A freeze-drying process for silica microsphere suspension, freeze-dried silica microsphere powder and application thereof

Abstract

The present invention belongs to the field of preparation of silica microspheres, and specifically, relates to a freeze-drying process of a silica microsphere suspension, a silica microsphere freeze-dried powder and an application thereof. The freeze-drying procedure includes a first section, a second section, a third section, a fourth section and a fifth stage, which are sequentially executed. From the first section to the fourth section, the vacuum degree is controlled, and the vacuum degree is gradually reduced. Through the freeze-drying process of the present invention, a silica microsphere reagent in a suspension state can be converted into a solid (powder) process through vacuum freeze-drying technology. The freeze-dried microspheres can be transported and stored in a room temperature environment, and the microspheres are stable during long-term storage and easy to use.

Description

Technical Field

[0001] The invention belongs to the field of preparation of silica microspheres, and specifically relates to a freeze-drying process of a silica microsphere suspension, a silica microsphere freeze-dried powder and an application thereof. Background Art

[0002] Blood is an easily accessible, stable, and minimally invasive biological fluid, and its composition can reveal the overall pathophysiological status of tissues and organs. The abundance of plasma proteins ranges widely, with the 22 most abundant proteins accounting for more than 99%, while low-abundance proteins account for less than 1%, but have important research value in early diagnosis of diseases, prognosis and treatment, and biomarker research. How to effectively remove high-abundance proteins in plasma or efficiently enrich low-abundance proteins has become a key issue in plasma proteomics.

[0003] The traditional pre-treatment strategy for blood proteome is to remove high-abundance proteins. However, some uncommon species cannot be adapted to commercially available high-abundance removal kits due to the specificity of immune binding, and many low-abundance proteins will be removed while removing high-abundance proteins, resulting in loss of protein information. For example, when albumin is removed, low-abundance proteins such as cytokines, peptide hormones, and lipoproteins that bind to albumin are likely to be removed. This pre-treatment method has obvious limitations. Enrichment of low-abundance proteins can effectively avoid the defects of traditional methods and increase the detection depth and quantitative accuracy of plasma proteome.

[0004] After performance testing of several commercially available reagents for removing high-abundance proteins from plasma and efficiently enriching low-abundance proteins, it was found that silica microsphere suspension meets the requirements for use in high-depth blood proteomes.

[0005] However, the use of silica microspheres to remove high-abundance proteins in plasma and efficiently enrich low-abundance proteins still has the following drawbacks:

[0006] Silica microspheres suitable for this function have relatively high requirements for particle size. Small-diameter silica microspheres are required, for example, below 600nm. Most of these silica microspheres are in a suspension state dispersed in a preservation solvent (20% ethanol solution), and need to be preserved at 2~8°C. The preservation and transportation costs are too high. There is a risk of their activity being destroyed due to excessively high temperatures during transportation. Due to the limitations of cold chain transportation, they cannot meet long-term transportation requirements.

[0007] The silica microspheres in the prior art are also in powder form, but they are all large-particle silica microspheres and cannot meet the requirements of removing high-abundance proteins in plasma and efficiently enriching low-abundance proteins. Summary of the invention

[0008] In order to solve the problem that the above-mentioned silica microsphere suspension cannot maintain 2-8°C conditions during long-term transportation (logistics time of one week or more) or the cost of cold chain transportation is too high, the present invention provides a freeze-drying process for the silica microsphere suspension, which can convert the silica microsphere reagent in a suspension state into a solid (powder) through vacuum freeze-drying technology. The freeze-dried microspheres can be transported and stored in a room temperature environment, and the microspheres are stable during long-term storage and are easy to use.

[0009] The present invention adopts the following technical solution:

[0010] A freeze-drying process for a silica microsphere suspension, wherein the freeze-drying procedure comprises a first section, a second section, a third section and a fourth section which are executed in sequence, and the vacuum degree is controlled from the first section to the fourth section, and the vacuum degree is gradually reduced.

[0011] Silica microsphere suspensions have been proven to have great application prospects in removing high-abundance proteins and enriching low-abundance proteins in plasma. However, due to the limitations of the preparation method of silica microspheres, most silica microspheres on the market are in the form of suspensions, usually dispersed in a storage solvent (20% ethanol solution). The solvent is removed during use to avoid the effect of the solvent on the results. In addition, silica microsphere suspensions are sensitive to temperature and need to be stored at 2~8°C. Transportation also requires the use of a cold chain. This brings great inconvenience to the storage, transportation, and use of silica microsphere suspensions, and the cost is also high.

