CN113088508A - Freeze-drying method of neuron specific enolase - Google Patents
Freeze-drying method of neuron specific enolase Download PDFInfo
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Abstract
The invention belongs to the technical field of freeze-drying, and particularly relates to a freeze-drying method of neuron specific enolase. The lyophilization process comprises the steps of: dissolving NSE in buffer solution to obtain NSE reagent, and freeze-drying in vacuum freeze-dryer; wherein, the buffer solution comprises the following components: trihydroxymethyl aminomethane, KCl, disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, and BSA. The freeze-drying method can effectively preserve neuron specific enolase and is beneficial to maintaining the maximum activity of the neuron specific enolase.
Description
Technical Field
The invention belongs to the technical field of freeze-drying, and particularly relates to a freeze-drying method of neuron specific enolase.
Background
Neuron-Specific Enolase (NSE) is an acid protease with a molecular weight of about 78000. The NSE is one of the important markers of the small cell lung cancer, and can be used for differential diagnosis and detection of the treatment effect of the small cell lung cancer after radiotherapy and chemotherapy. The NSE can also be used for differential diagnosis of neuroblastoma and nephroblastoma, and has higher clinical application value for early diagnosis of neuroblastoma.
NSE is an unstable bioactive substance that rapidly loses activity at ambient temperatures and in environments of 2-8 ℃. When NSE is used as a kit component, detection errors and kit failure can result if the lyophilization process is not performed. NSE is dissolved by using a special buffer system and is freeze-dried into dry powder, so that the activity of the NSE can be maintained for a long time, and the stability and the effectiveness of the kit are ensured.
General buffer solution is used for dissolving NSE, and the activity can be kept for 3 days at normal temperature (10-30 ℃), for 3 months at 2-8 ℃ and for 9 months at-20 ℃. The preservation time is short, which is not beneficial to the production, storage and application of the kit product.
Chinese patent application CN 106568976A discloses a neuron-specific enolase stabilizer and a preparation method thereof, relating to a neuron-specific enolase stabilizer, which is prepared from the following raw materials: Tris-HCl buffer solution, NaCl, sucrose, trehalose, casein, bovine serum albumin, PEG6000, lysine, ethylene diamine tetraacetic acid disodium, dithiothreitol, glycerol, Tween-20, TritonX-100, final concentration aprotinin, Proclin300 and Millipore ultrapure water are used as solvents, and the solutions are uniformly mixed, filtered by a 0.22 mu m filter membrane and stored at 0-10 ℃. The storage stability of the neuron-specific enolase can be improved, and the shelf life of the kit can be prolonged; the production process of the neuron specific enolase detection kit and the detection operation process of doctors are simplified; the capability of resisting qualitative changes in the process of preparation, transportation and storage of the neuron specific enolase is improved; the batch-to-batch variation caused by the lyophilization process is reduced. However, the stabilizer contains many kinds of active ingredients, and the cost is high, and the ingredients used in the present invention are complicated, and the interaction between different ingredients is not favorable for the stabilizer to improve the storage stability of NSE.
The invention aims to provide an effective preparation method of a freeze-dried product for keeping the activity of neuron-specific enolase to be maximum.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a freeze-drying method of neuron-specific enolase (NSE), which can effectively preserve the neuron-specific enolase and is beneficial to maintaining the maximum activity of the neuron-specific enolase.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a method for lyophilizing neuron-specific enolase, comprising the steps of: dissolving NSE in buffer solution to obtain NSE reagent, and freeze-drying in vacuum freeze-dryer; wherein, the buffer solution comprises the following components: tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, and Bovine Serum Albumin (BSA).
Preferably, the buffer solution comprises the following components in parts by weight: 4-20 parts of tris (hydroxymethyl) aminomethane, 6-25 parts of KCl, 1-6 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
Preferably, the buffer solution comprises the following components in parts by weight: 4.2-15.6 parts of trihydroxymethyl aminomethane, 8.5-24 parts of KCl, 1.2-5.8 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA;
preferably, the preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value, and fixing the volume;
(2) filtering with filter membrane.
Preferably, the pH value is between 6.8 and 8.5.
Preferably, the pore size of the filter is 0.22 μm.
Preferably, the NSE reagent is prepared from NSE antigen and buffer solution, and is used for a standard substance, a quality control substance or a reference substance.
Preferably, the lyophilization process comprises the steps of:
setting a vacuum freeze dryer into three stages, namely a pre-freezing section, a sublimation drying section and an analysis drying section; freeze-drying, and storing at 4-8 deg.C.
