Preparation method of protein freeze-drying preparation and high-concentration protein preparation

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a preparation method of a protein freeze-drying preparation and a high-concentration protein preparation. The preparation method of the protein freeze-dried preparation comprises the following steps: freeze-drying a protein solution with the protein concentration of more than or equal to 50mg / mL to obtain the protein freeze-dried preparation; the freeze drying comprises three stages of pre-freezing, primary drying and secondary drying. The high-concentration protein preparation is prepared by redissolving the protein freeze-drying preparation. According to the method, the freeze drying process is optimized, including the steps of improving the cooling / heating rate, adding the secondary annealing step, optimizing the temperature and time of the primary drying, directly heating the secondary drying and the like, so that key quality parameters such as the appearance, the redissolution time and the moisture of the cake of the high-concentration antibody preparation are remarkably improved, and clinical use is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing a freeze-dried protein preparation and a high-concentration protein preparation. Background Art

[0002] High-concentration protein drugs (>50 mg / mL) are usually administered by subcutaneous injection. Compared with intravenous administration, its advantage is that it reduces the treatment time and pain of patients. However, when the protein concentration is high, it leads to increased viscosity, solute concentration and phase separation, and the protein is more likely to aggregate particles and produce colloidal substances, which increases the instability of the preparation. Therefore, high-concentration protein drugs are usually developed in a lyophilized form to improve protein stability. However, the lyophilization process of high-concentration protein preparations faces many challenges, such as complex lyophilization process, poor cake structure, and long reconstitution time.

[0003] The prior art discloses a method for reducing the generation of visible bubbles when preparing a high-concentration monoclonal antibody preparation, comprising: (1) preparing a monoclonal antibody freeze-dried preparation: freeze-drying a monoclonal antibody solution containing a monoclonal antibody, a buffer salt, a protective agent and a surfactant to obtain a monoclonal antibody freeze-dried preparation; the concentration of the monoclonal antibody in the monoclonal antibody solution is ≥30 mg / mL; the freeze-drying comprises three stages: pre-freezing, primary drying and secondary drying, specifically comprising the following steps: S1 pre-freezing: equilibrating the monoclonal antibody solution at 5-10°C for 30-90 minutes; then cooling to 40°C for 0.5-3 hours. Keep frozen for 2 to 3 hours; then return to the temperature in 0.5 to 1 hour. Maintain for 2 to 3 hours; then cool down to Maintain freezing for 2 to 3 hours; S2 primary drying: Evacuate the vacuum to reduce the vacuum degree to 20-80 mTor; under the condition of vacuum degree of 20-80 mTor, heat to Maintain for 10 to 50 hours to complete the primary drying; S3 secondary drying: under the condition of a vacuum degree of 20 to 80 mTor, heat to 20 to 35 ° C in 2 to 8 hours, maintain for 5 to 10 hours for secondary drying, complete the freeze-drying process, and store for later use; (2) Preparation of high-concentration monoclonal antibody preparations: add water to the monoclonal antibody freeze-dried preparation to reconstitute the monoclonal antibody to prepare a high-concentration monoclonal antibody preparation with a monoclonal antibody concentration of ≥100 mg / mL. This method improves the freeze-drying process, adds an annealing step during the pre-freezing process, and combines it with specific freeze-drying process parameter settings to significantly reduce the volume of visible bubbles generated during the reconstitution of high-concentration monoclonal antibody preparations, greatly improving the efficiency of clinical medical staff. However, this method only mentions the problem of improving the problem of bubbles generated during the reconstitution of high-concentration preparations, and does not focus on the key quality parameters such as appearance, moisture content and reconstitution time. SUMMARY

[0004] In view of the above-mentioned disadvantages of the prior art, the present application aims to provide a preparation method of protein freeze-dried preparation and high-concentration protein preparation, so as to improve the quality of high-concentration protein freeze-dried preparation, reduce the reconstitution time of the product, and facilitate clinical use.

