Freeze-drying solution, freeze-drying method adopting freeze-drying solution and application

Abstract

The invention relates to a freeze-drying solution, a freeze-drying method adopting the freeze-drying solution and application of the freeze-drying solution. The formula of the freeze-drying solution comprises the following components in percentage by mass: 4-6% (w / v) of bovine serum albumin, 5-10% (w / v) of trehalose and 5-10% (w / v) of mannitol. The method for freeze-drying the material by adopting the freeze-drying solution comprises the following steps: S1, pre-freezing: firstly, putting the material to be freeze-dried into a freeze-drying box to freeze the material; s2, sublimation: raising the temperature to-10 DEG C to 0 DEG C, starting vacuum to sublimate the material under vacuum, and enabling water vapor to form ice crystals in a condenser; and S3, desorption drying: raising the temperature to 20-30 DEG C, and carrying out desorption drying on the material under vacuum to evaporate water, so as to obtain a freeze-dried product. By matching with a specific freeze-drying procedure, the bioactive substances are stored in the form of freeze-dried powder, so that the biological activity of the bioactive substances can be maintained for a longer time in a normal-temperature even high-temperature environment; and the freeze-drying time can be greatly shortened.

Description

Technical Field

[0001] The present invention relates to the field of freeze-drying technology, in particular to a freeze-dried solution and a freeze-drying method using the freeze-dried solution. Background Art

[0002] Freeze drying is to cool down the material containing a large amount of water and freeze it into a solid state in advance, and then sublime the solid water directly under vacuum conditions, while the material itself remains in the ice shelf when frozen, so its volume remains unchanged after drying. Solid water absorbs heat when sublimating, causing the temperature of the product itself to drop and slowing down the sublimation speed. In order to increase the sublimation speed and shorten the drying time, the product must be properly heated. The entire drying is carried out at a relatively low temperature.

[0003] The vacuum freeze drying technology is based on the principle of pressure reduction and boiling point reduction. As long as the pressure is below the triple point pressure, the water in the material can be sublimated directly from water to water vapor without passing through the liquid phase. According to this principle, the wet raw material can be frozen below the freezing point to turn the water in the raw material into solid ice, and then the ice can be directly converted into steam and removed under an appropriate vacuum environment, and then the water vapor can be condensed by a water vapor condenser in the vacuum system to dry the material. This method of using vacuum freezing to achieve drying is a process of physical state change and movement of water. This process occurs at low temperature and low pressure. Therefore, the basic principle of freeze drying is the mechanism of heat and mass transfer at low temperature and low pressure. Freeze drying is different from ordinary heating drying. The water in the material is basically sublimated on the frozen solid surface below 0°C to dry, and the material itself remains in the ice shelf when frozen. Therefore, the volume of the dried product remains unchanged and it is loose and porous.

[0004] Vacuum freeze drying technology is a drying technology that freezes wet materials or solutions into a solid state at a relatively low temperature (-10℃~-50℃), and then sublimates the water in it directly into a gaseous state without passing through a liquid state under a vacuum (1.3~13pa), and finally dehydrates the material. The freeze drying process of the product includes three stages: freezing, sublimation and re-drying. The existing freeze drying technology is generally time-consuming. The main reason is that the eutectic temperature of its formula solution during the sublimation process is too low, so it can only be sublimated and dried at a relatively low temperature. The lower temperature will cause the sublimation rate to be slower, thereby increasing the time required for sublimation drying.

[0005] Chinese patent document CN113930356A discloses a freeze-drying method for fermenting and producing CRM197 protein strains, which is a non-toxic form of diphtheria toxin, comprising the following steps: S1, spreading a bacterial solution containing a lyophilization protectant on a freeze-drying stainless steel plate, and placing the freeze-drying stainless steel plate on a vacuum freeze-drying partition; S2, sublimation drying; S2.1, first-order sublimation, the freeze-drying stainless steel plate is still placed on the vacuum freeze-drying machine partition; S2.2, second-order sublimation, the freeze-drying stainless steel plate is still placed on the vacuum freeze-drying machine partition; S3, analytical drying, the freeze-drying stainless steel plate is still placed on the vacuum freeze-drying machine partition to obtain CRM197 strain freeze-dried powder; the present application has the effect of increasing the freeze-dried bacterial concentration of the CRM197 strain freeze-dried powder.