[0012] The above technical scheme of the present invention is based on this prior art, in order to obtain solid powdered silica microspheres. Therefore, a freeze-drying process for a silica microsphere suspension is proposed. Through experimental research on this, the inventor found that in the freeze-drying process, in addition to the need to control the freeze-drying temperature, the vacuum degree is also controlled. On this basis, the degree is adjusted to four sections, and the temperature and vacuum degree of each section are controlled differently. The silica microsphere suspension can be efficiently freeze-dried without losing the activity of the silica microspheres in removing high-abundance proteins and enriching low-abundance proteins in plasma.

[0013] The freeze-dried powder of silica microspheres obtained by the freeze-drying process in this scheme is directly dissolved and used for the purpose of removing high-abundance proteins and enriching low-abundance proteins in plasma. Compared with the use of silica microsphere suspension, the step of removing the storage solvent (20% ethanol solution) is omitted.

[0014] Preferably, the parameters of the freeze-drying process are controlled as follows:

[0015] The first section: -30~-20℃, vacuum degree 1atm, time 3h~4h;

[0016] The second section: -20~-10℃, vacuum degree 20~100Pa, time 3h~4h;

[0017] The third section: -10~0℃, vacuum degree 20~100Pa, time 3h~4h;

[0018] The fourth section: 0~10℃, vacuum degree 0~10Pa, time 2h~3h;

[0019] The fifth section: 20~30℃, vacuum degree 0~10Pa, time 4h~6h.

[0020] In this scheme, the specific parameters of each section are optimized. Among them, the key points that have a greater impact on the freeze-drying results are: first, the first section, that is, the initial freeze-drying temperature. If the temperature is too low, below -30°C, it will have a great impact on the activity of the silica microspheres. If it is too high, above -20°C, it is not conducive to achieving efficient freeze-drying. Second, it is the parameter of the vacuum degree in each section. The control of the vacuum degree, combined with the control of the temperature, ensures that the activity of the silica microspheres is not affected while achieving efficient freeze-drying. Third, it is the control of the temperature amplitude of each section. The temperature increase amplitude between sections is preferably not more than 20°C, especially the first four sections, which have a great impact on the activity of the silica microspheres. If the amplitude is too large, it is easy to cause a decrease in activity. In short, in the freeze-drying process, it is necessary to ensure that the activity of the silica microspheres is not affected while achieving efficient freeze-drying, which requires the coordination of various parameters.

[0021] Preferably, no lyoprotectant is added during the freeze-drying process. In the process of debugging the freeze-drying parameters, one of the main purposes is to ensure that the activity of the silica microspheres is not affected. Adding lyoprotectant is usually a means to protect the activity of the freeze-dried object. However, the present invention is directed to a silica microsphere suspension. Considering that after adding the lyoprotectant, the lyoprotectant needs to be removed before use, which adds steps, and there will be a loss of silica microspheres during the removal process. By adopting the above-mentioned optimized freeze-drying parameters, the activity of the silica microspheres can be preserved to the maximum extent even without adding a lyoprotectant.

[0022] Preferably, the freeze-drying process comprises the following steps:

[0023] S1. Pour the silica microsphere suspension to be freeze-dried into a freeze-drying tray;

[0024] S2. Setting the freeze-drying program and starting freeze-drying;

[0025] S3. After the freeze-drying process is completed, the freeze-dried powder is collected.

[0026] Preferably, in step S1, the depth of the silica microsphere suspension in the freeze-drying tray does not exceed 6mm. The depth of the suspension determines the efficiency of freeze-drying on the one hand. If it is too deep, the freeze-drying is not thorough. On the other hand, the depth also affects the activity of the silica microspheres during freeze-drying. It is found that the depth is too large or too small, which is not conducive to the maintenance of activity, and the depth is too deep, and it is also easy to splash during freeze-drying, resulting in a reduction in the yield of silica microspheres. Taking into account the freeze-drying efficiency, activity and overall production efficiency of the silica microspheres, the depth of the silica microsphere suspension in the freeze-drying tray is selected at 3mm to 5mm.

[0027] Preferably, the operating environment humidity is below 30%, and the ambient temperature is between 18° C. and 28° C. In particular, the operating environment of step S3 needs to be strictly controlled within the above range.

[0028] The present invention also provides a freeze-dried powder of silicon dioxide microspheres, which is obtained by using the freeze-drying process of the silicon dioxide microsphere suspension.

