Human growth hormone freeze-drying protective agent

Abstract

The invention relates to the field of biological immunity and pharmaceutical preparations, and particularly discloses a human growth hormone (GH) freeze-drying protective agent. The invention further discloses a freeze-drying preparation prepared from the freeze-drying protective agent. The freeze-drying preparation mainly comprises the following components in parts by weight: 10-100 mM of a buffer solution, 1-30 parts of a protective agent, a stabilizer and an excipient, 0.01-1.5 parts of a surfactant, 0.1-0.8 part of an antioxidant and 0.05-3 parts of a preservative. The invention provides a specific preparation method of the freeze-dried preparation, the preparation has the advantages of long preservation period, short redissolution time compared with a common reagent, good form and good structural stability after freeze-drying, provides favorable protection conditions for preparation of ELISA standard substances and internal control substances, is beneficial to maintenance of the stability of proteins, and has good application prospects. The specificity and the sensitivity of the ELISA kit are improved.

Description

Technical Field

[0001] The present application relates to the fields of biotechnology and pharmaceutical preparations, and to the preparation and application of a lyophilized protein preparation, which provides a good foundation for the development and application of ELISA kits. Background Art

[0002] Human Growth Hormone (HGH) is a peptide hormone secreted by the anterior pituitary gland of the human body. It is composed of 191 amino acids and can promote the growth of bones, internal organs and the whole body. It promotes protein synthesis, affects fat and mineral metabolism, and plays a key role in human growth and development. However, HGH is easily inactivated during freeze-drying and storage, resulting in reduced biological activity. Therefore, the development of an effective freeze-drying protectant is crucial to maintaining the stability and activity of HGH.

[0003] Vacuum freeze-drying technology is a special drying method that freezes the solution at low temperatures and then directly evaporates the water in a vacuum to obtain a dry product. This technology can maintain the physical and chemical properties of the substance, as well as the integrity of its appearance and active ingredients, so that the product can be restored to its original state after rehydration and can be sealed and stored for a long time. Vacuum freeze-drying technology provides a stable storage method for various liquid test kits that have poor stability and require cold chain transportation and storage.

[0004] During the vacuum freeze-drying process, proteins will be damaged due to changes in low temperature, osmotic, mechanical and oxidative factors, resulting in the eventual loss of protein activity. Therefore, during the freeze-drying process, it is necessary to add appropriate freeze-dried protective components to maintain the stability and specificity of the protein and provide good protein preservation conditions. Freeze-dried protective agents can be classified according to their functions and properties: protective agents, fillers, antioxidants and acid-base regulators, etc.; according to the type of substance, they can be divided into sugars, alcohols, amino acids, salts and other components; according to the molecular weight, they can be divided into high molecular weight compounds and low molecular weight compounds, etc. Summary of the invention

[0005] The purpose of the present invention is to provide a highly stable and highly active freeze-dried protective agent for human growth hormone and its application, which provides powerful conditions for the preparation and storage of ELISA kit quality control products and standard products.

[0006] The stabilizer components are mainly sugars, including trehalose, sucrose, lactose, etc.; the excipient is mannitol; sugars are the most common and most frequently used freeze-drying protective agent, which can protect against low-temperature stress, freezing stress and drying stress generated during freeze-drying, and can also protect protein drug molecules at all stages of freeze-drying and during storage. The hydroxyl groups on polyhydroxy compounds such as sucrose, trehalose and mannitol can replace the structural water of some protein molecules, and form hydrogen bonds with the carbonyl and amino groups in the protein molecules, so that they can still maintain their original molecular structure under water-deficient conditions without changing their activity.

[0007] Amino acids are also a common lyophilization protective agent for protein drugs. They can stabilize protein drugs through a variety of mechanisms. Amino acids are amphoteric compounds. Their molecular structures contain not only acidic carboxyl groups but also basic amino groups. They have the function of regulating the pH value of the solution and can also inhibit the pH change of the lyophilized solution during low-temperature storage and freeze-drying of proteins. Amino acids with different structures have different hydrophilic functions. Amino acids with stronger hydrophilicity are more likely to form hydrogen bonds with proteins, which plays a vital protective role in the stability of proteins during the freeze-drying process.

[0008] The present invention provides a method for preparing a human growth hormone freeze-dried protective agent, and the technical scheme is as follows:

[0009] A human growth hormone freeze-dried protective agent, the freeze-dried protective agent comprises the following raw materials and usage amounts: 10-100 mM buffer, 1-30 parts of stabilizer, 1-30 parts of protective agent, 1-30 parts of excipient, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, 0.05-3 parts of preservative. Since the freeze-dried protective agent contains the protective agent, the freeze-dried preparation protected by the freeze-dried protective agent is yellow in color after reconstitution and can be used for color marking.

