A lyophilization protectant, a lyophilized reagent and a method for preparing the same

Abstract

The application provides a freeze-drying protective agent, a freeze-drying reagent and a preparation method thereof, and relates to the technical field of biology. The freeze-drying protective agent is composed of trehalose, mannitol or sorbitol, bovine serum albumin and PEG20000, and the composition is simple. The freeze-drying process parameters are optimized in the preparation method of the freeze-drying reagent by using the freeze-drying protective agent, the properties of the freeze-drying reagent are greatly improved, the stability of the freeze-drying reagent is ensured, the freeze-drying reagent obtained has the advantages of beautiful appearance, high integrity rate, stable enzyme activity, simple operation and the like, the preparation method of the freeze-drying reagent provided by the application is simple and feasible, the conditions are mild, the reproducibility is good, the controllability is strong, the method is suitable for large-scale production, and the freeze-drying reagent is more convenient for clinical use and storage.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a lyophilization protectant, a lyophilization reagent, and a method for preparing the same. Background Technology

[0002] Vacuum freeze-drying is a technique that freezes a solution at low temperatures and then directly sublimates and dries the water under vacuum without liquid extraction. The resulting substance retains essentially the same physical and chemical properties, exhibits minimal loss of active ingredients, has good rehydration properties, and can be stored for a long period. Vacuum freeze-drying provides a stable preservation method for various liquid nucleic acid detection kits that have poor stability and require cold chain transportation and storage.

[0003] In the freeze-drying process of bioproducts, three main effects can affect the stability of the active components and even lead to their inactivation: ① Freezing effect: During the freezing process of bioproducts, continuous crystallization leads to a rapid increase in solution concentration and ion concentration, which promotes chemical reactions. In some bioproduct solutions, the pH value also changes during freezing, causing physical aggregation and chemical denaturation of proteins. ② Dehydration effect: After proteins have undergone sufficient hydration in solution, a hydrated layer adheres to the surface of protein molecules. During freeze-drying, some of the bound water is removed. The removal of bound water may disrupt the native structure of the protein, ultimately leading to protein denaturation. ③ Low temperature effect: Active components in bioproducts can denature within a certain temperature range during the cooling and heating processes.

[0004] The freeze-drying process of biological products is a complex, multi-step process. In order to prevent the denaturation of key active components of pharmaceuticals and biological products during freeze-drying, appropriate freeze-drying protectants need to be added and formulated into a mixture before effective freeze-drying can be carried out. Summary of the Invention

[0005] The purpose of this application is to provide a freeze-drying protectant, a freeze-drying reagent and a method for preparing the same, in order to solve the problem of denaturation of key active components in biological products during freeze-drying.

[0006] To achieve the above objectives, this application provides a freeze-drying protectant comprising the following components by mass percentage: 5%–15% trehalose, 1%–10% mannitol or sorbitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0007] Preferably, the product comprises the following components by mass percentage: 5%–15% trehalose, 1%–2% mannitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0008] Preferably, the product comprises the following components by mass percentage: 5%–15% trehalose, 1%–10% sorbitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0009] This application also provides a method for preparing a lyophilized reagent, comprising:

[0010] The above-mentioned lyophilization protectant is mixed with the reagent to be lyophilized to obtain a mixture;

[0011] The mixture was freeze-dried to obtain the lyophilized reagent.

[0012] Preferably, the freeze-drying process includes, in sequence: pre-freezing, sublimation drying, and desorption drying; the pre-freezing temperature is -30℃ to -50℃, and the time is 10h to 20h; the sublimation drying temperature is -25℃ to 5℃, the time is 1h to 4h, and the vacuum degree is 5 to 30Pa; the desorption drying temperature is 5℃ to 30℃, the time is 5h to 15h, and the vacuum degree is ultimate vacuum.

[0013] Preferably, the pre-freezing includes:

[0014] First stage of pre-freezing: temperature -40℃, time 2 hours;

[0015] The second stage of pre-freezing: the temperature is -45℃ and the time is 12 hours.