Preferably, the pre-freezing section is: cooling the NSE reagent to 0 ℃ from normal temperature; the cooling time is 30 minutes;
after the temperature is kept at 0 ℃ for 10 minutes, the temperature is reduced to-30 ℃ for 30 minutes;
after the temperature is kept at minus 30 ℃ for 60 minutes, the temperature is reduced to minus 55 ℃ for 30 minutes; keeping the temperature at-55 ℃ for 240 minutes;
preferably, the sublimation drying section is: reducing the vacuum degree to 0-5 pascal (pa) within 1-5 minutes; setting 7 temperature points of-50 deg.C, -45 deg.C, -35 deg.C, -30 deg.C, -25 deg.C and-5 deg.C;
the temperature rise time from minus 55 ℃ to minus 50 ℃ is 10 minutes, and the heat preservation time at minus 50 ℃ is 60 minutes;
the temperature rise time from minus 50 ℃ to minus 45 ℃ is 60 minutes, and the heat preservation time at minus 45 ℃ is 120 minutes;
the temperature rise time from minus 45 ℃ to minus 35 ℃ is 60 minutes, and the heat preservation time at minus 35 ℃ is 240 minutes;
the temperature rise time from minus 35 ℃ to minus 30 ℃ is 60 minutes, and the heat preservation time at minus 30 ℃ is 480 minutes;
the temperature rise time from minus 30 ℃ to minus 25 ℃ is 60 minutes, and the heat preservation time at minus 25 ℃ is 240 minutes;
the temperature rise time from minus 25 ℃ to minus 15 ℃ is 60 minutes, and the heat preservation time at minus 15 ℃ is 120 minutes;
the temperature rise time at-15 ℃ to-5 ℃ is 30 minutes, and the heat preservation time at-5 ℃ is 60 minutes.
Preferably, the desorption drying section is: setting two temperature points at 20 ℃ and 25 ℃ respectively;
-a temperature rise time of 120 minutes at 5 ℃ to 20 ℃ and a holding time of 60 minutes at 20 ℃;
the temperature rise time at 20 ℃ to 25 ℃ is 15 minutes, and the heat preservation time at 25 ℃ is 460-620 minutes.
Compared with the prior art, the invention has the technical advantages that:
1. the freeze-drying method provided by the invention can effectively improve the preservation effect of the NSE by using the buffer system provided by the invention, and can ensure that the NSE can be stored for 12 months at 4-8 ℃ and 3 years at-20 ℃.
2. The freeze-drying method and the buffer solution provided by the invention can improve the detection sensitivity of the NSE detection reagent; the detection sensitivity reaches 0.5ng/ml (0.5 multiplied by 10)9g/mL)。
Detailed Description
The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 16g of trihydroxymethyl aminomethane, 12g of KCl, 3g of disodium hydrogen phosphate dodecahydrate, 6g of magnesium sulfate, 7g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value to 7.0, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) Preparation of lyophilized products
NSE recombinant protein is taken and dissolved by buffer solution, and 6 NSE reagents with the concentrations of 0ng/mL, 1ng/mL, 20ng/mL, 75ng/mL, 150ng/mL and 300ng/mL are prepared as calibration substances after being fully mixed.
(3) Freeze-drying products with different concentrations are placed in a freeze-drying bin of a vacuum freeze-drying machine, and freeze-drying is carried out according to the program designed in the table 1. Freeze-drying, and storing at 4-8 deg.C.
TABLE 1 Freeze drying procedure
Example 2
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 4g of trihydroxymethyl aminomethane, 25g of KCl, 1g of disodium hydrogen phosphate dodecahydrate, 0.1g of magnesium sulfate, 50g of trehalose and 1.0g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value to 8.5, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) Preparation of lyophilized products
NSE recombinant protein is taken and dissolved by buffer solution, and 6 NSE reagents with the concentrations of 0ng/mL, 1ng/mL, 20ng/mL, 75ng/mL, 150ng/mL and 300ng/mL are prepared as calibration substances after being fully mixed.
(3) Freeze-drying products with different concentrations are placed in a freeze-drying bin of a vacuum freeze-drying machine, and freeze-drying is carried out according to the program designed in the table 2. Freeze-drying, and storing at 4-8 deg.C.
TABLE 2 Freeze drying procedure
Example 3
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 20g of trihydroxymethyl aminomethane, 6g of KCl, 6g of disodium hydrogen phosphate dodecahydrate, 10g of magnesium sulfate, 5.0g of trehalose and 50g of BSA.
The preparation method of the buffer solution comprises the following steps:
(1) weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value to 6.8, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) Preparation of lyophilized products
NSE recombinant protein is taken and dissolved by buffer solution, and 6 NSE reagents with the concentrations of 0ng/mL, 1ng/mL, 20ng/mL, 75ng/mL, 150ng/mL and 300ng/mL are prepared as calibration substances after being fully mixed.
