Preparation process of high-purity and low IgA content human immunoglobulin for intravenous injection

Abstract

This invention belongs to the field of blood products, specifically relating to a preparation process for high-purity, low-IgA-content intravenous immunoglobulin. The preparation process of the intravenous immunoglobulin described in this invention mainly includes: preparation of component I+III or component III, S / D inactivation, first ultrafiltration, chromatography, second ultrafiltration, intermediate product preparation, pasteurization inactivation, third ultrafiltration, nanofiltration for virus removal, semi-finished product preparation, and sterile filling. The prepared intravenous immunoglobulin has high yield, high purity, and low IgA content. Simultaneously, a virus inactivation process combining S / D inactivation and pasteurization is used to inactivate both lipid-enveloped and non-lipid-enveloped viruses, further ensuring product safety.

Description

Technical Field

[0001] This invention belongs to the field of blood products, specifically relating to a preparation process for high-purity, low-IgA-content intravenous human immunoglobulin. Background Technology

[0002] Intravenous immunoglobulin is produced by separating and extracting immunoglobulin components from the plasma of healthy individuals, and then refining them through steps such as virus inactivation and removal. Intravenous immunoglobulin has dual therapeutic effects of immune replacement and immune regulation, and is widely used clinically, showing significant efficacy in the treatment of primary or acquired immunoglobulin deficiency, bacterial infections, viral infections, hematological diseases, Kawasaki disease, and other conditions.

[0003] As a blood product, intravenous immunoglobulin requires strict quality control to better manage clinical efficacy. Pharmacopoes of various countries have corresponding regulations for the quality control of intravenous immunoglobulin, including the following standards: ① The main component should be IgG, with a purity of no less than 95.0% of the total protein, and all IgG subclasses should be present, with their content similar to the distribution of IgG subclasses in normal human serum; ② Intravenous immunoglobulin products should contain as little or no non-IgG immunoglobulin components as possible, such as IgA and IgE (IgA can cause allergic reactions in congenital selective IgA deficiency, so its content should be as low as possible); ③ The biological activity, high titer, and high efficacy of antibodies in intravenous immunoglobulin should be guaranteed; ④ The viral safety of intravenous immunoglobulin products must also be guaranteed.

[0004] Therefore, controlling the preparation process of intravenous immunoglobulin is crucial. Currently, the preparation process of intravenous immunoglobulin basically adopts a low-temperature ethanol separation and extraction process. However, the IgA content in immunoglobulin preparations prepared by the current low-temperature ethanol process is relatively high, which can easily cause adverse reactions in patients with congenital selective IgA deficiency after infusion, requiring improvement. For example, the method disclosed in patent application CN104402993B produces human immunoglobulin products with an average IgA content of 9.3 μg / ml. Moreover, in the preparation process of intravenous immunoglobulin, S / D inactivation (some foreign blood product companies choose to inactivate the virus in intravenous immunoglobulin at 30°C for 6 hours) or nanofiltration is often used to remove the virus. The virus inactivation effect is generally not very good, and some viruses cannot be effectively removed, affecting the safety of clinical use of the product. For example, Zhang Zhan published a paper titled "The Influence of Different Virus Inactivation Processes on the Quality and Yield of Intravenous Immunoglobulin," which used low pH incubation, nanomembrane filtration, pasteurization, and S / D virus inactivation processes to treat intravenous human immunoglobulin stock solution. However, these methods had a certain impact on the quality of intravenous immunoglobulin and also affected its yield.

[0005] With the increasing market demand for intravenous immunoglobulin, the product yield and purity of existing intravenous immunoglobulin preparation technologies are no longer ideal. Therefore, finding a preparation process for intravenous immunoglobulin with high product yield and purity, low IgA content, and good safety is an urgent problem to be solved.

[0006] This invention discloses a preparation process for intravenous human immunoglobulin. The main steps of this invention include separating components I+III from components I+II+III, performing S / D inactivation, first ultrafiltration, chromatography, second ultrafiltration, intermediate product preparation, pasteurization inactivation, third ultrafiltration, nanofiltration for virus removal, semi-finished product preparation, and sterile filling. The resulting product has high yield, high purity, and low IgA content. Furthermore, the virus inactivation process combines S / D inactivation and pasteurization to achieve the purpose of inactivating lipid-enveloped and non-lipid-enveloped viruses, with good inactivation effect. Summary of the Invention

[0007] The purpose of this invention is to provide a preparation process for intravenously administered human immunoglobulin, the preparation process comprising the following steps:

[0008] (1) Prepare component I+III or component III;

[0009] (2) S / D inactivation: Add S / D solution to the filtrate of component I+III or component III, and treat at a constant temperature of 24-26℃ for 6h to obtain S / D inactivation solution, wherein the S / D solution is polyethylene glycol octylphenyl ether (Triton X-100) and tributyl phosphate (TNBP).

