformly spraying the amount of (area) on the solidified fibrinogen solid, and then freezing at-50 ℃ for 10 minutes; uniformly pouring the collagen solution which is cooled to 4 ℃ in advance on the solidified fibrinogen and thrombin solid to make the thickness of the collagen solution be 4mm, and then freezing the collagen solution at-50 ℃ for 45 minutes.

Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 60 minutes; starting a vacuum pump, and after the material pressure is reduced to 20Pa, raising the temperature of the shelf to-26 ℃; after the temperature of the material reaches-26 ℃ for 2.5 hours, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ for 2 hours, the temperature of the shelf is raised to 34 ℃; and after the temperature of the material reaches 34 ℃ for 1.5 hours, the freeze-drying is finished. The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 92.6 percent. And taking out the biological product DBT, shearing, sealing and packaging according to the specification of the product.

Packaging at 30deg.C/70% temperature/humidity, and storing at 15deg.C and 40% relative humidity for 12 months.

Example 6

The thrombin mixture solution was prepared at a concentration of 10. Mu.l/cm ² Uniformly spraying the (area) amount on the freeze-dried plasticFreezing in a material mould box at-50 ℃ for 10 minutes; uniformly spraying the fibrinogen mixture solution on the frozen thrombin solid in the freeze-drying mould box to make the thickness of the thrombin solid be 1.0mm, and freezing for 20 minutes at the temperature of minus 50 ℃; then the collagen solution which is cooled to 2 ℃ in advance is evenly poured on the solidified fibrinogen and thrombin solid to make the thickness of the collagen solution be 4mm, and then the collagen solution is frozen at the temperature of minus 50 ℃ for 45 minutes.

Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 45 minutes; starting a vacuum pump, and after the material pressure is reduced to 25Pa, raising the temperature of the shelf to-26 ℃; after the temperature of the material reaches-26 ℃ for 1.5 hours, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ for 2 hours, the temperature of the shelf is raised to 34 ℃; and after the temperature of the material reaches 34 ℃ for 1.5 hours, the freeze-drying is finished. The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 95.6 percent. And taking out the biological product DBT, shearing, sealing and packaging according to the specification of the product.

Packaging at 10deg.C/50% temperature/humidity, and storing at 4deg.C and 60% relative humidity for 12 months.

Example 7

Firstly, uniformly pouring the collagen solution into a freeze-dried plastic mould box to enable the thickness of the collagen solution to be 6mm, and freezing the collagen solution at the temperature of minus 50 ℃ for 45 minutes; the thrombin mixture solution, which had been previously cooled to 2℃was prepared at a concentration of 10. Mu.l/cm ² Uniformly spraying the amount of (area) on the frozen collagen solid in the freeze-dried plastic mould box, and freezing for 20 minutes at the temperature of minus 50 ℃; then uniformly spraying the fibrinogen mixture solution which is cooled to 2 ℃ in advance on the frozen collagen and thrombin solid in the freeze-drying mould box to ensure that the thickness of the fibrinogen mixture solution is 1.2mm, and freezing the collagen and thrombin solid at the temperature of-50 ℃ for 25 minutes.

Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 45 minutes; starting a vacuum pump, and after the material pressure is reduced to 25Pa, raising the temperature of the shelf to-26 ℃; after the temperature of the material reaches-26 ℃ for 1.5 hours, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ for 2 hours, the temperature of the shelf is raised to 34 ℃; and after the temperature of the material reaches 34 ℃ for 1.5 hours, the freeze-drying is finished. The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 91.3 percent. And taking out the biological product DBT, shearing, sealing and packaging according to the specification of the product.

Packaging at 20deg.C/65% temperature/humidity, and storing at 20deg.C and 60% relative humidity for 12 months.

Example 8

Firstly, uniformly pouring the collagen solution into a freeze-dried plastic mould box to enable the thickness of the collagen solution to be 6mm, and freezing the collagen solution at the temperature of minus 50 ℃ for 45 minutes; uniformly spraying the fibrinogen mixture solution which is cooled to 2 ℃ in advance on the frozen collagen solid in the freeze-drying mould box to ensure that the thickness of the collagen solid is 1.2mm, and freezing for 25 minutes at the temperature of minus 50 ℃; the thrombin mixture solution, which had been previously cooled to 2℃was prepared at a concentration of 12. Mu.l/cm ² The amount (area) was sprayed evenly on the frozen collagen and fibrinogen solids in the freeze-dried plastic mold box and frozen at-50 ℃ for 20 minutes.

Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 45 minutes; starting a vacuum pump, and after the material pressure is reduced to 25Pa, raising the temperature of the shelf to-26 ℃; after the temperature of the material reaches-26 ℃ for 1.5 hours, the temperature of the shelf is raised to 0 ℃; after the temperature of the material reaches 0 ℃ for 2 hours, the temperature of the shelf is raised to 34 ℃; and after the temperature of the material reaches 34 ℃ for 1.5 hours, the freeze-drying is finished. The DBT of the biological product after freeze-drying has complete and uniform appearance, no large cracks, delamination and other phenomena, and the yield reaches 92.1 percent. And taking out the biological product DBT, shearing, sealing and packaging according to the specification of the product.

Packaging at 15 deg.C/55% temperature/humidity, and storing at 4deg.C and 50% relative humidity for 12 months.

Example 9

After lyophilization was completed, the temperature between shear packs was set to 20 ℃ and relative humidity 25%. The lyophilized biological DBT is removed from the lyophilization nest and sheared to size according to product specifications. Then placing the mixture into a medicinal polypropylene hard box package for fastening; placing into aluminum foil bags for medicine packaging, placing into a pack of medicinal drying agent, heat sealing and sealing the aluminum foil bags, and finally placing the aluminum foil bags into paper box packages with proper sizes. And (5) placing the packaged biological product DBT finished product in a shade and dry place for storage.

The biological DBT has no obvious difference in color appearance and normal viscosity compared with the initial reference after being stored for 6 months at the shade and dry place (less than 20 ℃), has slight change in color appearance compared with the initial reference after being stored for 12 months, and has better performance in solidification time and adhesive strength when being used (Table 3).

TABLE 3 Property Change of biological DBT after storage at room temperature

Example 10

A fibrinogen mixture solution, a thrombin mixture solution, and a collagen solution were prepared as in example 1. Uniformly spraying the fibrinogen mixture solution precooled to 2 ℃ into a conical freeze-drying mold with the diameter of 15mm and the height of 18mm, so that the thickness of the fibrinogen mixture solution is 1.0mm, and freezing for 20 minutes at the temperature of-50 ℃; the thrombin mixture solution was prepared at 8. Mu.l/cm ² Uniformly spraying the amount of (area) on the solidified fibrinogen solid, and then freezing at-50 ℃ for 10 minutes; uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen and thrombin solid, filling the conical freeze-drying mould, and then freezing for 45 minutes at the temperature of minus 50 ℃. Then, vacuum freeze-drying was performed according to the method of example 1.

Packaging at 20deg.C/75% temperature/humidity, and storing at 4deg.C and 50% relative humidity for 12 months.

Example 11

A fibrinogen mixture solution, a thrombin mixture solution, and a collagen solution were prepared as in example 1. Coagulation pre-cooled to 2 DEG CEnzyme mixture solution according to 8. Mu.l/cm ² Uniformly spraying the amount of (area) into a cylindrical freeze-drying mold with the diameter of 15mm and the height of 18mm, and freezing for 20 minutes at the temperature of minus 50 ℃; uniformly spraying the fibrinogen mixture solution precooled to 2 ℃ on the coagulated thrombin solid to make the thickness of the fibrinogen mixture solution be 1.0mm, and then freezing the fibrinogen mixture solution at-50 ℃ for 30 minutes; uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen and thrombin solid, filling the cylindrical freeze-drying mould, and then freezing for 45 minutes at the temperature of minus 50 ℃. Then, vacuum freeze-drying was performed according to the method of example 1.

Packaging at 30deg.C/65% temperature/humidity, and storing at 20deg.C and 65% relative humidity for 12 months.

Example 12

Hemostasis for drug efficacy test

Hemostatic effect of biological product DBT in human liver partial resections.

20 liver sections were selected which failed to achieve "closed lip" suturing after resection of the right portion of the liver in general surgery, and were randomly divided into a biologic DBT group and a blank control group. The liver section of the biological product DBT group is firstly sewed with active bleeding, the biological product DBT with corresponding size is attached on the wound surface, dry gauze is respectively pressed on the biological product DBT for 5 minutes, and the hemostatic condition of the wound surface and the bonding condition of the dressing and the wound surface are observed. The liver section of the control group is sewed with active hemorrhage, and the residual blood and tissue fluid of the liver section are sucked and removed by the dry gauze without other treatment. The two groups are hemostatic by using a large compression liver section, and two abdominal drainage tubes are placed on the liver section (upper and lower right side).

