Recombinant human TGF-beta1 protein freeze-dried preparation and preparation method thereof

Abstract

The invention discloses a recombinant human TGF-beta1 protein freeze-dried preparation and a preparation method thereof, provides a brand-new recombinant human TGF-beta1 protein freeze-dried preparation for the field, and provides a related preparation method and medical application of the recombinant human TGF-beta1 protein freeze-dried preparation, and solves the problems that the TGF-beta1 protein is unstable, easy to aggregate and denature, low in activity and the like. The preparation is convenient to use, quick to absorb and convenient to store and transport, and has good industrial popularization and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of protein freeze-dried preparation, and specifically relates to a recombinant human TGF-β1 protein freeze-dried preparation and a preparation method thereof. Background Art

[0002] Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that plays a vital role in the body. TGF-β1 is a dimeric protein composed of two identical polypeptide chains connected by disulfide bonds. Each polypeptide chain contains 112 amino acids and has a molecular weight of about 25kDa. Its three-dimensional structure presents a unique "hand-like" structure, including an N-terminal "finger" domain and a C-terminal "palm" domain. This structure is essential for its binding to receptors and signal transduction. Recombinant human TGF-β1 protein is a protein with important biological functions synthesized by genetic engineering technology. Recombinant human TGF-β1 protein has multiple biological functions, including cell growth and differentiation regulation, extracellular matrix synthesis and regulation, immune regulation function, etc.

[0003] Since TGF-β plays a key role in the occurrence and development of many diseases, it has become a potential target for the treatment of many diseases. For example, in tissue fibrosis, by inhibiting the signal transduction or activity of TGF-β1, the synthesis and deposition of extracellular matrix can be reduced, thereby delaying the progression of the disease; in tumor treatment, drug development targeting the TGF-β1 signaling pathway is also underway, which is expected to provide new strategies and methods for the treatment of tumors. In the field of tissue engineering and regenerative medicine, TGF-β1 can be used as an important bioactive factor to promote tissue repair and regeneration. For example, after TGF-β1 is compounded with biomaterials, it can be made into a scaffold material with the function of inducing tissue regeneration, which is used to treat diseases such as bone defects, cartilage damage, and skin trauma.

[0004] In basic medical research, the lyophilized preparation of recombinant human TGF-β1 protein can be used as a stable and reliable research tool to study the biological function, mechanism of action, signaling pathway, etc. of TGF-β1 in depth. Its stable activity and quality can ensure the accuracy and repeatability of experimental results, providing strong support for life science research. However, there is a lack of research on lyophilized preparations of recombinant human TGF-β1 protein in this field. Summary of the invention

[0005] In order to make up for the above-mentioned technical gap in the prior art, the object of the present invention is to provide a recombinant human TGF-β1 protein lyophilized preparation and a preparation method and application thereof.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a lyophilized preparation of recombinant human TGF-β1 protein.

[0008] Furthermore, the recombinant human TGF-β1 protein lyophilized preparation is composed of a recombinant human TGF-β1 protein stock solution, a stock solution buffer, mannitol, and trehalose;

[0009] The stock solution buffer consists of NaAC, NaCl and glacial acetic acid.

[0010] Furthermore, the contents of the components in the stock solution buffer are: 10-30 mM NaAC, 100-200 mM NaCl, 0.5-1.5% glacial acetic acid, pH (2.5-4);

[0011] Optionally, the contents of the components in the stock solution buffer are: 20 mM NaAC, 150 mM NaCl, 1% glacial acetic acid, pH 3.3;

[0012] Optionally, the contents of mannitol and trehalose are: 3-9% mannitol, 2-8% trehalose respectively;

[0013] Optionally, the contents of mannitol and trehalose are 6% mannitol and 5% trehalose respectively.

[0014] Further, the concentration of the recombinant human TGF-β1 protein stock solution is 0.1-0.9 mg / mL;

[0015] Optionally, the concentration of the recombinant human TGF-β1 protein stock solution is 0.5 mg / mL.

[0016] Furthermore, the recombinant human TGF-β1 protein stock solution is prepared by the following method:

[0017] (1) HEK293 serum-free basal medium and sterile plasmid DNA were mixed at a ratio of 25 mL:1 mg, wherein the amino acid sequence expressed by the sterile plasmid DNA is shown in SEQ ID NO:1;

[0018] (2) adding the mixed solution in step (1) to a premixed transfection solution, wherein the premixed transfection solution comprises HEK293 serum-free basal medium and TA-293 transfection reagent, wherein the ratio of HEK293 serum-free basal medium to TA-293 transfection reagent is 25 mL:5 mL, to obtain a plasmid-vector complex;

[0019] (3) The plasmid-vector complex was allowed to stand at room temperature for 10 min;

[0020] (4) Take the cells to be transfected, add the prepared plasmid-vector complex while shaking to complete the transfection, and return the transfected cells to the cell culture constant temperature shaker for shaking culture, with the day being Day 0;

[0021] (5) On transfection day 1, add 0.6% KE-293 and 5% feed medium in the initial volume;

[0022] (6) On transfection day 3, add 5% of the initial volume of feed medium;

[0023] (7) On Day 5, the cells were harvested by centrifugation at 10,000 rpm for 10 min.