(3) Freeze-drying products with different concentrations are placed in a freeze-drying bin of a vacuum freeze-drying machine, and freeze-drying is carried out according to the program designed in the table 3. Freeze-drying, and storing at 4-8 deg.C.
TABLE 3 Freeze drying procedure
Comparative example 1
The difference compared to example 1 is only the composition of the buffer.
Part of trehalose was replaced with sucrose, and the rest steps were unchanged.
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 16g of trihydroxymethyl aminomethane, 12g of KCl, 3g of disodium hydrogen phosphate dodecahydrate, 6g of magnesium sulfate, 5g of sucrose, 2g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding sucrose, trehalose and bovine serum albumin into purified water, stirring until the sucrose, the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value to 7.0, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) The procedure of (1) to (3) is the same as in example 1.
Comparative example 2
The difference compared to example 1 is only the composition of the buffer.
Magnesium sulfate is replaced by disodium ethylene diamine tetraacetate, and the rest steps are unchanged.
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 16g of trihydroxymethyl aminomethane, 12g of KCl, 3g of disodium hydrogen phosphate dodecahydrate, 6g of disodium ethylene diamine tetraacetate, 7g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate, disodium ethylene diamine tetraacetate, trehalose and bovine serum albumin, adding into purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.0, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) The procedure of (1) to (3) is the same as in example 1.
Comparative example 3
The difference compared to example 1 is only the composition of the buffer.
Sodium phosphate dibasic dodecahydrate was replaced with NaCl.
A method for lyophilizing neuron-specific enolase, comprising the steps of:
(1) preparation of buffer
The buffer solution comprises the following components: 16g of tris (hydroxymethyl) aminomethane, 12g of KCl, 3g of NaCl, 6g of magnesium sulfate, 7g of trehalose and 15g of BSA.
The preparation method of the buffer solution comprises the following steps:
weighing Tris, KCl, NaCl and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value to 7.0, and fixing the volume to 1000 ml; filtering with 0.22 μm filter membrane.
(2) The procedure of (1) to (3) is the same as in example 1.
Comparative example 4
Compared with example 1, the difference is only that the freezing procedure is different, and other steps are not changed.
Table 4 lyophilization procedure
Comparative example 5
Embodiment 1 of chinese patent application CN 106568976 a.
NSE stabilizer preparation, composition is shown in the following table:
TABLE 5 specific example 1 Components of CN 106568976A
The preparation steps are as follows:
1. weighing a proper amount of Tris, adding Millipore ultrapure water for dissolving to prepare a 50mM Tris solution, and adjusting the pH value to 7.4 by hydrochloric acid;
2. sequentially weighing NaCl, sucrose, trehalose, casein, bovine serum albumin, PEG6000, lysine, disodium ethylene diamine tetraacetate and dithiothreitol, and dissolving in Tris & HCl buffer solution;
3. adding glycerol, Tween-20, Triton X-100, aprotinin and Proclin300 into the prepared solution, stirring thoroughly to dissolve, filtering with 0.22 μm filter membrane, and storing at 4 deg.C for use.
Examples of effects
1. Detection method
The detection is carried out by adopting a full-automatic chemiluminescence immunoassay analyzer self-developed by Beijing Meiliantaceae biotechnology limited company. The amount of sample required for the reaction was 30. mu.L, and the automatic assay procedure was:
immune reaction: and sequentially adding 30 mu L of sample, 50 mu L of reagent A, 50 mu L of reagent B and 50 mu L of reagent C into the reaction hole, and reacting at 37 ℃ for 20 min.
Magnetic separation and cleaning: adding 300 mu L of cleaning solution into the No. 1 cleaning hole, sucking the mixture containing the magnetic particles out of the reaction hole by magnetic force, and demagnetizing the No. 1 cleaning hole. After 2min of cleaning. And (3) respectively carrying out 1-time magnetic separation and cleaning at the No. 2 or No. 3 cleaning hole position.
Reading value: adding 150uL of luminescent substrate at the reading hole position, sucking the mixture containing magnetic particles out of the No. 3 cleaning hole position by magnetic force, and demagnetizing at the reading hole position. Relative luminescence intensity (RLU) was measured after luminescence of the ALP-catalyzed luminescent substrate.
Fourthly, obtaining an NSE concentration-luminous value standard curve according to the detected value of the calibrator. The curve was fitted using a four parameter Logistic equation.