[0016] Preferably, the sublimation drying includes:

[0017] First stage sublimation drying: temperature -20℃, time 1h, vacuum degree 10Pa;

[0018] The second stage of sublimation drying: temperature is 0℃, time is 1h, and vacuum degree is 10Pa.

[0019] Preferably, the analytical drying includes:

[0020] First stage of analytical drying: temperature 10℃, time 1h, vacuum degree is ultimate vacuum;

[0021] The second stage of drying was carried out at a temperature of 25°C for 8 hours under an ultimate vacuum.

[0022] Preferably, the volume ratio of the lyophilization protectant to the reagent to be lyophilized is 1:(10-30).

[0023] This application also provides a lyophilized reagent, which is prepared by the above-described method for preparing lyophilized reagents.

[0024] Compared with the prior art, the beneficial effects of this application include:

[0025] The lyophilization protectant provided in this application consists only of trehalose, mannitol or sorbitol, bovine serum albumin, and PEG20000, with a simple composition. Using this lyophilization protectant, the lyophilization process parameters in the preparation method of lyophilized formulations were optimized, greatly improving the properties of the lyophilized formulations and ensuring their stability. The resulting lyophilized formulations have advantages such as attractive appearance, high integrity rate, stable enzyme activity, and simple operation. Furthermore, the preparation method of the lyophilized formulations provided in this application is simple and feasible, with mild conditions, good reproducibility, and strong controllability, making it suitable for large-scale production and more convenient for clinical use and storage. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0027] Figure 1 The image shows the appearance of the lyophilized reagent prepared in Example 1.

[0028] Figure 2 The image shows the appearance of the lyophilized reagent prepared in Example 2.

[0029] Figure 3 The image shows the appearance of the lyophilized reagent prepared in Example 3;

[0030] Figure 4 The image shows the appearance of the lyophilized reagent prepared in Example 4;

[0031] Figure 5 The image shows the appearance of the lyophilized reagent prepared in Example 5;

[0032] Figure 6 The image shows the detection results of the lyophilized reagent prepared in Example 2;

[0033] Figure 7 The image shows the detection results of the lyophilized reagent prepared in Example 3;

[0034] Figure 8 The image shows the detection results of the lyophilized reagent prepared in Example 4;

[0035] Figure 9 The image shows the detection results of the lyophilized reagent prepared in Example 5;

[0036] Figure 10This is a graph showing the detection results of the freshly prepared liquid PCR reaction reagent. Detailed Implementation

[0037] As used in this article:

[0038] "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0039] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0040] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0041] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0042] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (K is any number representing a multiplier). It is important to understand that, unlike the number of parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0043] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0044] This application provides a freeze-drying protectant comprising the following components by weight percentage: 5%–15% trehalose, 1%–10% mannitol or sorbitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0045] The freeze-drying protectant consists only of trehalose, mannitol or sorbitol, bovine serum albumin, and PEG20000, making its composition very simple.

[0046] Trehalose functions as a cryoprotectant during freezing and as a dehydrating protectant during drying. As a natural sugar with a relatively high glass transition temperature, trehalose effectively prevents the plasticizing effect of water on the glassy state. During protein freeze-drying, proteins denature when they lose water; trehalose fills the gaps created by this water loss, thus effectively preventing protein denaturation during freeze-drying.

[0047] Mannitol or sorbitol have similar functions to trehalose. Mannitol is stable in sterile solutions and is not easily oxidized. In the freeze-drying process of biological products, mannitol is generally used as a support structure and filler, and it does not react with the active ingredient. Sorbitol is an isomer of mannitol, with higher solubility and hygroscopic properties, but it is unstable at high temperatures. In freeze-drying formulations, sorbitol is commonly used as a filler.

[0048] Bovine serum albumin (BSA) can raise the glass transition temperature of biological product mixtures, and is therefore often added to the formulations of some biological products. BSA generally acts as both a cryoprotectant and a dehydration protectant. Under normal circumstances, adding BSA during the freeze-drying process of biological products can increase the viscosity of the solution; inhibit the crystallization of small molecule excipients (such as sucrose); inhibit the decrease in solution pH; and create steric hindrance between protein molecules.