[0010] (3) First ultrafiltration: After adjusting the pH of the S / D inactivation solution in step (2) to 3.5-4.0, perform ultrafiltration concentration and dialysis to make the protein content >60g / L;

[0011] (4) Chromatography: Adjust the conductivity of the ultrafiltration concentrate in step (3) to 1.9-2.3 ms / cm, pH to 6.4-6.8, and protein concentration to ≥30 g / L. Load the concentrate onto an ion exchange chromatography column and collect the eluent.

[0012] (5) Second ultrafiltration: After adjusting the pH of the effluent from step (4) to 3.5-4.0, perform ultrafiltration concentration and dialysis to make the protein content ≥60g / L;

[0013] (6) Preparation of intermediate product: Add maltose to the dialysis concentrate described in step (5) to a final concentration of 100-110 g / L, filter with a sterile filter cartridge to obtain intermediate product;

[0014] (7) Pasteurization: Sorbitol was added to the intermediate product described in step (6) to a final concentration of 32-34% w / v, the pH was adjusted to 4.6-5.2, and the product was inactivated at a constant temperature of 59.5-60.5℃ for 10h to obtain the pasteurized solution.

[0015] (8) Third ultrafiltration: The pasteurization solution obtained in step (7) is concentrated by dialysis with 0.85% sodium chloride solution until the protein concentration is ≥60g / L, to obtain protein ultrafiltrate;

[0016] (9) Virus removal by nanofiltration: The protein solution described in step (8) is filtered using a nanofiltration membrane with a pore size of 20 nm to obtain the stock solution;

[0017] (10) Preparation of finished intravenous immunoglobulin: Maltose is added to the stock solution in step (9), the pH is adjusted to 3.9-4.3, the final protein content is 40-60 g / L, and the final maltose content is 100-110 g / L to obtain a semi-finished product. The semi-finished product is sterilized by a 0.45 μm filter and then filled to obtain the finished intravenous immunoglobulin.

[0018] Preferably, the preparation process of component I+III in step (1) is as follows: dissolve the precipitate of component I+II+III with 0.01mol / L sodium chloride at 0-2℃, adjust the pH to 5.05-5.15, add 95% ethanol at a temperature below -15℃ under conditions below 0℃, so that the final concentration of ethanol is 17% v / v, control the temperature to -6.0 to -4.0℃ for 2h, and separate the reaction solution to obtain component I+III filtrate.

[0019] Preferably, the ethanol addition rate is ≤80kg / h; the separation is pressure filtration separation, the pressure filtration pressure is 0.05~0.25MPa, and the outlet temperature is -5.5~-3.5℃.

[0020] Preferably, the final concentration of polyethylene glycol octylphenyl ether (Triton X-100) in step (2) is 1%, and the concentration of tributyl phosphate (TNBP) is 0.3%.

[0021] Preferably, in step (3), the S / D inactivation solution is filtered with a 0.45μm filter and then concentrated with a 30kDa ultrafiltration membrane. When the protein content is 30-50g / L, it is continuously dialyzed with an equal volume of 5 times the volume of the concentrated solution at 2-8℃ to remove alcohol, so that the protein content is >60g / L.

[0022] Preferably, in step (4), before placing the ion exchange chromatography column on the dialysate, the chromatography column is first equilibrated. The method for equilibrating the chromatography column is as follows: first treat the chromatography column with acetate buffer, and then equilibrate the chromatography column with phosphate equilibration buffer.

[0023] Preferably, the ion chromatography column is a DEAE Sepharose Fast Flow ion exchange chromatography column, and the loading rate of the dialysate is ≤3L / min.

[0024] Preferably, in step (5), the effluent is placed in a collection tank, and the rotation speed is adjusted to 10-40 rpm for ultrafiltration concentration. When the protein content is concentrated to 40-50 g / L, it is continuously dialyzed with an equal volume of dialysis water at 2-8℃ until the protein content is ≥60 g / L.

