Lyoprotectants, il-6 lyophilized powders, and methods of making same
By using a lyophilization protectant to prepare IL-6 into a lyophilized powder, the problem of instability of the liquid IL-6 reagent was solved, and a high-potency IL-6 lyophilized powder with long-term storage at room temperature was achieved, which is suitable for the biopharmaceutical field.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- DAAN GENE CO LTD
- Filing Date
- 2021-12-24
- Publication Date
- 2026-06-05
AI Technical Summary
Existing IL-6 liquid reagents are unstable at room temperature, easily deactivated, and their enzyme activity decreases during freeze-thaw cycles. They also have high storage costs and are difficult to preserve for long periods.
IL-6 lyophilized powder was prepared by freeze-drying using a lyophilization protectant including bovine serum albumin, polyethylene glycol, sodium chloride, surfactant and preservative, and adjusting the pH to 7.4-8.0.
IL-6 lyophilized powder can be stored stably at room temperature for a long time with minimal loss of potency and retains more than 80% of its activity. It is easy to use and has good reconstitution properties.
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Abstract
Description
Technical Field
[0001] The embodiments of this invention relate to the field of biomedicine, and particularly to lyophilization protectants, IL-6 lyophilized powder, and their preparation methods. Background Technology
[0002] Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in regulating immune responses, hematopoiesis, defense mechanisms, and acute-phase responses. It is primarily produced by various lymphocytes, including macrophages, T cells, and B cells, but can also be synthesized by non-lymphocytes such as monocytes / macrophages, fibroblasts, hepatocytes, keratinocytes, astrocytes, vascular endothelial cells, and various tumor cells. IL-6 synthesis increases in response to stimulation by various cytokines, mitotic factors, and antigens. IL-6 is a glycoprotein containing N- and O-linked carbohydrates with a molecular weight of 21-28 kDa. Its glycoprotein components are not essential for its function. The precursor of human IL-6 has 212 amino acids; after removing the N-terminal 28 amino acids, a mature protein of 184 amino acids is formed. It regulates the growth and differentiation of various cells, modulates immune responses, acute-phase responses, and hematopoiesis, and plays an important role in the body's anti-infective immune response. Therefore, interleukin-6 plays a crucial role in maintaining the body's immune response, bone marrow hematopoiesis, and inflammatory responses. IL-6 is widely used as a marker in clinical practice. Its use as an inflammatory indicator is becoming increasingly widespread in community hospitals, ICUs, and respiratory infection departments. Clinically, the detection of IL-6 levels in a patient's body fluids can reflect changes in the patient's condition.
[0003] Currently, the main methods for detecting interleukin include immunospot assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blotting, and reverse hemolytic plaque assay. However, all these methods use liquid IL-6 reagent for detection, which is easily affected by changes in its activity. Therefore, controlling the stability of IL-6 is crucial. However, liquid IL-6 reagent is unstable at room temperature and becomes inactive within a few weeks. Even at 2–8°C, its shelf life is only 4 to 6 months. Repeated freeze-thaw cycles can also easily reduce enzyme activity. Furthermore, even when frozen, cold chain transportation is required during transfer, which is costly.
[0004] Therefore, it is necessary to find a method that can stably store IL-6 for a long time. Summary of the Invention
[0005] The purpose of this invention is to provide an IL-6 freeze-drying protectant. Adding the protectant to the freeze-drying process of IL-6 results in IL-6 freeze-dried powder with good molding, high storage stability, and low potency loss.
[0006] Another object of the present invention is to provide an IL-6 lyophilized powder that allows IL-6 to be stored stably for a long period of time.
[0007] Another objective of this invention is to provide a method for preparing IL-6 lyophilized powder, which results in good shape, high stability, and minimal loss of potency of the IL-6 liquid reagent after lyophilization.
[0008] To solve the above-mentioned technical problems, a first aspect of the present invention provides an IL-6 lyophilization protectant, the IL-6 lyophilization protectant comprising: water and a non-aqueous component dissolved in the water, the non-aqueous component comprising bovine serum albumin, polyethylene glycol, sodium chloride, surfactant, preservative and inorganic acid, and the pH of the IL-6 lyophilization protectant being 7.4-8.0.
[0009] In some preferred embodiments, the inorganic acid is selected from Tris-HCl, hydrochloric acid, sulfuric acid, and phosphoric acid, with hydrochloric acid being more preferred.