[0005] To achieve the above-mentioned and other related purposes, the present application provides a preparation method of protein freeze-dried preparation, comprising the following steps:

[0006] Freeze-drying the protein solution with a protein concentration of ≥50 mg / mL to obtain a protein freeze-dried preparation;

[0007] The freeze-drying comprises three stages of pre-freezing, primary drying and secondary drying;

[0008] The pre-freezing stage comprises the following steps:

[0009] First, the protein solution is balanced;

[0010] Then, the temperature is lowered to -45℃ to -40℃, and maintained for 2h to 3h for the first freezing;

[0011] Then, the temperature is raised to -20℃ to -10℃, and maintained for 1h to 2h for the first annealing;

[0012] Then, the temperature is lowered to -45℃ to -40℃, and maintained for 30min to 90min for the second freezing;

[0013] Then, the temperature is raised to -20℃ to -10℃, and maintained for 30min to 90min for the second annealing;

[0014] Finally, the temperature is lowered to -45℃ to -40℃, and maintained for 30min to 120min for the third freezing;

[0015] The cooling rate of the first freezing, the second freezing and the third freezing, and the heating rate of the first annealing and the second annealing are all 0.2℃ / min to 1℃ / min;

[0016] The primary drying stage comprises the following steps: under the condition of -45℃ to -40℃, vacuum is extracted to a preset vacuum degree, and under the condition of the preset vacuum degree, the temperature is raised to -20℃ to -10℃, and maintained for 20h to 30h for primary drying;

[0017] The secondary drying stage comprises the following steps: under the condition of the preset vacuum degree, the temperature is raised to 25℃ to 30℃, and maintained for 10h to 20h for secondary drying.

[0018] Furthermore, the equilibrium temperature is 0°C to 10°C, and the equilibrium time is 30 min to 90 min.

[0019] Furthermore, the cooling rates of the first freezing, the second freezing and the third freezing, and the heating rates of the first annealing and the second annealing are all 0.3°C / min to 1°C / min.

[0020] Furthermore, the heating rate in the primary drying stage is 0.2°C / min to 1°C / min.

[0021] Furthermore, the heating rate in the secondary drying stage is 0.2°C / min to 1°C / min.

[0022] Furthermore, the preset vacuum degree is 75mTorr to 150mTorr.

[0023] Furthermore, the preparation method of the protein freeze-dried preparation further comprises: after completing the secondary drying, returning the temperature to the sample storage temperature and storing it for future use.

[0024] Furthermore, the sample storage temperature is 2°C to 8°C.

[0025] Furthermore, the protein includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, fusion proteins and polypeptides.

[0026] Furthermore, the protein solution further comprises at least one of the following components: a buffer, a protective agent, a viscosity reducer, and a surfactant.

[0027] Furthermore, the moisture content of the protein freeze-dried preparation is 0.5% to 2%.

[0028] The present invention also provides a method for preparing a high-concentration protein preparation, comprising: re-dissolving the lyophilized protein preparation prepared by the above method to prepare a high-concentration protein preparation with a protein concentration of ≥50 mg / mL.

[0029] Furthermore, the re-dissolution time is 2 minutes to 12 minutes.

[0030] As described above, the preparation methods of the freeze-dried protein preparation and the high-concentration protein preparation of the present invention have the following beneficial effects:

[0031] The present invention optimizes the freeze-drying process, including improving the cooling / heating rates, adding a secondary annealing step, optimizing the primary drying temperature and time, directly heating the secondary drying, shortening the secondary drying time, etc., thereby significantly improving key quality parameters such as cake appearance, reconstitution time and moisture content of high-concentration protein preparations, greatly improving product quality and the efficiency of clinical medical staff. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Fig. 1 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0033] Fig. 2 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0034] Fig. 3 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0035] Fig. 4 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0036] Fig. 5 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0037] Fig. 6 Figure A shows the appearance of the sample of the antibody lyophilized preparation obtained in the lyophilization process (1) in Example 1 of the present application, and Figure B shows the appearance of the sample after reconstitution.

[0038] It should be noted that the front, middle and rear samples refer to the different positions of the samples placed in the front, middle and rear of the plate layer of the freeze dryer during lyophilization. Due to the different heat radiation at different positions in the freeze dryer during lyophilization, the product temperature at the front, middle and rear positions will have certain differences, and such temperature differences can cause differences in the appearance and moisture of the samples, so the products at different positions will be observed during the study to reduce the differences in the same batch of products. DETAILED DESCRIPTION

[0039] The embodiments of the present application will be described in detail with specific examples. Those skilled in the art can easily understand other advantages and effects of the present application from the disclosure. The present application can also be implemented or applied in other different embodiments, and the details in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application.