[0025] Preferably, the front-end filtration pressure in the virus removal filtration process described in step (9) is ≤0.3MPa.

[0026] Preferably, the semi-finished product in step (10) has a protein content of 50.5 g / L and a maltose content of 100 g / L.

[0027] The beneficial effects of this invention are:

[0028] The preparation process described in this invention employs a combination of S / D inactivation and pasteurization inactivation to inactivate both lipid-enveloped and non-lipid-enveloped viruses, achieving excellent inactivation results. Furthermore, it does not affect the yield or purity of intravenous immunoglobulin. The intravenous immunoglobulin prepared according to this invention has a yield higher than 5.9 g / L (2271 vials / ton for 2.5 g / vial specification) and a purity higher than 99%. Simultaneously, the preparation process significantly reduces the IgA content of intravenous immunoglobulin and increases the content of IgG monomers and dimers. The prepared intravenous immunoglobulin contains less than 8 mg / mL of IgA and the sum of IgG monomer and dimer content is higher than 98%. Detailed Implementation

[0029] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further described below in conjunction with specific embodiments. However, the scope of protection of this invention is not limited to the embodiments described below.

[0030] Example 1: Preparation process of intravenous human immunoglobulin 1

[0031] (1) Separation of I+II+III precipitates from plasma

[0032] (2) Preparation and separation of components I and III

[0033] Dissolve the precipitates of components I+II+III in 0.01 mol / L sodium chloride solution at 0°C using 8 times their weight as the precipitate weight, stirring at 30 rpm. After complete dissolution, add pH 4.0 acetate buffer to adjust the pH to 5.10, cool to below 0°C, and then spray in 95% ethanol at -15°C to achieve a final ethanol concentration of 17% v / v at a spray rate of 80 kg / h. Control the final reaction solution temperature to -5.5°C. After the ethanol addition is complete, continue stirring for 2 hours, let stand for 6 hours, then turn on the stirrer at 30 rpm. After 15 minutes, perform pressure filtration separation, controlling the filtration pressure at 0.05 MPa and the outlet temperature at -5.5°C.

[0034] (3) S / D inactivation

[0035] Slowly add S / D solution at 10% of the filtrate volume (stirring speed 20 rpm), add polyethylene glycol octylphenyl ether (Triton X-100) to a final concentration of 1%, add tributyl phosphate (TNBP) to a concentration of 0.3%, and keep warm at 24°C for 6 hours.

[0036] (4) First ultrafiltration

[0037] Add 0.5 mol / L hydrochloric acid solution to the S / D inactivation solution to adjust the pH of the filtrate to 3.50. Then filter through a 0.45 μm filter cartridge and concentrate and dialyze using a 30 kDa ultrafiltration membrane. Dialyze with an equal volume of 8°C dialyzing water (water for injection) five times the volume of the concentrate until the protein concentration is >60 g / L. Collect the concentrate in an ultrafiltration vessel. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water (water for injection), and add the rinsing solution to the concentrate.

[0038] (5) Chromatography

[0039] Purification was performed using a DEAE Sepharose Fast Flow ion exchange chromatography column: The column (800 mm diameter, 180 mm height) was first treated with pH 4.0 acetate buffer; then equilibrated with phosphate equilibration buffer (0.02 mol / L, pH 6.4). The concentrate collected by ultrafiltration was adjusted to a conductivity of 1.9 ms / cm with 1 mol / L phosphate buffer, and then the pH was adjusted to 6.4 with 0.5 mol / L sodium hydroxide solution. The protein concentration was ≥30 g / L. For chromatographic loading, the loading rate was controlled below 3 L / min. Flow-through collection began when the UV detector of the chromatography system just showed a peak value. After loading, the column was washed with phosphate equilibration buffer until the UV detector value dropped to approximately 1 / 6 of the peak value, at which point flow-through collection was stopped. The column was then rinsed with pH 4.0 acetate buffer.