[0010] In some preferred embodiments, the surfactant is a nonionic surfactant selected from at least one of Tween-20 and Tween-80.
[0011] In some preferred embodiments, the polyethylene glycol is selected from at least one of PEG6000, PEG8000, PEG10000, PEG20000 and PEG40000; more preferably, the polyethylene glycol is selected from at least one of PEG6000 and PEG8000.
[0012] In some preferred embodiments, the preservative is selected from at least one of ProClin 150, ProClin 200 and ProClin 300, such as ProClin 300.
[0013] In some preferred embodiments, the bovine serum albumin (BSA) content in the IL-6 lyophilization protectant is 1-10% by mass.
[0014] In some preferred embodiments, the polyethylene glycol content is 0.2% to 2% by mass.
[0015] In some preferred embodiments, the sodium chloride content is 1-4% by mass.
[0016] In some preferred embodiments, the surfactant has a mass percentage content of 0.01 to 0.09%.
[0017] In some preferred embodiments, the preservative has a mass percentage content of 0.05% to 0.4%.
[0018] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0019] Bovine serum albumin (BSA) 1–10%;
[0020] Polyethylene glycol 0.2-2%;
[0021] Sodium chloride 1-4%;
[0022] Surfactant 0.01–0.09%;
[0023] Preservative 0.05-0.4%;
[0024] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0025] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0026] Bovine serum albumin (BSA) 2-6%;
[0027] Polyethylene glycol 0.2-1%;
[0028] Sodium chloride 1-3%;
[0029] Surfactant 0.02–0.04%;
[0030] Preservative 0.2-0.4%;
[0031] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0032] In some preferred embodiments, the non-aqueous component further includes sorbitol and / or sucrose.
[0033] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0034] Bovine serum albumin (BSA) 1–10%;
[0035] Polyethylene glycol 0.2-2%;
[0036] Sodium chloride 1-4%;
[0037] Tween-20 0.01~0.06%;
[0038] Tween-80 0.01%–0.03%
[0039] ProClin300 0.05-0.4%;
[0040] Sorbitol 1-5%;
[0041] 1-5% sucrose
[0042] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0043] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0044] Bovine serum albumin (BSA) 2–6%;
[0045] Polyethylene glycol 0.2-2%;
[0046] Sodium chloride 1-3%;
[0047] Tween-20 0.01~0.06%;
[0048] Tween-80 0.01%–0.03%
[0049] ProClin300 0.05-0.4%;
[0050] Sorbitol 1-5%;
[0051] 1-5% sucrose
[0052] The remainder is deionized water; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0053] In some preferred embodiments, the mass percentage ratio of bovine serum albumin (BSA), sorbitol, and sucrose is (0.2-5):1:1, more preferably (1-5):1:1.
[0054] A second aspect of the present invention also provides a method for preparing IL-6 lyophilized powder, the method comprising the steps of:
[0055] The IL-6 reaction buffer and the lyophilization protectant were mixed to obtain a pre-freezing solution;
[0056] The pre-frozen liquid was freeze-dried to obtain IL-6 lyophilized powder.
[0057] In some preferred embodiments, the IL-6 reaction buffer and the lyophilization protectant are mixed in a 1:1 volume ratio.
[0058] In some preferred embodiments, the pre-freezing solution contains 1-3% bovine serum albumin by mass.
[0059] The sodium chloride content is 0.5-1.5% by mass;
[0060] The surfactant has a mass percentage content of 0.01-0.03%;
[0061] The preservative has a mass percentage content of 0.1% to 0.2%.
[0062] In some preferred embodiments, the freeze-drying is performed using a freeze dryer.
[0063] In some preferred embodiments, the freeze-drying step includes:
[0064] The process includes precooling, prefreezing, annealing, sublimation, and desorption drying; wherein the sublimation is performed at least once, preferably three times.
[0065] In some preferred embodiments, the temperature of the shelf is 2-6°C during the precooling step, and the precooling time is 30-90 minutes.
[0066] In some preferred embodiments, the temperature of the shelf in the pre-freezing step is -55 to 40°C, and the pre-freezing time is 120 to 180 minutes.
[0067] In some preferred embodiments, during the annealing step, the shelf temperature is first maintained at -25 to -35°C for 120 to 180 minutes, and then cooled to -55 to -40°C and maintained for 120 to 180 minutes.