Visual inspection: the blood seepage density and speed of the liver section after DBT using biological product are obviously reduced compared with those before using. Diversion volume: compared with the self-hemostatic group, the drainage amount of the biological product DBT group in the previous 3 days after the operation is respectively reduced by 85%, 70% and 42%. Conclusion biological product DBT has obvious hemostatic effect on the bleeding of the liver injury section, and the blood loss (abdominal cavity drainage) of a patient is obviously reduced after the biological product DBT is used, so that the recovery after the operation is facilitated.

Claims

1. A freeze-drying production process of biological product DBT is characterized by adopting a method of carrying out layered gradual freezing and combining together 3 solutions for vacuum freeze-drying, wherein the solution (1) is fibrinogen mixture solution, the solution (2) is thrombin mixture solution and the solution (3) is collagen solution, at least one solution in the 3 solutions contains albumin, and at least one solution contains emulsifying agent.

2. A process for the lyophilization of biological product DBT according to claim 1, wherein the 1 st solution is fibrinogen mixture solution (1):

3. a process for the lyophilization of biological product DBT according to claim 1, wherein the 2 nd is thrombin mixture solution (2):

4. the process for the lyophilization of a biological product DBT according to claim 1, wherein the 3 rd is a collagen solution (3) containing 1-40 mg/ml collagen.

5. The process for the lyophilization of a biological DBT according to claim 1, wherein the layered freezing sequence is solution (1), solution (2), solution (3), or solution (3), solution (1), solution (2), or solution (3), solution (2), solution (1), or solution (2), solution (1), solution (3).

6. The process for freeze-drying biological DBT according to claim 5, wherein the freeze-drying process comprises the following steps:

(1) Preparing fibrinogen mixture solution, thrombin mixture solution and collagen solution respectively;

(2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.2-8 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

(3) The thrombin mixture solution is mixed according to 1 to 50 mu l/cm ² Uniformly spraying the amount of the mixture on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

(4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 0.5-15 mm, and then freezing for 5-60 minutes at-70-5 ℃;

(5) Cooling the cold trap of the freeze dryer water catcher to-70 to-40 ℃ and then preserving heat for 30-60 minutes;

(6) Starting a vacuum pump, and after the material pressure is reduced to 5-60 Pa, raising the temperature of the shelf to-30 to-15 ℃;

(7) After the temperature of the material reaches the set shelf temperature for 0.1-3 hours, the shelf temperature is raised to-20 to-10 ℃;

(8) After the temperature of the material reaches the set shelf temperature for 0.1-2 hours, the shelf temperature is raised to-5 ℃;

(9) After the temperature of the material reaches the set shelf temperature for 0.1-2 hours, the shelf temperature is raised to 30-35 ℃;

(10) And after the temperature of the material reaches the set shelf temperature for 0.1-2 hours, finishing freeze-drying.

7. The process for freeze-drying biological DBT according to claim 5, wherein the freeze-drying process comprises the following steps:

(2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.25-5 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

(3) The thrombin mixture solution is mixed according to the volume of 2 to 40 mu l/cm ² Uniformly spraying the mixture on the solidified fibrinogen layer solid, and freezing at-70 to-5 ℃ for 5 DEG CThe time is about 40 minutes;

(4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 1-12 mm, and then freezing for 5-60 minutes at-70-5 ℃;

(7) After the temperature of the material reaches the set shelf temperature for 0.25-3 hours, the shelf temperature is raised to-20 to-10 ℃;

(8) After the temperature of the material reaches the set shelf temperature for 0.25-2 hours, the shelf temperature is raised to-5 ℃;

(9) After the temperature of the material reaches the set shelf temperature for 0.25-2 hours, the shelf temperature is raised to 30-35 ℃;

(10) And after the temperature of the material reaches the set shelf temperature for 0.25-2 hours, finishing freeze-drying.