[0040] (6) Second ultrafiltration

[0041] After chromatography, start stirring in the product collection tank (30 rpm). After thorough stirring, add 0.5 mol / L hydrochloric acid solution to adjust the pH to 3.5. Adjust the stirring speed to 10 rpm and begin ultrafiltration concentration. When the protein content of the product reaches 40 g / L, begin dialysis. Perform continuous equal-volume dialysis with 6 times the volume of the concentrate at 8°C using dialyzing water (water for injection). After dialysis, perform final concentration and collect the concentrate. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water, and add the rinsing solution to the concentrate. The final concentrate has a protein content ≥60 g / L.

[0042] (7) Preparation of intermediate products

[0043] Turn on the agitator in the product collection tank (50 rpm), add maltose at a maltose content of 100 g / L and protein at a protein content of 50 g / L. After preparation, filter the solution using a 0.2 μm sterile filter cartridge.

[0044] (8) Inactivation of Parvovirus

[0045] The pH of the product was adjusted to 4.9±0.3, the sorbitol concentration was 33±1%, and the virus was inactivated at 60℃±0.5℃ for 10 hours.

[0046] (9) Third ultrafiltration

[0047] Dialyze with 5 times the volume of the ultrafiltrate in a 0.85% sodium chloride solution (8°C). After dialysis, concentrate the solution and collect the concentrate. Rinse the ultrafiltration membrane with an appropriate amount of dialysis water and add the rinsing solution to the concentrate. The final protein concentration in the collected solution should be ≥60 g / L to obtain the protein ultrafiltrate.

[0048] (10) Nanomembrane virus filtration

[0049] The ultrafiltrate is first pre-filtered with a 0.2μm filter cartridge, and then filtered for virus removal with a nano-membrane with a 20nm pore size. The front-end filtration pressure during the virus removal filtration process is 0.3MPa.

[0050] (11) Preparation of semi-finished products

[0051] Prepare the semi-finished product according to the following requirements: protein content 50g / L; maltose content 110g / L; pH value 3.9.

[0052] (12) Sterilization filling

[0053] The semi-finished product is sterilized and filled with a 0.45μm filter to obtain the finished product, intravenous immunoglobulin.

[0054] Example 2: Preparation process of intravenous human immunoglobulin 2

[0055] (1) Separation of I+II+III precipitates from plasma

[0056] (2) Preparation and separation of components I and III

[0057] Dissolve the precipitates of components I+II+III in 0.01 mol / L sodium chloride solution at 1°C using 10 times their weight as precipitates, stirring at 70 rpm. After complete dissolution, add pH 4.0 acetate buffer to adjust the pH to 5.0, cool to below 0°C, and then spray in 95% ethanol at -20°C to achieve a final ethanol concentration of 17% v / v at a spray rate of 70 kg / h. Maintain the final reaction solution temperature at -5.0°C. After the ethanol addition is complete, continue stirring for 2 hours, let stand for 6 hours, then turn on the stirrer at 35 rpm. After 15 minutes, perform pressure filtration separation at a pressure of 0.20 MPa and an outlet temperature of -4.5°C.

[0058] (3) S / D inactivation

[0059] Slowly add S / D solution at 10% of the filtrate volume (stirring speed 30 rpm), add polyethylene glycol octylphenyl ether (Triton X-100) to a final concentration of 1%, add tributyl phosphate (TNBP) to a concentration of 0.3%, and incubate at 25°C for 6 hours.

[0060] (4) First ultrafiltration

[0061] Add 0.5 mol / L hydrochloric acid solution to the S / D inactivation solution to adjust the pH of the filtrate to 3.8. Then filter through a 0.45 μm filter cartridge and concentrate and dialyze using a 30 kDa ultrafiltration membrane. Dialyze with an equal volume of 5 times the volume of the concentrate at 5°C using dialyzing water (water for injection) for de-alcoholization until the protein concentration is >60 g / L. Collect the concentrate in an ultrafiltration vessel. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water (water for injection), and add the rinsing solution to the concentrate.

[0062] (5) Chromatography

[0063] Purification was performed using a DEAE Sepharose Fast Flow ion exchange chromatography column: The column (800 mm diameter, 180 mm height) was first treated with pH 4.0 acetate buffer; then equilibrated with phosphate equilibration buffer (0.02 mol / L, pH 6.6). The concentrate collected by ultrafiltration was adjusted to a conductivity of 2.1 ms / cm with 1 mol / L phosphate buffer, and then the pH was adjusted to 6.6 with 0.5 mol / L sodium hydroxide solution. The protein concentration was ≥30 g / L. For chromatographic loading, the loading rate was controlled below 3 L / min. Flow-through collection began when the UV detector of the chromatography system just showed a peak value. After loading, the column was washed with phosphate equilibration buffer until the UV detector value dropped to approximately 1 / 6 of the peak value, at which point flow-through collection was stopped. The column was then rinsed with pH 4.0 acetate buffer.