[0068] In some preferred embodiments, the sublimation includes a first sublimation, a second sublimation, and a third sublimation;
[0069] During the first sublimation, the shelf temperature is -45 to -35°C and maintained for 300 to 600 minutes;
[0070] During the second sublimation, the shelf temperature is -30 to 10°C and maintained for 120 to 240 minutes;
[0071] During the third sublimation, the shelf temperature is 0-10℃ and maintained for 120-240 minutes.
[0072] In some preferred embodiments, during the analytical drying step, the temperature of the shelf is 10–25°C and maintained for 100–300 minutes.
[0073] A third aspect of the present invention also provides an IL-6 lyophilized powder, which is prepared by the method described in the second aspect of the present invention.
[0074] Compared with the prior art, the present invention has at least the following advantages:
[0075] (1) The IL-6 freeze-drying protectant provided by the present invention results in less loss of IL-6 valence during freeze-drying and better molding;
[0076] (2) The IL-6 freeze-drying protectant provided by the present invention can be used to prepare IL-6 freeze-dried powder that can be stored at room temperature for a long time of 1 to 2 years and retains more than 80% of its activity.
[0077] (3) The IL-6 lyophilized powder provided by the present invention is easy to use and can be directly reconstituted with deionized water.
[0078] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation
[0079] In existing technologies, IL-6 liquid reagent is prone to inactivation, difficult to store, and has high storage costs. Through research, the inventors discovered that converting IL-6 liquid reagent into a lyophilized powder can reduce its inactivation rate. However, the lyophilization process still causes a loss of IL-6 activity. To address this problem, the inventors, through extensive experimental research, invented a lyophilization protectant. The composition and ratio of this lyophilization protectant have been optimized through numerous experiments, ensuring that the potency loss of IL-6 after more than 12 months of storage at room temperature does not exceed 20%, thus completing this invention.
[0080] Some embodiments of the present invention provide an IL-6 lyophilization protectant, the IL-6 lyophilization protectant comprising: water and a non-aqueous component dissolved in the water, the non-aqueous component comprising bovine serum albumin, polyethylene glycol, sodium chloride, surfactant, preservative and inorganic acid, and the pH of the IL-6 lyophilization protectant being 7.4-8.0.
[0081] In some preferred embodiments, the inorganic acid is selected from Tris-HCl, hydrochloric acid, sulfuric acid, and phosphoric acid, with hydrochloric acid being more preferred.
[0082] In some preferred embodiments, the surfactant is a nonionic surfactant selected from at least one of Tween-20 and Tween-80.
[0083] In some preferred embodiments, the polyethylene glycol is selected from at least one of PEG6000, PEG8000, PEG10000, PEG20000 and PEG40000; more preferably, the polyethylene glycol is selected from at least one of PEG6000 and PEG8000.
[0084] In some preferred embodiments, the preservative is selected from at least one of ProClin 150, ProClin 200 and ProClin 300, such as ProClin 300.
[0085] In some preferred embodiments, the bovine serum albumin (BSA) content in the IL-6 lyophilization protectant is 1-10% by mass.
[0086] In some preferred embodiments, the polyethylene glycol content is 0.2% to 2% by mass.
[0087] In some preferred embodiments, the sodium chloride content is 1-4% by mass.
[0088] In some preferred embodiments, the surfactant has a mass percentage content of 0.01 to 0.09%.
[0089] In some preferred embodiments, the preservative has a mass percentage content of 0.05% to 0.4%.
[0090] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0091] Bovine serum albumin (BSA) 1–10%;
[0092] Polyethylene glycol 0.2-2%;
[0093] Sodium chloride 1-4%;
[0094] Surfactant 0.01–0.09%;
[0095] Preservative 0.05-0.4%;
[0096] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0097] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0098] Bovine serum albumin (BSA) 2–6%;
[0099] Polyethylene glycol 0.2-1%;
[0100] Sodium chloride 1-3%;
[0101] Surfactant 0.02–0.04%;
[0102] Preservative 0.2-0.4%;
[0103] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0104] In some preferred embodiments, the non-aqueous component further includes sorbitol and / or sucrose.