[0006] Therefore, it is necessary to propose a freeze-dried solution and a freeze-drying method and application using the freeze-dried solution, by coordinating a specific freeze-drying procedure, the biologically active substance can be preserved in the form of freeze-dried powder, so that its biological activity can be maintained for a longer time in a room temperature or even high temperature environment. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a freeze-dried solution, which can preserve the biologically active substance in the form of freeze-dried powder by cooperating with a specific freeze-drying procedure, so that its biological activity can be maintained for a longer time in a room temperature or even high temperature environment; the use of the freeze-dried solution can greatly shorten the freeze-drying time.

[0008] In order to solve the above technical solution, the technical solution of the present invention is: the formula of the freeze-dried solution includes: bovine serum albumin 4-6% (w / v), trehalose 5-10% (w / v) and mannitol 5-10% (w / v) by mass percentage.

[0009] It should be understood that w / v means weight / volume percentage.

[0010] Preferably, the formula of the freeze-dried solution comprises, by mass percentage: bovine serum albumin % (w / v), trehalose % (w / v) and mannitol % (w / v).

[0011] The technical problem that the present invention also solves is to provide a method for freeze-drying materials using a freeze-drying solution, and the freeze-drying time can be greatly shortened by using the freeze-drying solution.

[0012] In order to solve the above technical solution, the technical solution of the present invention is: the method of freeze-drying a material using a freeze-drying solution comprises the following steps:

[0013] S1 Pre-freezing: First, place the material to be freeze-dried in a freeze-drying chamber to freeze the material;

[0014] S2 sublimation: raise the temperature to -10~0℃ and start the vacuum to sublime the material under vacuum, so that the water vapor forms ice crystals in the condenser;

[0015] S3 desorption drying: Raise the temperature to 20-30°C, desorption dry the material under vacuum, evaporate the water, and obtain a freeze-dried product.

[0016] Preferably, the materials in step S1, step S2 and step S3 are all placed on a partition.

[0017] Preferably, the pre-freezing conditions in step S1 are: pre-freezing temperature -45°C, heating time 60 minutes, and maintaining time 120 to 240 minutes.

[0018] Preferably, step S2 also includes turning on the heating system to heat the partition first, thereby sublimating the material through the partition, wherein the partition is heated from -45°C to -10~0°C, and the heating time is 60min. The sublimation conditions are: the sublimation temperature is -10~0°C, and the maintenance time is 600~780min; the vacuum degree is controlled to be 2.2mbar.

[0019] Preferably, the step S3 also includes starting the heating system, heating the partition first, and performing analytical drying on the material through the insulation board, wherein the partition is heated from -10 to 0°C to 20 to 30°C, and the heating time is 120 minutes.

[0020] Preferably, the conditions for analytical drying in step S3 are: analytical drying temperature is 20-30° C., and the holding time is 60-120 min; and the vacuum degree is controlled to be 2.2 mbar.

[0021] Preferably, the pre-freezing conditions in step S1 are: pre-freezing temperature -45°C, heating time 60min, and maintenance time 240min; the sublimation conditions in step S2 are: sublimation temperature 0°C, heating time 60min, maintenance time 780min; the vacuum degree is controlled to be 2.2mbar; the analytical drying conditions in step S3 are: analytical drying temperature 30°C, heating time 120min, maintenance time 120min; the vacuum degree is controlled to be 2.2mbar.

[0022] The technical problem that the present invention needs to solve is to provide an application of a freeze-dried solution, which can greatly shorten the freeze-drying time.

[0023] In order to solve the above technical solution, the technical solution of the present invention is: the freeze-dried solution is applied to the biologically active substance to store the biologically active substance in the form of freeze-dried powder. The freeze-dried solution does not affect the activity of the substance and the morphology of the freeze-dried product, while greatly shortening the freeze-drying time.

[0024] Compared with the prior art, the present invention has the beneficial effect that by adjusting the freeze-dried solution formula and coordinating a specific freeze-drying procedure, the freeze-drying time can be greatly shortened without affecting the activity of the substance and the morphology of the freeze-dried product. Ideally, the total freeze-drying time can be shortened to less than 24 hours, thereby increasing the output and reducing the energy cost. DETAILED DESCRIPTION

[0025] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments. The embodiments are only used to explain the present invention and do not limit the protection scope of the present invention.

[0026] Embodiment: The formula of the freeze-dried solution comprises, by mass percentage, 4-6% (w / v) of bovine serum albumin, 5-10% (w / v) of trehalose and 5-10% (w / v) of mannitol.