[0040] In the present application, the term "a plurality of" means two or more, unless otherwise specified.

[0041] The character " / " represents an "or" relationship between the objects before and after it. For example, A / B means: A or B.

[0042] The term "and / or" is a description of the relationship between the objects, which means that there can be three relationships. For example, A and / or B means: A or B, or, A and B, the three relationships.

[0043] An embodiment of the present application provides a preparation method of a protein freeze-dried preparation, comprising the following steps:

[0044] Freeze-drying the protein solution with a protein concentration of ≥50 mg / mL to obtain the protein freeze-dried preparation;

[0045] The freeze-drying comprises three stages of pre-freezing, primary drying and secondary drying;

[0046] The pre-freezing stage comprises the following steps:

[0047] Firstly, the protein solution is balanced at 0-10°C for 30-90 min;

[0048] Then, the temperature is lowered to -45 to -40°C for 2-3 h for the first freezing, and the cooling rate is 0.2-1°C / min;

[0049] Then, the temperature is raised to -20 to -10°C for 1-2 h for the first annealing, and the heating rate is 0.2-1°C / min;

[0050] Then, the temperature is lowered to -45 to -40°C for 30-90 min for the second freezing, and the cooling rate is 0.2-1°C / min;

[0051] Then, the temperature is raised to -20 to -10°C for 30-90 min for the second annealing, and the heating rate is 0.2-1°C / min;

[0052] Finally, the temperature is lowered to -45 to -40°C for 30-120 min for the third freezing, and the cooling rate is 0.2-1°C / min;

[0053] The primary drying stage comprises the following steps: vacuumizing at -45 to -40°C until the vacuum degree reaches a preset vacuum degree, and then raising the temperature to -20 to -10°C for 20-30 h for the primary drying;

[0054] The secondary drying stage comprises the following steps: raising the temperature to 25-30°C for 10-20 h for the secondary drying under the preset vacuum degree.

[0055] The above-mentioned embodiments of the present application significantly improve the cake appearance, reconstitution time and moisture of high-concentration protein preparations, and greatly improve the product quality and the use efficiency of medical personnel by optimizing the freeze-drying process, including improving the cooling / warming rate, adding a secondary annealing step, optimizing the primary drying temperature and time, directly warming in the secondary drying, and shortening the secondary drying time. Among them, increasing the cooling / warming rate within the above-mentioned range and increasing the pre-freezing rate can improve the moisture of the sample to a certain extent; increasing the primary annealing temperature and shortening the primary annealing time within the above-mentioned range can reduce the reconstitution time of the product; adding a secondary annealing step can significantly improve the cracking of the sample appearance; reducing the primary drying temperature within the above-mentioned range can reduce the primary drying rate and improve the appearance to a certain extent; directly warming in the secondary drying is more conducive to the removal of residual moisture in the secondary drying process than the stepwise warming in the secondary drying, thereby reducing the moisture of the freeze-dried sample. In general, the combined action of the above-mentioned methods can help to uniformly freeze-dried preparation cake on the one hand, so as to reduce the problem of high moisture of high-concentration preparation freeze-dried sample and cracking of high-concentration preparation freeze-dried cake, thereby improving the product quality; on the other hand, it can significantly reduce the reconstitution time of the product, greatly improving the use efficiency of medical personnel.

[0056] In some embodiments, the cooling rate of the first freezing, the second freezing and the third freezing, and the warming rate of the first annealing and the second annealing are all 0.3℃ / min-1℃ / min, preferably 0.5℃ / min-1℃ / min, more preferably 0.8℃ / min-1℃ / min.

[0057] In some embodiments, the warming rate of the primary drying stage is 0.2℃ / min-1℃ / min, preferably 0.2℃ / min-0.5℃ / min, more preferably 0.2℃ / min-0.3℃ / min.

[0058] In some embodiments, the warming rate of the secondary drying stage is 0.2℃ / min-1℃ / min, preferably 0.2℃ / min-0.6℃ / min, more preferably 0.4℃ / min-0.6℃ / min.

