Omoflaibro antigen stock solution and preparation method of Omoflaibro antigen stock solution

Abstract

The invention discloses an emoflabizumab stock solution, and relates to the technical field of protein preparation prescription development, and the emoflabizumab stock solution comprises the following raw materials: emoflabizumab, a pH regulator, sucrose, polysorbate 80 and water; wherein the mass concentration of the emoflazumab is 10 g / L to 30 g / L; the pH value of the Omoflaibutin antigen stock solution is 5 to 6. The oral solution of the Omoflabizumab, provided by the invention, has good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of protein preparation prescription development, and in particular to an oemopoietin monoantigen solution and a preparation method thereof. Background Art

[0002] Omoopibizumab is an innovative long-acting recombinant humanized monoclonal antibody. The commonly used enzyme conjugate diluent cannot effectively maintain the activity of Omoopibizumab. The method to maintain the biological activity of protein is usually to store the protein solution in a low-temperature environment. However, long-term storage in a low-temperature environment will cause a significant loss of its biological activity. Therefore, how to store Omoopibizumab for a long time without affecting or less affecting its biological activity is a problem to be solved. Summary of the invention

[0003] The main purpose of the present invention is to provide an Omopoulin monoantigen solution and a preparation method thereof, aiming to improve the stability of the Omopoulin monoantigen solution.

[0004] To achieve the above object, the present invention provides an omoprobamate monoantigen solution, which comprises the following raw materials: omoprobamate, a pH regulator, sucrose, polysorbate 80 and water;

[0005] Wherein, the mass concentration of the Omopoubimab is 10 g / L to 30 g / L;

[0006] The pH of the Omopoulin single antigen solution is 5-6.

[0007] In one embodiment, the mass concentration of sucrose in the Omopoulin monoantigen solution is 0.02 g / ml to 0.08 g / ml.

[0008] In one embodiment, in the Omopoulin single antigen solution, the mass concentration of polysorbate 80 is 0.0001 g / ml to 0.0005 g / ml.

[0009] In one embodiment, the mass concentration of omoprofen is 18 g / L to 20 g / L.

[0010] In one embodiment, the pH adjuster comprises L-histidine and glacial acetic acid.

[0011] In one embodiment, in the Omopoulin monoantigen solution, the molar concentration of the pH adjuster is 10 mM to 30 mM.

[0012] In one embodiment, the pH of the Omopoulin single antigen solution is 5.5.

[0013] The present invention also provides a method for preparing the above-mentioned Omopoulin single antigen solution, comprising the following steps:

[0014] ATF harvesting the cell culture fluid to obtain a first intermediate product;

[0015] Purifying the first intermediate product by monoclonal antibody to obtain a second intermediate product;

[0016] Performing ultrafiltration and diafiltration on the second intermediate product to obtain a third intermediate product;

[0017] The third intermediate is mixed with sucrose solution, polysorbate 80 solution and pH regulator to obtain Omopoulin single antigen solution.

[0018] In one embodiment, the step of purifying the first intermediate product by monoclonal antibody to obtain the second intermediate product comprises:

[0019] The first intermediate is subjected to affinity chromatography, virus inactivation, filtration, anion exchange chromatography, cation exchange chromatography, and virus filtration to obtain a second intermediate.

[0020] In the technical solution provided by the present invention, the Omopubai monoantigen solution includes Omopubai monoantigen, pH adjusting agent, sucrose, polysorbate 80 and water, sucrose can adjust the osmotic pressure of the protein stock solution, the pH adjusting agent can adjust the pH of the Omopubai monoantigen solution, and polysorbate 80 is a non-ionic surfactant with a strong solubilizing ability, which can reduce the interfacial tension between protein molecules and water, so that the protein molecules that are not originally soluble in water can be evenly dispersed in the aqueous solution, thereby improving the solubility of the protein. Through the composite of the above-mentioned multiple substances and the control of the protein content in the Omopubai monoantigen solution, an appropriate amount of Omopubai monoantigen can be evenly dispersed in the stock solution, not easy to denature, thereby improving the stability of the Omopubai monoantigen solution. DETAILED DESCRIPTION

[0021] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0022] Omoopibizumab is an innovative long-acting recombinant humanized monoclonal antibody. The commonly used enzyme conjugate diluent cannot effectively maintain the activity of Omoopibizumab. The method to maintain the biological activity of protein is usually to store the protein solution in a low-temperature environment. However, long-term storage in a low-temperature environment will cause a significant loss of its biological activity. Therefore, how to store Omoopibizumab for a long time without affecting or less affecting its biological activity is a problem to be solved.