[0049] In the freeze-drying process of bioproducts, PEG20000 acts as a plasticizer, which can reduce the freezing and dehydration denaturation caused by the ice-water interfacial tension during freezing and dehydration, and can also act as a wetting agent and wrinkling agent for the active components during rehydration.

[0050] In one embodiment, the freeze-drying protectant comprises the following components by weight percentage: 5%–15% trehalose, 1%–2% mannitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0051] The dosage of trehalose can be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%; the dosage of mannitol can be, for example, 1%, 1.25%, 1.5%, 1.75%, or 2%; the dosage of bovine serum albumin can be, for example, 0.001%, 0.002%, 0.003%, 0.004%, or 0.005%; and the dosage of PEG20000 can be, for example, 0.05%, 0.75%, 1.5%, 3%, 5%, 6%, 7.5%, or 8%.

[0052] In another embodiment, the product comprises, by weight percentage: 5%–15% trehalose, 1%–10% sorbitol, 0.001%–0.005% bovine serum albumin, and 0.05%–8% PEG20000.

[0053] The dosage of trehalose can be, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%; the dosage of sorbitol can be, for example, 1%, 1.25%, 2.25%, 4.25%, or 8.25%; the dosage of bovine serum albumin can be, for example, 0.001%, 0.002%, 0.003%, 0.004%, or 0.005%; and the dosage of PEG20000 can be, for example, 0.05%, 0.75%, 1.5%, 3%, 5%, 6%, 7.5%, or 8%.

[0054] This application also provides a method for preparing a lyophilized reagent, comprising:

[0055] The above-mentioned lyophilization protectant is mixed with the reagent to be lyophilized to obtain a mixture;

[0056] The mixture was freeze-dried to obtain the lyophilized reagent.

[0057] In a preferred embodiment, the freeze-drying process includes, in sequence: pre-freezing, sublimation drying, and desorption drying; the pre-freezing temperature is -30℃ to -50℃, and the time is 10h to 20h; the sublimation drying temperature is -25℃ to 5℃, the time is 1h to 4h, and the vacuum degree is 5 to 30Pa; the desorption drying temperature is 5℃ to 30℃, the time is 5h to 15h, and the vacuum degree is ultimate vacuum, which means a vacuum degree of 0.

[0058] In one specific embodiment, the pre-freezing includes:

[0059] First stage of pre-freezing: temperature -40℃, time 2 hours;

[0060] The second stage of pre-freezing: the temperature is -45℃ and the time is 12 hours.

[0061] In one specific embodiment, the sublimation drying includes:

[0062] First stage sublimation drying: temperature -20℃, time 1h, vacuum degree 10Pa;

[0063] The second stage of sublimation drying: temperature is 0℃, time is 1h, and vacuum degree is 10Pa.

[0064] In one specific embodiment, the analytical drying includes:

[0065] First stage of analytical drying: temperature 10℃, time 1h, vacuum degree is ultimate vacuum;

[0066] The second stage of drying was carried out at a temperature of 25°C for 8 hours under an ultimate vacuum.

[0067] Preferably, the volume ratio of the freeze-drying protectant to the reagent to be freeze-dried is 1:(10-30), for example, it can be 1:10, or 1:15, or 1:18, or 1:20, or 1:25, or 1:30.

[0068] This application also provides a lyophilized reagent, which is prepared by the above-described method for preparing lyophilized reagents.

[0069] The lyophilization protectant of this application optimizes the lyophilization process parameters in the preparation method of lyophilized formulations, greatly improving the properties of the lyophilized formulations and ensuring their stability. The resulting lyophilized formulations have advantages such as attractive appearance, high integrity rate, stable enzyme activity, and simple operation. Furthermore, the preparation method of the lyophilized formulations provided in this application is simple and feasible, with mild conditions, good reproducibility, and strong controllability, making it suitable for large-scale production and more convenient for clinical use and storage.

