Protective agent for detecting freeze drying of enzyme preparation at constant temperature and application of protective agent

Abstract

The invention discloses a protective agent for detecting freeze drying of an enzyme preparation at a constant temperature and application of the protective agent, and belongs to the technical field of freeze drying, the protective agent is formed by mixing saccharides, polymers and amino acid according to the weight ratio of 5: 2: 1; the saccharides comprise the following raw materials in percentage by weight: 75-100% of trehalose and 0-30% of maltose; the polymer is prepared from the following raw materials in percentage by weight: 0 to 100 percent of polyethylene glycol and 0 to 100 percent of polyvinylpyrrolidone; the amino acid is glycine. The invention also discloses an application of the protective agent in detecting freeze drying of the enzyme preparation at a constant temperature, the application comprises the following steps: protein solution pretreatment and freeze drying, and the freeze drying is divided into three stages: pre-freezing, sublimation drying and desorption drying. The method finally solves the technical problem that the constant-temperature detection enzyme preparation depends on temperature in transportation and storage, the storage and transportation cost is reduced, and the storage life of the constant-temperature detection enzyme preparation product is prolonged.

Description

Technical Field

[0001] The invention belongs to the technical field of freeze drying, and in particular relates to a protective agent for freeze drying of a constant temperature detection enzyme preparation and application thereof. Background Art

[0002] Vacuum freeze drying technology is the most commonly used technology for preserving proteins in the research and production of biological products. It can not only maintain the biological characteristics of the product, but also extend the shelf life of biological products. Vacuum freeze drying is a drying technology that uses the principle of sublimation to dehydrate materials under appropriate vacuum conditions. It is a drying technology that freezes wet materials or solutions at a low temperature and then heats them in a vacuum (lower than the triple point pressure of water) environment, so that the water in the material sublimates directly to the gas state without passing through the liquid state, and finally dehydrates the material or solution.

[0003] Protein freeze drying is a multi-step process that will produce low temperature stress, freezing stress, drying stress and pH changes, all of which will affect the activity of the protein. Therefore, an appropriate amount of protective agent should be added before protein freeze drying to protect the protein from low temperature and freeze drying to ensure the activity of the protein after freeze drying. Summary of the invention

[0004] The purpose of the present invention is to provide a protective agent for freeze-drying of a constant temperature detection enzyme preparation and its application. The protective agent is used to protect the protein from freeze-drying. At the same time, the present invention provides a freeze-drying method using the protective agent to ensure the activity of the protein after freeze-drying.

[0005] One object of the present invention is to provide a protective agent for freeze-drying of a constant temperature detection enzyme preparation;

[0006] Another object of the present invention is to provide the use of the above protective agent.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A protective agent for freeze-drying of a constant temperature detection enzyme preparation, wherein the protective agent is a mixture of sugars, polymers and amino acids in a weight ratio of 5:2:1;

[0009] Among them, the sugars are composed of the following raw materials in weight percentage:

[0010] Trehalose 75%-100%;

[0011] Maltose 0%-30%;

[0012] Wherein, the polymer is composed of the following raw materials in weight percentage:

[0013] Polyethylene glycol 0-100%;

[0014] Polyvinylpyrrolidone 0-100%;

[0015] Among them, the amino acid is glycine.

[0016] Furthermore, the carbohydrate is composed of the following raw materials in weight percentage:

[0017] Trehalose 90%-100%;

[0018] Maltose 0%-10%.

[0019] Furthermore, the use of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0020] Protein solution pretreatment: fully mix the isothermal detection enzyme preparation, auxiliary reagents, freeze-drying protective agent, and double distilled water to obtain a mixed solution, place the mixed solution on ice for 20-30 minutes, and then divide it into eight strip tubes, and then freeze-dry. Specifically, freeze-drying is divided into three stages:

[0021] Prefreezing: First, prefreeze the mixed solution at -55-(-45)℃ for 6.5h;

[0022] Sublimation drying: then sublimation drying is carried out for 7.5 hours at a temperature of -35-(-25)°C and a pressure of 0.10-0.60 mbar;

[0023] Desorption and drying: Finally, desorption and drying were carried out for 7.5 hours at a temperature of 23-34°C and a pressure of 0.01-0.10 mbar to complete the freeze drying of the enzyme preparation for constant temperature detection.

[0024] Furthermore, the isothermal detection enzyme preparation is composed of the following components:

[0025] DNA polymerase 60-150 ng / μL;

[0026] Cofactor 10-50 ng / μL;

[0027] Single-chain binding protein 600-1200ng / μL;

[0028] Recombinant enzyme 60-150 ng / μL;

[0029] Exonuclease 15-20ng / μL.