[0024] (8) The cell feed solution in step (7) was subjected to a Ni purification step, specifically using 5 mL Ni-NTA Purose6FastFlow, and performing a 4%, 10%, and 50% equilibration-loading-equilibration-elution process, wherein the equilibration solution component was 50 mM Tris 0.1 M NaCl pH 8.0, and the elution solution component was 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0;

[0025] (9) The product of step (8) is further purified by cationic chromatography, specifically using 5 mL SP SepharoseTMFast Flow, adjusted to cond: 4.8 mS / cm with injection water, adjusted to pH 4.5 with 6 M HCl, and performing a process of equilibrium-loading-equilibrium-step elution, wherein the equilibrium solution components are 20 mM NaAc, 150 mM NaCl, 20 mM ammonium acetate, 0.5 M urea 1% glacial acetic acid, pH 4.5; the eluent components are 20 mM NaAc, 1 M NaCl, 0.5 M urea, 1% glacial acetic acid, pH 4.5, to obtain a purified high-activity recombinant human TGF-β1 protein, i.e., a recombinant human TGF-β1 protein stock solution.

[0026] Furthermore, the recombinant human TGF-β1 protein stock solution is treated by liquid replacement through a 3kD ultrafiltration tube.

[0027] In the present invention, the NaAC refers to sodium acetate, also known as sodium acetate, which is an organic substance with a molecular formula of CH3COONa and a molecular weight of 82.03. Sodium acetate trihydrate is a white crystal with a relative density of 1.45 and a melting point of 58°C. It weathers in dry air, loses crystal water at 120°C, and decomposes at higher temperatures; anhydrous sodium acetate is a colorless transparent crystal with a melting point of 324°C. It is easily soluble in water and can be used as a buffer and mordant.

[0028] In some embodiments, TGF-β1 protein is susceptible to denaturation or degradation in solution due to various factors, such as temperature change, pH fluctuation, protease action, etc. Lyophilized preparations can keep the protein in a dry, low-temperature stable state, reduce the damage to its structure and activity caused by these factors, and thus extend the shelf life of TGF-β1 protein.

[0029] In some embodiments, the recombinant human TGF-β1 protein lyophilized preparation is in the form of a solid powder with a small volume, light weight, and relatively low requirements for storage and transportation conditions. It does not require strict temperature control and vibration avoidance like liquid dosage forms, and is more convenient for long-term storage and long-distance transportation in different environments, which is beneficial to the timeliness of product marketing and clinical application.

[0030] In some embodiments, during clinical treatment, different patients and different disease stages may have different requirements for TGF-β1 protein. The lyophilized preparation can accurately reconstitute and adjust the concentration and dosage of the drug according to the specific situation, achieve precise drug administration, improve the therapeutic effect, and reduce the risk of side effects or insufficient treatment caused by inaccurate dosage.

[0031] In some embodiments, the lyophilized preparation has stable physical and chemical properties and is easy to be made into a variety of dosage forms, such as powder for injection, spray, implant, etc., which provides the possibility of exploring a variety of administration routes to meet the treatment needs of different diseases and individual differences of patients. For example, local injection or implantation can be used for local lesions, and inhalation can be tried for lung diseases, etc., to improve the clinical applicability and accessibility of the drug.

[0032] In some embodiments, the pharmaceutical composition or biological preparation comprising the aforementioned lyophilized preparation of the present invention is also included in the protection scope of the present invention.

[0033] In some embodiments, the lyophilized recombinant human TGF-β1 protein preparation according to the first aspect of the present invention has good activity. In a specific embodiment of the present invention, the activity is reflected by ED50.

[0034] In the present invention, the ED50 or median effective dose in the protein activity refers to the protein dose that can cause 50% of a group of experimental subjects to produce the expected specific biological effect under specific experimental conditions.

[0035] In some embodiments, the ED50 can be used to evaluate protein activity: ED50 is an important indicator for measuring the biological activity of a protein. The lower the value, the higher the activity of the protein, and only a smaller dose is needed to produce half the effective response; conversely, the higher the value, the lower the activity.

[0036] In some embodiments, the ED50 can also be used to compare the activities of different proteins or preparations: during the research and development process, the ED50 of proteins or protein preparations obtained from different batches and different production methods can be compared to evaluate whether their activities are consistent, thereby performing quality control and optimization.

[0037] In some embodiments, the ED50 can also be used to guide clinical drug dosage: for proteins with medicinal value, understanding their ED50 helps to preliminarily estimate the effective dosage range for clinical use, providing an important reference for subsequent clinical trials and drug development.

[0038] The second aspect of the present invention provides a method for preparing the lyophilized preparation of recombinant human TGF-β1 protein as described in the first aspect of the present invention.

