CN116549652A

CN116549652A - Freeze-drying protective agent, freeze-drying reagent and preparation method thereof

Info

Publication number: CN116549652A
Application number: CN202310602220.0A
Authority: CN
Inventors: 陈华云; 文素珍; 张达源; 黎秋华; 邱子莹
Original assignee: Guangzhou Heas Biotech Co ltd
Current assignee: Guangzhou Heas Biotech Co ltd
Priority date: 2023-05-25
Filing date: 2023-05-25
Publication date: 2023-08-08

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 only composed of trehalose, mannitol or sorbitol, bovine serum albumin and PEG20000, and the composition of the components is simple. The freeze-drying protecting agent is used for optimizing freeze-drying program parameters in a freeze-drying preparation method, greatly improving the characters of the freeze-drying preparation, ensuring the stability of the freeze-drying preparation, and simultaneously obtaining the freeze-drying preparation which has the advantages of attractive appearance, high perfection rate, stable enzyme activity, simplicity in operation and the like.

Description

Freeze-drying protective agent, freeze-drying reagent and preparation method thereof

Technical Field

The application relates to the field of biotechnology, in particular to a freeze-drying protective agent, a freeze-drying reagent and a preparation method thereof.

Background

The vacuum freeze drying technology is a technology that the solution is frozen at low temperature, then the water in the solution is directly sublimated and dried under vacuum without liquid extraction, the physical and chemical properties of the dried substance are basically unchanged, the properties are basically unchanged, the loss of active ingredients is small, the rehydration is good, and the sealed storage period is long. The vacuum freeze drying technology provides a stable preservation method for various liquid nucleic acid detection kits with poor stability and need for cold chain transportation and storage.

During the freeze-drying of biological products, there are mainly three effects that affect the stability of the active components therein and even lead to deactivation: (1) freezing effect: in the freezing process of biological products, the concentration of the solution is rapidly increased due to continuous crystallization, the ion concentration is increased, and the chemical reaction is promoted. During the freezing of some biological solutions, the pH of the solution also changes, resulting in physical aggregation and chemical denaturation of the protein. (2) Dehydration effect: after the protein in the solution is fully hydrated, a monohydrate layer is attached to the surface of the protein molecule, and part of bound water is removed in the freeze drying process, so that the removal of the bound water is likely to damage the natural structure of the protein, and finally the denaturation of the protein is caused. (3) Low temperature effect: the active components in the biological product can be denatured in a certain temperature range in the cooling and heating processes.

The freeze-drying process of biological products is a multi-step complex process, so that in order to prevent the denaturation of key active components in the freeze-drying process of medicines and biological products, proper freeze-drying protective agents are required to be added in the freeze-drying process of most medicines and biological products, and effective freeze-drying can be performed after mixed liquor is prepared.

Disclosure of Invention

The application aims to provide a freeze-drying protective agent, a freeze-drying reagent and a preparation method thereof, and aims to solve the problem of denaturation of key active components in a freeze-drying process of biological products.

In order to achieve the purpose, the application provides a freeze-drying protective agent which comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 10 percent of mannitol or sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

Preferably, the composition comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 2 percent of mannitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

Preferably, the composition comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 10 percent of sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

The application also provides a preparation method of the freeze-drying reagent, which comprises the following steps:

mixing the freeze-drying protective agent with a reagent to be freeze-dried to obtain a mixed solution;

and freeze-drying the mixed solution to obtain the freeze-dried reagent.

Preferably, the freeze-drying process comprises, in order: prefreezing, sublimation drying, and analytical drying; the pre-freezing temperature is minus 30 ℃ to minus 50 ℃ and the pre-freezing 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 Pa to 30Pa; the resolving and drying temperature is 5-30 ℃, the time is 5-15 h, and the vacuum degree is extreme vacuum.

Preferably, the prefreezing comprises:

pre-freezing in the first stage: the temperature is-40 ℃ and the time is 2 hours;

and (3) pre-freezing in the second stage: the temperature was-45℃and the time was 12 hours.

Preferably, the sublimation drying comprises:

sublimation drying in the first stage: the temperature is-20 ℃, the time is 1h, and the vacuum degree is 10Pa;

and (3) sublimation drying in the second stage: the temperature was 0℃for 1h and the vacuum was 10Pa.