[0070] The lyophilized reagents of this application exhibit greater stability and are less prone to deliquescence, improving the properties and stability of the lyophilized formulations while also resulting in aesthetically pleasing morphologies. Various liquid nucleic acid detection kits prepared into lyophilized formulations using the lyophilization process provided by this invention can completely eliminate the need for cold chain transportation, enabling long-distance transport and long-term storage at room temperature. This particularly solves the technical problem of ensuring a reliable low-temperature cold chain in remote areas.

[0071] Various liquid nucleic acid detection kits are prepared into lyophilized formulations using the lyophilization process of this invention. The preparation method is simple, reproducible, and easily scalable for large-scale production, while also being more convenient for clinical use and storage. After mixing the PCR amplification system for nucleic acid detection, the kit is lyophilized. For subsequent nucleic acid detection, the lyophilized formulation is simply reconstituted, and the extracted nucleic acid is added for PCR amplification and detection. Compared to liquid reagents, this method eliminates the cumbersome mixing and aliquoting process, reduces operational errors, simplifies operation, and fundamentally saves time and increases the detection capacity.

[0072] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0073] Example 1

[0074] The freeze-drying protectant mixture in Example 1 consisted of: 10% trehalose, 1.25% mannitol, 0.002% bovine serum albumin, 0.075% PEG20000, and sterile purified water.

[0075] The PCR reaction reagent in Example 1 is used for nucleic acid detection of reproductive tract pathogens and includes: PCR reaction buffer, dNTPs, enzyme system, sterile purified water, and primer mix; the primer mix includes primers for detecting Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum. The specific steps for preparing the PCR reaction reagent into a lyophilized reagent using the freeze-drying protectant in Example 1 are as follows:

[0076] (1) Prepare a mixture of freeze-drying protectants.

[0077] (2) Prepare the PCR reaction reagent mixture for nucleic acid detection.

[0078] (3) Mix the freeze-drying protectant mixture and the prepared nucleic acid amplification PCR reaction reagent mixture at a volume ratio of 1:15.

[0079] (4) Dispense the mixed liquid reagent into PCR reaction tubes.

[0080] (5) Place the PCR reaction tubes into a vacuum freeze dryer for freeze drying.

[0081] (6) Freeze-drying program parameter settings:

[0082] Pre-freezing settings:

[0083] The first stage of pre-freezing is carried out at -40℃ for 2 hours.

[0084] The second stage of pre-freezing is at -45℃ for 12 hours.

[0085] Sublimation drying settings:

[0086] The first stage of sublimation drying is carried out at -20°C for 1 hour under a vacuum of 10Pa.

[0087] The second stage of sublimation drying is carried out at 0°C for 1 hour under a vacuum of 10 Pa.

[0088] The drying process is set as follows:

[0089] The first stage of desorption and drying is carried out at 10°C for 1 hour, with the vacuum level being the ultimate vacuum.

[0090] The second stage of desorption and drying is carried out at 25°C for 8 hours, with the vacuum level set to the ultimate vacuum.

[0091] (7) After the freeze-drying is completed, argon gas is introduced, the chamber is sealed, and after the temperature and humidity of the environment meet the requirements, the freeze dryer door is opened and the nucleic acid detection PCR freeze-dried reagent reaction tube is taken out.

[0092] (8) Observe whether the appearance of the lyophilized preparation is qualified, such as Figure 1 As shown.

[0093] (9) Place the nucleic acid detection PCR lyophilized reagent reaction tube and desiccant together into an aluminum foil bag and vacuum seal it.

[0094] Example 2

[0095] The difference from Example 1 is that the lyophilization protectant in Example 2 is 1.5% PEG20000, while the other components are the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent are the same as in Example 1, and will not be repeated here. The appearance of the lyophilized formulation obtained in Example 2 is as follows. Figure 2 As shown.

[0096] Example 3

[0097] The difference from Example 1 is that the lyophilization protectant in Example 3 is 3% PEG20000, while the other components are the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent are the same as in Example 1, and will not be repeated here. The appearance of the lyophilized formulation obtained in Example 3 is as follows. Figure 3 As shown.