[0023] In view of this, the present invention proposes an oemopoxetine monoantigen solution and a preparation method thereof, which can improve the stability of the oemopoxetine monoantigen solution.

[0024] The omoprobamin antibody liquid provided by the present invention comprises the following raw materials: omoprobamin antibody, a pH regulator, sucrose, polysorbate 80 and water; wherein the mass concentration of the omoprobamin antibody is 10 g / L to 30 g / L.

[0025] In the technical solution provided by the present invention, the Omopubai monoantigen solution includes Omopubai monoantigen, pH adjusting agent, sucrose, polysorbate 80 and water, sucrose can adjust the osmotic pressure of the protein stock solution, the pH adjusting agent can adjust the pH of the Omopubai monoantigen solution so that the pH of the Omopubai monoantigen solution is 5 to 6, and polysorbate 80 is a non-ionic surfactant with a strong solubilizing ability, which can reduce the interfacial tension between protein molecules and water, so that protein molecules that are not originally soluble in water can be evenly dispersed in the aqueous solution, thereby improving the solubility of the protein. Through the combination of the above-mentioned multiple substances and the control of the protein content in the Omopubai monoantigen solution, an appropriate amount of Omopubai monoantigen can be evenly dispersed in the stock solution, not easy to denature, thereby improving the stability of the Omopubai monoantigen solution.

[0026] When the protein concentration in the Omopubai monoantigen solution is too high, the interaction between molecules is enhanced, and non-natural protein aggregates are easily formed. These aggregates may not be able to restore their natural protein structure, and may also cause the viscosity of the protein solution to increase, further promoting the occurrence of aggregation and precipitation. Low-concentration protein solutions are easily affected by hydration, resulting in a thinning of the water molecule layer on the surface of the protein molecules, thereby increasing the risk of protein denaturation. Low concentrations may also weaken the interaction between protein molecules, resulting in a loose protein structure, which is prone to depolymerization or degradation. Therefore, in the present application, the mass concentration of Omopubai monoantigen in the Omopubai monoantigen solution is 10g / L~30g / L, which can reduce the aggregation and precipitation of Omopubai monoantigen, and reduce the depolymerization or degradation of Omopubai monoantigen. Preferably, the mass concentration of Omopubai monoantigen in the Omopubai monoantigen solution is 18g / L~20g / L, which can better reduce the aggregation and precipitation of Omopubai monoantigen, reduce the depolymerization or degradation of Omopubai monoantigen, and improve the stability of the Omopubai monoantigen solution.

[0027] Furthermore, in the Omopobai single antigen solution, the mass concentration of the sucrose is 0.02g / ml to 0.08g / ml. Specifically, the mass concentration of the sucrose can be 0.02g / ml, 0.04g / ml, 0.05g / ml, 0.06g / ml, 0.08g / ml, etc. The hydroxyl groups of sucrose can form hydrogen bonds with polar groups in proteins, thereby forming a hydration film on the surface of the protein to protect the protein from damage by environmental factors. As the concentration of sucrose increases, the possibility of such hydrogen bond formation increases, and the protective effect on the protein is also enhanced accordingly. However, when the concentration of sucrose is too high, cell damage may occur due to excessive osmotic pressure. As an osmotic pressure regulator, the concentration of sucrose can greatly affect the osmotic pressure of the protein stock solution. Appropriate osmotic pressure helps maintain the normal morphology and function of cells and prevents cells from being damaged by osmotic pressure imbalance. When the sucrose concentration is too low, it may not provide sufficient osmotic pressure support; and when the sucrose concentration is too high, it may cause cell dehydration or rupture. In addition, the addition of sucrose will increase the viscosity of the protein stock solution, thereby improving its fluidity, and this improvement will help subsequent processing and storage operations. However, as the sucrose concentration increases, the viscosity of the solution will gradually increase. Therefore, in the Omopobai monoantigen solution provided in the present application, the mass concentration of the sucrose is 0.02g / ml to 0.08g / ml, which can balance the fluidity and viscosity of the solution while ensuring protein stability, and has a suitable osmotic pressure.