[0030] Furthermore, the auxiliary reagent is composed of the following components:

[0031] dNTPs 0.8-1.5mM;

[0032] ATP 2.0-10.5mM;

[0033] Tris 30-100mM;

[0034] DTT 2-10mM;

[0035] Potassium acetate 30-100 mM;

[0036] Creatine kinase 50-200ng / μL.

[0037] Furthermore, the weight ratio of the isothermal detection enzyme preparation, auxiliary reagent, lyophilization protective agent and double distilled water is 3-7:10-13:10-13:18-22.

[0038] Beneficial effects of the present invention:

[0039] (1) The protective agent in the present invention includes sugars, polymers and amino acids: First, maltose in sugars can prevent the change of protein secondary structure, and has a significant effect on the extension and aggregation of protein polypeptide chains during freeze-drying and storage; trehalose has a higher glass transition temperature, lower hygroscopicity, no reducing property, and can form a weak glass with good protective effect. At the same time, the inventors have found that the protective effect of sugar on protein is related to the concentration of sugar. The protective effect of sugar increases with the increase of concentration. When a certain concentration is reached, the protective effect reaches the maximum value. After that, the protective effect decreases instead of increasing when the concentration of sugar is increased. Therefore, the present invention strictly controls the amount of sugar in the protective agent. Then, the polymer can be preferentially precipitated from the protein surface, so that the viscosity of the protein solution increases, thereby inhibiting the drastic change of pH during freeze-drying. Finally, amino acids can improve the stability of freeze-dried products. After testing, the protective agent provided by the present invention can effectively protect proteins from freeze-drying.

[0040] (2) In view of the fact that the polyethylene glycol solution is relatively viscous and easily affects the uniformity of the mixed solution, the present invention reduces the amount of polyethylene glycol used to achieve uniform mixing of the mixed solution, thereby helping to improve the uniformity of the product.

[0041] (3) The components of the protective agent provided by the present invention are readily available and inexpensive, which helps to save production costs.

[0042] (4) The present invention provides an application of a protective agent in the freeze-drying of a constant temperature detection enzyme preparation. Drying the constant temperature detection enzyme preparation in a frozen state helps to maintain the original structure of the constant temperature detection enzyme preparation. The application of the present invention does not involve a concentration process. After adding a reconstitution solution, the constant temperature detection enzyme preparation can be quickly redissolved. At the same time, the application of the present invention solves the technical problem of the constant temperature detection enzyme preparation's dependence on temperature during transportation and storage, reduces storage and transportation costs, and extends the shelf life of the constant temperature detection enzyme preparation product. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The present invention discloses a protective agent for freeze-drying of a constant temperature detection enzyme preparation, which is prepared by mixing sugars, polymers and amino acids in a weight ratio of 5:2:1;

[0045] Among them, the sugars are composed of the following raw materials in weight percentage:

[0046] Trehalose 75%-100%;

[0047] Maltose 0%-30%;

[0048] Wherein, the polymer is composed of the following raw materials in weight percentage:

[0049] Polyethylene glycol 0-100%;

[0050] Polyvinylpyrrolidone 0-100%;

[0051] Among them, the amino acid is glycine.

[0052] Among them, trehalose is an analytical standard, HPLC ≥ 98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; maltose is an analytical standard, HPLC ≥ 98%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the weight average molecular weight of polyethylene glycol is 20000-40000, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the weight average molecular weight of polyvinyl pyrrolidone is 8000-40000, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; glycine is an analytical standard, HPLC ≥ 99%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Examples 1-5 specifically disclose the weight percentage ratio of the protective agent, as shown in Table 1 below:

[0054] Table 1 Protective agent ratio (unit: %)

[0055] project Example 1 Example 2 Example 3 Example 4 Example 5 Trehalose 35 35 45 50 37.5 maltose 15 15 5 0 12.5 Polyethylene glycol 10 15 0 20 5 Polyvinylpyrrolidone 10 5 20 0 15 Glycine 10 10 10 10 10

[0056] The present invention studies the freeze-drying process of a constant temperature detection enzyme preparation, which is composed of the following components:

[0057]

[0058] Among them, DNA polymerase, auxiliary factors, single-stranded binding proteins, recombinases, and exonucleases are all products of the company, and are all common components of isothermal detection enzyme preparations in the field, and no additional elaboration is required.

