Preparation method and application of human immune globulin preparation stock solution

Abstract

The invention relates to a preparation method and application of a human immune globulin preparation stock solution, the preparation method comprises the steps of pretreatment of an initial raw material, ultrafiltration concentration and dialysis, and virus inactivation and filtration, the ultrafiltration concentration and dialysis process is completed under the participation of a dialysis buffer solution composed of a main functional component L-proline, and the human immune globulin preparation stock solution is obtained. The L-proline is used for effectively protecting human immune globulin in the preparation process of the human immune globulin preparation stock solution and the preparation, so that the influence of various unstable factors possibly causing polymerization of the human immune globulin product in the preparation process of the human immune globulin product in the prior art is effectively overcome; therefore, the stability and the quality of the human immune globulin preparation stock solution and the prepared product are effectively improved, and compared with the prior art, the technical scheme provided by the invention is simple, convenient and practical, and can achieve an extremely good technical effect, so that the human immune globulin preparation has outstanding beneficial effects and remarkable progress, and has extremely high popularization and application values.

Description

Technical Field

[0001] The present invention relates to a method for preparing a preparation stock solution and its application, in particular to a method for preparing a human immunoglobulin preparation stock solution and its application, belonging to the technical field of production and manufacturing of biological preparations. Background Art

[0002] Human immunoglobulin has multiple immunomodulatory and disease prevention and treatment functions, including: prevention and treatment of infectious diseases such as measles and hepatitis A, neutralizing the toxicity of pathogens or toxins, and can also be used to treat immunodeficiency and autoimmune diseases, or using the Fc segment of human immunoglobulin to bind to complement or macrophage receptors to promote the clearance of pathogens and prevent the infection risk of high-risk groups such as premature infants, the elderly, and immunodeficiency patients.

[0003] Existing human immunoglobulin preparations are usually concentrates prepared by industrial purification and concentration of human immunoglobulin (abbreviated as Ig) extracted from healthy human plasma, including intravenous injection solutions, intramuscular injection solutions, and freeze-dried powder injections. The main component is human immunoglobulin G (abbreviated as IgG), and there are also trace amounts of human immunoglobulin A (abbreviated as IgA) and human immunoglobulin M (abbreviated as IgM).

[0004] The first large-scale industrial method for preparing human immunoglobulin preparation stock solution was developed by the Cohn team and the Oncley team at Harvard University in the 1940s. The Cohn team established the cold ethanol fractionation method to achieve the purpose of separating and purifying different proteins in human plasma; the Oncley team used the Cohn FII+III (abbreviated as FII+III) precipitate obtained by the cold ethanol fractionation method as the raw material, and through further refinement of the separation and purification method, Cohn FII (abbreviated as FII) was separated from Cohn FII+III, and then further purified to obtain the human immunoglobulin preparation stock solution.

[0005] In the prior art, the human immunoglobulin stock solution for preparing human immunoglobulin preparations is mainly prepared by further purifying and concentrating the FII precipitate containing human immunoglobulin components obtained after treatment by the cold ethanol extraction method and / or the crude human immunoglobulin obtained by ion exchange chromatography, and at the same time, using the low pH incubation and release or the S / D inactivation method combined with a nano-film to remove viruses. For example, in the invention patent "A Method for Extracting and Separating IgM and IgG from Plasma" (authorization announcement number: CN 111961130 B), the invention patent "Production Process of Intravenous Human Immunoglobulin" (authorization announcement number: CN 102552906 B), the invention patent application "A Method for Preparing Human Immunoglobulin" (application number: 201410259993.4) and "A Method for Preparing Intravenous Human Immunoglobulin" (application number: 202310559161.3), etc.

[0006] As a biological product directly injected into the human body, the preparation of human immunoglobulin preparations needs to strictly implement relevant technical specifications and meet the quality standards determined by the national pharmacopoeia and relevant regulations, and meet the stability requirements within its validity period.

[0007] However, in the human immunoglobulin preparations in the prior art, due to the presence of residual lipids or lipoproteins and denatured proteins, and because these lipids or lipoproteins and denatured proteins are unstable, they can become the centers of protein nucleation, leading to protein aggregation to form insoluble particles, thereby causing an increase in product turbidity and affecting the physical appearance and long-term stability.

[0008] In addition, during the production and preparation process of the human immunoglobulin preparation stock solution, its protein molecules are usually affected by various stresses, such as the shear force of the pump, surface tension, turbulence, etc. during the ultrafiltration process, and the pH change during the production process, the viscosity caused by the increase in product concentration, the temperature increase during the low pH incubation and release virus inactivation process, the dissolved oxygen and gas-liquid interface factors, etc., may also cause subtle changes in the conformation of protein molecules, thereby affecting the naturalness and integrity of the protein molecular structure, causing loss of biological function, and even leading to protein aggregation and denaturation, thus affecting the clinical safety, effectiveness and long-term stability of human immunoglobulin.

[0009] U.S. Patent US 8715652B2 describes a method of adding 0.2 - 0.3M of L-proline as a stabilizer to its formulation to improve the stability of IgG formulations. U.S. Patent US 9241897B2 describes a method of adding 0.22 - 0.3M of L-proline as a stabilizer to its formulation to reduce the viscosity of the formulation. Although these two methods can improve the stability of human immunoglobulin formulations or reduce their viscosity, since both of these methods achieve their purposes by adding L-proline at the formulation stage, obviously, such addition methods and formulation methods are not helpful or effective in overcoming various unstable factors that may cause human immunoglobulin aggregation and the like during the ultrafiltration concentration and virus inactivation operation steps of the human immunoglobulin formulation stock solution. Summary of the Invention

[0010] To overcome the deficiencies of the prior art, reduce and overcome the influence of various unstable factors that may cause its aggregation and the like during the ultrafiltration concentration and virus inactivation operation steps of human immunoglobulin, and improve the stability of its formulated products, the following technical solutions are provided.

[0011] A method for preparing a human immunoglobulin formulation stock solution, including a pretreatment step and a virus inactivation and filtration step for the initial raw materials. Among them, after the initial raw materials are processed through the pretreatment step, an initial treatment solution with a pH value of 4.0 - 5.0 is obtained. In addition, it further includes:

[0012] A processing step of subjecting the initial treatment solution to ultrafiltration concentration and dialysis to obtain a human immunoglobulin concentrate, and the ultrafiltration concentration and dialysis steps are carried out with the participation of a dialysis buffer solution, and the functional component of the dialysis buffer solution is L-proline.

[0013] Among them, the preparation method of the dialysis buffer solution is: adding normal-temperature injection water to L-proline to dissolve the L-proline, and then adjusting the pH with a 0.5 - 1M hydrochloric acid solution to finally form an aqueous solution containing L-proline with a molar concentration of 200 - 400 mmol / L and a pH value of 4.0 - 5.0, and the temperature of the dialysis buffer solution is controlled between 2 - 8°C.

[0014] Furthermore, the ultrafiltration concentration and dialysis steps include: subjecting the initial treatment solution to ultrafiltration concentration with a 30 - 100KD ultrafiltration membrane to obtain a ultrafiltrate, where the transmembrane pressure during ultrafiltration concentration is controlled between 0.1 - 0.4 Mpa; performing dialysis with a dialysis buffer solution not less than 5 times the volume of the ultrafiltrate to obtain a dialysate, and then adjusting the protein content of the dialysate to between 5.0% - 5.5% with the dialysis buffer solution to obtain a human immunoglobulin concentrate.

[0015] It should be noted that the initial raw materials are the FII precipitate containing human immunoglobulin components after being processed by the low-temperature ethanol process and / or the IgG flow-through fraction obtained by anion exchange chromatography, and the IgG elution fraction obtained by cation exchange chromatography.

[0016] Furthermore, the pretreatment step includes: dissolving the FII precipitate with injection water that is 3 to 5 times its mass and at a temperature of 0 to 5 °C to obtain a dissolution solution, then adding an adsorption filter aid to the dissolution solution at a ratio of 1 to 2 g / L by volume-weight ratio, and then performing deep filtration with a clarification filter plate to obtain a clarified filtrate. Next, adjusting the pH value of the clarified filtrate to 4.0 to 5.0 with a hydrochloric acid solution having a molar concentration of 0.5 to 1 M, and then filtering with a degreasing filter plate to obtain the initial treatment solution; and / or

[0017] In the IgG flow-through fraction obtained by anion exchange chromatography and the IgG elution fraction obtained by cation exchange chromatography, an adsorption filter aid is added respectively at a ratio of 1 to 2 g / L by volume-weight ratio. Then, deep filtration is respectively performed with a clarification filter plate to obtain respective clarified filtrates. Next, adjusting the pH value of each clarified filtrate to 4.0 to 5.0 with a hydrochloric acid solution having a molar concentration of 0.5 to 1 M, and then filtering with a degreasing filter plate to obtain the initial treatment solutions with the corresponding initial raw materials being the IgG flow-through fraction and the IgG elution fraction respectively;

[0018] In the above operations, the filtration pressure of the filtration does not exceed 0.3 Mpa.