[0039] Further, the method comprises the following steps:

[0040] (1) Preparation: Mix the stock solution buffer, mannitol and trehalose described in the first aspect of the present invention, and then add the recombinant human TGF-β1 protein stock solution described in the first aspect of the present invention;

[0041] (2) Packaging: Packaging the solution obtained in step (1);

[0042] (3) Freeze drying: compressor, set temperature -45°C, the temperature in the cold trap is -45°C, put the stock solution packaged in step (2) into the freeze dryer, pre-freeze for 30 minutes, set the "automatic mode" program parameters: total number of sections: 13, vacuum control section, specific freeze drying parameters are as follows:

[0043]

[0044] Furthermore, the amounts of the stock solution buffer, mannitol, trehalose, and recombinant human TGF-β1 protein stock solution added in step (1) are: 3.0-7.5 mL, 0.1-0.5 g, 0.1-0.5 g, and 0.1-0.5 mL, respectively;

[0045] Optionally, the amounts of the stock solution buffer, mannitol, trehalose, and recombinant human TGF-β1 protein stock solution added in step (1) are: 5.76 mL, 0.36 g, 0.3 g, and 0.24 mL, respectively;

[0046] Optionally, step (1) further comprises filtering and sterilizing the obtained solution using a 0.22 μm sterile filter.

[0047] The third aspect of the present invention provides a method for preparing a recombinant human TGF-β1 protein solution.

[0048] Furthermore, the method comprises the following steps: re-dissolving the lyophilized recombinant human TGF-β1 protein preparation according to the first aspect of the present invention to obtain a recombinant human TGF-β1 protein solution.

[0049] Furthermore, the reconstitution solvent used during the reconstitution is sterile water, sterile physiological saline, 0.3% sterile aqueous solution of lidocaine hydrochloride or 0.3% sterile physiological saline solution of lidocaine hydrochloride.

[0050] In some embodiments, the present invention has no particular restrictions on the reconstitution solvent used during the reconstitution, including but not limited to: aqueous solvents (water for injection, physiological saline, phosphate buffer, etc.), non-aqueous solvents (dimethyl sulfoxide, ethanol, etc.), mixed solvents (mixture of aqueous solvents and non-aqueous solvents, solvents with added protective agents, etc.), etc.

[0051] The fourth aspect of the present invention provides the use of the recombinant human TGF-β1 protein lyophilized preparation described in the first aspect of the present invention in the preparation of a drug for promoting wound healing, bone tissue repair, cartilage tissue repair, tumor treatment, corneal damage repair, treatment of retinopathy, myocardial repair after myocardial infarction, or repair and restenosis prevention after vascular damage.

[0052] In some embodiments, TGF-β1 plays an important role in various physiological and pathological processes, such as tissue repair, immune regulation, tumorigenesis, etc. The research and development of the recombinant human TGF-β1 protein lyophilized preparation of the present invention provides the possibility of treating various diseases, such as wound healing, osteoarthritis, autoimmune diseases, tumors and other fields, and has potential therapeutic application value, providing clinicians with more treatment options and means.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] The present invention provides a novel lyophilized preparation of recombinant human TGF-β1 protein in the art, and provides its related preparation method and medical use, which solves the problems of instability, easy aggregation and denaturation, and reduced activity of TGF-β1 protein itself. In addition, the preparation is easy to use, quickly absorbed, and convenient to store and transport, and has good value for industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 : Ni purification results of cell fermentation liquid;

[0056] Figure 2 :Ni 2+ Metal chelation chromatography;

[0057] Figure 3 :Cation chromatography purification result diagram;

[0058] Figure 4 : Cationic chromatogram;

[0059] Figure 5 : SEC-HPLC purity analysis chart;

[0060] Figure 6 :Statistical chart of activity test results of different batches of recombinant human TGF-β1 protein products;

[0061] Figure 7 : The morphology of the freeze-dried products prepared by each preparation method;

[0062] Figure 8 : The interface where freeze drying is performed;

[0063] Fig. 9 :Experimental steps for developing formulation samples. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0065] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually carried out according to conventional conditions in the art or according to conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0066] Example 1 Preparation of recombinant human TGF-β1 protein stock solution

[0067] 1. Design of expression plasmid

[0068] According to the differences in signal peptide sequences, tag positions, latent peptide sources, etc., the inventors of the present invention designed the following target proteins in preliminary studies, outsourced the corresponding expression gene synthesis, and constructed the expression genes on pCDNA3.4 to construct 5 expression plasmids.

[0069] JDS-22A (SEQ ID NO: 1):

[0070] MKWVTFLLLLFISGSAFSHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0071] JDS-22B(SEQ ID NO:2):

[0072] METDTLLLWVLLLWVPGSTGDAAHHHHHHHHALDTNYCFSSTEKNCCV RQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHN PGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0073] JDS-22C(SEQ ID NO:3):

[0074] METDTLLLWVLLLWVPGSTGDAAHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0075] JDS-22D(SEQ ID NO:4):

[0076] MKWVTFLLLLFISGSAFSHHHHHHHHALDTNYCFSSTEKNCCVRQLYIDF RKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAA PCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS。

[0077] JDS-22E(SEQ ID NO:5):

[0078] METDTLLLWVLLLWVPGSTGDAAHHHHHHHHLSTSKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFQTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSQNSWRYLSNRLLAPSDSPE WLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS.