[0028] Further, in the Omopobai single antigen solution, the mass concentration of the polysorbate 80 is 0.0001g / ml~0.0005g / ml. Specifically, the mass concentration of the polysorbate 80 can be 0.0001g / ml, 0.0002g / ml, 0.0004g / ml, 0.0005g / ml, etc. Polysorbate 80 is a nonionic surfactant with a strong solubilizing ability. It can reduce the interfacial tension between protein molecules and water, improve the dispersibility of protein molecules in aqueous solution, and then improve the solubility of protein. In addition, polysorbate 80 can form a stable emulsion in the protein stock solution, and can evenly disperse the oil phase or solid particles in the aqueous phase to form tiny emulsion droplets or particles, thereby preventing the aggregation and precipitation of protein molecules, and this emulsification and dispersion effect helps to maintain the stability and uniformity of the protein stock solution. In addition, by competing with the protein for the gas-liquid interface, the exposure of the protein is reduced, thereby reducing its interaction with nonspecific surfaces and increasing the stability of Omopoubimab. If the concentration of polysorbate 80 is too low, it may not provide sufficient protein stability, resulting in denaturation and aggregation of the protein during storage and transportation; while a high concentration of polysorbate 80 may lead to excessive stability, causing abnormal interactions between protein molecules, thereby affecting its normal structure and function, and may also increase the formation of particles in the solution. The formation of particles not only affects the clarity of the solution, but also may affect the purity and activity of the protein. Therefore, in the Omopoubimab antigen solution provided in the present application, the mass concentration of polysorbate 80 is 0.0001g / ml to 0.0005g / ml, which can provide suitable solubility for Omopoubimab and has a good effect of preventing denaturation and aggregation of Omopoubimab.

[0029] Further, the pH adjusting agent includes L-histidine and glacial acetic acid. L-histidine has good buffering capacity, especially near the pKa value of its side chain (about pH 6.0), which makes it an effective tool for maintaining the pH stability of biological preparations. In the Omopobai single antigen solution, this characteristic helps to ensure that the three-dimensional structure of the protein is not affected by pH changes, thereby maintaining its activity and stability. L-histidine can regulate the pH while maintaining the activity and stability of the protein. Glacial acetic acid can quickly and accurately adjust the pH value of the solution to meet the needs.

[0030] Furthermore, in the Omopoulin monoantigen solution, the molar concentration of the pH regulator is 10mM to 30mM. Specifically, the molar concentration of the pH regulator can be 10mM, 15mM, 20mM, 25mM, 30mM, etc. This setting can make the pH of the Omopoulin monoantigen solution 5 to 6.

[0031] The pH of the Omopoulin monoantigen solution is 5 to 6. This pH value is close to the isoelectric point (pI) of Omopoulin monoantigen, which makes the net charge on the surface of the protein molecule close to zero, reducing the electrostatic repulsion or attraction between protein molecules, which is beneficial for the protein to maintain its natural conformation and activity, thereby improving stability. Further, the pH of the Omopoulin monoantigen solution is 5.5, which can be more conducive to the protein maintaining its natural conformation and activity.

[0032] The present invention also provides a method for preparing the above-mentioned Omopoulin single antigen solution, comprising the following steps:

[0033] S10, harvesting the cell culture fluid by ATF, and obtaining a first intermediate after affinity chromatography;

[0034] During alternating tangential flow filtration (ATF harvesting), a diaphragm pump driven by compressed air is used to achieve rapid circulation of the culture medium between the bioreactor and the system, and hollow fiber tangential flow filtration technology is used to intercept cells, while allowing metabolic waste in the culture medium to pass through the membrane and out of the system, thereby effectively separating cells and metabolites.