[0029] By implementing the above technical solution, compared with the prior art, the present invention has the following advantages:

[0030] 1. The present invention freeze-dries the silica microsphere suspension through a specific freeze-drying procedure, and the detection performance of the obtained silica microsphere freeze-dried powder is basically not affected, and it can be stored at room temperature with basically no loss of activity, which greatly simplifies the storage and transportation conditions of the silica microspheres and reduces costs.

[0031] 2. The freeze-drying process of the present invention does not require the use of a freeze-drying protective agent during the freeze-drying process, and can achieve an excellent freeze-drying effect, with substantially no loss of activity of the silica microspheres, so the present invention omits the use of a freeze-drying protective agent.

[0032] 3. The freeze-drying process of the present invention is suitable for large-scale freeze-drying treatment on trays and can be mass-produced.

[0033] 4. The freeze-dried silica microsphere powder of the present invention can be directly dissolved and used, while the silica microsphere suspension needs to remove the storage solvent (usually 20% ethanol solution) before use, and there is loss in the solvent removal process, so the present invention simplifies the use steps and reduces the loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The freeze-dried powder of silicon dioxide microspheres (tray-packed) is obtained by the freeze-drying process of the present invention;

[0035] Figure 2 The present invention provides freeze-dried silicon dioxide microsphere powder (tube-packed) obtained by the freeze-drying process of the present invention. DETAILED DESCRIPTION

[0036] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the present invention is further described in detail below in conjunction with specific embodiments.

[0037] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

[0038] The silica microsphere suspension applicable to this specific embodiment can be a silica microsphere ethanol suspension product currently available on the market, or a silica microsphere ethanol suspension product prepared by an existing method. The particle size is between 400nm and 600nm. The same batch of commercially available monodisperse silica microsphere ethanol suspension with a particle size of 500nm is used in the following embodiments.

[0039] Example 1

[0040] This embodiment provides a freeze-drying process for a silica microsphere suspension, which freeze-dries a silica microsphere reagent (20% ethanol solution) in a suspension state through a vacuum freeze-drying process to convert it into a solid (powder) state. The process includes the following steps:

[0041] 1) Pour the silica microsphere suspension to be freeze-dried into a dedicated freeze-drying tray (liquid level 3 mm).

[0042] 2) Set the freeze-drying parameters, see Table 1.

[0043] Table 1 Freeze-drying procedure of Example 1

[0044] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -30 1 atm 4:00 2 -20 100 3:00 3 -10 100 3:00 4 0 10 2:00 5 30 10 4:00

[0045] 3) After the freeze-drying process is completed, collect the freeze-dried powder and bottle it for storage.

[0046] Comparative Example 1

[0047] The difference from Example 1 is that the freeze-drying parameters are different. The parameter conditions of this comparative example are shown in Table 2.

[0048] Table 2 Freeze-drying procedure of Comparative Example 1

[0049] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -40 0 3:00 2 -30 0 1:00 3 -20 15 3:00 4 -10 15 3:00 5 10 1 2:00 6 20 1 3:00 7 20 1 8:00

[0050] Comparative Example 2

[0051] In this comparative example, a vacuum concentration method is used to dry the silica microsphere suspension, and the parameter conditions are: 45°C, 6-7mBar centrifugal concentration for 1.5-2h.

[0052] In order to verify the effect of the freeze-drying process of the present invention on the performance of silica microspheres, in this specific embodiment, the silica microsphere suspension (reagent stock solution) and the freeze-dried powder obtained in Example 1, Comparative Example 1, and Comparative Example 2 were also tested and compared with the reagent stock solution under 2-8°C and room temperature storage conditions. This test was performed using a Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer, and the data results were obtained from DIA-NN software analysis.

[0053] The test results are shown in Table 3 below. The data corresponding to each sample is the average of three tests.

[0054]

[0055] From the test results shown in Table 3, it can be seen that the performance of the silica microsphere suspension (reagent stock solution) is significantly reduced when stored at room temperature. When stored for 5 days, the peptide and protein identification amount has been significantly reduced (the reduction is more than 20%) compared with the storage temperature of 2~8℃ specified by the manufacturer. After 30 days, the reduction is more than 60%, and its function is basically unable to be realized, which shows that the reagent stock solution cannot be preserved at room temperature.

[0056] However, Example 1 of the present invention still has relatively high detection activity at 2 days and 5 days under normal temperature conditions. Even at 30 days, the activity loss is still not obvious, which does not affect the normal use of the silica microspheres.

[0057] The freeze-drying process used in Comparative Example 1 has a greater impact on the activity of the silica microspheres during the freeze-drying process. The activity of the obtained freeze-dried silica microsphere powder is significantly reduced compared to the activity of the reagent stock solution at 0 days. Although the activity loss is not obvious during the subsequent room temperature storage process, this method is still not suitable for freeze-drying of silica microsphere suspensions.