[0079] 2. Plasmid extraction and transient expression test

[0080] The expression plasmid was transformed into E. coli DH5α competent cells to maximize the plasmid.

[0081] Use Zhuhai Kairui's HEK-293 transient expression kit (transfection reagent and enhancer) to express the target protein. The detailed steps are as follows:

[0082] a) Before transient transfection of cells, the cell density and viability must be determined. To ensure the transfection effect, it is recommended to use cells in the exponential growth phase (density of about 2-4×10 6 / mL) and the survival rate was greater than 95% of the cells transfected.

[0083] b) Cells do not need to be centrifuged. If the cell density is 3.0×10 6 If the cell density is >3.0×10 6 / mL, it is necessary to add HEK293 serum-free basal medium (Beijing Tongli Haiyuan, AS-11) and dilute the cell density to 3×10 6 Pieces / mL.

[0084] c) The shake flask was placed in a 5% CO2 constant temperature shaker, cultured at 37°C and 120 rpm, and then used for transfection.

[0085] d) Prepare two 50 mL sterile centrifuge tubes, add 25 mL HEK293 serum-free basal medium and 1 mg sterile plasmid DNA to one of them, and mix by gently pipetting; take another centrifuge tube, add 25 mL HEK293 serum-free basal medium and 5 mL TA-293 transfection reagent (Zhuhai Kairui, K20001), and mix by gently pipetting.

[0086] e) Transfer all the liquid in the centrifuge tube containing the plasmid to the centrifuge tube containing the transfection reagent and mix by gently pipetting.

[0087] f) Let stand at room temperature for 10 minutes.

[0088] g) Remove the cells for transfection from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and after transfection, take 1 mL of cell suspension for counting, record the cell density and viability, centrifuge and retain the supernatant, put the transfected cells back into the cell culture constant temperature shaker for shaking culture, and take that day as Day 0.

[0089] h) On transfection day 1, 1 mL of cell suspension was taken for counting, cell density and viability were recorded, and the supernatant was retained after centrifugation. The initial volume of 0.6% KE-293 (Zhuhai Kairui, K30001) and 5% feed medium (Tongli Haiyuan, AS-18) were added (i.e., 6 mL KE-293 and 50 mL feed medium were added to 1 L of cell culture medium).

[0090] i) On transfection day 3, 1 mL of cell suspension was taken for counting, cell density and viability were recorded, and the cell suspension was centrifuged to retain the supernatant, and 5% of the initial volume of feed medium was added again, i.e., 50 mL of feed medium.

[0091] j) On transfection day 4, take 1 mL of cell suspension and count, record the cell density and viability, centrifuge and save the supernatant. Day 5 or when the cell viability is less than 75% is the end point of harvest.

[0092] k) Centrifuge at 10,000 rpm for 10 min, harvest the cell solution, and carry out purification and detection of the target protein.

[0093] Transfection results: No target protein was expressed in JDS-22B and JDS-22D; the expression level of JDS-22A was higher than that of JDS-22C and JDS-22E.

[0094] 3. Ni purification in cell fermentation liquid

[0095] The transfected protein was Ni purified using Ni-NTA Purose 6Fast Flow (5 mL) and the process of equilibrium-loading-equilibrium-elution (4%, 10%, 50%) was performed, wherein the equilibrium solution was 50 mM Tris 0.1 M NaCl pH 8.0, and the elution solution was 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0. The results are shown in Figure 1 , Figure 2 shown.

[0096] 4. Cationic chromatography purification

[0097] SP SepharoseTM Fast Flow (5mL) was used for cationic chromatography purification. The sample source was the eluted sample of the above Ni purification. Sample treatment: adjusted to cond: 4.8mS / cm with injection water, adjusted to 4.5 with 6MHCl, and carried out the process of equilibrium-loading-equilibrium-step elution. Among them, the equilibrium liquid composition was 20mM NaAc, 150mM NaCl, 20mM ammonium acetate, 0.5M urea, 1% glacial acetic acid, pH 4.5, and the eluent composition was 20mM NaAc, 1M NaCl, 0.5M urea, 1% glacial acetic acid, pH4.5. The results are as follows Figure 3 , Figure 4 shown.

[0098] 5. SEC-HPLC analysis and activity detection

[0099] The purity of the purified recombinant human TGF-β1 protein was 99.40% as analyzed by SEC-HPLC. Figure 5 shown.

[0100] Activity detection: After repeated testing of multiple batches, the purification process is stable, and the activity of the obtained TGF-β1 protein is significantly better than the products sold by R* Company and Kai* Company. The specific results are shown in Table 1, and the detailed information is shown in Figure 6 .

[0101] Table 1 Activity test results

[0102]

[0103] Example 2 Process development and research of lyophilized preparation of recombinant human TGF-β1 protein

[0104] The recombinant protein solution is first frozen in a low temperature environment, and then dried under vacuum conditions to form a recombinant protein solid preparation. The drying process can be divided into preliminary drying to remove crystal water and secondary drying to remove bound water. However, since the freeze-drying process is essentially a process of material and energy conversion, the protein quality may change to a certain extent. The purpose of formulation development is to reduce the impact on protein quality during protein freeze-drying by adding types of freeze-drying protectants, adjusting freeze-drying protectant concentrations, buffer pH range, etc. Therefore, this embodiment optimizes the preparation process and product activity of recombinant human TGF-β1 protein (JDS-22A) in order to improve product quality and competitiveness.