[0035] S20, purifying the first intermediate product to obtain a second intermediate product;

[0036] By performing monoclonal antibody purification on the first intermediate, efficient and high-purity purification of the first intermediate can be achieved.

[0037] S30, performing ultrafiltration and diafiltration on the second intermediate product to obtain a third intermediate product;

[0038] Ultrafiltration technology uses pressure difference as a driving force. Through a membrane with a certain pore size, it can retain proteins while allowing small molecules in the solution, such as nucleic acids, endotoxins, salts, alcohols, etc. to pass through. Through the selective retention of the ultrafiltration membrane, a large number of small molecule impurities released by cell rupture can be effectively removed during the protein purification process, thereby improving the purity of the protein and concentrating the protein solution. In the later stages of ultrafiltration, the permeability of proteins through the membrane can be increased by adding diafiltration water, thereby further increasing the concentration of proteins in the retentate.

[0039] S40, mixing the third intermediate with sucrose solution, polysorbate 80 solution and pH regulator to obtain Omopoulin single antigen solution.

[0040] The sterilized and filtered Omopoulin monoantigen solution can be dispensed into a container system and stored at -60°C to -80°C for a long time.

[0041] Furthermore, the step of purifying the first intermediate product by monoclonal antibody to obtain the second intermediate product comprises:

[0042] The first intermediate is subjected to affinity chromatography, virus inactivation, filtration, anion exchange chromatography, cation exchange chromatography, and virus filtration to obtain a second intermediate. The first intermediate is subjected to affinity chromatography to capture the product, followed by low pH virus inactivation and neutralization, deep filtration, and then anion exchange chromatography, cation exchange chromatography, and virus removal filtration to obtain a second intermediate. Affinity chromatography has the advantages of high efficiency and high selectivity, which can ensure the effective capture of the target monoclonal antibody while removing a large amount of impurities. Virus inactivation and filtration can significantly improve the purity of the product, laying the foundation for subsequent purification steps. Using anion exchange chromatography and cation exchange chromatography to treat the first intermediate after deep filtration can remove remaining trace impurities and unwanted target product structure variants.

[0043] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0044] Example 1

[0045] Provided is an oemopoxetine monoantigen solution, comprising 20 mg / mL oemopoxetine monoantigen, 20 mM pH regulator, 0.08 g / ml sucrose, 0.0005 g / ml polysorbate 80 and water, wherein the pH of the oemopoxetine monoantigen solution is 5.5;

[0046] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0047] Example 2

[0048] Provided is an omoprobamate monoantigen solution, comprising 10 mg / mL omoprobamate monoantigen, 10 mM pH regulator, 0.02 g / ml sucrose, 0.0001 g / ml polysorbate 80 and water;

[0049] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0050] Example 3

[0051] Provided is an omoprobamate monoantigen solution, comprising 30 mg / mL omoprobamate monoantigen, 30 mM pH regulator, 0.06 g / ml sucrose, 0.0004 g / ml polysorbate 80 and water;

[0052] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0053] Example 4

[0054] Provided is an omoprobamate monoantigen solution, comprising 20 mg / mL omoprobamate monoantigen, 32 mM pH regulator, 0.10 g / ml sucrose, 0.0006 g / ml polysorbate 80 and water;

[0055] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0056] Example 5

[0057] Provided is an omoprobamate monoantigen solution, comprising 18 mg / mL omoprobamate monoantigen, 8 mM pH regulator, 0.01 g / ml sucrose, 0.0005 g / ml polysorbate 80 and water;

[0058] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0059] Example 6

[0060] Provided is an omoprobamate monoantigen solution, comprising 18 mg / mL omoprobamate monoantigen, 20 mM pH regulator, 0.08 g / ml sucrose, 0.0005 g / ml polysorbate 80 and water;