[0019] Furthermore, the adsorption filter aid is refined bentonite, and the preparation method of the refined bentonite is: adding 5 to 10 L of injection water to each kilogram of commercial bentonite, then adding an acetate buffer solution with a pH value of 4.0, stirring evenly and then performing suction filtration with a Buchner funnel to obtain a primary bentonite filter cake. Then, adding 5 to 10 L of injection water to the primary bentonite filter cake, stirring evenly to form a suspension, and then adding sodium citrate buffer solution at a ratio of 10 to 15 mmol / L per liter of the suspension. After stirring evenly, performing vacuum suction filtration with a Buchner funnel to obtain a secondary bentonite filter cake. Then, adding an equal amount of injection water to the secondary bentonite filter cake, stirring evenly and then performing vacuum suction filtration with a Buchner funnel to obtain the refined bentonite.

[0020] It should be noted that the virus inactivation and filtration step includes low-pH incubation inactivation and nano-filtration, wherein:

[0021] The specific operation of the low-pH incubation inactivation includes: filtering and sterilizing the human immunoglobulin concentrate with a 0.2 µm filter element to obtain a sterilized filtrate, and then placing the sterilized filtrate in a container at 24 ± 1°C with a nitrogen gas headspace pressure not lower than 0.15 Mpa for incubation for at least 21 consecutive days to inactivate the residual lipid-enveloped viruses in the sterilized filtrate and obtain an incubated and inactivated solution;

[0022] The specific operation of the nanofiltration removal includes: filtering and removing viruses from the incubated and inactivated solution with a 20-nanometer nanofilm to obtain the stock solution of the human immunoglobulin preparation.

[0023] Furthermore, based on the above method for preparing the stock solution of the human immunoglobulin preparation, the present invention also provides an application method for the obtained stock solution of the human immunoglobulin preparation, which specifically includes:

[0024] Preparing the stock solution of the human immunoglobulin preparation obtained by the above method for preparing the stock solution of the human immunoglobulin preparation into a human immunoglobulin preparation that meets the relevant quality standards of the current "Chinese Pharmacopoeia" and has a labeled protein content that meets the regulations, including:

[0025] Intravenous human immunoglobulin with a labeled protein content of 5% and / or intravenous human immunoglobulin with a labeled protein content of 10%, human immunoglobulin, freeze-dried human immunoglobulin, and subcutaneous injection human immunoglobulin with a labeled protein content of 20%, where:

[0026] The specific preparation method of intravenous human immunoglobulin with a labeled protein content of 5% includes: filtering and sterilizing the stock solution of the human immunoglobulin preparation with a 0.2 µm sterilizing filter element, and then filling it into a container with a suitable capacity under sterile / sterile nitrogen filling conditions and sealing it to obtain the product;

[0027] The preparation methods of intravenous human immunoglobulin with a labeled protein content of 10% and / or human immunoglobulin, freeze-dried human immunoglobulin, and subcutaneous injection human immunoglobulin with a labeled protein content of 20% include a concentration and pH readjustment step and a preparation production step, where:

[0028] The specific operation of the concentration and pH readjustment step includes: ultrafiltrating and reconcentrating the stock solution of the human immunoglobulin preparation with a 30-100 KD ultrafiltration membrane, where the transmembrane pressure during ultrafiltration and reconcentration is controlled between 0.1 and 0.4 Mpa, and then readjusting the concentration and pH of it with the dialysis buffer solution and a 0.5-1 M hydrochloric acid solution to make it a human immunoglobulin preparation solution with a mass percentage content of protein reaching the requirements of the pre-prepared preparation label amount and a pH value of 4.0-5.0;

[0029] The preparation steps of the preparation include: preparing the human immunoglobulin preparation solution for intravenous injection of human immunoglobulin and / or human immunoglobulin, freeze-dried human immunoglobulin with a labeled protein content of 10% and subcutaneous injection of human immunoglobulin with a labeled protein content of 20% by adapting the mass percentage content of its protein respectively, filtering and sterilizing them with a 0.2 µm sterilizing filter element respectively, and then separately carrying out aseptic / aseptic nitrogen filling and packaging into their respective adapted containers. After sealing, the intravenous injection of human immunoglobulin and / or human immunoglobulin, subcutaneous injection of human immunoglobulin with the labeled amount meeting the requirements can be obtained respectively, or the freeze-dried human immunoglobulin obtained after freeze-drying and then sealing. Compared with the prior art, the outstanding beneficial effects and remarkable progress of the present invention are as follows:

[0030] 1) In the technical solution of the preparation method and application of the human immunoglobulin preparation stock solution provided by the present invention, the preparation method of the human immunoglobulin preparation stock solution includes pretreatment, ultrafiltration concentration, dialysis, virus inactivation and filtration steps for its initial raw materials, that is, the FII precipitate containing human immunoglobulin components after treatment by the cold ethanol process and / or the IgG flow-through component and IgG elution component obtained respectively after anion and cation exchange chromatography. Among them, the ultrafiltration concentration and dialysis process are completed with the participation of dialysis buffer, and the main functional component of the dialysis buffer is L-proline. That is, the technical solution provided by the present invention enables L-proline to effectively protect human immunoglobulin during the preparation process of the human immunoglobulin preparation stock solution and the preparation, rather than, as in the prior art, only protecting human immunoglobulin through the formulation method in the preparation product. Thus, it more effectively overcomes various possible factors that may cause its aggregation and other instability factors during the preparation process of human immunoglobulin products in the prior art, thereby improving the stability of the preparation stock solution and further improving the stability of its preparation.

[0031] 2) During the preparation process of human immunoglobulin products, when performing operations such as ultrafiltration concentration and dialysis on human immunoglobulin, human immunoglobulin will be affected by various stresses, including the shear force of the pump, surface tension, transmembrane pressure, turbulence, and factors such as pH change and increased viscosity caused by the increase in product concentration, which will cause changes in the conformation of protein molecules and thus affect its biological function. In the technical solution provided by the present invention, the dialysis buffer can effectively protect the stability of protein molecules through its functional component L-proline, reduce the influence of stresses such as shear force, surface tension, transmembrane pressure, and turbulence on human immunoglobulin molecules, and at the same time avoid the pH drift phenomenon that easily occurs when only using injection water to participate in dialysis in the prior art, thereby effectively reducing the influence of these stresses and influencing factors on the molecular structure of human immunoglobulin and being able to effectively maintain the natural complete structure and biological function of human immunoglobulin molecules.

[0032] 3) In the human immunoglobulin products obtained by the prior art, the residual lipids or lipoproteins and denatured proteins will become the nucleation centers of proteins, leading to protein aggregation, forming insoluble particles, resulting in an increase in the turbidity of the product, thus affecting the physical appearance and long-term stability of the stock solution of the human immunoglobulin preparation and its preparation products. However, the technical solution provided by the present invention adsorbs and filters the initial raw materials for preparing the stock solution of the human immunoglobulin preparation by using an adsorption and filtration aid composed of refined bentonite, that is, removes the residual lipids or lipoproteins and denatured proteins from the very beginning, reduces the nucleation effect of these unstable substances, thereby effectively reducing the unstable factors causing protein aggregation in the stock solution of the human immunoglobulin preparation, improving the stability of the stock solution of the human immunoglobulin preparation and its preparation products, reducing its turbidity, and improving the appearance of the product;

[0033] 4) During the preparation process of human immunoglobulin products, the low-pH incubation and inactivation operation needs to be continuously incubated at 24 ± 1 °C for at least 21 days, and the long-term exposure of proteins to relatively high temperatures is extremely unfavorable for maintaining their stability. However, the technical solution provided by the present invention can effectively reduce the adverse effects of high temperature on the protein structural molecules by utilizing the stabilizing and protecting effect of L-proline on protein molecules, reducing the interaction between proteins - proteins, thereby effectively improving the stability of the product;

[0034] 5) During the low-pH incubation and inactivation process, since the oxygen in the air will contact the surface of the human immunoglobulin concentrate or dissolve in the concentrate to oxidize the proteins, thus affecting the stability of the protein molecules, and the carbon dioxide gas in the air will dissolve in the human immunoglobulin concentrate to affect the pH value of the concentrate, thus affecting the virus inactivation effect. However, the technical solution provided by the present invention effectively overcomes the adverse effects of oxygen and carbon dioxide in the low-pH incubation and inactivation of the human immunoglobulin concentrate by filling nitrogen in the headspace of the low-pH incubation container, improving the stability of human immunoglobulin during the preparation process of its product and the effect of inactivating its virus;

[0035] In summary, the technical solution provided by the present invention overcomes the deficiencies of the prior art, can effectively improve the stability of human immunoglobulin products, improve their quality and quality, and compared with the prior art, the preparation method is not complicated, but can achieve excellent technical effects. Therefore, it has outstanding beneficial effects and significant progress, and has great value for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the technical solution of the present invention and the technical effects of implementing the present invention, hereinafter, a brief introduction will be made to the drawings used in the embodiments of the present invention.