Preferably, the analytical drying comprises:

first stage resolution drying: the temperature is 10 ℃, the time is 1h, and the vacuum degree is extreme vacuum;

and (3) analyzing and drying in the second stage: the temperature is 25 ℃, the time is 8 hours, and the vacuum degree is extreme vacuum.

Preferably, the volume ratio of the lyoprotectant to the reagent to be lyophilized is 1 (10-30).

The application also provides a freeze-drying reagent, which is prepared by the preparation method of the freeze-drying reagent.

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

the freeze-drying protective agent provided by the application only comprises trehalose, mannitol or sorbitol, bovine serum albumin and PEG20000, and the composition of the components is simple. The freeze-drying protecting agent is used for optimizing freeze-drying program parameters in a freeze-drying preparation method, greatly improving the characters of the freeze-drying preparation, ensuring the stability of the freeze-drying preparation, and simultaneously obtaining the freeze-drying preparation which has the advantages of attractive appearance, high perfection rate, stable enzyme activity, simplicity in operation and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate certain embodiments of the present application and therefore should not be considered as limiting the scope of the present application.

FIG. 1 is an external view of a lyophilized reagent prepared in example 1;

FIG. 2 is an external view of the lyophilized reagent prepared in example 2;

FIG. 3 is an external view of the lyophilized reagent prepared in example 3;

FIG. 4 is an external view of the lyophilized reagent prepared in example 4;

FIG. 5 is an external view of the lyophilized reagent prepared in example 5;

FIG. 6 is a graph showing the detection results of the lyophilized reagent prepared in example 2;

FIG. 7 is a graph showing the results of the freeze-dried reagent prepared in example 3;

FIG. 8 is a graph showing the detection results of the lyophilized reagent prepared in example 4;

FIG. 9 is a graph showing the detection results of the lyophilized reagent prepared in example 5;

FIG. 10 is a graph showing the detection results of the liquid PCR reagents prepared in advance.

Detailed Description

The term as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified element, step or component. If used in a claim, such phrase will cause the claim to be closed, such that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the claim body, rather than immediately following the subject, it is limited to only the elements described in that clause; other elements are not excluded from the stated claims as a whole.

When an equivalent, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "1 to 5" are disclosed, the described ranges should be construed to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"parts by mass" means a basic unit of measurement showing the mass ratio of a plurality of components, and 1 part may be any unit mass, for example, 1g may be expressed, 2.689g may be expressed, and the like. If we say that the mass part of the a component is a part and the mass part of the B component is B part, the ratio a of the mass of the a component to the mass of the B component is represented as: b. alternatively, the mass of the A component is aK, and the mass of the B component is bK (K is an arbitrary number and represents a multiple factor). It is not misunderstood that the sum of the parts by mass of all the components is not limited to 100 parts, unlike the parts by mass.

"and/or" is used to indicate that one or both of the illustrated cases may occur, e.g., a and/or B include (a and B) and (a or B).

The application provides a freeze-drying protective agent, which comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 10 percent of mannitol or sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

The freeze-drying protective agent is only composed of trehalose, mannitol or sorbitol, bovine serum albumin and PEG20000, and the composition of the components is very simple.

The trehalose can play a role of a low-temperature protective agent in the freezing process and a role of a dehydration protective agent in the drying and dehydration process. Trehalose is a natural saccharide with a relatively high glass transition temperature, and can effectively prevent plasticization of the glassy state by water. In the protein freeze-drying process, denaturation can occur when the protein is dehydrated, and trehalose can fill the gap generated by the dehydration, so that the denaturation of the protein in the freeze-drying process is effectively prevented.

Wherein mannitol or sorbitol has the same effect as trehalose. Mannitol is stable in neutral state in sterile solution and is not easy to oxidize. Mannitol generally provides a support structure, acts as a bulking agent, and does not react with the active ingredient during lyophilization of the bioproduct. Sorbitol is an isomer of mannitol that has greater solubility than mannitol, is hygroscopic but is not stable at high temperatures, and is commonly used as a bulking agent in freeze-dried formulations.

Among them, bovine serum albumin can raise the glass transition temperature of mixed solutions of biological products, so that bovine serum albumin is often added in the formulation of some biological products. Bovine serum albumin generally acts as both a cryoprotectant and a dehydration protectant. In general, the addition of bovine serum albumin during the freeze-drying process in biological products can increase the viscosity of the solution; inhibiting crystallization of small molecule excipients (e.g., sucrose); inhibiting the pH value of the solution from decreasing; steric hindrance between protein molecules, etc.