[0064] (6) Second ultrafiltration

[0065] After chromatography, start stirring in the product collection tank (50 rpm). After thorough stirring, add 0.5 mol / L hydrochloric acid solution to adjust the pH to 3.8. Adjust the stirring speed to 10-40 rpm and begin ultrafiltration concentration. When the protein content of the product reaches 45 g / L, begin dialysis. Perform continuous equal-volume dialysis with 6 times the volume of the concentrate at 5°C using dialyzing water (water for injection). After dialysis, perform final concentration and collect the concentrate. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water, and add the rinsing solution to the concentrate. The final concentrate has a protein content ≥60 g / L.

[0066] (7) Preparation of intermediate products

[0067] Turn on the agitator in the product collection tank (70 rpm), add maltose at a maltose content of 100 g / L and a protein content of 55 g / L. After preparation, filter the solution using a 0.45 μm sterile filter cartridge.

[0068] (8) Inactivation of Parvovirus

[0069] The pH of the product was adjusted to 4.9, the sorbitol concentration was set to 33%, and the virus was inactivated at 60°C for 10 hours.

[0070] (9) Third ultrafiltration

[0071] Dialyze with 5 times the volume of the ultrafiltrate in a 0.85% sodium chloride solution (5°C). After dialysis, concentrate the solution and collect the concentrate. Rinse the ultrafiltration membrane with an appropriate amount of dialysis water and add the rinsing solution to the concentrate. The final collected solution should have a protein concentration ≥60 g / L, which is the protein ultrafiltrate.

[0072] (10) Nanomembrane virus filtration

[0073] The ultrafiltrate is first pre-filtered with a 0.2μm filter cartridge, and then filtered for virus removal with a nano-membrane with a 20nm pore size. The front-end filtration pressure during the virus removal filtration process is 0.2MPa.

[0074] (11) Preparation of semi-finished products

[0075] Based on the test results of the original solution, the semi-finished product should be prepared according to the following requirements: protein content 50.5g / L; maltose content 100g / L; pH value 4.0.

[0076] (12) Sterilization filling

[0077] The semi-finished product is sterilized and filled with a 0.45μm filter to obtain the finished product, intravenous immunoglobulin.

[0078] Example 3: Preparation process of intravenous human immunoglobulin 3

[0079] (1) Separation of I+II+III precipitates from plasma

[0080] (2) Preparation and separation of components I and III

[0081] Dissolve the precipitates of components I+II+III in 0.01 mol / L sodium chloride solution at 2°C using 12 times their weight as precipitates, stirring at 30 rpm. After complete dissolution, add pH 4.0 acetate buffer to adjust the pH to 5.15, cool to below 0°C, and then spray in 95% ethanol at -20°C to achieve a final ethanol concentration of 17% v / v at a spray rate of 50 kg / h. Maintain the final reaction solution temperature at -5.5°C. After the ethanol addition is complete, continue stirring for 2 hours, let stand for 6 hours, then turn on the stirrer at 50 rpm. After 15 minutes, perform pressure filtration separation at a pressure of 0.25 MPa and an outlet temperature of -4.0°C.

[0082] (3) S / D inactivation

[0083] Slowly add S / D solution at 10% of the filtrate volume (stirring speed 40 rpm), add polyethylene glycol octylphenyl ether (Triton X-100) to a final concentration of 1%, add tributyl phosphate (TNBP) to a concentration of 0.3%, and incubate at 26°C for 6 hours.

[0084] (4) First ultrafiltration

[0085] Add 0.5 mol / L hydrochloric acid solution to the S / D inactivation solution to adjust the pH of the filtrate to 4.0. Then filter through a 0.45 μm filter cartridge and concentrate and dialyze using a 30 kDa ultrafiltration membrane. Dialyze with an equal volume of 5 times the volume of the concentrate at 2°C using dialyzing water (water for injection) for de-alcoholization until the protein concentration is >60 g / L. Collect the concentrate in an ultrafiltration tank. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water (water for injection), and add the rinsing solution to the concentrate.