[0058] The freeze-drying process adopted in Comparative Example 2 causes a certain loss of activity during the freeze-drying process. Although the degree is less than that of Comparative Example 1, the activity of the silica microspheres will still be further reduced during the subsequent room temperature storage process, and is not suitable for room temperature storage.

[0059] Note: The activity of the reagent stock solution at day 0 is lower than that of Example 1. This is because the reagent stock solution needs to be washed before being used for detection to remove the ethanol in it so as not to affect the detection results, and some of it is lost during the washing process.

[0060] Example 2

[0061] This embodiment provides a freeze-drying process for a silica microsphere suspension, which freeze-dries a silica microsphere reagent (20% ethanol solution) in a suspension state through a vacuum freeze-drying process to convert it into a solid (powder) state. The process includes the following steps:

[0062] 1) Pour the silica microsphere suspension to be freeze-dried into a dedicated freeze-drying tray (liquid level 3 mm).

[0063] 2) Set the freeze-drying parameters, see Table 4.

[0064] Table 4 Freeze-drying procedure of Example 2

[0065] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -25 1 atm 3.0 2 -15 50 3.0 3 -5 50 3.5 4 5 2 2.5 5 20 2 4.0

[0066] 3) After the freeze-drying process is completed, collect the freeze-dried powder and bottle it for storage.

[0067] Example 3

[0068] This embodiment provides a freeze-drying process for a silica microsphere suspension, which freeze-dries a silica microsphere reagent (20% ethanol solution) in a suspension state through a vacuum freeze-drying process to convert it into a solid (powder) state. The process includes the following steps:

[0069] 1) Pour the silica microsphere suspension to be freeze-dried into a dedicated freeze-drying tray (liquid level 4 mm).

[0070] 2) Set the freeze-drying parameters, see Table 5.

[0071] Table 5 Freeze-drying procedure of Example 3

[0072] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -30 1 atm 3.5 2 -20 60 4.0 3 -10 60 3.5 4 10 10 2.5 5 20 10 6.0

[0073] 3) After the freeze-drying process is completed, collect the freeze-dried powder and bottle it for storage.

[0074] Example 4

[0075] This embodiment provides a freeze-drying process for a silica microsphere suspension, which freeze-dries a silica microsphere reagent (20% ethanol solution) in a suspension state through a vacuum freeze-drying process to convert it into a solid (powder) state. The process includes the following steps:

[0076] 1) Pour the silica microsphere suspension to be freeze-dried into a dedicated freeze-drying tray (liquid level 5 mm).

[0077] 2) Set the freeze-drying parameters, see Table 6.

[0078] Table 6 Freeze-drying procedure of Example 4

[0079] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -20 1 atm 04:00 2 -15 20 04:00 3 -10 20 04:00 4 0 5 02:00 5 30 5 06:00

[0080] 3) After the freeze-drying process is completed, collect the freeze-dried powder and bottle it for storage.

[0081] Example 5

[0082] This embodiment provides a freeze-drying process for a silica microsphere suspension, which freeze-dries a silica microsphere reagent (20% ethanol solution) in a suspension state through a vacuum freeze-drying process to convert it into a solid (powder) state. The process includes the following steps:

[0083] 1) Pour the silica microsphere suspension to be freeze-dried into a dedicated freeze-drying tray (liquid level should not exceed 5 mm).

[0084] 2) Set the freeze-drying parameters, see Table 7.

[0085] Table 7 Freeze-drying procedure of Example 5

[0086] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -25 1 atm 03:00 2 -10 80 04:00 3 -5 80 04:00 4 5 1 02:00 5 25 1 05:00

[0087] 3) After the freeze-drying process is completed, collect the freeze-dried powder and bottle it for storage.

[0088] Comparative Example 3

[0089] The difference from Example 1 is that the parameter control table 8 (vacuum degree is not controlled):

[0090] Table 8 Freeze-drying procedure of Comparative Example 3

[0091] Section Temperature (℃) Time (h) 1 -30 4:00 2 -20 3:00 3 -10 3:00 4 0 2:00 5 30 4:00

[0092] Comparative Example 4

[0093] The difference from Example 1 is that the parameter control is as shown in Table 9 (minus two sections):

[0094] Table 9 Freeze-drying procedure of Comparative Example 4

[0095] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -30 1 atm 4:00 2 -20 100 3:00 5 30 10 4:00

[0096] Comparative Example 5

[0097] The difference from Example 1 is that the parameter control is shown in Table 10 (the temperature of the first section is lowered):

[0098] Table 10 Freeze-drying procedure of Comparative Example 5

[0099] Section Temperature (℃) Vacuum degree (Pa) Time (h) 1 -40 1 atm 4:00 2 -30 100 3:00 3 -10 100 3:00 4 0 10 2:00 5 30 10 4:00

[0100] Comparative Example 6

[0101] The difference from Example 1 is that the depth of the suspension in the freeze-drying tray is 6 mm.