[0010] Preferably, based on the above technical solution, the lyophilization protective agent is composed of the following components in parts by weight:

[0011] 10-100 parts of buffer, 1-30 parts of sucrose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0012] The stabilizer and the excipient may be used alternatively. Based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0013] 10-100 parts of buffer, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0014] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0015] 10-100 parts of buffer, 1-30 parts of lactose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0016] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0017] Buffer 10-100 parts, sucrose 1-30 parts, surfactant 0.01-1.5 parts, bovine serum albumin 0.01-1 parts, antioxidant 0.1-0.8 parts, preservative 0.05-3 parts. Since the addition of bovine serum albumin to the freeze-dried preparation will turn yellow, it can be added or not.

[0018] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0019] 10-100 parts of buffer, 1-30 parts of sucrose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0020] Furthermore, the buffer is one of citric acid-sodium citrate buffer, succinic acid-sodium succinate buffer, Tris-HCl buffer, glycine buffer, histidine buffer, and phosphate buffer, with a pH of 6.0-8.0, and a specific dosage of 10-100 mM.

[0021] Furthermore, the surfactant is Tween 20, Tween 80, ethylene glycol or Triton X-100, and its weight part is 0.01-1.5 parts.

[0022] Furthermore, the antioxidant is vitamin D, vitamin E or sodium thiosulfate, and its weight portion is 0.1-1.0 parts.

[0023] Furthermore, the preservative is potassium azide, sodium azide or Proclin-300, and its weight proportion is 0.05-3 parts.

[0024] Preferably, the surfactant is Tween 20.

[0025] Preferably, the antioxidant is sodium thiosulfate.

[0026] Preferably, the preservative is Proclin-300.

[0027] Furthermore, after the components are dissolved, the pH value is adjusted to 6.0-8.0; preferably 7.2-7.4.

[0028] More preferably, the buffer is a phosphate buffer, and the mass ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 2.5±0.5:1.

[0029] A human growth hormone freeze-dried preparation is provided, wherein any of the above-mentioned freeze-dried protective agents is mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; and the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0030] In the present invention, growth hormone is preferably GH or GH variant, which can be obtained by a variety of suitable methods, such as by recombinant means, or further separated or purified from natural sources, or chemically synthesized, or any combination thereof. "GH variant" refers to a molecule that has different amino acid sequences, glycosylation patterns or inter-subunit connections from human GH, but exhibits GH activity.

[0031] Weigh each component according to its weight, dissolve each component in a suitable buffer, adjust the pH value to 6.0-8.0 and perform freeze-drying.

[0032] Compared with the prior art, the beneficial effects of this application include:

[0033] The lyophilized protective agent invented in the present application is composed of only sucrose / trehalose, mannitol, Tween 20, sodium thiosulfate, Proclin 300, etc. The composition is relatively simple and does not interfere with the target protein. The lyophilized preparation prepared therefrom has a good reconstitution effect. The lyophilized protective agent effectively ensures the stability of the lyophilized preparation. At the same time, the obtained lyophilized preparation is in the shape of a round cake, which has the advantages of beautiful appearance, high intact rate, stable enzyme activity, simple operation, etc.

[0034] Mannitol has a great influence on the morphology of the reconstituted lyophilized preparation. The morphology, color and reconstitution effect of mannitol are better at 10-30. After adding BSA (bovine serum albumin) to the lyophilized preparation, the color becomes yellowish, and it can be used in lyophilized preparations that need to be color-marked.

[0035] In addition, the preparation process of the lyophilized preparation provided in the present application is stable, the conditions are mild, the reproducibility is good, and it is suitable for large-scale production. The present application greatly improves the preparation efficiency and storage period of the standard and internal control products of the liquid detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The color and morphology of the lyophilized lyoprotectants of Examples 2, 14 and 18 after lyophilization. DETAILED DESCRIPTION

[0037] A human growth hormone freeze-dried protective agent, the freeze-dried protective agent comprises the following raw materials and usage amounts: 10-100 mM buffer, 1-30 parts of stabilizer, 1-30 parts of protective agent, 1-30 parts of excipient, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, 0.05-3 parts of preservative. Since the freeze-dried protective agent contains the protective agent, the freeze-dried preparation protected by the freeze-dried protective agent is yellow in color after reconstitution and can be used for color marking.

