Freeze-drying process and application thereof

Abstract

The invention relates to the technical field of medicine production, and discloses a freeze-drying process and application thereof. The freeze-drying process comprises the step of enabling a sample to sequentially pass through a pre-freezing stage, a primary drying stage and a secondary drying stage to obtain a freeze-dried product, in the pre-freezing stage, a cooling-heating-cooling mode is adopted, and the temperature change range is-50 DEG C to-10 DEG C; the primary drying stage comprises three times of slope heating, and the temperature change range is-50 DEG C to-2 DEG C; the secondary drying stage comprises three times of slope heating, and the temperature change range is-2 DEG C to 30 DEG C. According to the freeze-drying process provided by the invention, by adopting a cooling-heating-cooling pre-freezing mode, the crystallization rate of the sample is effectively improved, the water solubility of the freeze-dried sample is good, the sublimation drying time is effectively shortened, and the freeze-drying efficiency is accelerated; and in the primary drying stage and the secondary drying stage, the heating temperature parameter and the vacuum degree are controlled, so that the freeze-dried sample shows a relatively good appearance and has a proper water content.

Description

Technical Field

[0001] The present invention relates to the technical field of drug production, and in particular to a freeze-drying process and application thereof. Background Art

[0002] As a new type of gene therapy drug, oligonucleotide drugs have a wide range of clinical applications. Due to the instability of oligonucleotides, they usually need to be converted into lyophilized powder before production or use, which places high demands on the lyophilization process of oligonucleotides.

[0003] At present, there are few studies on the freeze-drying process of oligonucleotide APIs, and the parameters of the freeze-drying process will affect the key quality indicators of oligonucleotide APIs such as solubility, water content, purity, etc. Therefore, developing a freeze-drying process and optimizing the process parameters are of great significance for improving the quality of oligonucleotide APIs and ensuring drug safety. Summary of the invention

[0004] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, one of the purposes of the present invention is to provide a freeze-drying process; the second purpose of the present invention is to provide an application of the freeze-drying process.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A first aspect of the present invention provides a freeze-drying process, comprising subjecting a sample to a pre-freezing stage, a primary drying stage, and a secondary drying stage in sequence to obtain a freeze-dried product;

[0007] The pre-freezing stage adopts a cooling-heating-cooling method, and the temperature variation range is -50°C to -10°C;

[0008] The primary drying stage includes three slope temperature increases, and the temperature variation range is -50°C to -2°C;

[0009] The secondary drying stage includes three slope temperature increases, and the temperature variation range is -2°C to 30°C.

[0010] Preferably, the pre-freezing stage adopts a cooling-heating-cooling method, and the temperature variation range is -42°C to -10°C.

[0011] Preferably, the primary drying stage includes three slope temperature increases, and the temperature variation range is -42°C to -3°C.

[0012] Preferably, the secondary drying stage includes three slope temperature increases, and the temperature variation range is -3°C to 26°C.

[0013] Preferably, the thickness of the sample is 0.5 to 1.5 cm; further preferably, the thickness of the sample is 1.0 to 1.2 cm.

[0014] Preferably, the concentration of the sample is 5-45 mg / L; further preferably, the concentration of the sample is 6-15 mg / L.

[0015] Preferably, the pre-freezing stage specifically comprises the following steps:

[0016] S11, cooling to -50℃~-40℃ once;

[0017] S12, raise the temperature to -15℃~-10℃, keep warm for 50~130min;

[0018] S13, cool down to -50℃~-40℃ for the second time, and keep warm for 120~200min.

[0019] Further preferably, the pre-freezing stage specifically comprises the following steps:

[0020] S11, once cooling to -42℃~-40℃;

[0021] S12, raise the temperature to -12℃~-10℃, keep warm for 60~120min;

[0022] S13, cool down to -42℃~-40℃ for the second time and keep warm for 120~180min.

[0023] Preferably, the time for the cooling down once in step S11 is 40 to 90 minutes; further preferably, the time for the cooling down once in step S11 is 50 to 80 minutes.

[0024] Preferably, the heating time in step S12 is 50 to 100 minutes; further preferably, the heating time in step S12 is 50 to 90 minutes.

[0025] Preferably, the time for the secondary cooling in step S13 is 20 to 60 minutes; further preferably, the time for the secondary cooling in step S13 is 30 to 50 minutes.

[0026] Preferably, the primary drying stage specifically comprises the following steps:

[0027] S21, heating to -35°C to -30°C at a rate of 0.1 to 0.5°C / min;

[0028] S22, heating to -15°C to -10°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 minutes;

[0029] S23. Raise the temperature to -5°C to -2°C at a rate of 0.1 to 0.6°C / min and keep warm for 20 to 35 hours.