[0105] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0106] Bovine serum albumin (BSA) 1–10%;
[0107] Polyethylene glycol 0.2-2%;
[0108] Sodium chloride 1-4%;
[0109] Tween-20 0.01~0.06%;
[0110] Tween-80 0.01%–0.03%
[0111] ProClin300 0.05-0.4%;
[0112] Sorbitol 1-5%;
[0113] 1-5% sucrose
[0114] The remainder is deionized water containing inorganic acids; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0115] In some preferred embodiments, the IL-6 lyophilization protectant comprises:
[0116] Bovine serum albumin (BSA) 2–6%;
[0117] Polyethylene glycol 0.2-2%;
[0118] Sodium chloride 1-3%;
[0119] Tween-20 0.01~0.06%;
[0120] Tween-80 0.01%–0.03%
[0121] ProClin300 0.05-0.4%;
[0122] Sorbitol 1-5%;
[0123] 1-5% sucrose
[0124] The remainder is deionized water; the pH of the system is adjusted to 7.4-8.0 using inorganic acids.
[0125] In some preferred embodiments, the mass percentage ratio of bovine serum albumin (BSA), sorbitol, and sucrose is (0.2-5):1:1, more preferably (1-5):1:1.
[0126] The present invention also provides a method for preparing IL-6 lyophilized powder, the method comprising the steps of:
[0127] The IL-6 reaction buffer and the lyophilization protectant were mixed to obtain a pre-freezing solution;
[0128] The pre-frozen liquid was freeze-dried to obtain IL-6 lyophilized powder.
[0129] In some preferred embodiments, the IL-6 reaction buffer and the lyophilization protectant are mixed in a 1:1 volume ratio.
[0130] In some preferred embodiments, the pre-freezing solution contains 1-3% bovine serum albumin by mass.
[0131] The sodium chloride content is 0.5-1.5% by mass;
[0132] The surfactant has a mass percentage content of 0.01-0.03%;
[0133] The preservative has a mass percentage content of 0.1% to 0.2%.
[0134] In some preferred embodiments, the freeze-drying is performed using a freeze dryer.
[0135] In some preferred embodiments, the freeze-drying step includes:
[0136] The process includes precooling, prefreezing, annealing, sublimation, and desorption drying; wherein the sublimation is performed at least once, preferably three times.
[0137] In some preferred embodiments, the temperature of the shelf is 2-6°C during the precooling step, and the precooling time is 30-90 minutes.
[0138] In some preferred embodiments, the temperature of the shelf in the pre-freezing step is -55 to 40°C, and the pre-freezing time is 120 to 180 minutes.
[0139] In some preferred embodiments, during the annealing step, the shelf temperature is first maintained at -25 to -35°C for 120 to 180 minutes, and then cooled to -55 to -40°C and maintained for 120 to 180 minutes.
[0140] In some preferred embodiments, the sublimation includes a first sublimation, a second sublimation, and a third sublimation;
[0141] During the first sublimation, the shelf temperature is -45 to -35°C and maintained for 300 to 600 minutes;
[0142] During the second sublimation, the shelf temperature is -30 to 10°C and maintained for 120 to 240 minutes;
[0143] During the third sublimation, the shelf temperature is 0-10℃ and maintained for 120-240 minutes.
[0144] In some preferred embodiments, during the analytical drying step, the temperature of the shelf is 10–25°C and maintained for 100–300 minutes.
[0145] The embodiments of the present invention also provide an IL-6 lyophilized powder, which is prepared by the method described in the second convenience of the present invention.
[0146] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.
[0147] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.
[0148] Example 1: Preparation of Lyophilization Protectant
[0149] Bovine serum albumin (BSA), PEG6000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0150] Bovine serum albumin (BSA) 10%
[0151] PEG6000 0.2%
[0152] 4% sodium chloride
[0153] Tween 20 0.01%
[0154] ProClin300 0.4%
[0155] The rest is deionized water.
[0156] Example 2: Preparation of lyophilization protectant
[0157] Bovine serum albumin (BSA), PEG6000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0158] Bovine serum albumin (BSA) 1%
[0159] PEG6000 2%
[0160] 1% sodium chloride
[0161] Tween 20 0.04%
[0162] ProClin300 0.05%
[0163] The rest is deionized water.
[0164] Example 3: Preparation of Lyophilization Protectant
[0165] Bovine serum albumin (BSA), PEG8000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0166] Bovine serum albumin (BSA) 5%
[0167] PEG8000 2.0%
[0168] Sodium chloride 2.5%
[0169] Tween 20 0.02%
[0170] ProClin300 0.3%
[0171] The rest is deionized water.