[0059] Examples 6-10 specifically disclose the dosage of the isothermal detection enzyme preparation, as shown in Table 2 below:

[0060] Table 2 Dosage of enzyme preparation for constant temperature detection (unit: ng / μL)

[0061] project Example 6 Example 7 Example 8 Example 9 Example 10 DNA polymerase 60 90 120 150 100 Cofactors 10 20 40 50 30 Single-chain binding protein 600 800 1000 1200 900 Recombinase 60 90 120 150 100 Exonuclease 15 16 18 20 18

[0062] The present invention studies the freeze-drying process of the isothermal detection enzyme preparation, which requires the use of auxiliary reagents for the isothermal detection enzyme. The auxiliary reagents are composed of the following components:

[0063]

[0064] Among them, dNTPs (Aladdin), ATP (Aladdin), Tris (Sangon), DTT (Aladdin), potassium acetate (Aladdin), and creatine kinase (Sigma) are all common raw materials for isothermal detection enzyme preparations in the art and no additional elaboration is required.

[0065] Examples 11-15 specifically disclose the dosage of auxiliary reagents, as shown in Table 3 below:

[0066] Table 3 Amount of auxiliary reagents (unit: ng / μL)

[0067] project Embodiment 11 Example 12 Example 13 Embodiment 14 Embodiment 15 dNTPs 0.8 1.0 1.3 1.5 1.2 ATP 2.0 4.5 8.0 10.5 6.5 Tris 30 50 80 100 70 DTT 2 5 7 10 6 Potassium acetate 30 50 80 100 70 Creatine kinase 50 100 150 200 120

[0068] Example 16

[0069] The application of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0070] Protein solution pretreatment: The constant temperature detection enzyme preparation in Example 6, the auxiliary reagent in Example 11, the lyophilization protective agent in Example 1, and double distilled water were fully mixed in a weight ratio of 3:10:10:18 to obtain a mixed solution, the mixed solution was placed on ice for 20 minutes, and then divided into eight strips of tubes, each tube was filled with 50 μL of the mixed solution, the eight strips of tubes were divided into a freeze dryer, the freeze drying program was set, and freeze drying was started according to the set program. Specifically, freeze drying is divided into three stages:

[0071] Prefreezing: First, prefreeze the mixed solution at -45°C for 6.5h;

[0072] Sublimation drying: then perform sublimation drying for 7.5 hours at -25°C and 0.10 mbar to remove free water in the mixed solution (the sublimation drying is concluded when the vacuum values ​​measured by the capacitance vacuum gauge and the Pirani vacuum gauge are consistent);

[0073] Desorption and drying: Finally, desorption and drying were carried out for 7.5 hours at a temperature of 23°C and a pressure of 0.01 mbar to remove bound water (the end of desorption was determined by the temperature probe method) and complete the freeze-drying of the enzyme preparation for constant temperature detection.

[0074] Embodiment 17

[0075] The application of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0076] Protein solution pretreatment: The constant temperature detection enzyme preparation in Example 7, the auxiliary reagent in Example 12, the lyophilization protective agent in Example 2, and double distilled water were fully mixed in a weight ratio of 4:11:11:24 to obtain a mixed solution, the mixed solution was placed on ice for 25 minutes, and then divided into eight strips of tubes, each tube was filled with 50 μL of the mixed solution, the eight strips of tubes were divided into a freeze dryer, the freeze drying program was set, and freeze drying was started according to the set program. Specifically, freeze drying is divided into three stages:

[0077] Prefreezing: First, prefreeze the mixed solution at -50°C for 6.5h;

[0078] Sublimation drying: then perform sublimation drying for 7.5 hours at -30°C and 0.30 mbar to remove free water in the mixed solution (the sublimation drying is concluded when the vacuum values ​​measured by the capacitance vacuum gauge and the Pirani vacuum gauge are consistent);

[0079] Desorption and drying: Finally, desorption and drying were carried out at a temperature of 30°C and a pressure of 0.05 mbar for 7.5 hours to remove bound water (the end of desorption was determined by the temperature probe method) and complete the freeze-drying of the enzyme preparation for constant temperature detection.