[0030] Further preferably, the primary drying stage specifically comprises the following steps:

[0031] S21, heating to -35°C to -33°C at a rate of 0.1 to 0.3°C / min;

[0032] S22, raise the temperature to -15°C to -12°C at a rate of 0.1 to 0.4°C / min, and keep warm for 90 to 120 minutes;

[0033] S23. Raise the temperature to -5°C to -3°C at a rate of 0.1 to 0.3°C / min and keep warm for 20 to 30 hours.

[0034] Preferably, the vacuum degree of the primary drying stage is 10-30 Pa; further preferably, the vacuum degree of the primary drying stage is 15-30 Pa.

[0035] Preferably, the secondary drying stage specifically comprises the following steps:

[0036] S31, heating to 3°C to 7°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 min;

[0037] S32, heating to 12°C to 17°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 min;

[0038] S33. Raise the temperature to 23°C-30°C at a rate of 0.1-0.6°C / min and keep warm for 10-20 hours.

[0039] Further preferably, the secondary drying stage specifically comprises the following steps:

[0040] S31, heating to 4°C to 6°C at a rate of 0.1 to 0.3°C / min, and keeping warm for 90 to 120 min;

[0041] S32, heating to 14°C to 16°C at a rate of 0.1 to 0.3°C / min, and keeping warm for 90 to 120 min;

[0042] S33. Raise the temperature to 24-26°C at a rate of 0.1-0.3°C / min and keep warm for 10-15 hours.

[0043] Preferably, the vacuum degree of the secondary drying stage is 1-7Pa; further preferably, the vacuum degree of the secondary drying stage is 1-5Pa.

[0044] The second aspect of the present invention provides the application of the freeze-drying process described in the first aspect of the present invention in the preparation of raw materials.

[0045] Preferably, the API comprises an oligonucleotide API.

[0046] The basic principle of the present invention is described as follows:

[0047] The freeze-drying process provided by the present invention is divided into a pre-freezing stage, a primary drying stage and a secondary drying stage. An annealing process is adopted in the pre-freezing stage, and the method of first cooling, then heating and then cooling again can improve the crystallization rate of the sample, shorten the time of sublimation drying, and improve the solubility of the freeze-dried product.

[0048] The primary drying stage and the secondary drying stage are the sublimation drying stage and the analytical drying stage respectively. By controlling the temperature parameters and the vacuum degree, the control over the appearance and moisture of the sample can be enhanced; by controlling the heating rate, the ice crystals are heated more evenly, and the moisture in the sample can be better removed.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1) The freeze-drying process provided by the present invention effectively improves the crystallization rate of the sample by adopting a pre-freezing method of cooling-heating-cooling, so that the water solubility of the freeze-dried sample is better, and the sublimation drying time is effectively shortened, thereby accelerating the freeze-drying efficiency; in the primary drying stage and the secondary drying stage, the heating temperature parameters and the vacuum degree are controlled, so that the freeze-dried sample presents a better appearance and has an appropriate water content;

[0051] 2) When the freeze-drying process provided by the present invention is applied to the preparation of raw materials such as oligonucleotide raw materials, compared with the prior art, it can obtain high-quality raw materials with good water solubility, high purity, low water content and no shrinkage, which can ensure the quality of the product within the validity period, make the medication safer, and is suitable for industrial mass production. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0053] Example 1

[0054] In this example, the oligonucleotide API was freeze-dried, the sample thickness was 1.0 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0055] Pre-freezing stage: adopt the method of cooling-heating-cooling, the specific steps are:

[0056] S111, first cooling to -40℃, cooling time is 50min;

[0057] S121, heat to -10℃, heating time is 60min, heat preservation time is 60min;

[0058] S131, cooling to -40℃ for the second time, cooling time is 50min, keeping temperature for 120min;

[0059] Primary drying stage: three-slope heating, maintaining the vacuum degree at 30Pa, the specific steps are:

[0060] S211, heating to -35°C at a rate of 0.16°C / min;

[0061] S221, heating to -15°C at a rate of 0.33°C / min, keeping warm for 90 min;

[0062] S231, raise the temperature to -5°C at a rate of 0.16°C / min, and keep warm for 24h;

[0063] Secondary drying stage: Use three-slope heating to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0064] S311, heating to 5°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0065] S321, heating to 15°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0066] S322. Raise the temperature to 25°C at a rate of 0.16°C / min and keep warm for 10 hours.