[0038] Preferably, based on the above technical solution, the lyophilization protective agent is composed of the following components in parts by weight:

[0039] 10-100 parts of buffer, 1-30 parts of sucrose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0040] The stabilizer and the excipient may be used alternatively. Based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0041] 10-100 parts of buffer, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0042] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0043] 10-100 parts of buffer, 1-30 parts of lactose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0044] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0045] Buffer 10-100 parts, sucrose 1-30 parts, surfactant 0.01-1.5 parts, bovine serum albumin 0.01-1 parts, antioxidant 0.1-0.8 parts, preservative 0.05-3 parts. Since the addition of bovine serum albumin to the freeze-dried preparation will turn yellow, it can be added or not.

[0046] Preferably, based on the above technical solution, preferably, the lyophilization protective agent is composed of the following components in parts by weight:

[0047] 10-100 parts of buffer, 1-30 parts of sucrose, 1-30 parts of mannitol, 0.01-1.5 parts of surfactant, 0.1-0.8 parts of antioxidant, and 0.05-3 parts of preservative.

[0048] Furthermore, the buffer is one of citric acid-sodium citrate buffer, succinic acid-sodium succinate buffer, Tris-HCl buffer, glycine buffer, histidine buffer, and phosphate buffer, with a pH of 6.0-8.0, and a specific dosage of 10-100 mM.

[0049] Furthermore, the surfactant is Tween 20, Tween 80, ethylene glycol or Triton X-100, and its weight part is 0.01-1.5 parts.

[0050] Furthermore, the antioxidant is vitamin D, vitamin E or sodium thiosulfate, and its weight portion is 0.1-1.0 parts.

[0051] Furthermore, the preservative is potassium azide, sodium azide or Proclin-300, and its weight proportion is 0.05-3 parts.

[0052] Preferably, the surfactant is Tween 20.

[0053] Preferably, the antioxidant is sodium thiosulfate.

[0054] Preferably, the preservative is Proclin-300.

[0055] Furthermore, after the components are dissolved, the pH value is adjusted to 6.0-8.0; preferably 7.2-7.4.

[0056] More preferably, the buffer is a phosphate buffer, and the mass ratio of disodium hydrogen phosphate to sodium dihydrogen phosphate is 2.5±0.5:1.

[0057] A human growth hormone freeze-dried preparation is provided, wherein any of the above-mentioned freeze-dried protective agents is mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; and the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0058] In the present invention, growth hormone is preferably GH or GH variant, which can be obtained by a variety of suitable methods, such as by recombinant means, or further separated or purified from natural sources, or chemically synthesized, or any combination thereof. "GH variant" refers to a molecule that has different amino acid sequences, glycosylation patterns or inter-subunit connections from human GH, but exhibits GH activity.

[0059] Weigh each component according to its weight, dissolve each component in a suitable buffer, adjust the pH value to 6.0-8.0 and perform freeze-drying.

[0060] Source of raw materials: Unless otherwise specified, the raw materials required for this application can be purchased commercially.

[0061] Examples 1 to 18, preparation of GH freeze-dried preparation, single factor experiment to screen the proportion of each component.

[0062] The formulas of GH freeze-dried preparations prepared in Examples 1 to 18 of the present invention are shown in Table 1 below.

[0063] Example 1

[0064] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 1 part of sucrose, 10 parts of mannitol, 0.1 part of Tween 20, 0.1 part of sodium thiosulfate, and 0.1 part of Proclin-300.

[0065] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0066] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0067] Example 2

[0068] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0069] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0070] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0071] Example 3

[0072] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 30 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0073] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0074] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0075] Example 4

[0076] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100mM PBS buffer, 1 part of trehalose, 10 parts of mannitol, 0.1 part of Tween 20, 0.1 part of sodium thiosulfate, and 0.1 part of Proclin-300.

[0077] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0078] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0079] Example 5

[0080] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of trehalose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0081] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0082] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0083] Example 6

[0084] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 30 parts of trehalose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0085] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0086] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0087] Example 7

[0088] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 1 part of mannitol, 0.1 part of Tween 20, 0.1 part of sodium thiosulfate, and 0.1 part of Proclin-300.

[0089] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0090] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0091] Example 8

[0092] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0093] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0094] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0095] Example 9

[0096] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 30 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0097] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0098] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is white.

[0099] Example 10

[0100] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 1 part of BSA, 0.1 part of Tween 20, 0.1 part of sodium thiosulfate, and 0.1 part of Proclin-300.