[0037] Obviously:

[0038] The following attached drawings are only partial drawings in the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings, and these other drawings also belong to the scope of the drawings required for the embodiments of the present invention, where:

[0039] Figure 1 It is a comparison chart of the change curves of the monomer content of the human immunoglobulin preparation stock solutions obtained from Case 1.1 and Comparative Example 1.1 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0040] Figure 2 It is a comparison chart of the change curves of the dimer content of the human immunoglobulin preparation stock solutions obtained from Case 1.1 and Comparative Example 1.1 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0041] Figure 3 It is a comparison chart of the change curves of the polymer content of the human immunoglobulin preparation stock solutions obtained from Case 1.1 and Comparative Example 1.1 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0042] Figure 4 It is a comparison chart of the change curves of the fragment content of the human immunoglobulin preparation stock solutions obtained from Case 1.1 and Comparative Example 1.1 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0043] Figure 5 It is a comparison chart of the change curves of the monomer content of the human immunoglobulin preparation stock solutions obtained from Case 1.2 and Comparative Example 1.2 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0044] Figure 6 It is a comparison chart of the change curves of the dimer content of the human immunoglobulin preparation stock solutions obtained from Case 1.2 and Comparative Example 1.2 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0045] Figure 7 It is a comparison chart of the change curves of the polymer content of the human immunoglobulin preparation stock solutions obtained from Case 1.2 and Comparative Example 1.2 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0046] Figure 8 It is a comparison chart of the change curves of the fragment content of the human immunoglobulin preparation stock solutions obtained from Case 1.2 and Comparative Example 1.2 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C;

[0047] Figure 9This is a comparison graph of the turbidity change curves of the undiluted human immunoglobulin preparations obtained from Example 1.3 and Comparative Example 1.3 of the present invention during the 6-month accelerated stability test at 30 ± 2°C.

[0048] In the figure: 1 # is the detection curve of the undiluted human immunoglobulin preparation obtained from Example 1.1, 1 b is the detection curve of the undiluted human immunoglobulin preparation obtained from Comparative Example 1.1, 2 # is the detection curve of the undiluted human immunoglobulin preparation obtained from Example 1.2, 2 b is the detection curve of the undiluted human immunoglobulin preparation obtained from Comparative Example 1.2, 3 # is the detection curve of the undiluted human immunoglobulin preparation obtained from Example 1.3, 3 b is the detection curve of the undiluted human immunoglobulin preparation obtained from Comparative Example 1.3. Detailed implementation mode

[0049] To describe the technical solutions, beneficial effects, and significant improvements of the present invention more clearly and comprehensively, the technical solutions provided by the present invention will be clearly and completely described below through specific examples and their comparative examples. Obviously, all the examples and comparative examples described below are only partial examples and comparative examples of the present invention, rather than all of them.

[0050] Based on the examples and comparative examples provided by the present invention, all other examples and comparative examples obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0051] It should be noted that the terms "first", "second", etc. in the claims, the description of the present invention, and the examples and comparative examples of its embodiments are only used to distinguish different objects, rather than to describe a specific order; in addition, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device, etc. that includes a series of listed steps or units not only includes the steps or units listed, but also optionally includes steps or units not listed, or optionally includes other operating steps or units inherent to these processes, methods, products, or devices.

[0052] It should be understood that in the description of the embodiments of the present invention, some basic operation terms commonly used in the art are used, such as "mixing", "stirring", "dissolving" and "filtering", etc. For these terms, a broad understanding should be made, that is, it can be the conventional operations performed by various conventional equipment and instruments in the art, or it can be operations such as programmed operations and unmanned automatic operations performed by using the latest equipment. Unless otherwise clearly defined, those of ordinary skill in the art should understand the specific meanings of the above terms in the present invention according to specific circumstances and adopt specific operation methods to achieve their operation purposes.

[0053] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in the cases and comparative examples in some embodiments; in addition, various raw materials, their auxiliary materials and reagents, production equipment, facilities, etc. involved in the following specific embodiments of the cases and comparative examples are commercially available unless otherwise specified, where:

[0054] The water involved in the following operations is all self-made and meets the quality standards of the current "Chinese Pharmacopoeia" for injection water;

[0055] The raw material plasma for preparing the initial raw materials must meet the relevant requirements of the current "Chinese Pharmacopoeia" for human plasma used in the production of blood products during its collection process and quality requirements, where:

[0056] The FII precipitate refers to the crude human immunoglobulin containing IgG prepared by the existing low-temperature ethanol process;

[0057] The IgG flow-through fraction refers to the chromatographic flow-through fraction containing IgG obtained after anion exchange chromatography;

[0058] The IgG elution fraction refers to the chromatographic elution fraction containing IgG obtained after cation exchange chromatography;

[0059] The production equipment, facilities, environment, etc. involved in the cases and comparative examples in the embodiments of this specification should all meet the requirements of GMP specifications, and the chemical reagents used, including L-proline, hydrochloric acid, commercial bentonite, acetate, and sodium citrate, etc. are all of pharmaceutical grade.

[0060] Next, the technical solutions of the present invention will be described in detail with specific embodiments. Example 1

[0061] This example provides a method for preparing the stock solution of a human immunoglobulin preparation.

[0062] A method for preparing the stock solution of a human immunoglobulin preparation, which includes pretreating the initial raw material to obtain an initial treatment solution with a pH value of 4.0 - 5.0, and then, with the participation of a dialysis buffer solution, subjecting the initial treatment solution to ultrafiltration concentration and dialysis to obtain a human immunoglobulin concentrated solution, and then performing virus inactivation and filtration treatment on the human immunoglobulin concentrated solution to obtain the stock solution of the human immunoglobulin preparation, wherein:

[0063] The dialysis buffer solution is prepared by dissolving L-proline in normal-temperature injection water, adjusting the pH with a 0.5 - 1M hydrochloric acid solution to finally obtain an aqueous solution containing L-proline with a molar concentration of 200 - 400 mmol / L and a pH value of 4.0 - 5.0, and then storing it in an environment of 2 - 8°C for standby;

[0064] The specific operations of ultrafiltration concentration and dialysis are as follows: subject the initial treatment solution to ultrafiltration concentration with a 30 - 100KD ultrafiltration membrane, wherein the transmembrane pressure during ultrafiltration concentration is controlled between 0.1 - 0.4 Mpa to obtain a ultrafiltrate;

[0065] Perform dialysis with a dialysis buffer solution not less than 5 times the volume of the ultrafiltrate to obtain a dialysate, and then adjust the protein content of the dialysate to between 5.0% - 5.5% with the dialysis buffer solution to obtain a human immunoglobulin concentrated solution.

[0066] During the above treatment process, the initial raw material is the FII precipitate containing human immunoglobulin components after treatment by the cold ethanol process and / or the IgG flow-through fraction obtained by anion exchange chromatography, and the IgG elution fraction obtained by cation exchange chromatography.