[0067] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0068] Example 2

[0069] In this example, the oligonucleotide API was freeze-dried, the sample thickness was 1.2 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0070] Pre-freezing stage: adopt the method of cooling-heating-cooling, the specific steps are:

[0071] S111, first cooling to -40℃, cooling time is 50min;

[0072] S121, heating to -10℃, heating time is 60min, keeping temperature for 100min;

[0073] S131, cooling to -40℃ for the second time, cooling time is 50min, and heat preservation time is 150min;

[0074] Primary drying stage: three-slope heating, maintaining the vacuum degree at 30Pa, the specific steps are:

[0075] S211, heating to -35°C at a rate of 0.2°C / min;

[0076] S221, heating to -15°C at a rate of 0.38°C / min, keeping warm for 90 min;

[0077] S231, raise the temperature to -5°C at a rate of 0.2°C / min, and keep warm for 30h;

[0078] Secondary drying stage: Use three-slope heating to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0079] S311, heating to 5°C at a rate of 0.21°C / min, keeping warm for 120 min;

[0080] S321, heating to 15°C at a rate of 0.21°C / min, keeping warm for 120 min;

[0081] S322. Raise the temperature to 25°C at a rate of 0.21°C / min and keep warm for 12 hours.

[0082] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0083] Comparative Example 1

[0084] In this comparative example, the oligonucleotide API was freeze-dried, the sample thickness was 1.0 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0085] Pre-freezing stage: adopt one-time cooling method, the specific steps are:

[0086] S111, cool to -40℃, cool for 50min, keep warm for 180min;

[0087] Primary drying stage: three-slope heating, maintaining the vacuum degree at 30Pa, the specific steps are:

[0088] S211, heating to -35°C at a rate of 0.16°C / min;

[0089] S221, heating to -15°C at a rate of 0.33°C / min, keeping warm for 90 min;

[0090] S231, raise the temperature to -5°C at a rate of 0.16°C / min, and keep warm for 30h;

[0091] Secondary drying stage: Use three-slope heating to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0092] S311, heating to 5°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0093] S321, heating to 15°C at a rate of 0.33°C / min, keeping temperature for 90 min;

[0094] S322. Raise the temperature to 25°C at a rate of 0.33°C / min and keep warm for 12 hours.

[0095] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0096] Comparative Example 2

[0097] In this comparative example, the oligonucleotide API was freeze-dried, the sample thickness was 1.0 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0098] Pre-freezing stage: four cooling methods are adopted, and the specific steps are as follows:

[0099] S111, once cooling to -10℃, cooling time is 30min;

[0100] S112, cooling down to -20℃ for 30min;

[0101] S113, cooling to -30°C for three times, cooling time is 30min;

[0102] S114, cooling to -40℃ for four times, cooling time is 30min, keeping temperature for 120min;

[0103] Primary drying stage: three-slope heating, maintaining the vacuum degree at 30Pa, the specific steps are:

[0104] S211, heating to -35°C at a rate of 0.16°C / min;

[0105] S221, heating to -15°C at a rate of 0.33°C / min, keeping warm for 90 min;

[0106] S231, raise the temperature to -5°C at a rate of 0.16°C / min, and keep warm for 30h;

[0107] Secondary drying stage: Use three-slope heating to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0108] S311, heating to 5°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0109] S321, heating to 15°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0110] S322. Raise the temperature to 25°C at a rate of 0.16°C / min and keep warm for 13 hours.

[0111] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0112] Comparative Example 3

[0113] In this comparative example, the oligonucleotide API was freeze-dried, the sample thickness was 1.0 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0114] Pre-freezing stage: adopt one-time cooling method, the specific steps are:

[0115] S111, cool to -40℃, keep warm for 180min;

[0116] Primary drying stage: three-slope heating, maintaining the vacuum degree at 30Pa, the specific steps are:

[0117] S211, heating to -35°C at a rate of 0.16°C / min;

[0118] S221, heating to -15°C at a rate of 0.33°C / min, keeping warm for 90 min;

[0119] S231, raise the temperature to -5°C at a rate of 0.16°C / min, and keep warm for 31h;

[0120] Secondary drying stage: Use three-slope heating to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0121] S311, heating to 5°C at a rate of 0.16°C / min, keeping warm for 90 min;

[0122] S321, heating to 15°C at a rate of 0.33°C / min, keeping temperature for 90 min;

[0123] S322. Raise the temperature to 25°C at a rate of 0.33°C / min and keep warm for 12 hours.

[0124] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0125] Comparative Example 4

[0126] In this comparative example, the oligonucleotide API was freeze-dried, the sample thickness was 1.2 cm, the concentration was 10 mg / mL, and the freeze-drying process was as follows:

[0127] Pre-freezing stage: adopt the method of cooling-heating-cooling, the specific steps are:

[0128] S111, the first cooling is to -40℃, the cooling time is 50min, and the heat preservation time is 80min;

[0129] S121, heating to -10℃, heating time is 60min, keeping temperature for 100min;

[0130] S131, cooling to -40℃ for the second time, cooling time is 50min, and heat preservation time is 150min;

[0131] Primary drying stage: using two slope heating steps to maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0132] S211, heating to -35°C at a rate of 0.16°C / min;

[0133] S221, raise the temperature to -5°C at a rate of 0.5°C / min, and keep warm for 40h;

[0134] Secondary drying stage: adopt a slope heating method and maintain the vacuum degree at 2Pa. The specific steps are as follows:

[0135] S311. Raise the temperature to 25°C at a rate of 0.5°C / min and keep warm for 18 hours.