[0059] In some embodiments, the preset vacuum degree is 75mTorr-150mTorr, more preferably 80mTorr-120mTorr. During the freeze-drying process, appropriate vacuum degree helps to accelerate the sublimation process of ice crystals. Adjusting the vacuum degree within the above-mentioned range according to the actual situation is conducive to achieving the best drying effect. Generally, the higher the vacuum degree, the faster the sublimation speed; but too high vacuum degree may cause the product surface to be over-dried, affecting the product quality; too low vacuum degree will cause incomplete sublimation of ice crystals, resulting in too high residual moisture and affecting the product quality.

[0060] In some embodiments, the method for preparing the protein lyophilized preparation further comprises: after the secondary drying is completed, the temperature is adjusted back to a sample storage temperature, and the sample is stored for standby. The sample storage temperature is, for example, 2-8°C.

[0061] In some embodiments, the protein comprises a monoclonal antibody, a polyclonal antibody, a bispecific antibody, a fusion protein, and a polypeptide.

[0062] In some embodiments, the protein solution further comprises a buffer, a protective agent, a viscosity-reducing agent, and a surfactant. The buffer comprises, but is not limited to, a histidine / histidine hydrochloride buffer, an acetic acid buffer, a phosphate buffer, a citric acid buffer, a succinic acid buffer, and the like. The protective agent comprises, but is not limited to, trehalose, mannitol, sucrose, sorbitol, and the like. The viscosity-reducing agent comprises, but is not limited to, glutamic acid, proline, arginine hydrochloride, sodium chloride, glycine, and a combination of any two or more of the above, and the like. The surfactant comprises, but is not limited to, a Tween series, poloxamer P188, and the like.

[0063] Further, in some embodiments, the concentration of the buffer in the protein solution is 5-50 mM, the pH is 5.0-7.0, the concentration of the protective agent is 2%-12%, the concentration of the viscosity-reducing agent is 10-200 mM, and the concentration of the surfactant is 0.01-2 mg / mL.

[0064] In some embodiments, the protein lyophilized preparation has a moisture content of 0.5%-2%, preferably 0.5%-1%. The lyophilization process provided by the present application can significantly reduce the moisture content of the product and improve the quality of the product.

[0065] Another embodiment of the present application provides a method for preparing a high-concentration protein preparation, comprising: reconstituting the protein lyophilized preparation prepared by the method described above to prepare a high-concentration protein preparation with a protein concentration of ≥50 mg / mL. In the technical field, a concentration greater than 50 mg / mL is generally considered to be a high-concentration protein preparation, and there is no upper limit. Due to the limitations of actual production, the concentration of an antibody product generally does not exceed 220 mg / mL. Therefore, the protein concentration of the high-concentration protein preparation of the present application is preferably 50-220 mg / mL.

[0066] In some embodiments, the reconstitution time is 2-12 min, preferably 2-3 min. The lyophilization process provided by the present application can significantly shorten the reconstitution time of the product and greatly improve the use efficiency of clinical medical staff.

[0067] The following specific examples are provided to further illustrate the present application. It is also to be understood that the following examples are only used to illustrate the present application and should not be construed as limiting the scope of the present application. Any modification and adjustment made by those skilled in the art according to the above description of the present application are within the scope of the present application. The following examples are only one example in the suitable range, i.e. those skilled in the art can make suitable selection within the range according to the description herein, and are not limited to the specific values in the following examples.

[0068] Example 1

[0069] 1. Preparation of the chamber and the laboratory: clean the freeze-drying machine room and the freeze-drying machine, and reduce the temperature of the laboratory environment, preferably below 21°C, to reduce the radiant heat.

[0070] 2. Preparation of the sample: filter the sample through a 0.22 μm filter membrane in a clean bench, and then dispense into clean 2 mL vials, half-capped. Add 0.5 mL of the antibody solution to each vial. The antibody solution contains 150 mg / mL of monoclonal antibody, 20 mM of histidine-histidine hydrochloride buffer, 40 mg / mL of trehalose, 20 mg / mL of mannitol, 0.1 mg / mL of polysorbate 80, and 30 mM of glutamic acid and 200 mM of proline.

[0071] 3. Freeze-dry the antibody solution according to the freeze-drying processes (I) to (VI) shown in Tables 1 to 6, respectively, to obtain the antibody lyophilized preparation. Seal the vials by pressing the stopper under a vacuum of 100 mTorr, and store in a refrigerator at 2 to 8°C for later use.