[0086] (5) Chromatography

[0087] Purification was performed using a DEAE Sepharose Fast Flow ion exchange chromatography column: The column (800 mm diameter, 180 mm height) was first treated with pH 4.0 acetate buffer; then equilibrated with phosphate equilibration buffer (0.02 mol / L, pH 6.5). The concentrate collected by ultrafiltration was first adjusted to a conductivity of 2.3 mS / cm with 1 mol / L phosphate buffer, and then the pH was adjusted to 6.5 with 0.5 mol / L sodium hydroxide solution. The protein concentration was ≥30 g / L. For chromatographic loading, the loading rate was controlled below 3 L / min. Flow-through collection began when the UV detector of the chromatography system just showed a peak value. After loading, the column was washed with phosphate equilibration buffer until the UV detector value dropped to approximately 1 / 6 of the peak value, at which point flow-through collection was stopped. The column was then rinsed with pH 4.0 acetate buffer.

[0088] (6) Second ultrafiltration

[0089] After chromatography, start the product collection tank and stir (70 rpm). After thorough stirring, add 0.5 mol / L hydrochloric acid solution to adjust the pH to 4.0. Adjust the stirring speed to 40 rpm and begin ultrafiltration concentration. Perform continuous equal-volume dialysis with 6 times the volume of the concentrate in 2–8℃ dialyzing water. After dialysis, perform final concentration and collect the concentrate. Rinse the ultrafiltration membrane with an appropriate amount of dialyzing water, and add the rinsing solution to the concentrate. The final concentrate has a protein content ≥60 g / L.

[0090] (7) Preparation of intermediate products

[0091] Turn on the agitator in the product collection tank (60 rpm), add maltose at a maltose content of 100 g / L and protein at a protein content of 60 g / L. After preparation, filter the solution using a 0.2 μm sterile filter cartridge.

[0092] (8) Inactivation of Parvovirus

[0093] The pH of the product was adjusted to 5.0, the sorbitol concentration was set to 32%, and the virus was inactivated at 60°C for 10 hours.

[0094] (9) Third ultrafiltration

[0095] Dialysis was performed using 5 times the volume of the solution to be ultrafiltrated with 0.85% sodium chloride solution (2℃). After dialysis, the solution was concentrated, and the concentrate was collected. The ultrafiltration membrane was rinsed with an appropriate amount of dialysis water, and the rinsing solution was added to the concentrate. The final protein concentration of the collected solution should be ≥60 g / L to obtain the protein ultrafiltrate.

[0096] (10) Nanomembrane virus filtration

[0097] The ultrafiltrate is first pre-filtered with a 0.1μm filter cartridge, and then filtered for virus removal with a nano-membrane with a pore size of 20nm. The front-end filtration pressure during the virus removal filtration process is 0.3MPa.

[0098] (11) Preparation of semi-finished products

[0099] Based on the test results of the original solution, the semi-finished product should be prepared according to the following requirements: protein content 60g / L, maltose content 90g / L, pH value 4.3.

[0100] (12) Sterilization filling

[0101] The semi-finished product is sterilized and filled with a 0.45μm filter to obtain the finished product, intravenous immunoglobulin.

[0102] Example 4: Quality Index Detection of Finished Intravenous Human Immunoglobulin

[0103] The purity, yield, IgA content, and molecular size distribution (sum of IgG monomer and dimer content) of the finished intravenous immunoglobulin prepared according to steps 1-3 were determined. The standard stipulates that the purity of the intravenous immunoglobulin should be no less than 95.0%, and the molecular size distribution should be no less than 95.0%. The results are shown in Table 1 below. The finished intravenous immunoglobulin prepared according to the process described in this invention has a purity higher than 99.2%, a yield as high as 5.9 g / L (2271 vials / ton for 2.5g vials), an IgA content lower than 8 mg / mL, and a sum of IgG monomer and dimer content higher than 98.2%. Compared with finished intravenous immunoglobulin prepared by existing technologies, the finished intravenous immunoglobulin prepared by the process described in this invention has a significantly lower IgA content, a significantly higher sum of IgG monomer and dimer content (significantly exceeding the national standard), and a significantly higher purity than the national standard, with a higher yield.

[0104] Table 1. Quality indicators of finished intravenous human immunoglobulin.