[0105] 1. Experimental Materials

[0106] The equipment and instruments, and main reagents and consumables involved in this embodiment are shown in Tables 2 and 3 below, respectively.

[0107] Table 2 Equipment and instruments

[0108]

[0109] Table 3 Main reagents and consumables

[0110]

[0111] 2. Pre-freezing experiment

[0112] It is preferred to use the existing fixed freeze-drying process of 6% mannitol for the first freeze-drying to reduce the work of production preparation conversion.

[0113] a) Purification: Two batches of stock solutions, No. 3 and No. 4, are provided, see Table 4 below for details. Among them, No. 3 and No. 4 are the stock solutions of No. 1 and No. 2 respectively after the corresponding batches were replaced, and there is no activity data, and the stock solution of No. 3 is turbid.

[0114] Table 4

[0115]

[0116] b) Freeze-dry according to the minimum specification of the product on sale, 10 μg, as required. See Table 5 below for details.

[0117] Table 5

[0118]

[0119] Results: Due to the later change of re-dissolution conditions, there was no comparison between the two batches, and the overall activity was poor. The next step was to explore the formulation.

[0120] 3. Development stage

[0121] (1) Optimization was performed based on the special nature of TGF-β1 protein, which requires low pH buffer for storage, and the special nature of ethanol. First, ethanol has a lower boiling point than water and evaporates faster than water, so ethanol may evaporate during the freeze-drying process. This may cause changes in sample concentration and a reduction in sample volume, affecting the accuracy of the experimental results. Second, ethanol tends to form a glassy state at extremely low temperatures rather than completely crystallize, which may affect the effect and time of freeze-drying. Therefore, during the freeze-drying process of ethanol solutions, it is recommended to use a lower freezing temperature and a larger cooling area to minimize ethanol volatilization and sample capacity loss. At the same time, it is also necessary to control the air flow rate and vacuum degree during the drying process, and maintain the appropriate heating temperature and time to ensure that the sample can be fully freeze-dried and achieve the required quality and stability. Since the freeze-drying machine parameter process will not be changed, the formula that removes ethanol is given priority.

[0122] a) Protein solution replacement. See Table 6 below for details. Among them, serial numbers 3 and 4 are the stock solutions produced by mixing serial numbers 1 and 2 and replacing the solutions respectively. Serial numbers 3 and 4 are sent for activity inspection at the same time as the freeze-dried product 03-16 below. Serial numbers 1 and 2 are TGF-β1 protein stock solutions prepared by the method described in Example 1, among which JDS-22A-240719-SBC is the protein stock solution obtained in the purification process exploration stage; JDS-22A-240719-NS50 is the protein stock solution obtained after the purification process is stabilized, and the difference between it and JDS-22A-240719-NS100 is the different concentrations. Both are prepared by the method described in Example 1.

[0123] Table 6

[0124]

[0125] Results: From the comparison of the activities of the stock solutions No. 3 and 4, glycine is preferred in the buffer system.

[0126] b) Freeze-dried formula methods 1-7 are 8% trehalose, 5% trehalose, 5% trehalose + 6% mannitol, 5% sucrose + 6% mannitol, 5% sucrose + 0.02% Tween 80, 5% sucrose, 6% mannitol + 0.01% Tween 80, 03-09 use the stock solution of sequence number 3 (protein in glycine buffer), 10-16 use the stock solution of sequence number 4 (protein in sodium citrate buffer). See Table 7 below.

[0127] Table 7

[0128]

[0129] Results: According to the activity data of the lyophilized product, glycine buffer with 4mM HCL and sodium citrate buffer with H2O were the better combinations for reconstitution. According to the activity comparison between the stock solution and the lyophilized product, it was confirmed that the formula of the lyophilized product matched with the appropriate reconstitution conditions could effectively improve the protein activity. The key point was the protein buffer formula to ensure that the protein activity did not decrease after the solution was changed.

[0130] (2) Adjust the buffer solution formula based on the above experimental data.

[0131] a) Liquid exchange: The amount of stock solution required to customize the protein buffer is relatively large. Comprehensive consideration is given to make adjustments based on the purification buffer. A small amount of stock solution is first used for liquid exchange verification. If the activity is qualified, a large amount of liquid is exchanged with the freeze-dried formula for experiment. Serial numbers 3, 4, and 5 are the stock solutions after liquid exchange. The three batches of serial numbers 3, 4, and 5 are the TGF-β1 protein stock solutions prepared in Example 1, respectively, using a G25 desalting column to exchange the buffer to the "buffer" column in Table 8. See Table 8 below.

[0132] Table 8

[0133]

[0134] Results: The activity was qualified according to the results, and the next step was to prepare the freeze-dried formula.

[0135] b) Freeze drying: Due to insufficient stock solution, multiple combinations were not made. 17, 18, and 19 correspond to the stock solutions of No. 3, 4, and 5, respectively. Methods 1-3 are 6% mannitol + 5% sucrose, 6% mannitol + 0.01% Tween 80, and 5% sucrose, respectively. See Table 9 below.