[0067] The specific operations of the pretreatment step are as follows:

[0068] Dissolve the FII precipitate in injection water with a mass 3 - 5 times that of the FII precipitate and at a temperature of 0 - 5°C to obtain a dissolution solution, then add an adsorption filter aid to the dissolution solution at a volume-weight ratio of 1 - 2 g / L, and then perform deep filtration with a clarification filter plate to obtain a clarified filtrate, then adjust the pH value of the clarified filtrate to 4.0 - 5.0 with a hydrochloric acid solution with a molar concentration of 0.5 - 1M, and then perform filtration with a degreasing filter plate to obtain an initial treatment solution; and / or

[0069] In the IgG flow-through fraction or IgG elution fraction obtained by anion or cation exchange chromatography respectively, an adsorption filter aid is added at a ratio of 1-2 g / L by volume weight ratio respectively, and then, deep filtration is carried out with a clarification filter plate to obtain respective clarified filtrates, and then the pH value of each clarified filtrate is adjusted to 4.0-5.0 with a hydrochloric acid solution with a molar concentration of 0.5-1 M, and then filtration is carried out with a degreasing filter plate, so as to obtain initial treatment liquids corresponding to the initial raw materials being the IgG flow-through fraction and the IgG elution fraction respectively;

[0070] In the above operation, the filtration pressure does not exceed 0.3 Mpa.

[0071] In addition, in the above operation, the adsorption filter aid used is refined bentonite, and the preparation method of the refined bentonite is as follows:

[0072] Injectable water is added to a certain amount of commercial bentonite at a ratio of 5-10 L of injectable water per kilogram of commercial bentonite, and then an acetate buffer solution with a pH value of 4.0 is added. After stirring evenly, vacuum filtration is carried out with a Buchner funnel to obtain a primary bentonite filter cake. Then, injectable water is added to the primary filter cake at a ratio of 5-10 L of injectable water per kilogram of the primary commercial bentonite filter cake, stirred evenly to form a suspension, and then sodium citrate buffer solution is added at a ratio of 10-15 mmol / L of sodium citrate buffer solution per liter of the suspension. After stirring evenly, vacuum filtration is carried out with a Buchner funnel to obtain a secondary bentonite filter cake. Then, an equal amount of injectable water is added to the secondary bentonite filter cake, stirred evenly, and vacuum filtration is carried out with a Buchner funnel to obtain refined bentonite.

[0073] In the above virus inactivation and filtration steps, low pH incubation inactivation and nanofilm filtration operations are included, among which:

[0074] The specific operation of low pH incubation inactivation includes: filtering and sterilizing the human immunoglobulin concentrate with a 0.2 µm filter element to obtain a sterilized filtrate, and then placing the sterilized filtrate in a container at 24±1 °C and with its headspace filled with nitrogen with a pressure not lower than 0.15 Mpa for incubation, and continuously incubating for at least 21 days, so as to inactivate the residual lipid envelope virus in the sterilized filtrate to obtain an incubated and inactivated liquid;

[0075] The specific operation of nanofilm filtration includes: filtering the virus from the incubated and inactivated liquid with a 20-nanometer nanofilm to obtain the stock solution of the human immunoglobulin preparation.

[0076] From the above description, it can be seen that:

[0077] First, the method for preparing the bulk solution of human immunoglobulin provided in this embodiment includes the pretreatment of the initial raw materials, ultrafiltration concentration, dialysis, virus inactivation, and filtration steps. The ultrafiltration concentration and dialysis steps are carried out in the presence of a dialysis buffer with L-proline as its main functional component, that is, L-proline effectively protects human immunoglobulin during the preparation of the bulk solution, rather than, as in the prior art, only adding L-proline to the formulated product for formula protection. Thus, it overcomes various unstable factors that may cause polymerization and other problems during the preparation of human immunoglobulin in the prior art, thereby improving the stability of the bulk solution and further enhancing the stability of its formulated product;

[0078] Secondly, when performing operations such as ultrafiltration concentration and dialysis on human immunoglobulin, human immunoglobulin is affected by various stresses, such as the shear force of the pump, surface tension, transmembrane pressure, turbulence, as well as factors such as pH changes and increased viscosity due to the increase in product concentration, which can cause changes in the conformation of protein molecules and thus affect their biological functions. In response to this situation, the technical solution provided in this embodiment is to carry out the ultrafiltration concentration and dialysis steps in a dialysis buffer, so that the dialysis buffer stably protects the protein molecules through its functional component L-proline, thereby reducing the impact of stresses such as shear force, surface tension, transmembrane pressure, and turbulence on human immunoglobulin molecules. At the same time, it avoids the pH drift phenomenon that is likely to occur when only using injection water for ultrafiltration concentration and dialysis in the prior art, and further effectively reduces the impact of these stresses and influencing factors on the molecular structure of human immunoglobulin, ensuring its natural intact structure and biological functions;

[0079] In addition, in the human immunoglobulin products obtained by the prior art, residual lipids or lipoproteins and denatured proteins will become the nucleation centers of proteins, leading to protein aggregation and the formation of insoluble particles, resulting in an increase in the turbidity of the product, thereby affecting the physical appearance and long-term stability of the bulk solution of human immunoglobulin and its formulated product. However, the technical solution provided in this embodiment adsorbs and filters the initial raw materials for preparing the bulk solution of human immunoglobulin using an adsorption and filtration aid composed of refined bentonite, that is, removing residual lipids or lipoproteins and denatured proteins from the very beginning, thereby reducing the nucleation effect of these unstable substances and effectively reducing the unstable factors causing protein aggregation in the bulk solution of human immunoglobulin, improving the stability of the bulk solution of human immunoglobulin and its formulated product;

[0080] In the preparation process of the human immunoglobulin preparation stock solution, a low-pH incubation inactivation operation needs to be carried out continuously for at least 21 days at 24 ± 1 °C. Since proteins are extremely unfavorable for maintaining their stability when exposed to high temperatures for a long time, in this embodiment, a technical solution is provided that uses L-proline to have a stabilizing and protective effect on protein molecules, thereby effectively reducing the impact of high temperature on protein structural molecules, reducing the interaction between proteins, and improving the stability of the human immunoglobulin preparation stock solution;

[0081] On this basis, in order to effectively reduce the adverse effects of oxygen and carbon dioxide in the low-pH incubation inactivation of human immunoglobulin concentrate, this embodiment provides a technical solution of filling nitrogen gas in the headspace of its container during the low-pH incubation inactivation process, thereby effectively isolating the contact between the oxygen in the headspace of the low-pH incubation container and the surface of the human immunoglobulin concentrate or the oxidation of the proteins in the concentrate by dissolved oxygen, thus ensuring the stability of protein molecules and eliminating the hidden danger that carbon dioxide gas in the air dissolves in the human immunoglobulin concentrate and affects the pH value of the concentrate, thereby affecting the virus inactivation effect. Thereby, the stability of human immunoglobulin and the virus inactivation effect are improved.

[0082] To further help understand the specific implementation manner and achievable technical effects of the technical solution of the preparation method of the human immunoglobulin preparation stock solution provided in this embodiment, hereinafter, specific cases and comparative examples are used to further illustrate this, but for the sake of brevity, some identical steps are only briefly described. Case 1.1

[0083] Preparation of dialysis buffer: Under stirring conditions, add room-temperature injection water to L-proline to dissolve L-proline, and then adjust the acidity with a 0.75 M hydrochloric acid solution to finally obtain an aqueous solution containing L-proline with a molar concentration of 300 mmol / L and a pH value of 4.5, which is the dialysis buffer of this case. Then, lower its temperature to 5 °C and store it sealed for later use;

[0084] Preparation of adsorption filter aid: Add 7.5 L of injection water to 1 kg of commercial bentonite, and then add 7.5 ml of acetate buffer with a pH value of 4.0. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the primary bentonite filter cake. Then, add 7.5 L of injection water to the primary bentonite filter cake, stir evenly to form a suspension, and then add sodium citrate buffer in a ratio of 12.5 mmol / L per liter of suspension. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the secondary bentonite filter cake. Then, add an equal amount of injection water to this secondary bentonite filter cake, stir evenly, and perform vacuum filtration with a Buchner funnel to obtain refined bentonite, which is the adsorption filter aid of this case;

[0085] Pretreatment of the initial raw material: Take the IgG eluate fraction containing human immunoglobulin after cation exchange chromatography process as the initial raw material, and add the adsorption filter aid prepared in this case to this eluate fraction at a ratio of 2 g / L of adsorption filter aid per liter of IgG eluate fraction. Stir and adsorb for 45 minutes, then perform clarification filtration with a clarification filter plate, and the filtration pressure does not exceed 0.3 Mpa to obtain a clarified filtrate. Then, adjust the pH of the clarified filtrate to 4.5 with 0.5 M hydrochloric acid solution, and then perform degreasing filtration with a degreasing filter plate, and the filtration pressure also does not exceed 0.3 Mpa to obtain the initial treatment solution;

[0086] Ultrafiltration concentration and dialysis operations:

[0087] Ultrafilter and concentrate the obtained initial treatment solution with a 100 KD ultrafiltration membrane until the protein content is above 5.0%. Among them, the transmembrane pressure during ultrafiltration concentration is 0.4 Mpa to obtain an ultrafiltrate; Dialyze the ultrafiltrate with 8 times the volume of the dialysis buffer prepared in this case to obtain a dialysate, and then adjust the protein content of the dialysate to 5.5% with the dialysis buffer prepared in this case to obtain a human immunoglobulin concentrate;

[0088] Virus inactivation and filtration operations: Filter the human immunoglobulin concentrate obtained in this case with a 0.2 μm filter element to obtain a sterilized filtrate. During sterilization filtration, the pressure does not exceed 0.2 Mpa. Then, place the obtained sterilized filtrate in a low pH incubation container, and fill the headspace of the low pH incubation container with nitrogen not less than 0.15 Mpa. Then, heat the sterilized filtrate to 24 ± 1 °C and perform continuous incubation for 21 days to inactivate the residual lipid envelope virus in the sterilized filtrate to obtain an incubated and inactivated solution. The incubated and inactivated solution is then filtered through a 20-nanometer nanomembrane to remove viruses, and the filtration pressure does not exceed 0.3 Mpa to obtain a human immunoglobulin preparation stock solution 1.1 with a pH value of 4.5 and a protein content of 5.5% # 。 Comparative Example 1.1

[0089] The initial raw material used in this comparative example and all treatment operations performed on the initial raw material, including operation steps and process conditions, are the same as those in Example 1.1. The difference is only that in this comparative example, the preparation of the dialysis buffer is not carried out, and during the dialysis operation of the ultrafiltrate, instead of using the dialysis buffer prepared in Example 1.1, dialysis is performed with 3 °C injection water with the same volume as the dialysis buffer in Example 1.1, and then the subsequent operations are the same as those in Example 1.1. Finally, a human immunoglobulin preparation stock solution 1.1 with a pH value of 4.5 and a protein content of 5.5% is obtained b 。

[0090] Table 1 lists the bulk solutions of human immunoglobulin preparations 1.1 obtained from Case 1.1 and Comparative Example 1.1 respectively # and 1.1 b When they were each subjected to a 6-month accelerated stability test at 30 ± 2°C, their stability was characterized by high performance liquid chromatography (HPLC), including the mass percentage data of monomers, dimers, polymers, and fragments.

[0091] According to the detection data listed in Table 1, with the detection time as the abscissa and the mass percentage of the detected substances in the bulk solutions of human immunoglobulin preparations obtained from Case 1.1 and Comparative Example 1.1 respectively in their respective bulk solutions as the ordinate, the attached Figures 1 to 4 can be obtained, that is, the comparative graph of the change curves of the mass percentages of monomers, dimers, polymers, and fragments of the bulk solutions of human immunoglobulin preparations obtained from Case 1.1 and Comparative Example 1.1 of the present invention respectively in the 6-month accelerated stability test at 30 ± 2°C.

[0092] From the data listed in Table 1 and the attached Figures 1 to 4 it can be clearly seen that:

[0093] The preparation method of the bulk solution of human immunoglobulin preparation provided by the embodiment of the present invention can effectively reduce the influence of factors such as the shear force of the pump, surface tension, transmembrane pressure, turbulence, pH change, and viscosity increase caused by concentration increase during ultrafiltration concentration and dialysis operations on the molecular conformation of human immunoglobulin by adding a dialysis buffer solution with L-proline as the main functional component during the ultrafiltration concentration and dialysis operation steps, effectively reduce the polymerization and cleavage of human immunoglobulin molecules, and significantly improve the stability of the bulk solution of human immunoglobulin preparation. Table 1

[0094] Case 1.2

[0095] Preparation of dialysis buffer solution: Under stirring conditions, add room-temperature injection water to L-proline to dissolve L-proline, and then adjust the acidity with a 1.0 M hydrochloric acid solution to make it finally an aqueous solution containing L-proline with a molar concentration of 400 mmol / L and a pH value of 5.0, that is, the dialysis buffer solution of this case. Then lower its temperature to 8°C and store it sealed for later use;

[0096] Preparation of adsorbent filter aid: Add 10 L of injection water to 1 kg of commercial bentonite, then add 20 ml of acetate buffer solution with a pH value of 4.0. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the primary bentonite filter cake. Then, add 10 L of injection water to the primary bentonite filter cake, stir evenly to form a suspension, and add sodium citrate buffer solution in the proportion of 15 mmol / L sodium citrate buffer solution per liter of suspension. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the secondary bentonite filter cake. Then, add an equal amount of injection water to this secondary bentonite filter cake, stir evenly, and perform vacuum filtration with a Buchner funnel to obtain refined bentonite, which is the adsorbent filter aid in this case;

[0097] Pretreatment of initial raw materials:

[0098] Take the IgG flow-through fraction containing human immunoglobulin after anion exchange chromatography process as the initial raw material, and add the adsorbent filter aid prepared in this case to this flow-through fraction in the proportion of 1 g / L adsorbent filter aid per liter of chromatographic IgG flow-through fraction. After stirring and adsorbing for 60 minutes, perform clarification filtration with a clarification filter plate, and the filtration pressure does not exceed 0.3 Mpa to obtain a clarified filtrate. Then, adjust the pH of the clarified filtrate to 5.0 with 1 M hydrochloric acid solution, and then perform degreasing filtration with a degreasing filter plate, and the filtration pressure also does not exceed 0.3 Mpa to obtain the initial treatment solution;

[0099] Ultrafiltration and concentration operation: Ultrafilter and concentrate the obtained initial treatment solution with a 50 KD ultrafiltration membrane until the protein content is above 5.0%. Among them, the transmembrane pressure during ultrafiltration and concentration is 0.3 Mpa to obtain an ultrafiltrate;

[0100] Dialysis operation: Dialyze with 7 times the volume of the ultrafiltrate of the dialysis buffer solution prepared in this case to obtain a dialysate, and then adjust the protein content of the dialysate to 5.0% with the dialysis buffer solution prepared in this case to obtain a human immunoglobulin concentrate;

[0101] Virus inactivation: Filter the human immunoglobulin concentrate obtained in this case with a 0.2 μm filter element to obtain a sterilized filtrate. Among them, during sterilization filtration, the pressure does not exceed 0.2 Mpa. Then, place the obtained sterilized filtrate in a low pH incubation container, and fill the headspace of the low pH incubation container with nitrogen not less than 0.15 Mpa. Then, heat the sterilized filtrate to 24 ± 1 °C and perform continuous incubation for 21 days to inactivate the residual lipid envelope virus to obtain an incubated and inactivated solution;

[0102] Virus filtration: Filter the above incubated and inactivated solution with a 20-nanometer nanomembrane, and the filtration pressure does not exceed 0.3 Mpa, that is, obtain the original solution of human immunoglobulin preparation with a pH value of 5.0 and a protein content of 5.0% 1.2 # . Comparative Example 1.2

[0103] The initial raw materials used in this comparative example and all the processing operations performed on the initial raw materials, including steps and process conditions, are the same as those in Example 1.2, except that when virus inactivation is performed on the sterile filtrate in this comparative example, nitrogen is no longer filled into the headspace of its pH incubation container, but only virus incubation inactivation is carried out while maintaining it in clean compressed air, and then through subsequent processing operations, a human immunoglobulin preparation stock solution 1.2 with a pH value of 5.0 and a protein content of 5.0% is obtained. b 。

[0104] Table 2 lists the human immunoglobulin preparation stock solutions 1.2 obtained from Example 1.2 and Comparative Example 1.2 respectively. # and 1.2 b When each of them undergoes a 6-month accelerated stability test at 30 ± 2 °C, the mass percentage data of related substances characterizing their stability are detected by high performance liquid chromatography (HPLC).