[0172] Example 4: Preparation of lyophilization protectant
[0173] Bovine serum albumin (BSA), PEG6000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0174] Bovine serum albumin (BSA) 2%
[0175] PEG6000 2%
[0176] Sodium chloride 1.0%
[0177] Tween 20 0.06%
[0178] ProClin300 0.2%
[0179] The rest is deionized water.
[0180] Example 5: Preparation of lyophilization protectant
[0181] Bovine serum albumin (BSA), PEG6000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0182] Bovine serum albumin (BSA) 6%
[0183] PEG6000 2%
[0184] Sodium chloride 3.0%
[0185] Tween 20 0.02%
[0186] ProClin300 0.4%
[0187] The rest is deionized water.
[0188] Example 6: Preparation of Lyophilization Protectant
[0189] Bovine serum albumin (BSA), sodium chloride, Tween 20, ProClin 300, Tween 80, sorbitol, sucrose, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0190] Bovine serum albumin (BSA) 10%
[0191] PEG6000 0.2%
[0192] 4% sodium chloride
[0193] Tween 20 0.01%
[0194] ProClin300 0.4%
[0195] Tween 80 0.01%
[0196] Sorbitol 1%
[0197] 1% sucrose
[0198] The rest is deionized water.
[0199] Example 7: Preparation of Lyophilization Protectant
[0200] Bovine serum albumin (BSA), sodium chloride, Tween 20, ProClin 300, Tween 80, sorbitol, sucrose, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0201] Bovine serum albumin (BSA) 10%
[0202] PEG6000 0.2%
[0203] 4% sodium chloride
[0204] Tween 20 0.01%
[0205] ProClin300 0.4%
[0206] Tween 80 0.03%
[0207] 5% Sorbitol
[0208] 5% sucrose
[0209] The rest is deionized water.
[0210] Example 8: Preparation of IL-6 lyophilized powder
[0211] Step 1, Preparation of IL-6 pre-freezing solution
[0212] The IL-6 antigen was diluted to a suitable working concentration using a buffer solution to obtain the reaction reagent.
[0213] The lyophilization protectant prepared in Example 1 was added to the above reaction reagents and mixed evenly at a volume ratio of 1:1. The pH was adjusted to 7.4-7.8 with acid to obtain the IL-6 pre-freezing solution. The obtained IL-6 pre-freezing solution contained 1-3% bovine serum albumin (BSA), 0.5-1.5% sodium chloride, 0.01-0.03% Tween-20, and 0.1-0.2% ProClin300.
[0214] Step 2: Preparation of IL-6 lyophilized powder
[0215] The prepared pre-freezing solution is added to a container (vial or eight-tube set) according to the preset loading volume, and then transferred to the freeze dryer shelf for freeze drying. The specific method is as follows:
[0216] 1) Pre-cooling: Place the sample in the container and lower the shelf temperature to 2-6°C. Maintain this temperature for 30-90 minutes to ensure the temperature consistency of the sample solution before freeze-drying.
[0217] 2) Pre-freezing: Turn off the pre-cooling setting, lower the shelf temperature to -40 to -55°C at a rate of 1 to 3 min / °C, and maintain the temperature for 120 to 180 min;
[0218] 3) Annealing: Heat the shelf to -25 to -35°C at a rate of 1 to 3 min / °C and hold for 120 to 180 min; then cool it down to -40 to -55°C at a rate of 1 to 3 min / °C and hold for 120 to 180 min.
[0219] 4) Sublimation: First stage: Turn on the vacuum, set the vacuum degree to 0.05~0.5mbar, the cold hydrazine temperature to -60~-77℃, raise the shelf temperature to -35~-45℃, the heating rate is 2~4min / ℃, and hold for 300~600min;
[0220] Secondary heating stage: vacuum degree is 0.05~0.5mbar, cold hydrazine temperature is -60~-80℃, shelf temperature is raised to -10~-30℃, heating rate is 2~3min / ℃, and held for 120~240min;
[0221] Three stages of heating: vacuum degree is 0.05~0.5mbar, cold hydrazine temperature is -60~-80℃, shelf temperature is raised to 0~-10℃, heating rate is 2~3min / ℃, and held for 120~240min;
[0222] 5) Desorption and drying: Set the vacuum degree to 0.01~0.05mbar, the cold hydrazine temperature to -60~-80℃, raise the shelf temperature to 10~25℃, the heating rate is 1~2min / ℃, and hold for 100~300min.