[0136] Table 9

[0137]

[0138] Results: The activity was qualified, but the freeze-dried forms of 18 and 19 were not good. The formulas in this experiment were all single-item combinations, so multiple combinations experiments were still needed for freeze-drying, and the data also needed to be repeated.

[0139] (3) The modified plan is shown in Table 10 below.

[0140] Table 10

[0141]

[0142] (4) Results (H2O re-dissolution)

[0143] a) Morphology: Morphological results are shown in Figure 7 .

[0144] b) Activity: The activity results are shown in Table 11 below.

[0145] Table 11

[0146]

[0147] Results: From the morphology, 2, 3, and 4 were all acceptable. Combined with the activity results, the formula was confirmed to be 6% mannitol + 5% trehalose.

[0148] 4. Final Solution

[0149] (1) Stock buffer: 20 mM NaAC, 150 mM NaCl, 1% glacial acetic acid, pH 3.3.

[0150] (2) Preparation formula: 6% mannitol + 5% trehalose.

[0151] (3) Freeze-drying curve Figure 8 shown.

[0152] Example 3 Preparation of Lyophilized Recombinant Human TGF-β1 Protein

[0153] 1. Environmental confirmation: room temperature.

[0154] 2. Preparation: Clean the laboratory table and clean bench, remove all items except the preparation and freeze-dried materials, clean the table with alcohol, and prepare the required materials.

[0155] 3. Solution preparation: see Table 12 below.

[0156] Table 12

[0157]

[0158] 4. Experimental steps: Fig. 9 shown.

[0159] 5. Experimental methods

[0160] (1) Preparation work: main instruments and equipment and main materials are shown in Table 2 and Table 3.

[0161] 1) Clean the lab bench

[0162] Clean the laboratory table and clean bench, remove all items except the preparation and freeze-dried materials, and irradiate the clean bench with ultraviolet light for 30 minutes for later use;

[0163] 2) Clean the vials and sterilize the stoppers

[0164] Take 100 2mL vials and place them in a glass beaker, fill it with sterile water for injection, put it into an ultrasonic cleaner, and sonicate for 30 minutes;

[0165] Take out the vial, drain the water in the vial, put it into the freeze-drying tray, and wrap it with two layers of aluminum foil;

[0166] Put it into the electric heating blast drying oven, remove the heat source and set it aside, and turn it off immediately after use. Set the parameters: temperature 250℃, time 30min;

[0167] Take 100 2 mL vials of freeze-dried rubber stoppers, put them into a glass beaker, and seal the beaker mouth with a breathable membrane;

[0168] Place in a vertical high-pressure steam sterilizer for sterilization and set aside for future use. Sterilize immediately before use. Set parameters: temperature 121°C, time 30 min.

[0169] (2) Preparation

[0170] 1) Dilution solution: 20 mM NaAC, 150 mM NaCL, 1% glacial acetic acid, pH 3.3;

[0171] 2) Formula: 6% mannitol + 5% trehalose;

[0172] 3) Specifications: 0.01 mg / tube, 0.5 mL / tube, 10 tubes;

[0173] 4) Stock solution pretreatment 1

[0174] Stock solution: batch number JDS-22A-240922-NS100, concentration: 0.52 mg / mL;

[0175] After 3kD ultrafiltration tube liquid replacement treatment: batch number JDS-22A-241022 (solution obtained by replacing the JDS-22A-240922-NS100 with 3kD ultrafiltration tube liquid), concentration: 0.5 mg / mL;

[0176] The calculation is as follows:

[0177] Total volume 6mL;

[0178] Formula addition amount: 6*6%=0.36g mannitol powder + 6*5%=0.3g trehalose powder;

[0179] Theoretical packaging concentration 0.02mg / mL: 0.01 / 0.5 (mass / volume);

[0180] Amount of stock solution added: 6*0.02 / 0.5≈0.24mL stock solution;

[0181] Amount of diluent added: 6-0.24=5.76mL;

[0182] Dilution: Add 0.36g mannitol and 0.3g trehalose powder to a 15mL sterile centrifuge tube, then add 5.76mL diluent, mix well and add 0.24mL stock solution. The freeze-dried batch number is FD-TGF-β1-241025-22;

[0183] 5) Stock solution pretreatment 2

[0184] Stock solution: batch number JDS-22A-240929-NS100, concentration: 0.57 mg / mL;

[0185] After 3kD ultrafiltration tube liquid replacement treatment: Batch No. JDS-22A-241104-2 (solution obtained by replacing the JDS-22A-240929-NS100 with 3D ultrafiltration tube liquid), concentration: 2.08 mg / mL;

[0186] The calculation is as follows:

[0187] Total volume 6mL;

[0188] Formula addition amount: 6*6%=0.36g mannitol powder + 6*5%=0.3g trehalose powder;

[0189] Theoretical packaging concentration 0.02mg / mL: 0.01 / 0.5 (mass / volume);

[0190] Amount of stock solution added: 6*0.02 / 2.08≈0.058mL stock solution;