[0027] The method for freeze-drying a material using a freeze-drying solution comprises the following steps:

[0028] S1 Pre-freezing: First, place the material to be freeze-dried in a freeze-drying chamber to freeze the material;

[0029] In step S1, the material is first placed on the partition, and the pre-freezing conditions in step S1 are: the pre-freezing temperature is -45°C, the heating time is 60 minutes, and the maintenance time is 120 to 240 minutes;

[0030] S2 sublimation: raise the temperature to -10~0℃ and start the vacuum to sublime the material under vacuum, so that the water vapor forms ice crystals in the condenser;

[0031] The step S2 also includes starting the heating system, first heating the partition, so as to sublime the material through the partition, wherein the partition is heated from -45°C to -10-0°C, and the heating time is 60 minutes. The sublimation conditions are: sublimation temperature is -10-0°C, heating time is 60 minutes, and maintenance time is 600-780 minutes; the vacuum degree is controlled to be 2.2 mbar;

[0032] S3 analytical drying: heating to 20-30°C, analytical drying the material under vacuum, evaporating water, and obtaining a freeze-dried product; the step S3 also includes turning on the heating system, first heating the partition, and analytical drying the material through the insulation board, wherein the partition is heated from -10-0°C to 20-30°C, and the heating time is 120 minutes; the analytical drying conditions in the step S3 are: analytical drying temperature is 20-30°C, and the maintenance time is 60-120 minutes; the vacuum degree is controlled to be 2.2 mbar;

[0033] The freeze-dried solution is applied to the biologically active substance to preserve the biologically active substance in the form of freeze-dried powder. Specific embodiment:

[0035] The formula of the freeze-dried solution comprises, by mass percentage, 4% (w / v) of bovine serum albumin, 10% (w / v) of trehalose and 10% (w / v) of mannitol.

[0036] The method for freeze-drying a material using a freeze-drying solution comprises the following steps:

[0037] S1 Pre-freezing: First, place the material to be freeze-dried on the partition and place it in the freeze-drying box to freeze the material;

[0038] The pre-freezing conditions in step S1 are: pre-freezing temperature -45°C, heating time 60min, and maintenance time 240min;

[0039] S2 sublimation: the temperature is raised to -10~0℃, and the vacuum is started. The material placed on the partition is sublimated under vacuum, so that the water vapor forms ice crystals in the condenser;

[0040] The step S2 also includes starting the heating system to heat the partition first, so as to sublime the material through the partition, wherein the partition is heated from -45°C to 0°C, and the heating time is 60 minutes. The sublimation conditions are: sublimation temperature is 0°C, heating time is 60 minutes, and maintenance time is 780 minutes; vacuum degree is controlled to be 2.2 mbar; vacuum degree is controlled to be 2.2 mbar; and the heating system is started to heat the partition;

[0041] S3 analytical drying: heating to 20-30°C, analytical drying the material on the partition under vacuum, evaporating water to obtain a freeze-dried product; the step S3 also includes turning on the heating system, first heating the partition, and analytical drying the material through the insulation board, wherein the partition is heated from 0°C to 30°C, and the heating time is 120 minutes; the analytical drying conditions in the step S3 are: analytical drying temperature is 30°C, and the maintenance time is 120 minutes; the vacuum degree is controlled to be 2.2 mbar; the vacuum degree is controlled to be 2.2 mbar.

[0042] A freeze-dried product of human pituitary-derived thyroid stimulating hormone (TSH) was obtained using the freeze-dried solution formula and freeze-drying method of the specific embodiment, and subjected to a homogeneity test, an in-machine stability test, and a transportation stability test.

[0043] (I) Homogeneity test

[0044] The human pituitary-derived thyrotropin TSH freeze-dried product obtained by the freeze-dried solution formula and freeze-drying method of the specific embodiment was tested for homogeneity. The detection instrument was a fully automatic chemiluminescence analyzer (manufactured by Nanjing Lanyu Biotechnology Co., Ltd.), and the specific steps were as follows:

[0045] First, the experiment was divided into two levels, level 1 and level 2, and 10 bottles of freeze-dried product samples were taken from each group;

[0046] Add 1 mL of purified water respectively, let stand to dissolve for 30 minutes, test the samples, and repeat 3 times respectively; to obtain the experimental results, each bottle was measured 3 times, and the measurements were performed in the following order: first time: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10; second time: 10, 9, 8, 7, 6, 5, 4, 3, 2, 1; third time: 2, 4, 6, 8, 10, 1, 3, 5, 7, 9; the test results are shown in Tables 1 and 2 below.