[0080] Embodiment 18

[0081] The application of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0082] Protein solution pretreatment: The constant temperature detection enzyme preparation in Example 8, the auxiliary reagent in Example 13, the lyophilization protective agent in Example 3, and double distilled water were fully mixed in a weight ratio of 5:12:12:21 to obtain a mixed solution, the mixed solution was placed on ice for 25 minutes, and then divided into eight strips of tubes, each tube was filled with 50 μL of the mixed solution, the eight strips of tubes were divided into a freeze dryer, the freeze drying program was set, and freeze drying was started according to the set program. Specifically, freeze drying is divided into three stages:

[0083] Prefreezing: First, prefreeze the mixed solution at -50°C for 6.5h;

[0084] Sublimation drying: then perform sublimation drying for 7.5 hours at -30°C and 0.30 mbar to remove free water in the mixed solution (the sublimation drying is concluded when the vacuum values ​​measured by the capacitance vacuum gauge and the Pirani vacuum gauge are consistent);

[0085] Desorption and drying: Finally, desorption and drying were carried out at a temperature of 30°C and a pressure of 0.05 mbar for 7.5 hours to remove bound water (the end of desorption was determined by the temperature probe method) and complete the freeze-drying of the enzyme preparation for constant temperature detection.

[0086] Embodiment 19

[0087] The application of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0088] Protein solution pretreatment: The constant temperature detection enzyme preparation in Example 9, the auxiliary reagent in Example 14, the lyophilization protective agent in Example 4, and double distilled water were fully mixed in a weight ratio of 6:12:12:20 to obtain a mixed solution, the mixed solution was placed on ice for 25 minutes, and then divided into eight strips of tubes, each tube was filled with 50 μL of the mixed solution, the eight strips of tubes were divided into a freeze dryer, the freeze drying program was set, and freeze drying was started according to the set program. Specifically, freeze drying is divided into three stages:

[0089] Prefreezing: First, prefreeze the mixed solution at -50°C for 6.5h;

[0090] Sublimation drying: then perform sublimation drying for 7.5 hours at -30°C and 0.30 mbar to remove free water in the mixed solution (the sublimation drying is concluded when the vacuum values ​​measured by the capacitance vacuum gauge and the Pirani vacuum gauge are consistent);

[0091] Desorption and drying: Finally, desorption and drying were carried out at a temperature of 30°C and a pressure of 0.05 mbar for 7.5 hours to remove bound water (the end of desorption was determined by the temperature probe method) and complete the freeze-drying of the enzyme preparation for constant temperature detection.

[0092] Embodiment 20

[0093] The application of the protective agent in the freeze-drying of the isothermal detection enzyme preparation comprises the following steps:

[0094] Protein solution pretreatment: The constant temperature detection enzyme preparation in Example 10, the auxiliary reagent in Example 15, the lyophilization protective agent in Example 5, and double distilled water were fully mixed in a weight ratio of 7:13:13:17 to obtain a mixed solution, the mixed solution was placed on ice for 30 minutes, and then divided into eight strips of tubes, each tube was filled with 50 μL of the mixed solution, the eight strips of tubes were divided into a freeze dryer, the freeze drying program was set, and freeze drying was started according to the set program. Specifically, freeze drying is divided into three stages:

[0095] Prefreezing: First, prefreeze the mixed solution at -55°C for 6.5h;

[0096] Sublimation drying: then perform sublimation drying for 7.5 hours at a temperature of -35°C and a pressure of 0.60 mbar to remove free water in the mixed solution (the sublimation drying is concluded when the vacuum values ​​measured by the capacitance vacuum gauge and the Pirani vacuum gauge are consistent);

[0097] Desorption and drying: Finally, desorption and drying were carried out for 7.5 hours at a temperature of 34°C and a pressure of 0.10 mbar to remove bound water (the end of desorption was determined by the temperature probe method) and complete the freeze-drying of the enzyme preparation for constant temperature detection.

[0098] Comparative Example 1

[0099] Comparative Example 1 is the control group of Example 18, in which the polymer and amino acid in the lyophilizing protectant in Example 3 in Example 18 are removed, that is, the lyophilizing protectant in Example 3 in Example 18 is replaced by: 70% alginate + 30% maltose (weight percentage), and the other materials and processes remain unchanged as in Example 18, completing the freeze-drying of the isothermal detection enzyme preparation.

[0100] Comparative Example 2

[0101] Comparative Example 2 is the control group of Example 18, in which the sugars and amino acids in the lyophilization protectant in Example 3 in Example 18 are removed, that is, the lyophilization protectant in Example 3 in Example 18 is replaced by polyvinyl pyrrolidone, and the other materials and processes remain unchanged as in Example 18, completing the freeze-drying of the isothermal detection enzyme preparation.

[0102] Comparative Example 3

[0103] Comparative Example 3 is the control group of Example 18, in which the sugars and polymers in the lyophilization protectant in Example 3 in Example 18 are removed, that is, the lyophilization protectant in Example 3 in Example 18 is replaced by glycine, and the other materials and processes remain unchanged as in Example 18, completing the freeze-drying of the isothermal detection enzyme preparation.