[0098] Example 4

[0099] The difference from Example 1 is that the lyophilization protectant in Example 4 is 6% PEG20000, while the other components are the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent are the same as in Example 1, and will not be repeated here. The appearance of the lyophilized formulation obtained in Example 4 is as follows. Figure 4 As shown.

[0100] Example 5

[0101] The difference from Example 1 is that the lyophilization protectant in Example 5 is 7.5% PEG20000, while the other components are the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent are the same as in Example 1, and will not be repeated here. The appearance of the lyophilized formulation obtained in Example 5 is as follows. Figure 5 As shown.

[0102] Example 6

[0103] The difference from Example 1 is that the freeze-drying protectant in Example 6 consists of: 10% trehalose, 1.25% 4-O-alpha-pyranoyl-D-sorbitol, 0.002% bovine serum albumin, 1.5% PEG20000, and sterile purified water.

[0104] The specific steps for using the lyophilization protectant in Example 6 to prepare PCR reaction reagents into lyophilized reagents are the same as in Example 1, and will not be repeated here.

[0105] Example 7

[0106] The difference from Example 6 is that the freeze-drying protectant in Example 7 is 2.25% 4-O-alpha-pyranoyl-D-sorbitol, and the other components are the same as in Example 6. The steps for preparing the PCR reaction reagent into a freeze-dried reagent are the same as in Example 1, and will not be repeated here.

[0107] Example 8

[0108] The difference from Example 6 is that the freeze-drying protectant in Example 8 is 4.25% 4-O-alpha-glucanyl-D-sorbitol, and the other components are the same as in Example 6. The steps for preparing the PCR reaction reagent into a freeze-dried reagent are the same as in Example 1, and will not be repeated here.

[0109] Example 9

[0110] The difference from Example 6 is that the freeze-drying protectant in Example 9 is 8.25% 4-O-alpha-glucopyranosyl-D-sorbitol, and the other components are the same as in Example 6. The steps for preparing the PCR reaction reagent into a freeze-dried reagent are the same as in Example 1, and will not be repeated here.

[0111] Comparative Example 1

[0112] The difference from Example 1 is that the lyophilization protectant in Comparative Example 1 was 0.075% PEG2000, while the other components were the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent were the same as in Example 1, and will not be repeated here. The use of PEG2000 as the lyophilization protectant in Comparative Example 1 resulted in a poorly shaped lyophilized reagent.

[0113] Comparative Example 2

[0114] The difference from Example 1 is that the lyophilization protectant in Comparative Example 2 is 0.075% PEG8000, while the other components are the same as in Example 1. The steps for preparing the PCR reaction reagent into a lyophilized reagent are the same as in Example 1, and will not be repeated here. The use of PEG8000 as the lyophilization protectant in Comparative Example 2 resulted in a poorly shaped lyophilized reagent.

[0115] Comparative Examples 1 and 2 illustrate that the freeze-drying protectant of this application requires PEG20000 as a plasticizer, and other polyethylene glycols cannot achieve the same effect.

[0116] Comparative Example 3

[0117] Unlike Example 1, the freeze-drying conditions were changed, and there was no second stage of pre-freezing temperature. The specific freeze-drying conditions were as follows:

[0118] Pre-freezing settings:

[0119] The first stage of pre-freezing is carried out at -40℃ for 2 hours.

[0120] Sublimation drying settings:

[0121] The first stage of sublimation drying is carried out at -20°C for 1 hour under a vacuum of 10Pa.

[0122] The second stage of sublimation drying is carried out at 0°C for 1 hour under a vacuum of 10 Pa.

[0123] The drying process is set as follows:

[0124] The first stage of desorption and drying is carried out at 10°C for 1 hour, with the vacuum level being the ultimate vacuum.

[0125] The second stage of desorption and drying is carried out at 25°C for 8 hours, with the vacuum level set to the ultimate vacuum.

[0126] The resulting lyophilized reagent was loosened.