[0047] Table 1 Test results of freeze-dried samples at level 1

[0048] Level 1 first Second time The third time 1 1730 1839 2088 2 1842 1855 1897 3 1809 1684 2095 4 1773 1889 1935 5 1794 1757 2090 6 1732 1821 1854 7 1647 1834 1935 8 1820 1824 1837 9 1760 1682 1973 10 1876 1782 1964

[0049] Table 2 Test results of freeze-dried samples at level 2

[0050] Level 2 first Second time The third time 1 21956 22483 22013 2 21749 21228 24140 3 21645 20984 22286 4 22199 22438 23696 5 22031 23425 23427 6 21844 22079 23861 7 21175 21985 22171 8 22019 22034 23220 9 23290 23648 23879 10 19749 20599 20269

[0051] The bottle-to-bottle uniformity CV is calculated according to equations (1) to (12).

[0052]

[0053] SS 瓶内 =SS 总和 -SS 瓶间 …………(3);

[0054]

[0055] v 1 =a-1……………………(7);

[0056] v 2 =Na………………………………(8);

[0057]

[0058] Where: SS represents variance; x i Indicates the i-th measurement value or calculation result of the specified parameter; represents the average value; n i Indicates the number of repeated measurements of sample i; x ijrepresents the jth result of sample i; MS represents mean square; v represents degrees of freedom; F represents the F test value; n 0 represents the number of effective measurements; a represents the number of samples drawn; N represents the total number of tests; S bb represents the standard deviation between bottles; S r represents the standard deviation within the bottle; n represents the number of measurements.

[0059] When F≤1, S r Instead of S bb Calculate CV 瓶间 ;

[0060] when When the test results show that there is no significant difference in uniformity between bottles, the calculated result CV 瓶间 ;

[0061] when S bb When ≤0.3σ, the uniformity between bottles is considered good, and the calculated result CV 瓶间 ;

[0062] when S bb When >0.3σ, the uniformity between bottles is considered to be poor.

[0063] Note: σ is the target standard deviation.

[0064] According to the data in Table 1 and Table 2, CV 瓶间 They are 7.17% and 3.55% respectively, which meet the requirements.

[0065] (II) On-machine stability test

[0066] The human pituitary-derived thyrotropin TSH freeze-dried product obtained by the freeze-dried solution formula and freeze-drying method of the specific embodiment was subjected to an on-machine stability test. The detection instrument was a fully automatic chemiluminescence analyzer (manufactured by Nanjing Lanyu Biotechnology Co., Ltd.), and the specific steps were as follows:

[0067] First, the experiment was divided into two levels, namely Level 1 and Level 2. Three bottles of freeze-dried samples were taken from each group. 1 mL of purified water was added and the samples were allowed to stand and dissolve for 30 minutes. Then, the samples were mixed evenly. After the test at 0h, the samples were tested once at 1, 3, 4, 5, 6, 8h and on the 2nd day. The samples were numbered Z0, Z1, Z3, Z4, Z5, Z6, Z8 and Z9 respectively. The test results are shown in Tables 3 and 4.

[0068] Table 3 Test results of freeze-dried samples at level 1

[0069] sample Luminous value Deviation from the mean value at 0h Z0 1,736 / Z1 1,766 1.75％ Z3 1778 2.42％ Z4 1674 3.59％ Z5 1732 0.21％ Z6 1685 2.96％ Z8 1652 4.84％ Z9 1909 9.94％

[0070] Table 4 Test results of freeze-dried samples at level 2

[0071] sample Luminous value Deviation from the mean value at 0h Z0 22,020 / Z1 21,401 2.81％ Z3 21,322 3.17％ Z4 22,131 0.51％ Z5 21,821 0.91％ Z6 21,703 1.44％ Z8 21,159 3.91％ Z9 21,064 4.34％

[0072] According to the test, the TSH freeze-dried product remains stable in the instrument for 24 hours after freeze-drying.

[0073] (III) Transportation stability test

[0074] The human pituitary-derived thyrotropin TSH freeze-dried product obtained by the freeze-dried solution formula and freeze-drying method of the specific embodiment was tested for transportation stability. The detection instrument was a fully automatic chemiluminescence analyzer (manufactured by Nanjing Lanyu Biotechnology Co., Ltd.), and the specific steps were as follows:

[0075] First, the experiment was divided into two groups, level 5 and level 6. Three bottles of freeze-dried product samples were taken from each group. They were placed in a constant temperature oscillator at 60°C and 200rpm to simulate transportation conditions for 7 days. On the 0th, 1st, 4th, 6th, and 7th days of transportation, all samples were taken out on the 8th day.