The detection value of the sample can correspond to the unique concentration value obtained on the curve, thereby realizing the concentration detection of the unknown sample.
2. Detecting the index
2.1 appearance
The freeze-dried product is white loose body without impurities. The whole freeze-dried substance should be a column with the same width of upper and lower parts, the upper surface should not be cracked, and the lower surface should not shrink. No significant porosity should appear throughout. The redissolved liquid was uniform (no visible particles, no precipitate) and was considered to be acceptable.
2.2 deviation in concentration
The freeze-dried products with six concentrations were tested using a neuron-specific enolase detection kit (magnetic particle chemiluminescence method) and a double parallel test was performed. And (4) running according to the set program, reading the luminous value after the program is finished, and inputting the concentration and the luminous value into an instrument for fitting. The calculation was performed according to equation (1). The concentration deviation should satisfy: the concentration of the B point is within the range of +/-12% of the bottle label concentration; the concentrations of spots C, D, E and F were within. + -. 8% of the concentrations of the vials (wherein A: 0 ng/mL; B: 1 ng/mL; C: 20 ng/mL; D: 75 ng/mL; E: 150 ng/mL; F: 300 ng/mL). The product was considered as qualified.
Concentration deviation (fitted concentration-standard concentration)/standard concentration … … … … … … … (1)
2.3 Water content
Taking 10 lyophilized products with different concentrations. The freeze-dried product was weighed after removing the cap and plug and recorded as M1n(n is 1, 2, 3 … … 10). Drying at 200 deg.C until the mass no longer changes, weighing again, and recording as M2n(n is 1, 2, 3 … … 10). The vials containing the sample were washed clean in sequence, oven dried at 200 ℃ until the mass no longer changed, and the vials were weighed in sequence and recorded as M3n(n is 1, 2, 3 … … 10). The calculation is performed according to equation (2). The water content of the lyophilized product should be less than 2%. Viewed as aAnd (4) passing.
Water content [ (M1)n-M3n)/(M2n-M3n)]-1×100％…………………(2)
2.4 accuracy
Neuron-specific enolase (NSE) solution (A) with a concentration of about 20ng/mL (tolerance. + -. 10%) was added to a sample B with a concentration ranging from 0ng/mL to 0.5ng/mL, at a volume ratio of 1:9 between the added NSE antigen and the sample B, and the assay was performed using a lyophilizate and a kit for detection (magnetic particle chemiluminescence assay). And (4) calculating the recovery rate R according to the formula (3), wherein the recovery rate is in a range of 85-115%. The product was considered as qualified.
In the formula:
r-recovery rate;
v is the volume of the sample A liquid;
V0-volume of serum sample B fluid;
c is the average value of 3 measurements after the serum sample B liquid is added into the liquid A;
C0-mean of 3 measurements of serum sample B fluid;
CS-concentration of sample a liquid.
2.5 Linear region
Mixing a high value sample close to the upper limit of the linear region and a low value sample close to the lower limit of the linear region or a zero concentration sample to obtain not less than 5 dilution concentrations, wherein the low value concentration sample is close to the lower limit of the linear region. The test was repeated 3 times for each concentration of the sample to obtain the luminescence value, the measurement result of each sample was recorded, and the average value (y) of the 3 measurements of each sample was calculatedi). In diluted concentration (x)i) As independent variable, the mean value (y) of the results is determinedi) Linear regression equations were solved for the dependent variables. And (3) calculating a correlation coefficient (r) of the linear regression according to the formula (2), wherein the correlation coefficient r is not less than 0.990 within a linear interval of 0.5-300 ng/mL. The product was considered as qualified.
In the formula:
r- — -correlation coefficient;
xi-dilution ratio;
yi-mean value of individual sample measurements;
2.6 repeatability
Repeating the test of the C point of the freeze-dried product for 10 times, and calculating the average value of the 10 test results
And standard deviation SD. The Coefficient of Variation (CV) was calculated according to equation (5) and the result CV was less than or equal to 8%. The product was considered as qualified.