[0127] Comparative Example 4

[0128] Unlike Example 1, the freeze-drying conditions were changed; the desorption drying temperature in the second stage of the desorption drying process was shortened to 2 hours, and argon gas was not used for purging. The specific freeze-drying conditions were:

[0129] Pre-freezing settings:

[0130] The first stage of pre-freezing is carried out at -40℃ for 2 hours.

[0131] The second stage of pre-freezing is at -45℃ for 12 hours.

[0132] Sublimation drying settings:

[0133] The first stage of sublimation drying is carried out at -20°C for 1 hour under a vacuum of 10Pa.

[0134] The second stage of sublimation drying is carried out at 0°C for 1 hour under a vacuum of 10 Pa.

[0135] The drying process is set as follows:

[0136] The first stage of desorption and drying is carried out at 10°C for 1 hour, with the vacuum level being the ultimate vacuum.

[0137] The second stage of desorption and drying is carried out at 25°C for 2 hours, with the vacuum level set to the ultimate vacuum.

[0138] After the freeze-drying process is complete and the ambient temperature and humidity meet the requirements, open the freeze dryer door and remove the nucleic acid detection PCR freeze-dried reagent reaction tubes. The resulting freeze-dried reagents will then loosen and solidify.

[0139] The lyophilized reagents obtained in Comparative Examples 3 and 4 were loosely formed, unlike the tightly formed lyophilized reagent in Example 1. This indicates that the freeze-drying procedure of this application, combined with the freeze-drying protectant of this application, can yield the best lyophilized reagent.

[0140] Test case

[0141] The PCR reaction lyophilized reagents obtained in Examples 1-5 and the comparative examples were subjected to stability testing after being accelerated at 50°C for 6 months. Stability testing was conducted by pathogen detection. Meanwhile, the control group consisted of freshly prepared liquid PCR reaction reagents.

[0142] The results of Example 2 are as follows Figure 6 As shown; the results of Example 3 are as follows Figure 7 As shown; the results of Example 4 are as follows Figure 8 As shown; the results of Example 5 are as follows. Figure 9 As shown; Figure 10 The results are from the freshly prepared liquid PCR reaction reagent. This demonstrates that the lyophilized reagent of this application, after being stored at 50°C for 6 months, yields results comparable to those from the freshly prepared liquid PCR reaction reagent.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0144] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are intended to be within the scope of this application and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for preparing a lyophilized reagent, characterized in that, include: The lyophilization protectant mixture is mixed with the reagent to be lyophilized to obtain the mixture; The mixture was freeze-dried to obtain the lyophilized reagent. The freeze-drying process includes, in sequence: pre-freezing, sublimation drying, and desorption drying; The freeze-drying protectant mixture consists of the following components by mass percentage: 5%~15% trehalose, 1%~10% mannitol, 0.001%~0.005% bovine serum albumin, 0.05%~8% PEG20000, and sterile purified water; The pre-freezing includes: First stage of pre-freezing: temperature -40℃, time 2 hours; Second stage pre-freezing: temperature -45℃, time 12h; The sublimation drying includes: First stage sublimation drying: temperature -20℃, time 1 h, vacuum degree 10 Pa; Second stage sublimation drying: temperature 0℃, time 1h, vacuum degree 10Pa; The analytical drying includes: First stage of analytical drying: temperature 10℃, time 1 h, vacuum degree is ultimate vacuum; The second stage of desorption and drying: the temperature is 25℃, the time is 8 hours, and the vacuum degree is ultimate vacuum.

2. The method for preparing the lyophilized reagent according to claim 1, characterized in that, The freeze-drying protectant mixture consists of the following components by mass percentage: 5%~15% trehalose, 1%~2% mannitol, 0.001%~0.005% bovine serum albumin, 0.05%~8% PEG20000, and sterile purified water.

3. The method for preparing the lyophilized reagent according to claim 1, characterized in that, The volume ratio of the freeze-drying protectant mixture to the reagent to be freeze-dried is 1:10~30.

4. A lyophilized reagent, characterized in that, It is prepared by the method for preparing the lyophilized reagent according to any one of claims 1 to 3.

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