The PEG20000 is used as a shaping agent in the freeze-drying process of biological products, can reduce freezing and dehydration denaturation caused by ice water interfacial tension in the freezing and dehydration process, and can play the roles of a wetting agent and a heavy wrinkling agent for active components in the rehydration process.

In one embodiment, the lyoprotectant comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 2 percent of mannitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

The trehalose may be used in an amount of, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%; mannitol may be used in an amount of, for example, 1%, 1.25%, 1.5%, 1.75% or 2%; bovine serum albumin may be used, for example, in an amount of 0.001%, 0.002%, 0.003%, 0.004% or 0.005%; the amount of PEG20000 may be, for example, 0.05%, 0.75%, 1.5%, 3%, 5%, 6%, 7.5% or 8%.

In another embodiment, the composition comprises the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 10 percent of sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

The trehalose may be used in an amount of, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%; sorbitol may be used in an amount of, for example, 1%, 1.25%, 2.25%, 4.25% or 8.25%; bovine serum albumin may be used, for example, in an amount of 0.001%, 0.002%, 0.003%, 0.004% or 0.005%; the amount of PEG20000 may be, for example, 0.05%, 0.75%, 1.5%, 3%, 5%, 6%, 7.5% or 8%.

and freeze-drying the mixed solution to obtain the freeze-dried reagent.

In a preferred embodiment, the freeze-drying process comprises, in order: prefreezing, sublimation drying, and analytical drying; the pre-freezing temperature is minus 30 ℃ to minus 50 ℃ and the pre-freezing 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 Pa to 30Pa; the resolving and drying temperature is 5-30 ℃, the time is 5-15 h, the vacuum degree is extreme vacuum, and the extreme vacuum refers to the vacuum degree of 0.

In a specific embodiment, the pre-freezing comprises:

In a specific embodiment, the sublimation drying comprises:

In a specific embodiment, the analytical drying comprises:

Preferably, the volume ratio of the lyoprotectant to the reagent to be lyophilized is 1: (10-30), for example, may be 1:10, or 1:15, or 1:18, or 1:20, or 1:25, or 1:30.

The freeze-drying protecting agent optimizes freeze-drying program parameters in the preparation method of the freeze-drying preparation, greatly improves the properties of the freeze-drying preparation, ensures the stability of the freeze-drying preparation, and has the advantages of attractive appearance, high integrity, stable enzyme activity, simplicity in operation and the like.

The freeze-dried reagent has more stable performance, is not easy to deliquesce, improves the property of the freeze-dried preparation, ensures the stability of the freeze-dried preparation, and simultaneously ensures that the obtained freeze-dried preparation has attractive shape. After various liquid nucleic acid detection kits are prepared into freeze-dried preparations through the freeze-drying process provided by the invention, the transportation of a cold chain can be completely eliminated, the long-distance transportation at normal temperature and the long-term storage at normal temperature are realized, and the technical problem that the low-temperature cold chain in remote areas is difficult to guarantee is solved.

The preparation method is simple, has good reproducibility, is easy to realize large-scale production, and is more convenient for clinical use and preservation. And uniformly mixing a PCR amplification system for nucleic acid detection, and freeze-drying, wherein in the later-stage nucleic acid detection, the PCR amplification detection can be performed by adding the extracted nucleic acid after re-dissolving the freeze-dried preparation. Compared with liquid reagent, the method has the advantages of no complicated mixing and split charging processes, less operation error, simpler operation, and capability of fundamentally saving time and increasing detection quantity.

Embodiments of the present application will be described in detail below with reference to specific examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

The lyoprotectant mixture of example 1 comprises the following components: 10% trehalose, 1.25% mannitol, 0.002% bovine serum albumin, 0.075% PEG20000 and sterile purified water.

The PCR reagents of example 1 are used to perform genital pathogen nucleic acid detection, comprising: PCR reaction Buffer, dNTPs, enzyme system, sterilized purified water and primer mix; primer mix includes primers for detecting chlamydia trachomatis, neisseria gonorrhoeae and ureaplasma urealyticum. The specific procedure for preparing the PCR reaction reagents into the lyoprotectant of example 1 is as follows:

(1) Preparing a freeze-drying protective agent mixed solution.

(2) Preparing a mixed solution of nucleic acid detection PCR reaction reagents.