In the formula: s-standard deviation of sample test values;
2.7 stability Effect
The experimental method comprises the following steps: tests were carried out under different conditions (five time points of 0 month, 3 months, 6 months, 9 months and 12 months at 4-8; -six time points of 0 month, 6 months, 12 months, 18 months, 24 months and 36 months at-20 ℃) using the index detection method described in the above 2.1-2.6, and the stability of examples 1-3 and comparative examples 1-5 was tested, wherein out of six indexes of 2.1-2.6, 1 point was counted for each qualified index, and 6 points were counted for all six indexes, i.e., the condition of satisfying the validity period. The results of the tests are given in the following table:
table 6 stability effect data
Therefore, the buffer solution and the freeze-drying method provided by the invention can effectively store NSE for 12 months at 4-8 ℃ and 36 months at-20 ℃. When the buffer system or the freeze-drying method is changed, the performance stability is influenced to different degrees. Meanwhile, it is understood from the experimental results of comparative example 5 that the addition of an excessive amount of the effective ingredient to the buffer solution is disadvantageous in that the stabilizer improves the storage stability of the NSE.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A method for lyophilizing neuron-specific enolase, comprising the steps of: dissolving NSE in buffer solution to obtain NSE reagent, and freeze-drying in vacuum freeze-dryer; wherein, the buffer solution comprises the following components: trihydroxymethyl aminomethane, KCl, disodium hydrogen phosphate dodecahydrate, magnesium sulfate, trehalose, and BSA.
2. The lyophilization process according to claim 1, wherein the buffer comprises the following components in parts by weight: 4-20 parts of trihydroxymethyl aminomethane, 6-25 parts of KCl, 1-6 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
3. The lyophilization process according to claim 1, wherein the buffer comprises the following components in parts by weight: 4.2-15.6 parts of trihydroxymethyl aminomethane, 8.5-24 parts of KCl, 1.2-5.8 parts of disodium hydrogen phosphate dodecahydrate, 0.1-10 parts of magnesium sulfate, 5.0-50 parts of trehalose and 1.0-50 parts of BSA.
4. The lyophilization process according to claim 1, wherein the buffer is prepared by a process comprising the steps of:
(1) weighing Tris, KCl, disodium hydrogen phosphate dodecahydrate and MgSO4Adding trehalose and bovine serum albumin into purified water, stirring until the trehalose and the bovine serum albumin are completely dissolved, adjusting the pH value, and fixing the volume;
(2) filtering with filter membrane.
5. The lyophilization process according to claim 4, wherein the pH is between 6.8 and 8.5.
6. The lyophilization process according to claim 4, wherein the pore size of the filter membrane is 0.22 μm.
7. The lyophilization process according to claim 1, wherein the NSE reagent is formulated from NSE antigen and buffer for use in a standard, quality control or reference.
8. The lyophilization process of claim 1, wherein the lyophilization process comprises the steps of:
setting a vacuum freeze dryer into three stages, namely a pre-freezing section, a sublimation drying section and an analysis drying section; freeze-drying, and storing at 4-8 deg.C.
9. The lyophilization process of claim 8, wherein the pre-freezing stage is: cooling the NSE reagent to 0 ℃ from normal temperature; the cooling time is 30 minutes;
after the temperature is kept at 0 ℃ for 10 minutes, the temperature is reduced to-30 ℃ for 30 minutes;
after the temperature is kept at minus 30 ℃ for 60 minutes, the temperature is reduced to minus 55 ℃ for 30 minutes; incubate at-55 ℃ for 240 minutes.
10. The lyophilization process of claim 8, wherein the sublimation drying section is: reducing the vacuum degree to 0-5 pascal (pa) within 1-5 minutes; setting 7 temperature points of-50 deg.C, -45 deg.C, -35 deg.C, -30 deg.C, -25 deg.C and-5 deg.C;
the temperature rise time from minus 55 ℃ to minus 50 ℃ is 10 minutes, and the heat preservation time at minus 50 ℃ is 60 minutes;
the temperature rise time from minus 50 ℃ to minus 45 ℃ is 60 minutes, and the heat preservation time at minus 45 ℃ is 120 minutes;
the temperature rise time from minus 45 ℃ to minus 35 ℃ is 60 minutes, and the heat preservation time at minus 35 ℃ is 240 minutes;
the temperature rise time from minus 35 ℃ to minus 30 ℃ is 60 minutes, and the heat preservation time at minus 30 ℃ is 480 minutes;
the temperature rise time from minus 30 ℃ to minus 25 ℃ is 60 minutes, and the heat preservation time at minus 25 ℃ is 240 minutes;
the temperature rise time from minus 25 ℃ to minus 15 ℃ is 60 minutes, and the heat preservation time at minus 15 ℃ is 120 minutes;
the temperature rise time from-15 ℃ to-5 ℃ is 30 minutes, and the heat preservation time at-5 ℃ is 60 minutes;
the analysis drying section comprises: setting two temperature points at 20 ℃ and 25 ℃ respectively;
-a temperature rise time of 120 minutes at 5 ℃ to 20 ℃ and a holding time of 60 minutes at 20 ℃;
the temperature rise time at 20 ℃ to 25 ℃ is 15 minutes, and the heat preservation time at 25 ℃ is 460-620 minutes.
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