[0104] The constant temperature detection enzyme preparations after freeze drying in Example 16 to Example 20 and Comparative Example 1 to Comparative Example 3, the present invention records the appearance, 4°C storage life, rehydration rate and on-machine detection performance of the constant temperature detection enzyme preparations after freeze drying, wherein:

[0105] In Examples 16-20, the product has a crisp and porous white powder with a full appearance. The performance does not decrease significantly after being stored at 4°C for 6 months, and it can be rehydrated within 1 minute. In Comparative Examples 1-3, the product has a collapsed appearance, and the performance decreases significantly after being stored at 4°C for 6 months, and the rehydration time is prolonged.

[0106] In Example 16, the appearance is a loose and porous white powder with slightly shrunk shape, and the performance is slightly reduced after being stored at 4°C for 6 months; in Example 17, the appearance is a loose and porous white powder with a plump shape, and the performance is slightly reduced after being stored at 4°C for 6 months; in Example 18, the appearance is a loose and porous white powder with a plump shape, and the performance remains unchanged after being stored at 4°C for 6 months; in Example 19, the appearance is a loose and porous white powder with a plump shape, and the performance remains unchanged after being stored at 4°C for 6 months; in Example 20, the appearance is a loose and porous white powder with shrunk shape, and the performance is significantly reduced after being stored at 4°C for 6 months.

[0107] After sublimation drying and analytical drying, the constant temperature detection enzyme preparation dry powder can remove more than 95% of the moisture, which plays a vital role in extending the shelf life of the constant temperature detection enzyme preparation. The removal of moisture brings great convenience to the storage and transportation of the enzyme preparation. Storage and transportation do not require overly strict low temperature requirements, and the cost is greatly reduced.

[0108] After freeze drying, the enzyme preparation product was tested at constant temperature and it was white fluffy powder, full in appearance, good in shape and good in rehydration. At the same time, the addition of trehalose, maltose, polyethylene glycol, polyvinyl pyrrolidone and glycine also played a good role in protecting and supporting the freeze drying process of the enzyme preparation, greatly reducing the damage of freeze drying of the enzyme preparation to its effective ingredients (functional protein activity).

[0109] It should be noted that, in this article, terms such as "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or equipment that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment.

[0110] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A protective agent for isothermal detection of freeze-drying of enzyme preparations, characterized in that: The protective agent is prepared by mixing sugars, polymers and amino acids in a weight ratio of 5:2:1; Among them, the sugars are composed of the following raw materials in weight percentage: Trehalose 75%-100%; Maltose 0%-30%; Wherein, the polymer is composed of the following raw materials in weight percentage: Polyethylene glycol 0-100%; Polyvinylpyrrolidone 0-100%; Among them, the amino acid is glycine.

2. A protective agent for freeze-drying of a constant temperature detection enzyme preparation according to claim 1, characterized in that: The sugars are composed of the following raw materials in weight percentage: Trehalose 90%-100%; Maltose 0%-10%.

3. Use of the protective agent according to any one of claims 1-2 in isothermal detection of freeze-drying of enzyme preparations, characterized in that: The following steps are involved: Protein solution pretreatment: fully mix the isothermal detection enzyme preparation, auxiliary reagents, freeze-drying protective agent, and double distilled water to obtain a mixed solution, place the mixed solution on ice for 20-30 minutes, and then divide it into eight strip tubes, and then freeze-dry. Specifically, freeze-drying is divided into three stages: Prefreezing: First, prefreeze the mixed solution at -55-(-45)℃ for 6.5h; Sublimation drying: then sublimation drying is carried out for 7.5 hours at a temperature of -35-(-25)°C and a pressure of 0.10-0.60 mbar; Desorption and drying: Finally, desorption and drying were carried out for 7.5 hours at a temperature of 23-34°C and a pressure of 0.01-0.10 mbar to complete the freeze drying of the enzyme preparation for constant temperature detection.

4. The use according to claim 3, characterized in that: The isothermal detection enzyme preparation is composed of the following components:

5. The use according to claim 3, characterized in that: The auxiliary reagent is composed of the following components:

6. The use according to claim 3, characterized in that: The weight proportions of the constant temperature detection enzyme preparation, auxiliary reagents and freeze-dried protective agent are: 25%-45%, 10%-15%, 20%-28% respectively, and the rest is supplemented to 100% with double distilled water.

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