(3) And uniformly mixing the freeze-drying protective agent mixed solution and the prepared nucleic acid amplification PCR reaction reagent mixed solution according to the volume ratio of 1:15.

(4) And subpackaging the uniformly mixed liquid reagent into PCR reaction tubes.

(5) The PCR reaction tube was placed in a vacuum freeze dryer for freeze drying.

(6) Freeze drying procedure parameter settings:

prefreezing is set as:

the pre-freezing temperature in the first stage reaches-40 ℃ and is kept for 2 hours;

the second stage pre-freezing temperature is-45 ℃, and the temperature is kept for 12 hours.

The sublimation drying is set as follows:

the sublimation drying temperature is kept at-20 ℃ for 1h, and the vacuum degree is 10Pa;

and in the second stage, the sublimation drying temperature is kept at 0 ℃ for 1h, and the vacuum degree is 10Pa.

Analytical drying was set as:

the first stage analyzes the drying temperature to 10 ℃, keeps for 1h, and the vacuum degree is extreme vacuum;

and in the second stage, the desorption drying temperature is 25 ℃, the temperature is kept for 8 hours, and the vacuum degree is extreme vacuum.

(7) After freeze-drying is finished, argon is filled into the freeze-drying machine, the box is plugged into the box, and after the temperature and the humidity of the environment meet the requirements, the box door of the freeze-drying machine is opened, and the nucleic acid detection PCR freeze-drying preparation reaction tube is taken out.

(8) The lyophilized formulation was observed for acceptable appearance as shown in fig. 1.

(9) And (3) placing the nucleic acid detection PCR freeze-dried preparation reaction tube and a drying agent into an aluminum foil bag together for vacuumizing and sealing.

Example 2

The difference from example 1 is that: the lyoprotectant of example 2 is 1.5% PEG20000, the other components are the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant is the same as in example 1, and is not described here again. The appearance of the lyophilized preparation obtained in example 2 is shown in FIG. 2.

Example 3

The difference from example 1 is that: the lyoprotectant of example 3 is 3% PEG20000, the other components are the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant is the same as in example 1, and is not described here again. The appearance of the lyophilized preparation obtained in example 3 is shown in FIG. 3.

Example 4

The difference from example 1 is that: the lyoprotectant of example 4 is 6% PEG20000, the other components are the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant is the same as in example 1, and is not described here again. The appearance of the lyophilized preparation obtained in example 4 is shown in FIG. 4.

Example 5

The difference from example 1 is that: the lyoprotectant of example 5 is 7.5% PEG20000, the other components are the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant is the same as in example 1, and is not described here again. The appearance of the lyophilized preparation obtained in example 5 is shown in FIG. 5.

Example 6

The difference from example 1 is that: the lyoprotectant ingredients of example 6 were: 10% trehalose, 1.25% 4-O-alpha-glucopyranosyl-D-sorbitol, 0.002% bovine serum albumin, 1.5% PEG20000 and sterile purified water.

The specific steps of the lyoprotectant of example 6 for preparing the PCR reaction reagent into the lyoprotectant are the same as those of example 1, and will not be repeated here.

Example 7

The difference from example 6 is that: the lyoprotectant of example 7 was 2.25% 4-O-alpha-glucopyranosyl-D-sorbitol, and the other components were the same as in example 6, and the procedure for preparing the PCR reaction reagent as the lyoprotectant was the same as in example 1, and will not be repeated here.

Example 8

The difference from example 6 is that: the lyoprotectant of example 8 was 4.25% 4-O-alpha-glucopyranosyl-D-sorbitol, and the other components were the same as in example 6, and the procedure for preparing the PCR reaction reagent as the lyoprotectant was the same as in example 1, and will not be repeated here.

Example 9

The difference from example 6 is that: the lyoprotectant of example 9 was 8.25% 4-O-alpha-glucopyranosyl-D-sorbitol, and the other components were the same as in example 6, and the procedure for preparing the PCR reaction reagent as the lyoprotectant was the same as in example 1, and will not be repeated here.

Comparative example 1

The difference from example 1 is that: the lyoprotectant of comparative example 1 was 0.075% PEG2000, and the other components were the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant was the same as in example 1, and will not be repeated here. The freeze-drying protective agent of comparative example 1 uses PEG2000, and the obtained freeze-drying agent has poor form and is not molded.