[0105] According to the data listed in Table 2, with the detection time as the abscissa and the mass percentage of the detected substances in the respective human immunoglobulin preparation stock solutions obtained from Example 1.2 and Comparative Example 1.2 as the ordinate, the attached Figures 5 to 8 can be obtained respectively, that is, a comparative graph of the change curves of the monomer, dimer, polymer, and fragment contents of the human immunoglobulin preparation stock solutions obtained from Example 1.2 and Comparative Example 1.2 of the present invention during the 6-month accelerated stability test at 30 ± 2 °C is obtained. Table 2

[0106]

[0107] From the data listed in Table 2 and the attached Figures 5 to 8 it can be clearly seen that:

[0108] The method for preparing a human immunoglobulin preparation stock solution provided by the embodiment of the present invention can effectively reduce the polymerization and cleavage of human immunoglobulin molecules and significantly improve the stability of the human immunoglobulin preparation stock solution by filling nitrogen into the headspace of its pH incubation container when virus inactivation is performed on the sterile filtrate. Example 1.3

[0109] Preparation of dialysis buffer: Under stirring conditions, add room temperature injection water to L-proline to dissolve L-proline, and after dissolution, adjust the acidity with a 0.5 M hydrochloric acid solution to make it finally an aqueous solution containing L-proline with a molar concentration of 200 mmol / L and a pH value of 4.0, that is, the dialysis buffer, and then lower its temperature to 2 °C and store it sealed for later use;

[0110] Preparation of adsorbent filter aid: Add 5 L of injection water to 1 kg of commercial bentonite, then add 20 ml of acetate buffer solution with a pH value of 4.0. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the primary bentonite filter cake. Then, add 5 L of injection water to the primary bentonite filter cake, stir evenly to form a suspension, and add sodium citrate buffer solution in a proportion of 10 mmol / L per liter of the suspension. After stirring evenly, perform vacuum filtration with a Buchner funnel to obtain the secondary bentonite filter cake. Then, add an equal amount of injection water to this secondary bentonite filter cake, stir evenly, and perform vacuum filtration with a Buchner funnel to obtain refined bentonite, which is the adsorbent filter aid in this case;

[0111] Pretreatment of initial raw materials:

[0112] Take the FII precipitate containing human immunoglobulin components after treatment by the low-temperature ethanol process as the initial raw material, dissolve the FII precipitate with injection water at 5 times the weight of the FII precipitate and a temperature of 0 °C to obtain a dissolved solution. Add the above-mentioned adsorbent filter aid to this dissolved solution at a proportion of 1 g / L, stir and adsorb for 30 minutes, then perform clarification filtration with a clarification filter plate, and the filtration pressure does not exceed 0.3 Mpa to obtain a clarified filtrate. Then, adjust the pH of the clarified filtrate to 4.0 with a 0.5 M hydrochloric acid solution, and then perform defatting filtration with a defatting filter plate, and the filtration pressure also does not exceed 0.3 Mpa to obtain the initial treatment solution;

[0113] Ultrafiltration and concentration operation: Ultrafilter and concentrate the obtained immunoglobulin filtrate with a 30 KD ultrafiltration membrane until the protein content is above 5.0%. Among them, the transmembrane pressure during ultrafiltration and concentration is 0.1 Mpa to obtain an ultrafiltrate;

[0114] Dialysis operation: Dialyze with 6 times the volume of the above-mentioned dialysis buffer solution of the ultrafiltrate to obtain a dialysate, and then adjust the protein content of the dialysate to 5.2% with the above-mentioned dialysis buffer solution to obtain a human immunoglobulin concentrate;

[0115] Virus inactivation: Filter the human immunoglobulin concentrate through a 0.2 μm filter element to obtain a sterile filtrate. During sterile filtration, the pressure does not exceed 0.2 Mpa. Then, place the obtained sterile filtrate in a low-pH incubation container, and fill the headspace of the low-pH incubation container with nitrogen not less than 0.15 Mpa. Then, heat the sterile filtrate to 24 ± 1 °C and perform continuous incubation for 21 days to inactivate the remaining lipid envelope virus to obtain an incubated and inactivated solution;

[0116] Virus filtration: Filter the above-mentioned incubated and inactivated solution with a 20-nanometer nanofilm, and the filtration pressure does not exceed 0.3 Mpa, that is, obtain the original solution of human immunoglobulin preparation with a pH value of 4.0 and a protein content of 5.2% 1.3 # . Comparative Example 1.3

[0117] The initial raw materials used in this comparative example and all processing operations performed on the initial raw materials, including steps and process conditions, are the same as those in Example 1.3. The only difference is that in this comparative example, the preparation of the adsorption filter aid is not carried out, and when performing the pretreatment operation on the FII precipitate of the initial raw materials, no adsorption filter aid is added for adsorption and filtration. Instead, only water for injection is added to the FII precipitate to dissolve the FII precipitate, and then clarification filtration and other subsequent processing operations are directly carried out. Finally, a human immunoglobulin preparation stock solution 1.3 with a pH value of 4.0 and a protein content of 5.2% is obtained. b 。

[0118] Table 3 lists the human immunoglobulin preparation stock solutions 1.3 obtained from Example 1.3 and Comparative Example 1.3 respectively. # and 1.3 b When performing the 6-month stability accelerated test at 30 ± 2°C, the turbidity data of each of them are detected by the turbidimeter method. Table 3

[0119]

[0120] According to the data listed in Table 3, that is, with the detection time as the abscissa and the turbidity values (NTU) obtained by the turbidimeter method for the human immunoglobulin preparation stock solutions obtained from Example 1.3 and Comparative Example 1.3 respectively as the ordinate, an attachment Figure 9 can be obtained, that is, a comparative graph of the turbidity change curves of the human immunoglobulin preparation stock solutions obtained from Example 1.3 and Comparative Example 1.3 of the present invention respectively during the 6-month stability accelerated test at 30 ± 2°C is obtained.

[0121] From the data listed in Table 3 and the attachment Figure 9 it can be clearly seen that:

[0122] The method for preparing the human immunoglobulin preparation stock solution provided by the embodiment of the present invention can effectively reduce the turbidity of the human immunoglobulin preparation stock solution and improve the appearance quality by adding an adsorption filter aid composed of refined bentonite for adsorption and filtration during the pretreatment operation of the initial raw materials of the human immunoglobulin preparation.

[0123] It should be particularly noted that: Although this embodiment only describes the comparative test of the influence of the adsorption filter aid on the turbidity of the human immunoglobulin preparation stock solutions obtained from Example 1.3 and Comparative Example 1.3 respectively, in fact, through detection, similar results to the comparison between Example 1.3 and Comparative Example 1.3 can also be obtained for Example 1 and Comparative Example 1 as well as Example 2 and Comparative Example 2, indicating that in this embodiment, the adsorption filter aid composed of refined bentonite has a significant effect on improving the turbidity of the human immunoglobulin preparation stock solution. Example 2

[0124] This example provides an application of the human immunoglobulin preparation stock solution obtained through Example 1, specifically including:

[0125] The human immunoglobulin preparation stock solution obtained through the above Example 1 is made into human immunoglobulin preparations that meet the relevant quality standards of the current "Chinese Pharmacopoeia" and have the protein contents marked respectively in line with the regulations, including:

[0126] Intravenous human immunoglobulin with a marked protein content of 5% and / or intravenous human immunoglobulin with a marked protein content of 10%, human immunoglobulin, freeze-dried human immunoglobulin, and subcutaneous injection human immunoglobulin with a marked protein content of 20%, wherein:

[0127] The specific preparation method of intravenous human immunoglobulin with a marked protein content of 5% includes: filtering the human immunoglobulin preparation stock solution obtained through the preparation method provided in the above Example 1 with a 0.2 µm sterilizing filter element for sterilization filtration, and then filling it into a container with a suitable capacity in a sterile / sterile nitrogen-filled manner, and sealing it to obtain;

[0128] The preparation methods of intravenous human immunoglobulin with a marked protein content of 10% and / or human immunoglobulin, freeze-dried human immunoglobulin, and subcutaneous injection human immunoglobulin with a marked protein content of 20% include a concentration and pH readjustment step and a preparation step, wherein:

[0129] The specific operations of the concentration and pH readjustment step include: ultrafiltrating and reconcentrating the human immunoglobulin preparation stock solution obtained through Example 1 with a 30 - 100 KD ultrafiltration membrane, wherein the transmembrane pressure during ultrafiltration and reconcentration is controlled between 0.1 - 0.4 Mpa, and then readjusting its concentration and pH with the dialysis buffer solution prepared in Example 1 and a 0.5 - 1 M hydrochloric acid solution to make it a human immunoglobulin preparation solution with the mass percentage content of protein respectively reaching the requirements of the pre-prepared preparation marked amount and a pH value of 4.0 - 5.0;

[0130] The preparation step includes: respectively using the preparation solutions of human immunoglobulin with the mass percentage content of protein respectively adapted to prepare intravenous human immunoglobulin with a marked protein content of 10% and / or human immunoglobulin, freeze-dried human immunoglobulin, and subcutaneous injection human immunoglobulin with a marked protein content of 20%, filtering them respectively with a 0.2 µm sterilizing filter element for sterilization filtration, and then respectively filling them into their respective adapted containers in a sterile / sterile nitrogen-filled manner, and after sealing, intravenous human immunoglobulin and / or human immunoglobulin, subcutaneous injection human immunoglobulin with the marked amount meeting the requirements can be obtained respectively, or freeze-dried human immunoglobulin obtained after freeze-drying and then sealing.