[0076] The placement method is as follows:

[0077] Day 1: Take out one set and put it in, transport it for 7 days, that is, take it out after 7 days, numbered as D0;

[0078] Day 2: Take out one set and put it in, transport it for 6 days, that is, take it out after 6 days; numbered D1;

[0079] Day 5: Take out one set and put it in, transport it for 3 days, and then take it out after 3 days; numbered D3;

[0080] Day 7: Take out one set and put it in, transport it for one day, and take it out after one day; numbered D6;

[0081] Day 8: Take 1 set, transport for day 0, i.e. take it out after day 0 (take it directly from 2-8℃); numbered as D7;

[0082] Finally, all samples were added with 1 mL of purified water at one time, and allowed to stand for 30 minutes to dissolve (inverted and mixed during this period), and the samples were tested to obtain the experimental results, as shown in Tables 5 and 6 below.

[0083] Table 5 Test results of freeze-dried samples at level 1

[0084] sample Luminous value Deviation from the mean on day 0 D0 1,630 4.47％ D1 1,761 3.22％ D3 1,713 0.38％ D6 1,684 1.31％ D7 1,706 /

[0085] Table 6 Test results of freeze-dried samples at level 2

[0086] sample Luminous value Deviation from the mean on day 0 D0 20,559 2.19％ D1 21,370 1.67％ D3 21,458 2.08％ D6 21,343 1.54％ D7 21,020 /

[0087] Based on the test, it was concluded that TSH freeze-dried samples were stable within 7 days after transportation.

[0088] For ordinary technicians in this field, the specific embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A freeze-dried solution, characterized in that The formula of the freeze-dried solution comprises, by mass percentage, 4-6% (w / v) of bovine serum albumin, 5-10% (w / v) of trehalose and 5-10% (w / v) of mannitol.

2. The freeze-dried solution according to claim 1, characterized in that The formula of the freeze-dried solution comprises, by mass percentage, 4% (w / v) of bovine serum albumin, 10% (w / v) of trehalose and 10% (w / v) of mannitol.

3. A method for freeze-drying a material using the freeze-drying solution according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1 Pre-freezing: First, place the material to be freeze-dried in a freeze-drying chamber to freeze the material; S2 sublimation: raise the temperature to -10~0℃ and start the vacuum to sublime the material under vacuum, so that the water vapor forms ice crystals in the condenser; S3 desorption drying: Raise the temperature to 20-30°C, desorption dry the material under vacuum, evaporate the water, and obtain a freeze-dried product.

4. The method for freeze-drying a material using a freeze-drying solution according to claim 3, characterized in that: The materials of step S1, step S2 and step S3 are all placed on the partition.

5. The method for freeze-drying a material using a freeze-drying solution according to claim 4, characterized in that: The pre-freezing conditions in step S1 are: pre-freezing temperature -45°C, heating time 60 minutes, and maintaining time 120 to 240 minutes.

6. The method for freeze-drying a material using a freeze-drying solution according to claim 5, characterized in that: The step S2 also includes turning on the heating system to heat the partition first, thereby sublimating the material through the partition, wherein the partition is heated from -45°C to -10~0°C, and the heating time is 60 minutes. The sublimation conditions are: the sublimation temperature is -10~0°C, and the maintenance time is 600~780 minutes; the vacuum degree is controlled to be 2.2mbar.

7. The method for freeze-drying a material using a freeze-drying solution according to claim 6, characterized in that: The step S3 also includes starting the heating system, first heating the partition, and performing analytical drying on the material through the insulation board, wherein the partition is heated from -10 to 0°C to 20 to 30°C, and the heating time is 120 minutes.

8. The method for freeze-drying a material using a freeze-drying solution according to claim 7, characterized in that: The conditions for the analytical drying in step S3 are: the analytical drying temperature is 20-30° C., the holding time is 60-120 min; and the vacuum degree is controlled to be 2.2 mbar.

9. The method for freeze-drying a material using a freeze-drying solution according to claim 7, characterized in that: The pre-freezing conditions in step S1 are: pre-freezing temperature -45°C, heating time 60min, and maintenance time 240min; the sublimation conditions in step S2 are: sublimation temperature 0°C, heating time 60min, maintenance time 780min; the vacuum degree is controlled to be 2.2mbar; the analytical drying conditions in step S3 are: analytical drying temperature 30°C, heating time 120min, maintenance time 120min; the vacuum degree is controlled to be 2.2mbar.

10. An application of a freeze-dried solution, characterized in that: The freeze-dried solution is applied to the biologically active substance to preserve the biologically active substance in the form of freeze-dried powder.

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