[0072] Table 1. Freeze-drying process (I)

[0073]

[0074] Table 2. Freeze-drying process (II)

[0075]

[0076]

[0077] Table 3. Freeze-drying process (III)

[0078]

[0079] Table 4. Freeze-drying process (IV)

[0080]

[0081] Table 5. Freeze-drying process (V)

[0082]

[0083]

[0084] Table 6. Lyophilization process (six)

[0085]

[0086] 4. Analysis of lyophilization results:

[0087] The antibody lyophilized preparations obtained by using lyophilization processes (one) ~ (six) were subjected to moisture detection, observation of appearance after lyophilization, detection of reconstitution time and observation of appearance after reconstitution, and the results are shown in Table 7 and Figs. 1~6 .

[0088] Table 7. Sample information and lyophilization results of antibody lyophilized preparations obtained by using lyophilization processes (one) ~ (six)

[0089]

[0090]

[0091] According to the analysis of Tables 1 ~ 7 and Figs. 1~6 , it can be known that:

[0092] The sample appearance of lyophilization process (one) had obvious cracking phenomenon, which may be due to the slow pre-freezing rate, high local concentration of the sample during pre-freezing, non-uniform cake shape, poor sublimation channel formation, leading to sample cracking; the sample had high moisture, which may be due to the unsmooth sublimation channel leading to the inability of water to escape during the first drying process; the sample reconstitution time was about 17 min, and the reconstitution time was relatively long.

[0093] Lyophilization process (two) was optimized based on the test results of lyophilization process (one), considering increasing the cooling rate, fast pre-freezing to make the sample more uniform during pre-freezing, and considering that the second drying time was relatively long, which may increase the cracking of the cake, so the second drying time was also shortened. According to the moisture results, increasing the pre-freezing rate had certain improvement on the sample moisture; moreover, the sample reconstitution time was about 12 min, and the reconstitution time was obviously shortened; but the sample appearance had obvious cracking phenomenon, which may be due to the high temperature during the first drying process, leading to fast sublimation rate, damage to the sublimation channel, and increase of the moisture and cracking of the cake.

[0094] The freeze-drying process (three) was optimized based on the results of the freeze-drying process (two), and the primary drying temperature was reduced to reduce the primary drying rate. According to the calculation of the sample Tg' = -27℃, the primary drying temperature of the freeze-drying process (three) was set to -20℃, and the primary drying time was adjusted to 1800 min according to the actual freeze-drying process. According to the sample appearance results, the primary drying temperature was slightly increased because the appearance of the sample at the front of the box was significantly better than that at the back of the box. The moisture results showed that the sample moisture increased significantly, indicating that the primary drying temperature of the freeze-drying process (three) was too low, and in addition, the stepwise temperature increase in the secondary drying stage may affect the removal of residual moisture during the secondary drying process. The sample reconstitution time was about 17 min 21 s, and the reconstitution time was relatively long.

[0095] The freeze-drying process (four) was optimized based on the results of the freeze-drying process (three), and the primary drying temperature was increased to -15℃ and the stepwise temperature increase in the secondary drying stage was cancelled. The moisture results showed that the secondary drying temperature significantly improved the product moisture. According to the sample appearance results, the freeze-dried sample still had slight cracking, and since the sample concentration was high, the annealing process was modified to improve the sample appearance. The sample reconstitution time was about 11 min 43 s, which was significantly improved compared to the previous process.

[0096] Based on the results of the freeze-drying process (four), the moisture and reconstitution time were significantly improved, but the sample appearance still had cracking. The annealing process can improve the cracking of the sample, so the freeze-drying process (five) tried to modify the annealing process in the pre-freezing stage to improve the sample appearance. The specific modification was to increase the annealing temperature and shorten the annealing time. The sample reconstitution time of the freeze-drying process (five) was about 3 min, which was significantly improved compared to the previous process. The reconstitution time results showed that the freeze-drying process (five) significantly improved the product reconstitution time; however, according to the sample appearance results, it did not significantly improve the sample appearance, and the freeze-dried sample still had slight cracking.

[0097] Based on the results of the freeze-drying process (five), the sample reconstitution time was significantly improved, but the sample appearance still had cracking. The freeze-drying process (six) still tried to improve the sample appearance by changing the pre-freezing stage process, and the specific modification was to add a second annealing step. The sample reconstitution time of the freeze-drying process (six) was about 2 min 54 s, which was relatively fast. According to the sample appearance results, the sample had no obvious cracks, indicating that the freeze-drying process (six) significantly improved the sample appearance.