Comparative example 2

The difference from example 1 is that: the lyoprotectant of comparative example 2 was 0.075% PEG8000, and the other components were the same as in example 1, and the procedure for preparing the PCR reaction reagent into the lyoprotectant was the same as in example 1, and will not be described here. The freeze-drying protective agent of comparative example 2 uses PEG8000, and the obtained freeze-drying agent has bad form and is not molded.

Comparative example 1 and comparative example 2 demonstrate that the lyoprotectant of the present application requires PEG20000 as a shaping agent and other polyethylene glycols do not achieve the same effect.

Comparative example 3

Unlike example 1, the lyophilization conditions were changed, the pre-lyophilization temperature was without the second stage, and the specific lyophilization conditions were:

prefreezing is set as:

the sublimation drying is set as follows:

Analytical drying was set as:

The resulting lyophilized reagent was formed loosely.

Comparative example 4

Unlike example 1, the lyophilization conditions were changed and the analytical drying temperature in the second stage of the analytical drying stage was reduced to 2 hours without argon aeration. The specific lyophilization conditions were:

prefreezing is set as:

The sublimation drying is set as follows:

Analytical drying was set as:

and in the second stage, the desorption drying temperature is 25 ℃, the temperature is kept for 2 hours, and the vacuum degree is extreme vacuum.

After the freeze-drying is finished, opening a freeze dryer door after the temperature and the humidity of the environment meet the requirements, and taking out a nucleic acid detection PCR freeze-drying preparation reaction tube. The resulting lyophilized reagent was formed loosely.

The freeze-dried reagent obtained in comparative example 3 and comparative example 4 was more relaxed in shape, and the freeze-dried reagent of example 1 was not compact in shape, indicating that the freeze-drying procedure of the present application combined with the lyoprotectant of the present application, the best-performing freeze-dried reagent was obtained.

Test examples

The lyophilized reagents for PCR reactions obtained in examples 1 to 5 and comparative example were subjected to stability test after accelerating at a high temperature of 50℃for 6 months, and stability test was performed by performing pathogen detection, while the control group was an freshly prepared liquid PCR reaction reagent.

The results of example 2 are shown in FIG. 6; the results of example 3 are shown in FIG. 7; the results of example 4 are shown in FIG. 8; the results of example 5 are shown in FIG. 9; FIG. 10 shows the detection results of the now-prepared liquid PCR reagents. The freeze-dried reagent disclosed by the application is equivalent to the detection result of the existing liquid PCR reaction reagent after being stored at the high temperature of 50 ℃ for 6 months.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features but not others included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the present application and form different embodiments. For example, in the claims below, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims

1. The freeze-drying protective agent is characterized by comprising the following components in percentage by mass: 5 to 15 percent of trehalose, 1 to 10 percent of mannitol or sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

2. The lyoprotectant of claim 1, comprising the following components in mass percent: 5 to 15 percent of trehalose, 1 to 2 percent of mannitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

3. The lyoprotectant of claim 1, comprising the following components in mass percent: 5 to 15 percent of trehalose, 1 to 10 percent of sorbitol, 0.001 to 0.005 percent of bovine serum albumin and 0.05 to 8 percent of PEG20000.

4. A method of preparing a lyophilized reagent comprising:

mixing the lyoprotectant of any one of claims 1 to 3 with a reagent to be lyophilized to obtain a mixed solution;

and freeze-drying the mixed solution to obtain the freeze-dried reagent.

5. The method for preparing a lyophilized reagent according to claim 4, wherein the freeze-drying process sequentially comprises: prefreezing, sublimation drying, and analytical drying; the pre-freezing temperature is minus 30 ℃ to minus 50 ℃ and the pre-freezing 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 Pa to 30Pa; the resolving and drying temperature is 5-30 ℃, the time is 5-15 h, and the vacuum degree is extreme vacuum.

6. The method of preparing a lyophilized reagent according to claim 5, wherein the pre-freezing comprises:

7. The method of preparing a lyophilized reagent according to claim 5, wherein the sublimation drying comprises:

8. The method of preparing a lyophilized reagent according to claim 5, wherein the analytical drying comprises:

9. The method for preparing a freeze-dried reagent according to claim 4, wherein the volume ratio of the freeze-drying protecting agent to the reagent to be freeze-dried is 1 (10-30).

10. A lyophilized reagent prepared by the method of any one of claims 4 to 9.