[0101] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0102] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolution color is yellow.

[0103] Embodiment 11

[0104] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 10 parts of BSA, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0105] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0106] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is yellow.

[0107] Example 12

[0108] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 30 parts of BSA, 0.1 part of Tween 20, 0.1 part of sodium thiosulfate, and 0.1 part of Proclin-300.

[0109] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0110] The freeze-dried preparation prepared by the freeze-dried protective agent of this example has a poor morphology after freeze-drying, is easy to collapse, and is easy to re-dissolve. The re-dissolution time is 1 S, and the re-dissolved color is yellow.

[0111] Embodiment 13

[0112] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.01 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0113] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0114] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0115] Embodiment 14

[0116] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.5 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0117] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0118] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0119] Embodiment 15

[0120] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 1.5 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0121] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0122] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0123] Example 16

[0124] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.1 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0125] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0126] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0127] Embodiment 17

[0128] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.5 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0129] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0130] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0131] Embodiment 18

[0132] A human growth hormone freeze-dried protective agent comprises the following raw materials and dosages: 10-100 mM PBS buffer, 10 parts of sucrose, 10 parts of mannitol, 0.1 parts of Tween 20, 0.8 parts of sodium thiosulfate, and 0.1 parts of Proclin-300.

[0133] The above components are mixed with 0.05 parts of human growth hormone recombinant protein to obtain a mixed solution; the mixed solution is freeze-dried to obtain the freeze-dried reagent.

[0134] The lyophilized protective agent prepared in this example has a good shape, a round cake shape, is easy to re-dissolve, the re-dissolution time is 1 S, and the re-dissolved color is white.

[0135] Table 1: Formulations of GH freeze-dried preparations of Examples 1 to 18

[0136]

[0137]

[0138] 0.05 parts of GH, Tween 20, sucrose or trehalose, mannitol, sodium thiosulfate and buffer solution were added into the container according to the formula of Examples 1-18, mixed, stirred to completely dissolve, adjusted the pH value to 7.2, filtered, and the filtered solution was placed in a sealed glass bottle and freeze-dried.

[0139] By observing the morphology, color and re-dissolution effect of Examples 1-18, it can be seen that the morphology, color and re-dissolution effect are all good when the ratio of sucrose is 10, trehalose is 30, and mannitol is 10-30; after adding BSA (bovine serum albumin) to the lyophilized preparation, the color is yellowish. When the color of the lyophilized preparation after re-dissolution is required to be white, the addition of BSA is cancelled. On the other hand, if the lyophilized preparation needs to be color-marked, BSA can be added thereto. Since the lyophilized preparation has a good morphology after re-dissolution and is in the shape of a round cake, Examples 2, 6, 8, 13-18 were selected for subsequent stability and activity tests.

[0140] Stability and activity testing of GH freeze-dried preparations

[0141] The finished reagents of Examples 1-18 were stored in a -20° refrigerator, and the stability and resolubility of the lyophilized preparations were analyzed and tested using ELISA kits.

[0142] Testing the stability and activity of GH freeze-dried preparations

[0143] 1. Preparation of solid phase carrier: 96-well microplate is usually used as the solid phase carrier.

[0144] 2. Absorption of antibodies: adding antibodies to a microplate and incubating under specific conditions to allow them to be adsorbed onto a solid phase carrier; the antibody is an anti-human growth hormone antibody.

[0145] 3. Blocking: Use 0.5% BSA buffer (prepared in PBS, containing 0.1% sodium azide) to block the unadsorbed sites to reduce nonspecific binding.

[0146] 4. Add 100 μL of the diluted standard (lyophilized human growth hormone protein), cover the ELISA plate, and incubate at 37°C for 90 minutes. Tips: When adding samples, add the samples to the bottom of the ELISA plate, try not to touch the well wall, and gently shake to mix to avoid bubbles. The addition time should be controlled within 10 minutes.

[0147] 5. Shake off the liquid in the wells without washing. Add 100 μL of biotinylated antibody working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 1 hour.

[0148] 6. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μL of washing solution to each well, soak for 1 minute, suck or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. Tip: You can use a plate washer here and other washing steps (refer to the parameter settings of Beijing Top DEM-3 plate washer: 2-point aspiration, add 350μL of washing solution to each well, vibrate the plate for 5 seconds, and aspirate for 0.5 seconds). Please proceed to the next step immediately after washing the plate, and do not let the microplate dry.

[0149] 7. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes.