8. The process for freeze-drying biological DBT according to claim 5, wherein the freeze-drying process comprises the following steps:

(2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.5-3 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

(3) The thrombin mixture solution is mixed according to 3 to 30 mu l/cm ² Uniformly spraying the amount of the mixture on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

(4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 2-10 mm, and then freezing for 5-60 minutes at-70-5 ℃;

(7) After the temperature of the material reaches the set shelf temperature for 0.5-2 hours, the shelf temperature is raised to-20 to-10 ℃;

(8) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to-5 ℃;

(9) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to 30-35 ℃;

(10) And after the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, finishing freeze-drying.

9. The process for freeze-drying biological DBT according to claim 5, wherein the freeze-drying process comprises the following steps:

(2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to ensure that the thickness of the fibrinogen mixture solution is 0.75-2 mm, and freezing the fibrinogen mixture solution for 5-60 minutes at the temperature of-70 to-5 ℃;

(3) The thrombin mixture solution is mixed according to 5 to 20 mu l/cm ² Uniformly spraying the amount of the mixture on the solidified fibrinogen layer solid, and then freezing for 5-40 minutes at the temperature of-70 to-5 ℃;

(4) Uniformly pouring collagen solution which is cooled to 2-8 ℃ in advance on the solidified fibrinogen layer and thrombin layer solid, enabling the thickness of the collagen solution to be 3-9 mm, and then freezing for 5-60 minutes at-70-5 ℃;

(7) After the temperature of the material reaches the set shelf temperature for 0.5-1.5 hours, the shelf temperature is raised to-20 to-10 ℃;

(8) After the temperature of the material reaches the set shelf temperature for 0.5-1 hour, the shelf temperature is raised to-5 ℃;

(9) After the temperature of the material reaches the set shelf temperature for 0.5-1 hour, the shelf temperature is raised to 30-35 ℃;

(10) And after the temperature of the material reaches the set shelf temperature for 0.5-1 hour, finishing freeze-drying.

10. The process for freeze-drying biological DBT according to claim 5, wherein the freeze-drying process comprises the following steps:

(2) Pouring the fibrinogen mixture solution into a freeze-dried plastic mould box to make the thickness of the fibrinogen mixture solution be 1mm, and freezing the fibrinogen mixture solution at the temperature of-45 ℃ for 35 minutes;

(3) The thrombin mixture solution was prepared at a concentration of 10. Mu.l/cm ² Uniformly spraying the obtained mixture on the solidified fibrinogen layer solid, and then freezing at-45 ℃ for 25 minutes;

(4) Uniformly pouring the collagen solution which is cooled to 2 ℃ in advance on the solidified fibrinogen layer and thrombin layer solids to enable the thickness of the collagen solution to be 5mm, and then freezing the collagen solution at-45 ℃ for 40 minutes;

(5) Cooling the cold trap of the freeze dryer water catcher to-60 ℃ and then preserving heat for 30 minutes;

(6) Starting a vacuum pump, and after the material pressure is reduced to 30Pa, raising the temperature of the shelf to-26 ℃;

(7) After the temperature of the material reaches the set shelf temperature for 1 hour, the shelf temperature is raised to-15 ℃;

(8) After the temperature of the material reaches the set shelf temperature for 1 hour, the shelf temperature is raised to 0 ℃;

(9) After the temperature of the material reaches the set shelf temperature for 0.5 hour, the shelf temperature is raised to 35 ℃;

(10) And after the temperature of the material reaches the set shelf temperature for 0.5 hour, finishing freeze-drying.

11. The process for freeze-drying biological product DBT according to claim 1, wherein the temperature of the freeze-dried product shearing package environment is 4-35 ℃ and the relative humidity is 10-90%.

12. The process for freeze-drying biological product DBT according to claim 1, wherein the freeze-dried DBT is internally packaged by a medicinal plastic hard box, then is packaged in a composite aluminum foil bag for packaging medicine, is sealed, is filled with a medicinal drying agent, and is stored in an environment with a temperature of 2-37 ℃ and a relative humidity of 8-90%.

13. The process for the lyophilization of a DBT biological product according to claim 1, wherein the DBT is formed into a shape of tablet, tube, cone, cylinder, sphere.

14. Biological product DBT produced according to the lyophilization process as claimed in claim 1, characterized by an auxiliary hemostasis for surgery and emergency: includes hemostasis of various wounds and substantial organ sections, hemostasis of surgical bleeding during operation, sealing of defective tissues, preventing tissue adhesion and promoting wound healing.