[0191] Amount of diluent added: 6-0.058=5.942mL;

[0192] Dilution: Add 0.36g mannitol and 0.3g trehalose powder to a 15mL sterile centrifuge tube, then add 5.942mL diluent, mix well and add 0.058mL stock solution. The freeze-dried batch number is FD-TGF-β1-241106-25;

[0193] 6) Stock solution pretreatment 3

[0194] Stock solution: batch number JDS-22A-241016-NS100, concentration: 1.32 mg / mL;

[0195] After 3kD ultrafiltration tube liquid replacement treatment: batch number JDS-22A-241104-3 (solution obtained by replacing the JDS-22A-241016-NS100 with 3kD ultrafiltration tube liquid), concentration: 1.32 mg / mL;

[0196] The calculation is as follows:

[0197] Total volume 6mL;

[0198] Formula addition amount: 6*6%=0.36g mannitol powder + 6*5%=0.3g trehalose powder;

[0199] Theoretical packaging concentration 0.02mg / mL: 0.01 / 0.5 (mass / volume);

[0200] Amount of stock solution added: 6*0.02 / 1.32≈0.091mL stock solution;

[0201] Amount of diluent added: 6-0.091=5.909mL;

[0202] Dilution: Add 0.36g mannitol and 0.3g trehalose powder to a 15mL sterile centrifuge tube, then add 5.909mL diluent, mix well and add 0.091mL stock solution. The freeze-dried batch number is FD-TGF-β1-241106-26;

[0203] 7) The solutions obtained in 4), 5) and 6) were sterilized by filtration using a 0.22 μm sterile filter in a clean bench and stored in 15 mL sterile centrifuge tubes for later use.

[0204] (3) Packaging

[0205] 1) Take out the treated vials and freeze-dried rubber stoppers and transfer them to the clean bench, and package them according to the specification of 0.01 mg / vial, with a packaging volume of 0.5 mL / vial, for a total of 30 vials;

[0206] 2) Half-cover the mouth of the vial with the rubber stopper, leaving a gap to allow air to be extracted during freeze-drying.

[0207] (4) Freeze-drying

[0208] 1) Before starting the machine, make sure that the "gas ballast valve" on the vacuum pump is closed and the "air inlet valve" on the side of the chassis is closed;

[0209] 2) After powering on

[0210] Enter the system menu and confirm the freeze-drying process;

[0211] On the operation screen, select manual mode, turn on the compressor, set the temperature to -45°C, click manual start, and pre-freeze the empty machine for 30 minutes to ensure that the temperature in the cold trap reaches -45°C;

[0212] 3) Carefully place the packaged stock solution into the freeze dryer, tighten the hatch valve, and pre-freeze again for 30 minutes to ensure that the sample is frozen;

[0213] 4) Set the "Automatic Mode" program parameters: total number of sections: 13, vacuum control section: 7. See Table 13 below;

[0214] Table 13

[0215]

[0216]

[0217] 5) Run Figure 8 The interface shown;

[0218] 6) Click the operation screen, click manual stop, change the control selection to "automatic mode", click automatic start, and return to the main screen to check whether the current section is correct;

[0219] 7) Harvest: After the freeze-drying process is completed and runs for 46 hours and 25 minutes, first cover the rubber stopper tightly, then open the "inlet valve" on the side of the chassis, and then take out the vial;

[0220] 8) Capping: Open the capping machine, place the aluminum cap on the rubber plug, press down the handrail to complete the capping;

[0221] 9) Randomly select four samples after capping for testing of quantity, moisture, activity and purity;

[0222] 10) The results are shown in Table 14. It can be seen that the recombinant human TGF-β1 protein stock solution prepared by the method described in Example 1 is combined with the following final solution obtained by screening and verification in Example 2: ① Stock solution buffer: 20mM NaAC, 150mM NaCl, 1% glacial acetic acid, pH 3.3; ② Preparation formula: 6% mannitol + 5% trehalose; ③ Freeze-drying curve is as shown in Figure 8 As shown, the prepared lyophilized preparation of recombinant human TGF-β1 protein has higher activity and is significantly better than the commercial product. This result is a technical effect that was unexpected by those skilled in the art based on the contents disclosed in the prior art.

[0223] Table 14

[0224]

Claims

1. A recombinant human TGF-β1 protein lyophilized preparation, characterized in that: The recombinant human TGF-β1 protein lyophilized preparation is composed of recombinant human TGF-β1 protein stock solution, stock solution buffer, mannitol and trehalose; The stock solution buffer consists of NaAC, NaCl and glacial acetic acid.

2. The recombinant human TGF-β1 protein lyophilized preparation according to claim 1, characterized in that: The contents of the components in the stock solution buffer are: 10-30 mM NaAC, 100-200 mM NaCl, 0.5-1.5% glacial acetic acid, pH (2.5-4); Optionally, the contents of the components in the stock solution buffer are: 20 mM NaAC, 150 mM NaCl, 1% glacial acetic acid, pH 3.3; Optionally, the contents of mannitol and trehalose are: 3-9% mannitol, 2-8% trehalose respectively; Optionally, the contents of mannitol and trehalose are 6% mannitol and 5% trehalose respectively.