[0131] To further assist in understanding the preparation method of the human immunoglobulin preparation provided in this embodiment, its specific implementation manners and achievable technical effects, the following will further illustrate through specific cases. For the sake of brevity, some identical steps will only be briefly described. Case 2.1

[0132] This case provides an intravenous human immunoglobulin labeled with a protein content of 5%.

[0133] Take 1.2 of the human immunoglobulin preparation stock solution with a pH value of 5.0 and a protein content of 5.0%. # , perform aseptic filtration with a 0.2 µm sterilizing filter element, and then fill it into a glass bottle with a suitable capacity under aseptic / aseptic nitrogen filling, seal it, and that's it. Case 2.2

[0134] This case provides an intravenous human immunoglobulin and a human immunoglobulin labeled with a protein content of 10%.

[0135] Preparation of the intravenous human immunoglobulin labeled with 10% protein: Rinse and balance the ultrafiltration concentration and dialysis device with the dialysis buffer solution prepared in Example 1, and then put 1.1 of the human immunoglobulin preparation stock solution with a pH value of 4.5 and a protein content of 5.5%. # , perform ultrafiltration and reconcentration with a 30KD ultrafiltration membrane, control the transmembrane pressure at 0.1 Mpa during ultrafiltration and reconcentration, and then readjust the concentration and pH of the liquid after ultrafiltration and reconcentration with the same dialysis buffer solution and 0.5 M hydrochloric acid solution to make it a human immunoglobulin preparation solution with a mass percentage content of protein reaching the requirement of the prefabricated preparation label and a pH value of 4.5; sterilize and filter this human immunoglobulin preparation solution with a 0.2 µm sterilizing filter element, and then perform aseptic / aseptic nitrogen filling and packaging into glass bottles with a suitable capacity, seal it, and that's it.

[0136] Using the same method as preparing the above-mentioned intravenous human immunoglobulin labeled with 10%, a human immunoglobulin labeled with a protein content of 10% can be prepared. Case 2.3

[0137] This case provides a subcutaneous human immunoglobulin labeled with a protein content of 20%.

[0138] Rinse and balance the ultrafiltration concentration and dialysis device with the dialysis buffer solution in Example 3, and then put 1.3 of the human immunoglobulin preparation stock solution with a pH value of 4.0 and a protein content of 5.2%. #, ultrafiltration and reconcentration were carried out using a 50KD ultrafiltration membrane, and the transmembrane pressure during ultrafiltration and reconcentration was controlled at 0.3 Mpa. Then, the ultrafiltered and reconcentrated liquid was readjusted in concentration and pH using the same dialysis buffer and 0.5 M hydrochloric acid solution to make a human immunoglobulin preparation solution with a protein mass percentage reaching the requirement of the preformulation label and a pH value of 4.0; this human immunoglobulin preparation solution was sterilized by filtration using a 0.2 µm sterilizing filter element, and then aseptically / aseptically nitrogen-filled and subpackaged into glass bottles with appropriate capacities, and sealed to obtain the product. Case 2.4

[0139] This case provides a freeze-dried human immunoglobulin.

[0140] The ultrafiltration concentration and dialysis device were rinsed and balanced with the dialysis buffer prepared in Example 3, and then 1.3 of the stock solution of human immunoglobulin preparation with a pH value of 4.0 and a protein content of 5.2% was put in. # , ultrafiltration and reconcentration were carried out using a 100KD ultrafiltration membrane, and the transmembrane pressure during ultrafiltration and reconcentration was controlled at 0.4 Mpa. Then, the ultrafiltered and reconcentrated liquid was readjusted in concentration and pH using the same dialysis buffer and 1 M hydrochloric acid solution to make a human immunoglobulin preparation solution with a protein mass percentage reaching the requirement of the preformulation label and a pH value of 4.0;

[0141] This human immunoglobulin preparation solution was sterilized by filtration using a 0.2 µm sterilizing filter element, and then filled into appropriate vials according to the content specifications of the preformulated freeze-dried injection. Freeze-drying was carried out according to the set freeze-drying curve. After the freeze-drying was completed, vacuum was pumped and the vials were sealed to obtain the freeze-dried human immunoglobulin.

[0142] According to the relevant quality standards of the current "Chinese Pharmacopoeia", key item quality tests were carried out on the subcutaneous injection human immunoglobulin with a labeled protein content of 20% obtained in Case 2.3 above, and the test result data are listed in Table 4 below. Table 4

[0143]

[0144] From the data listed in Table 4, it can be seen that:

[0145] A subcutaneous injection human immunoglobulin with a labeled protein of 20% prepared from the stock solution of human immunoglobulin obtained in Case 1.3 of Application Example 1 in Case 2.3, after testing, all its indicators fully meet the relevant quality standards of the current "Chinese Pharmacopoeia".

[0146] It should be noted specifically that:

[0147] Although Table 4 only lists the relevant test data obtained by testing the subcutaneous human immunoglobulin with a labeled protein content of 20% prepared from the stock solution of human immunoglobulin obtained in Case 1.3 of Application Example 1 provided in Case 2.3 in accordance with the current Chinese Pharmacopoeia, in fact, through experiments and tests, the stock solutions of human immunoglobulin obtained from Cases 1.1, 1.2, and 1.3 of Example 1, through the preparation method of the preparation described in Example 2, result in intravenous human immunoglobulin and / or human immunoglobulin with different labeled dosage specifications, freeze-dried human intravenous immunoglobulin. For the freeze-dried human immunoglobulin products, their relevant quality indicators all meet the quality standards of the current Chinese Pharmacopoeia and can be applied to clinical treatment. This specification only simplifies the content and does not list their respective test data, and only makes a brief statement here.

[0148] From the above description, it can be seen that: through the application method provided in this example, it is convenient to use the stock solution of human immunoglobulin obtained in Example 1 to prepare relevant preparations, which is simple, convenient, and practical, and all indicators of the product can meet the quality standards of the current Chinese Pharmacopoeia, thus ensuring the safety and effectiveness of clinical medication.

[0149] In summary, it can be seen that:

[0150] The technical solution of the preparation method and its application of the stock solution of human immunoglobulin provided by the present invention effectively protects human immunoglobulin during the preparation process of the stock solution of human immunoglobulin and its preparation through a dialysis buffer solution with L-proline as the main functional component, rather than, as in the prior art, only protecting human immunoglobulin through the formulation method in the preparation product. Thus, it more effectively overcomes various unstable factors that may affect the polymerization of human immunoglobulin products during their preparation process in the prior art, thereby improving the stability of the stock solution of the preparation and its preparation product, effectively maintaining the natural complete structure and biological function of human immunoglobulin molecules, and improving their quality and efficacy;

[0151] Secondly, the present invention effectively overcomes the adverse effects of oxygen and carbon dioxide on the human immunoglobulin concentrate during low-pH incubation inactivation by filling nitrogen in the headspace of the low-pH incubation container, improving the stability of human immunoglobulin during the preparation process of its product and the effect of virus inactivation on it;

[0152] Again, the present invention adsorbs and filters the initial raw materials for preparing the stock solution of human immunoglobulin preparation by using an adsorption and filtration aid composed of refined bentonite, removing residual lipids or lipoproteins and denatured proteins therein from the very beginning, reducing the nucleation of these unstable substances, thereby effectively reducing the unstable factors causing protein aggregation in the product, improving the stability of the stock solution of human immunoglobulin preparation and its preparation products, reducing its turbidity, and improving the appearance of the product.

[0153] In summary, the technical solution provided by the present invention overcomes the deficiencies of the prior art, can effectively improve the stability of human immunoglobulin products, improve their quality and quality, and compared with the prior art, the preparation method is not complicated, but can achieve excellent technical effects. Therefore, it has outstanding beneficial effects and significant progress, and has great promotion and application value.

[0154] During the description process of the above specification:

[0155] The descriptions of terms such as "this embodiment", "the embodiment of the present invention", "this case", "this comparative example", "as shown in...", "further", etc. mean that the specific features, structures, materials or characteristics described in the embodiment or case, comparative example are included in at least one embodiment or case and comparative example of the present invention;

[0156] In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or case, comparative example, and moreover, the specific features, structures, materials or characteristics described can be combined or combined in a suitable manner in any one or more embodiments or cases, comparative examples; in addition, on the premise of not generating contradictions, those of ordinary skill in the art can also combine or combine the different embodiments or cases, comparative examples described in this specification and the features in different embodiments or cases, comparative examples.