[0136] After the above pre-freezing stage, primary drying stage and secondary drying stage, freeze-dried oligonucleotide raw materials are obtained.

[0137] Performance Testing

[0138] The water solubility, purity and water content of the lyophilized oligonucleotide APIs obtained in Examples 1 and 2 and Comparative Examples 1-4 were tested, and the appearance properties were compared to evaluate the effects of the lyophilization processes in Examples 1 and 2 and Comparative Examples 1-4.

[0139] Table 1 Performance test results of freeze-dried oligonucleotide APIs in Examples 1, 2 and Comparative Examples 1-4

[0140]

[0141]

[0142] Table 1 shows the performance test results of the freeze-dried oligonucleotide APIs in Examples 1, 2 and Comparative Examples 1-4. It can be seen from Table 1 that the freeze-drying process provided by the present invention is used to treat the oligonucleotide APIs in Examples 1 and 2, and the resulting products are white loose blocks in appearance, with good solubility, high purity, low water content, and good quality. There is no annealing process in Comparative Example 1, and the primary drying time is long, with a total time consumption of 34 hours; Comparative Example 2 adopts a slow freezing method, resulting in a long sample re-dissolution time; Comparative Example 3 adopts a fast freezing method, resulting in a long primary drying time, with a total time consumption of 34 hours; Comparative Example 4 does not control the heating rate during the drying stage, and the temperature rises quickly, resulting in a faster escape of water, affecting the structure of the sample skeleton, and then affecting the appearance of the sample, resulting in granular protrusions on the surface.

[0143] It can be seen that by adopting the freeze-drying process provided by the present invention, annealing is adopted in the pre-freezing stage, and the crystallization rate of the sample can be improved through a cooling-heating-cooling treatment process, so that the solubility of the freeze-dried product is good, and the sublimation drying time can be shortened; in the drying stage, the parameters such as the heating rate, heating temperature and vacuum degree are strictly controlled, so that the ice crystals can be heated evenly, which is manifested in the good appearance and water solubility of the product, and the purity of the sample after freeze-drying is still high.

Claims

1. A freeze-drying process, characterized in that: The method comprises subjecting the sample to a pre-freezing stage, a primary drying stage and a secondary drying stage in sequence to obtain a freeze-dried product; The pre-freezing stage adopts a cooling-heating-cooling method, and the temperature variation range is -50°C to -10°C; The primary drying stage includes three slope temperature increases, and the temperature variation range is -50°C to -2°C; The secondary drying stage includes three slope temperature increases, and the temperature variation range is -2°C to 30°C.

2. The freeze-drying process according to claim 1, characterized in that The thickness of the sample is 0.5 to 1.5 cm; And / or, the concentration of the sample is 5-45 mg / L.

3. The freeze-drying process according to claim 1, characterized in that: The pre-freezing stage specifically comprises the following steps: S11, cooling to -50℃~-40℃ once; S12, raise the temperature to -15℃~-10℃, keep warm for 50~130min; S13, cool down to -50℃~-40℃ for the second time, and keep warm for 120~200min.

4. The freeze-drying process according to claim 3, characterized in that: The time of cooling once in step S11 is 40 to 90 minutes; And / or, the heating time in step S12 is 50 to 100 minutes; And / or, the time of the secondary cooling in step S13 is 20 to 60 minutes.

5. The freeze-drying process according to claim 1, characterized in that: The primary drying stage specifically comprises the following steps: S21, heating to -35°C to -30°C at a rate of 0.1 to 0.5°C / min; S22, heating to -15°C to -10°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 minutes; S23. Raise the temperature to -5°C to -2°C at a rate of 0.1 to 0.6°C / min and keep warm for 20 to 35 hours.

6. The freeze-drying process according to claim 5, characterized in that: The vacuum degree in the primary drying stage is 10-30Pa.

7. The freeze-drying process according to claim 1, characterized in that: The secondary drying stage specifically comprises the following steps: S31, heating to 3°C to 7°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 min; S32, heating to 12°C to 17°C at a rate of 0.1 to 0.6°C / min, and keeping warm for 80 to 130 min; S33. Raise the temperature to 23°C-30°C at a rate of 0.1-0.6°C / min and keep warm for 10-20 hours.

8. The freeze-drying process according to claim 7, characterized in that: The vacuum degree in the secondary drying stage is 1-7Pa.

9. Use of the freeze-drying process according to any one of claims 1 to 8 in the preparation of bulk drugs.

10. The use according to claim 9, characterized in that: The APIs include oligonucleotide APIs.

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