[0150] 8. Shake off the liquid in the wells and wash the plate 5 times.

[0151] 9. Add 90 μL of substrate solution (TMB) to each well, cover the plate with film, and incubate at 37°C in the dark for about 15 minutes. Tips: shorten or extend the time as appropriate according to the actual color development, but do not exceed 30 minutes. When a clear gradient appears in the standard well (clear blue gradient appears in the first 4 color development wells), it can be terminated. Turn on the microplate reader 15 minutes in advance to preheat.

[0152] 10. Add 50 μL of stop solution (concentrated sulfuric acid) to each well to terminate the reaction. Tip: The order of adding the stop solution should be the same as that of adding the substrate solution.

[0153] 11. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme-labeled instrument.

[0154] 12. Data analysis: Perform qualitative analysis based on OD values.

[0155] Experimental Results

[0156] As shown in Table 2, the OD values ​​obtained by ELISA test verified the 30-day stability and activity of Example 2, Example 6, Example 8, and Examples 13-18.

[0157]

[0158] like Figure 1 As shown, the picture shows the color and morphology of the lyophilized lyophilized protectants of Examples 2, 14 and 18 after lyophilization. It can be seen that the color is white and does not collapse.

[0159] As shown in Table 1, A, B, C, D, E, F, G, and H are serial numbers, respectively representing the concentration gradients of 8 points on the standard curve. By comparing the OD values ​​of Examples 2, 6, 8, and 13 to 18, it can be found that when the proportion of sodium thiosulfate is 0.8 parts, the stability and activity of the standard curve are better.

[0160] It can be seen that when the ratio of sodium thiosulfate is 0.8 parts and Tween 20 is 0.01 parts, the stability and activity of the standard song are better. Finally, sucrose 10 parts, sodium thiosulfate 0.8 parts, mannitol 10 parts and Tween 20 0.01 parts are selected as the freeze-drying protective solution with the best effect.

[0161] In summary, the present invention successfully prepared a human growth hormone lyophilized protectant containing the lyophilized protectant by screening the lyophilized protectant components, which can quantitatively detect human growth hormone with high detection sensitivity, wide detection range and high accuracy, and has good promotion and application value.

[0162] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A human growth hormone lyophilization protective agent, which specifically comprises human growth hormone recombinant protein, a buffer suitable for the protein, a stabilizer, an excipient, a surfactant, a preservative and an antioxidant, wherein the pH of the buffer component is 6.0-8.

0.

2. A human growth hormone freeze-dried protective agent according to claim 1, characterized in that: The buffer is one of citric acid-sodium citrate buffer, succinic acid-sodium succinate buffer, Tris-HCl buffer, glycine buffer, histidine buffer, and phosphate buffer, with a pH of 6.0-8.0 and a specific dosage of 10-100 mM.

3. A human growth hormone freeze-dried protective agent according to claim 1, characterized in that: The stabilizer is one or more of sucrose, trehalose and lactose, and the amount is 1-30 parts by weight.

4. A human growth hormone freeze-dried protective agent according to claim 1, characterized in that: The excipient is mannitol, which is 1-30 parts by weight.

5. The human growth hormone lyophilized protective agent according to claim 1, characterized in that: The invention also comprises a protective agent, which is one or more of bovine serum albumin, casein, peptone, and serum plasma, and the protective agent is 1-30 parts by weight.

6. The human growth hormone lyophilization protectant according to claim 1, characterized in that: The surfactant is one or more of Tween 20, Tween 80, ethylene glycol, and Triton X-100, and the amount thereof is 0.01-1.5 parts by weight.

7. The human growth hormone lyophilization protectant according to claim 1, characterized in that: The antioxidant is one or more of vitamin D, vitamin E, and sodium thiosulfate, and the amount thereof is 0.1-1.0 parts by weight.

8. The human growth hormone lyophilization protectant according to claim 1, characterized in that: The preservative is one or more of potassium azide, sodium azide, and Proclin-300, and the preservative is 0.05-3 parts by weight.

9. The human growth hormone lyophilization protectant according to claim 5, characterized in that: The protective agent is bovine serum albumin, and the freeze-dried preparation prepared by the freeze-dried protective agent is yellow in color after being reconstituted.

10. A freeze-dried preparation of human growth hormone, characterized in that: The lyophilized protective agent according to any one of claims 1 to 9 is mixed with human growth hormone recombinant protein to obtain a mixed solution; and the mixed solution is freeze-dried to obtain the lyophilized reagent.

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