[0105]

[0106] According to the pharmacopoeia, the pyrogen and virus content of the finished intravenous immunoglobulin prepared by procedures 1-3 all met the national standards (0.5g of protein was injected per 1kg of rabbit body weight, which met the requirements).

[0107] According to the pharmacopoeia, the residual fire extinguishing agent in the finished intravenous human immunoglobulin prepared according to steps 1-3 was determined, and all met the requirements (tributyl phosphate less than 10 μg / ml).

[0108] The above description is merely a few exemplary embodiments of the present invention. For those skilled in the art, the present invention can be modified and varied in practice depending on specific preparation conditions, and is not intended to limit the present invention. Everything within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preparation process for intravenously administered human immunoglobulin, characterized in that, The preparation process includes the following steps: (1) Preliminary separation of component I+III precipitate from plasma: Dissolve component I+II+III precipitate with 0.01mol / L sodium chloride at 1℃, adjust pH to 5.0, add 95% ethanol at -20℃ under conditions below 0℃ to make the final ethanol concentration 17%v / v, control the temperature at -5.0℃ for 2h, let stand for 6h, stir at 35rpm for 15min and then filter to obtain component I+III filtrate. The filtration pressure is 0.20MPa and the outlet temperature is -4.5℃; the ethanol addition rate is ≤80kg / h; (2) S / D inactivation: Add 10% of the filtrate volume of S / D solution to the filtrate of component I+III, and treat at 25℃ for 6 hours to obtain S / D inactivation solution, wherein the S / D solution is polyethylene glycol octylphenyl ether (Triton X-100) and tributyl phosphate (TNBP); the final concentration of polyethylene glycol octylphenyl ether (Triton X-100) is 1%, and the final concentration of tributyl phosphate (TNBP) is 0.3%; (3) First ultrafiltration: After adjusting the pH of the S / D inactivation solution in step (2) to 3.8, filter it with a 0.45μm filter element and then concentrate it with a 30kDa ultrafiltration membrane. When the protein content is 30~50g / L, dialyze it continuously with 5 times the volume of concentrated solution and 5℃ dialyzing water to remove alcohol, so that the protein content is >60g / L; (4) Chromatography: Adjust the conductivity of the ultrafiltration concentrate in step (3) to 2.1 ms / cm, pH to 6.6, and protein concentration to ≥30 g / L. Load the ion exchange chromatography column and collect the eluent. Before loading the ion exchange chromatography column with the dialysate, equilibrate the chromatography column. The method for equilibrating the chromatography column is as follows: first treat the chromatography column with acetate buffer, and then equilibrate the chromatography column with phosphate equilibration buffer. (5) Second ultrafiltration: After adjusting the pH of the effluent in step (4) to 3.8, perform ultrafiltration concentration and dialysis. When the protein content is concentrated to 45 g / L, use 5℃ dialysis water for continuous equal volume dialysis until the protein content is ≥60 g / L. (6) Preparation of intermediate product: Add maltose to the dialysis concentrate described in step (5) to a final concentration of 100 g / L, filter with a sterile filter cartridge to obtain the intermediate product; (7) Pasteurization: Add sorbitol to the intermediate product described in step (6) to a final concentration of 33% w / v, adjust the pH to 4.9, and inactivate at 60°C for 10 h to obtain pasteurization solution; (8) Third ultrafiltration: The pasteurization solution obtained in step (7) is concentrated by dialysis with 0.85% sodium chloride solution until the protein concentration is ≥60g / L, to obtain protein ultrafiltrate; (9) Virus removal by nanofiltration: The protein ultrafiltrate described in step (8) is filtered with a nanofiltration membrane with a pore size of 20 nm to obtain the stock solution; the front-end filtration pressure in the virus removal filtration process is 0.2 MPa; (10) Preparation of finished intravenous immunoglobulin: Maltose is added to the stock solution in step (9), the pH is adjusted to 4.0, the final protein content is 50.5 g / L, and the final maltose content is 100 g / L to obtain a semi-finished product. The semi-finished product is sterilized by a 0.45 μm filter and then filled to obtain the finished intravenous immunoglobulin.

2. The preparation process according to claim 1, characterized in that, The ion chromatography column is a DEAE Sepharose Fast Flow ion exchange chromatography column, and the loading rate of the dialysate is ≤3L / min.

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