[0061] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0062] Example 7

[0063] Provided is an omoprobamate monoantigen solution, comprising 19 mg / mL omoprobamate monoantigen, 20 mM pH regulator, 0.08 g / ml sucrose, 0.0005 g / ml polysorbate 80 and water;

[0064] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0065] Comparative Example 1

[0066] Provided is an omoprobamate antigen solution, comprising 20 mg / mL omoprobamate, 20 mM pH regulator, 0.08 g / ml sucrose, 0.0005 g / ml poloxamer 188 and water;

[0067] The pH regulator includes L-histidine and glacial acetic acid, and the mass ratio of L-histidine to glacial acetic acid is 2.95:1.

[0068] Performance Testing

[0069] The above Example 1 was tested for appearance, pH, protein concentration, subvisible particles, SEC-HPLC, iCIEF, sodium dodecyl sulfate capillary electrophoresis test under non-reducing conditions, sodium dodecyl sulfate capillary electrophoresis test under reducing conditions, and biological activity test when the preparation was completed, after stirring at 100 rpm and 25°C for five days, after five freeze-thaw cycles (-20°C / RT), after being placed at 35°C for two weeks, after being placed at 35°C for three weeks, after being placed at 25°C for two weeks, after being placed at 25°C for four weeks, and after being placed at 25°C for twelve weeks.

[0070] The test results are shown in Table 1.

[0071] Table 1 Performance test results of the Omopubai single antigen solution provided in Example 1

[0072]

[0073]

[0074] As can be seen from Table 1, after the samples were placed at 25°C and 35°C for 12 weeks and 3 weeks, respectively, there was no obvious change in appearance, pH, protein concentration, insoluble particles, purity (non-reduced & reduced CE-SDS), biological activity, purity (SEC-HPLC), and charge heterogeneity (iCIEF), indicating that the Omopobai single antigen solution provided by the present invention has good stability.

[0075] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An Omopoulin single antigen solution, characterized in that The omoprobamate antigen solution comprises the following raw materials: omoprobamate, pH regulator, sucrose, polysorbate 80 and water; Wherein, the mass concentration of the Omopoubimab is 10 g / L to 30 g / L; The pH of the Omopoulin single antigen solution is 5-6.

2. The Omopoulin single antigen solution according to claim 1, characterized in that In the Omopoulin monoantigen solution, the mass concentration of sucrose is 0.02 g / ml to 0.08 g / ml.

3. The Omopoulin single antigen solution according to claim 1, characterized in that In the Omopoulin single antigen solution, the mass concentration of polysorbate 80 is 0.0001 g / ml to 0.0005 g / ml.

4. The Omopoulin single antigen solution according to claim 1, characterized in that The mass concentration of the omoprofen is 18 g / L to 20 g / L.

5. The Omopoulin single antigen solution according to claim 1, characterized in that The pH adjuster includes L-histidine and glacial acetic acid.

6. The Omopoulin single antigen solution according to claim 5, characterized in that In the Omopoulin monoantigen solution, the molar concentration of the pH regulator is 10mM to 30mM.

7. The Omopoulin single antigen solution according to claim 1, characterized in that The pH of the Omopoulin single antigen solution is 5.

5.

8. A method for preparing the Omopoulin single antigen solution as claimed in claims 1 to 7, characterized in that: The following steps are involved: ATF harvesting the cell culture fluid to obtain a first intermediate product; Purifying the first intermediate product by monoclonal antibody to obtain a second intermediate product; Performing ultrafiltration and diafiltration on the second intermediate product to obtain a third intermediate product; The third intermediate is mixed with sucrose solution, polysorbate 80 solution and pH regulator to obtain Omopoulin single antigen solution.

9. The method for preparing the Omopoulin single antigen solution according to claim 8, characterized in that: The step of purifying the first intermediate product by monoclonal antibody to obtain the second intermediate product comprises: The first intermediate is subjected to affinity chromatography, virus inactivation, filtration, anion exchange chromatography, cation exchange chromatography, and virus filtration to obtain a second intermediate.

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