[0223] In the other examples and comparative examples, the preparation process of IL-6 lyophilized powder was largely the same as in Example 8, except that different lyophilization protectants were used. The prepared IL-6 lyophilized powder was then tested.
[0224] Comparative Example 1
[0225] Without adding any freeze-drying protectant, a diluted solution of IL-6 of the same concentration was freeze-dried, and the freeze-drying process is described in Example 8.
[0226] Comparative Example 2
[0227] Bovine serum albumin (BSA), sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0228] Bovine serum albumin (BSA) 10%
[0229] 4% sodium chloride
[0230] Tween 20 0.01%
[0231] ProClin300 0.4%
[0232] The rest is deionized water.
[0233] Using the lyophilization protectant prepared above, the IL-6 dilution was lyophilized according to the lyophilization process in Example 8 to obtain IL-6 lyophilized powder.
[0234] Comparative Example 3
[0235] Bovine serum albumin (BSA), PEG20000, sodium chloride, Tween 20, ProClin 300, and deionized water were mixed in the following mass ratio, and hydrochloric acid was added to adjust the pH to 7.4–8.0:
[0236] Bovine serum albumin (BSA) 10%
[0237] PEG20000 0.2%
[0238] 4% sodium chloride
[0239] Tween 20 0.01%
[0240] ProClin300 0.4%
[0241] The rest is deionized water.
[0242] Using the lyophilization protectant prepared above, the IL-6 dilution was lyophilized according to the lyophilization process in Example 8 to obtain IL-6 lyophilized powder.
[0243] [Stability Test of IL-6 Lyophilized Powder at Room Temperature]
[0244] The lyophilized stability of IL-6 lyophilized powders prepared in Examples 8 to 14 and Comparative Examples 1 to 3 was tested. The specific steps were as follows: the IL-6 lyophilized powders prepared in the examples and comparative examples were placed at 37°C for 7 days and 30 days, respectively. Equal volumes of deionized powder were then used for reconstitution, followed by reaction with hypersensitized anti-IL-6 antibody latex particles to form an immune complex. The turbidity was measured using a UV spectrophotometer, and its absorbance at 280 nm was measured. The potency loss was calculated compared to that of IL-6 in its liquid state before lyophilization. The results are shown in Table 1.
[0245] Table 1
[0246]
[0247] As shown in Table 1, the potency loss of the freeze-dried product is less than 8% compared with the liquid control, and the potency loss does not exceed 15% after being stored at 37°C for one month. The IL-6 freeze-drying protectant formulation provided by this invention can effectively avoid IL-6 freeze-drying loss, maintain IL-6 activity, and maintain high stability after 12 months of storage at room temperature (30 days at 37°C is equivalent to 12 months at room temperature).
[0248] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.
Claims
1. An IL-6 lyophilization protectant, characterized in that, The composition of the IL-6 lyophilization protectant, by weight percentage, is as follows: Bovine serum albumin (BSA) 1-10%; Polyethylene glycol 0.2-2%; Sodium chloride 1-4%; Surfactant 0.01~0.09%; Preservatives: 0.05-0.4%; The remainder is deionized water containing inorganic acids; The pH of the IL-6 lyophilization protectant is 7.4-8.0; Among them, polyethylene glycol is selected from at least one of PEG6000 and PEG8000; The preservative is selected from at least one of ProClin 150, ProClin 200 and ProClin 300; The surfactant is Tween 20 and / or Tween 80.
2. The freeze-drying protectant according to claim 1, characterized in that, The inorganic acid is selected from any one of Tris-HCl, hydrochloric acid, sulfuric acid, and phosphoric acid.
3. The freeze-drying protectant according to claim 2, characterized in that, The IL-6 lyophilization protectant has the following composition: Bovine serum albumin (BSA) 2-6%; Polyethylene glycol 0.2-1%; Sodium chloride 1-3%; Surfactant 0.02~0.04%; Preservative 0.2~0.4%; The remainder is deionized water containing inorganic acids; The pH of the IL-6 lyophilization protectant is 7.4-8.
0.
4. A method for preparing IL-6 lyophilized powder, characterized in that, The method includes the following steps: The IL-6 reaction buffer and the lyophilization protectant according to any one of claims 1 to 3 are mixed to obtain a pre-freezing solution; The pre-frozen liquid was freeze-dried to obtain IL-6 lyophilized powder.
5. An IL-6 lyophilized powder, characterized in that, The IL-6 lyophilized powder is prepared by the method described in claim 4.