[0098] After six rounds of freeze-drying process development, the sample moisture decreased from 5.79% to 1.08%, the reconstitution time decreased from 17 min to 3 min, and the cracking of the sample appearance was significantly improved. The freeze-drying process (six) was the optimal process for this sample.

[0099] In summary, by optimizing the freeze-drying process parameters, including increasing the cooling / heating rate, adding a secondary annealing step, reducing the primary drying temperature, directly increasing the secondary drying temperature, and shortening the secondary drying time, on the one hand, it is helpful to uniform the pore size of the freeze-dried cake and make the structure uniform, thereby reducing the problem of high moisture in high-concentration preparation freeze-dried samples and cracking of high-concentration preparation freeze-dried cakes, thereby improving product quality; on the other hand, it can significantly reduce the product reconstitution time and greatly improve the use efficiency of clinical medical staff.

[0100] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for preparing a freeze-dried protein preparation, characterized in that: The steps include: freeze-drying a protein solution having a protein concentration of ≥50 mg / mL to obtain a lyophilized protein preparation; The freeze drying comprises three stages: pre-freezing, primary drying and secondary drying; The pre-freezing stage comprises the following steps: First, the protein solution is balanced; Then cool to -45℃~-40℃ and maintain for 2h~3h for the first freezing; Then return the temperature to -20℃~-10℃ and maintain for 1h~2h for the first annealing; Then cool it down to -45℃~-40℃ and keep it for 30min~90min for the second freezing; Then return the temperature to -20℃~-10℃ and maintain for 30min~90min for the second annealing; Finally, cool to -45℃~-40℃ and maintain for 30min~120min for the third freezing; The cooling rates of the first freezing, second freezing and third freezing, as well as the heating rates of the first annealing and second annealing are all 0.2°C / min to 1°C / min; The primary drying stage comprises the following steps: vacuuming at -45°C to -40°C to reduce the vacuum degree to a preset vacuum degree, heating to -20°C to -10°C under the preset vacuum degree condition, and maintaining the temperature for 20h to 30h for primary drying; The secondary drying stage comprises the following steps: under a preset vacuum degree, heating to 25° C. to 30° C. and maintaining for 10 hours to 20 hours for secondary drying.

2. The method for preparing a freeze-dried protein preparation according to claim 1, wherein: The equilibrium temperature is 0℃~10℃, and the equilibrium time is 30min~90min.

3. The method for preparing a freeze-dried protein preparation according to claim 1, wherein: The cooling rates of the first freezing, the second freezing and the third freezing, and the heating rates of the first annealing and the second annealing are all 0.3°C / min to 1°C / min.

4. The method for preparing a freeze-dried protein preparation according to claim 1, wherein: The heating rate of the primary drying stage is 0.2°C / min to 1°C / min; And / or, the heating rate in the secondary drying stage is 0.2° C. / min to 1° C. / min.

5. The method for preparing a freeze-dried protein preparation according to claim 1, wherein: The preset vacuum degree is 75 mTorr to 150 mTorr.

6. The method for preparing a freeze-dried protein preparation according to any one of claims 1 to 5, characterized in that: The preparation method of the protein freeze-dried preparation further includes: after completing the secondary drying, returning the temperature to the sample storage temperature and storing it for future use.

7. The method for preparing a freeze-dried protein preparation according to any one of claims 1 to 5, characterized in that: The proteins include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, fusion proteins and polypeptides; And / or, the protein solution further comprises at least one of the following components: a buffer, a protective agent, a viscosity reducer, and a surfactant.

8. The method for preparing a freeze-dried protein preparation according to any one of claims 1 to 5, characterized in that: The moisture content of the protein freeze-dried preparation is 0.5% to 2%.

9. A method for preparing a high-concentration protein preparation, characterized in that: include: The lyophilized protein preparation prepared by the method according to any one of claims 1 to 8 is reconstituted to prepare a high-concentration protein preparation with a protein concentration of ≥50 mg / mL.

10. The method for preparing a high-concentration protein preparation according to claim 9, wherein: The re-dissolution time is 2 minutes to 12 minutes.

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