3. The lyophilized preparation of recombinant human TGF-β1 protein according to claim 1, characterized in that: The concentration of the recombinant human TGF-β1 protein stock solution is 0.1-0.9 mg / mL; Optionally, the concentration of the recombinant human TGF-β1 protein stock solution is 0.5 mg / mL.

4. The lyophilized preparation of recombinant human TGF-β1 protein according to claim 1, characterized in that: The recombinant human TGF-β1 protein stock solution is prepared by the following method: (1) HEK293 serum-free basal medium and sterile plasmid DNA were mixed at a ratio of 25 mL:1 mg, wherein the amino acid sequence expressed by the sterile plasmid DNA is shown in SEQ ID NO:1; (2) adding the mixed solution in step (1) to a premixed transfection solution, wherein the premixed transfection solution comprises HEK293 serum-free basal medium and TA-293 transfection reagent, wherein the ratio of HEK293 serum-free basal medium to TA-293 transfection reagent is 25 mL:5 mL, to obtain a plasmid-vector complex; (3) The plasmid-vector complex was allowed to stand at room temperature for 10 min; (4) Take the cells to be transfected, add the prepared plasmid-vector complex while shaking to complete the transfection, and return the transfected cells to the cell culture constant temperature shaker for shaking culture, with the day being Day 0; (5) On transfection day 1, add 0.6% KE-293 and 5% feed medium in the initial volume; (6) On transfection day 3, add 5% of the initial volume of feed medium; (7) On Day 5, the cells were harvested by centrifugation at 10,000 rpm for 10 min. (8) The cell feed solution in step (7) is subjected to a Ni purification step, specifically using 5 mL Ni-NTA Purose6 Fast Flow, and performing a equilibration-loading-equilibration-elution process at a ratio of 4%, 10%, and 50%, wherein the equilibration solution component is 50 mM Tris 0.1 M NaCl pH 8.0, and the elution solution component is 50 mM Tris 0.1 M NaCl 0.5 M imidazole pH 8.0; (9) The product of step (8) is further purified by cationic chromatography, specifically using 5 mL SP SepharoseTM FastFlow, adjusted to cond: 4.8 mS / cm with injection water, adjusted to pH 4.5 with 6 M HCl, and performing a process of equilibrium-loading-equilibrium-step elution, wherein the equilibrium solution components are 20 mM NaAc, 150 mM NaCl, 20 mM ammonium acetate, 0.5 M urea 1% glacial acetic acid, pH 4.5; the eluent components are 20 mM NaAc, 1 M NaCl, 0.5 M urea, 1% glacial acetic acid, pH 4.5, to obtain a purified high-activity recombinant human TGF-β1 protein, i.e., a recombinant human TGF-β1 protein stock solution.

5. The lyophilized preparation of recombinant human TGF-β1 protein according to claim 4, characterized in that: The recombinant human TGF-β1 protein stock solution is treated by liquid replacement through a 3kD ultrafiltration tube.

6. A method for preparing a lyophilized preparation of recombinant human TGF-β1 protein according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) Preparation: Mix the stock solution buffer, mannitol and trehalose described in claim 2, and then add the recombinant human TGF-β1 protein stock solution described in claim 3; (2) Packaging: Packaging the solution obtained in step (1); (3) Freeze drying: compressor, set temperature -45°C, the temperature in the cold trap is -45°C, put the stock solution packaged in step (2) into the freeze dryer, pre-freeze for 30 minutes, set the "automatic mode" program parameters: total number of sections: 13, vacuum control section, specific freeze drying parameters are as follows:

7. The method according to claim 6, characterized in that The added amounts of the stock solution buffer, mannitol, trehalose, and recombinant human TGF-β1 protein stock solution in step (1) are: 3.0-7.5 mL, 0.1-0.5 g, 0.1-0.5 g, and 0.1-0.5 mL, respectively; Optionally, the amounts of the stock solution buffer, mannitol, trehalose, and recombinant human TGF-β1 protein stock solution added in step (1) are: 5.76 mL, 0.36 g, 0.3 g, and 0.24 mL, respectively; Optionally, step (1) further comprises filtering and sterilizing the obtained solution using a 0.22 μm sterile filter.

8. A method for preparing a recombinant human TGF-β1 protein solution, characterized in that: The method comprises the following steps: re-dissolving the lyophilized recombinant human TGF-β1 protein preparation according to any one of claims 1 to 5 to obtain a recombinant human TGF-β1 protein solution.

9. The method according to claim 8, characterized in that The reconstitution solvent used during the reconstitution is sterile water, sterile physiological saline, 0.3% sterile aqueous solution of lidocaine hydrochloride or 0.3% sterile physiological saline solution of lidocaine hydrochloride.

10. Use of the lyophilized recombinant human TGF-β1 protein preparation according to any one of claims 1 to 5 in the preparation of a drug for promoting wound healing, bone tissue repair, cartilage tissue repair, treating tumors, corneal damage repair, treating retinopathy, myocardial repair after myocardial infarction, or repair and prevention of restenosis after vascular damage.

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