[0102] Comparative Example 7

[0103] The difference from Example 1 is that the depth of the suspension in the freeze-drying tray is 8 mm.

[0104] The performance of the freeze-dried silica microsphere powder obtained in Examples 2-5 and Comparative Examples 1-7 was tested. The test results are shown in Table 11 below.

[0105]

[0106] From the results shown in Table 11, it can be seen that in Examples 2 to 5 using the freeze-drying process of the present invention, not only is the activity of the silica microspheres essentially unchanged during the process, but the freeze-dried powder can also maintain the activity of the silica microspheres without significant decrease when stored at room temperature.

[0107] In the freeze-drying process of Comparative Example 3, the vacuum degree was not controlled. The results showed that the activity of the silica microspheres had already decreased significantly during the freeze-drying process, and during the subsequent storage at room temperature, the activity also decreased significantly with the passage of time.

[0108] In the freeze-drying process of the freeze-drying process adopted in Comparative Example 4, only three sections are set. Under this program, the activity of the silica microspheres has already shown a significant decrease during the freeze-drying process. During the subsequent storage process at room temperature, the activity is relatively stable with the passage of time, and there is no significant decrease.

[0109] In Comparative Example 5, the freeze-drying temperature of the first section was lowered. From the results, the activity of the silica microspheres had already dropped significantly during the freeze-drying process. During the subsequent storage at room temperature, the activity was relatively stable with the passage of time, without a significant drop.

[0110] In Comparative Example 6, during the freeze-drying process, the depth of the silica microsphere suspension in the freeze-drying dish was controlled at 6 mm. The results showed that not only did the activity of the silica microspheres decrease significantly during the freeze-drying process, but also during the subsequent storage at room temperature, the activity decreased significantly over time.

[0111] In Comparative Example 7, during the freeze-drying process, the depth of the silica microsphere suspension in the freeze-drying dish was controlled at 8 mm. The results showed that not only did the activity of the silica microspheres decrease significantly during the freeze-drying process, but also during the subsequent storage at room temperature, the activity decreased significantly over time.

Claims

1. A freeze-drying process for a silica microsphere suspension, characterized in that: The freeze-drying program includes the first section, the second section, the third section, the fourth section and the fifth section which are executed in sequence, and the specific parameters of each section are controlled as follows; The first section: -30~-20℃, vacuum degree 1atm, time 3h~4h; The second section: -20~-10℃, vacuum degree 20~100Pa, time 3h~4h; The third section: -10~0℃, vacuum degree 20~100Pa, time 3h~4h; The fourth section: 0~10℃, vacuum degree 0~10Pa, time 2h~3h; The fifth section: 20~30℃, vacuum degree 0~10Pa, time 4h~6h.

2. The freeze-drying process according to claim 1, characterized in that From the first section to the fourth section, the temperature difference between adjacent sections does not exceed 20°C at most.

3. The freeze-drying process according to claim 1 or 2, characterized in that: No lyoprotectants were added during the freeze-drying process.

4. The freeze-drying process according to claim 1, characterized in that: The freeze-drying process comprises the following steps: S1. Pour the silica microsphere suspension to be freeze-dried into a freeze-drying tray; S2. Setting the freeze-drying program and starting freeze-drying; S3. After the freeze-drying process is completed, the freeze-dried powder is collected.

5. The freeze-drying process according to claim 4, characterized in that: In step S1, the depth of the silica microsphere suspension in the freeze-drying tray does not exceed 5 mm.

6. The freeze-drying process according to claim 4, characterized in that: The operating environment humidity is below 30% and the ambient temperature is between 18℃ and 28℃.

7. A freeze-dried powder of silicon dioxide microspheres, characterized in that: Obtained by the freeze-drying process described in any one of claims 1 to 6.

8. The freeze-dried silica microsphere powder according to claim 7, characterized in that: The particle size of the freeze-dried silica microsphere powder is 400nm to 600nm.

9. The use of the freeze-dried silica microsphere powder according to any one of claims 7 to 8, characterized in that: Used for enrichment of low-abundance proteins.

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