[0157] Finally, it should be noted that:

[0158] The above embodiments and each comparative example are only used to illustrate the technical solutions and technical effects of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments and cases, comparative examples, those of ordinary skill in the art should understand that they can still modify or supplement the technical solutions or technical effects recorded in the foregoing embodiments and cases, comparative examples, or perform equivalent substitution on some or all of the technical features, and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions described in the embodiments of the present invention. The non-essential improvements, adjustments or substitutions made by those skilled in the art according to the content of this specification all fall within the scope protected by the present invention.

Claims

1. A method for preparing a human immunoglobulin preparation stock solution, comprising the steps of pretreatment of the initial raw material, virus inactivation and filtration, wherein: After the initial raw material is processed by the pretreatment step, an initial treatment liquid having a pH value of 4.0 to 5.0 is obtained, characterized in that: The method also includes a step of ultrafiltration concentration and dialysis of the initial treatment liquid to obtain a human immunoglobulin concentrate, and the ultrafiltration concentration and dialysis steps are performed with the participation of a dialysis buffer, the functional component of which is L-proline.

2. The method for preparing a human immunoglobulin preparation stock solution according to claim 1, characterized in that: The preparation method of the dialysis buffer is as follows: adding injection water at room temperature to L-proline to dissolve the L-proline, and then adjusting the pH with a 0.5-1M hydrochloric acid solution to finally form an aqueous solution with a molar concentration of L-proline of 200-400 mmol / L and a pH value of 4.0-5.0, and the temperature of the dialysis buffer is controlled between 2 and 8°C.

3. The method for preparing a human immunoglobulin preparation stock solution according to claim 1, characterized in that: The ultrafiltration concentration and dialysis steps include: The initial treatment liquid is ultrafiltered and concentrated using a 30-100KD ultrafiltration membrane to obtain an ultrafiltrate, wherein the transmembrane pressure during the ultrafiltration and concentration is controlled between 0.1 and 0.4 MPa; The dialysis is performed with the dialysis buffer solution in an amount not less than 5 times the volume of the ultrafiltrate to obtain a dialysis fluid, and the protein content of the dialysis fluid is adjusted to between 5.0% and 5.5% with the dialysis buffer solution, thereby obtaining a human immunoglobulin concentrate.

4. The method for preparing a human immunoglobulin preparation stock solution according to claim 1, characterized in that: The initial raw material is an FII precipitate containing a human immunoglobulin component after being treated with a low-temperature ethanol process and / or an IgG flow-through component obtained by anion exchange chromatography and an IgG elution component obtained by cation exchange.

5. The method for preparing a human immunoglobulin preparation stock solution according to claim 4, characterized in that: The pre-processing step comprises: The FII precipitate is dissolved in water for injection with a volume ratio of 3 to 5 times the mass of the FII precipitate and a temperature of 0 to 5°C to obtain a solution, and then an adsorption filter aid is added to the solution at a volume weight ratio of 1 to 2 g / L, and then deep filtration is performed using a clarifying filter plate to obtain a clarified filtrate, and then the pH value of the clarified filtrate is adjusted to 4.0 to 5.0 using a hydrochloric acid solution with a molar concentration of 0.5 to 1 M, and then filtered using a degreasing filter plate to obtain the initial treatment solution; and / or Adding adsorption filter aids to the IgG flow-through fraction obtained by anion exchange chromatography and the IgG elution fraction obtained by cation exchange chromatography at a volume weight ratio of 1 to 2 g / L, respectively, and then performing deep filtration with a clarifying filter plate to obtain respective clarified filtrates, and then adjusting the pH value of each clarified filtrate to 4.0 to 5.0 with a hydrochloric acid solution having a molar concentration of 0.5 to 1 M, and then filtering with a fat removal filter plate, thereby obtaining the initial treatment liquids whose corresponding initial raw materials are the IgG flow-through fraction and the IgG elution fraction, respectively; In the above operation, the filtering pressure does not exceed 0.3Mpa.

6. The method for preparing a human immunoglobulin preparation stock solution according to claim 5, characterized in that: The adsorption filter aid is refined bentonite, and the preparation method of the refined bentonite is as follows: Add 5 to 10 L of water for injection to each kilogram of commercial bentonite, then add acetate buffer with a pH value of 4.0, stir evenly, and use a Buchner funnel to vacuum filter to obtain a primary bentonite filter cake, then add 5 to 10 L of water for injection to the primary bentonite filter cake, stir evenly to form a suspension, then add sodium citrate buffer at a ratio of 10 to 15 mmol / L sodium citrate buffer per liter of suspension, stir evenly, and use a Buchner funnel to vacuum filter to obtain a secondary bentonite filter cake, then add an equal amount of water for injection to the secondary bentonite filter cake, stir evenly, and use a Buchner funnel to vacuum filter to obtain the refined bentonite.

7. The method for preparing a human immunoglobulin preparation stock solution according to claim 1, characterized in that: The virus inactivation and filtration steps include low pH incubation inactivation and nano-membrane filtration, wherein: The specific operation of the low pH incubation inactivation comprises: filtering and sterilizing the human immunoglobulin concentrate with a 0.2 µm filter element to obtain a sterilized filtrate, and then placing the sterilized filtrate in a container at 24±1°C and having a headspace filled with nitrogen gas with a pressure of not less than 0.15 MPa for incubation, and incubating continuously for at least 21 days to inactivate the lipid enveloped viruses remaining in the sterilized filtrate, and obtaining an incubation inactivation solution; The specific operation of the nano-membrane filtration includes: using a 20-nanometer nano-membrane to filter out viruses from the incubation and inactivation liquid to obtain the human immunoglobulin preparation stock solution.

8. An application of a human immunoglobulin preparation stock solution, characterized in that: The application is to prepare the human immunoglobulin preparation stock solution obtained by the preparation method of the human immunoglobulin preparation stock solution described in any one of claims 1 to 7 into a human immunoglobulin preparation that meets the relevant quality standards of the current "Chinese Pharmacopoeia" and has a labeled protein content that meets the regulations.

9. The use according to claim 8, characterized in that: The human immunoglobulin preparation includes: intravenous human immunoglobulin with a labeled protein content of 5% and / or intravenous human immunoglobulin with a labeled protein content of 10%, human immunoglobulin, freeze-dried human immunoglobulin and subcutaneous human immunoglobulin with a labeled protein content of 20%.

10. The use according to claim 9, characterized in that: The specific preparation method of intravenous human immunoglobulin with a labeled protein content of 5% comprises: sterilizing and filtering the human immunoglobulin preparation stock solution with a 0.2µm sterilizing filter element, and then aseptically / sterilely filling it with nitrogen into a container with suitable capacity, and sealing it to obtain the product; The preparation method of intravenous human immunoglobulin and / or human immunoglobulin with a labeled protein content of 10%, freeze-dried human immunoglobulin and subcutaneous human immunoglobulin with a labeled protein content of 20% comprises a concentration and pH readjustment step and a preparation preparation step, wherein: The specific operation of the concentration and pH readjustment step includes: ultrafiltration and re-concentration of the human immunoglobulin preparation stock solution using a 30-100KD ultrafiltration membrane, wherein the transmembrane pressure during ultrafiltration and re-concentration is controlled between 0.1 and 0.4Mpa, and then, the concentration and pH are readjusted using the dialysis buffer and 0.5-1M hydrochloric acid solution to obtain a human immunoglobulin preparation preparation liquid having a protein mass percentage that meets the labeled amount requirements of the preformed preparation and a pH value of 4.0-5.0; The preparation preparation steps include: respectively adapting the mass percentage of the protein to prepare the human immunoglobulin preparation solution of intravenous human immunoglobulin and / or human immunoglobulin with a labeled protein content of 10%, freeze-dried human immunoglobulin and subcutaneous human immunoglobulin with a labeled protein content of 20%, respectively performing sterile filtration with a 0.2µm sterilizing filter element, and then respectively performing aseptic / sterile nitrogen filling and packaging into respectively adapted containers, and after sealing, the intravenous human immunoglobulin and / or human immunoglobulin, subcutaneous human immunoglobulin with a labeled amount that meets the requirements, or the freeze-dried human immunoglobulin obtained after freeze-drying and then sealing can be obtained.

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