Method for preparing human immunoglobulin for intravenous injection by sodium caprylate precipitation process

Abstract

The invention provides a method for preparing human immunoglobulin for intravenous injection by a sodium caprylate precipitation process. Comprising the following steps: raw material plasma preparation, FI reaction, FI + II + III separation, FI + II + III filter pressing, FI + II + III precipitation and dissolution, sodium caprylate precipitation reaction, filter pressing filtration, ultrafiltration concentration, DEAE chromatography, TMAE chromatography, ultrafiltration dialysis, nanofiltration, low-pH incubation, ultrafiltration concentration (stock solution), semi-finished product solution preparation, degerming and split charging to obtain the human immunoglobulin finished product for intravenous injection. According to the process, plasma is adopted as an initial raw material, FI + II + III is prepared through an ethanol reaction, and albumin, IgA, IgM and other impure proteins are removed more thoroughly through a method of sodium caprylate precipitation primary purification and multi-step chromatography refining purification, so that the risk of hemolysis and adverse reaction after infusion is reduced. Meanwhile, saccharides are not added into the preparation as a stabilizer, and safer glycine is used as the stabilizer, so that the kidney load of a patient is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals and blood products, and particularly relates to a method for preparing intravenous human immunoglobulin by using a sodium caprylate precipitation process. Background Art

[0002] Intravenous human immunoglobulin (IVIG) products are blood products made from more than 1000 plasma units. They are composed of complete IgG molecules, which are protein complexes composed of four polypeptide chains and therefore cannot display the amino acid sequence. 3 γ heavy (H) chain and two identical 25×10 3 κ or λ light (L) chains are linked together by disulfide bonds between polypeptide chains. IgG is the main immunoglobulin component in serum, accounting for approximately 75% of the total immunoglobulin content in serum, with a normal concentration of 9.5 to 12.5 mg / ml. Of this, 40 to 50% is distributed in serum, with the remainder distributed in tissues. Its molecular weight is approximately 150,000 Daltons. IgG in human serum is primarily monomeric, and normal human IgG consists of four subtypes: IgG1 accounts for 60 to 70%, IgG2 for 15 to 20%, IgG3 for 5 to 10%, and IgG4 for 1 to 7%. These subtypes differ in their ability to bind to the classical pathway of complement activation.

[0003] Intravenous human immunoglobulin (IVIG) is a human immunoglobulin product derived from human plasma. Its primary component is human immunoglobulin G (IgG), along with smaller amounts of IgA and IgM. It is widely used as an effective treatment for primary and secondary immunodeficiency disorders and autoimmune diseases (such as Kawasaki disease, idiopathic thrombocytopenic purpura, and chronic inflammatory demyelinating polyneuropathy), playing a role that is irreplaceable by other medications. In recent years, with the continuous expansion of clinical indications and the application of new technologies, the demand for IV human immunoglobulin has maintained a high growth momentum among plasma protein products, and its production and preparation has attracted widespread attention.

[0004] Currently, most domestic blood product companies use the low-temperature ethanol method to extract component II from components I+II+III, then purify it through a single step of chromatography to obtain IVIG. They then use low-pH incubation, low-pH incubation followed by 50nm nano-membrane filtration, or pasteurization followed by low-pH incubation to inactivate viruses and remove any viruses that may be present in the product. They also use varying concentrations of sugar as a stabilizer. This process has the following disadvantages:

[0005] 1. The products prepared by this process have high levels of IgA and IgM impurities, which can easily cause allergic reactions and side effects;

[0006] 2. Most of the sugars used in this process are stabilizers at varying concentrations, with a tolerance of only 0.4g / kg body weight. This directly impacts the therapeutic efficacy of patients who require high-dose (1-2g / kg body weight) IVIG infusions (e.g., for Kawasaki disease, bone marrow transplantation, ACID, etc.). Furthermore, the inclusion of sugars as stabilizers in formulations may not only cause acute renal failure, but also burden the heart and other organs, leading to complications such as cardiomegaly, pulmonary edema, and liver rupture.

[0007] 3. The most common viral inactivation process combinations, both domestically and internationally, utilize S / D + nanomembrane or pasteurization + nanomembrane + S / D. The S / D method requires the use of an IgG-adsorbing gel to effectively remove the S / D. Large-scale production inevitably requires the use of large-diameter chromatography columns, which complicates the process and increases costs. Traditional pasteurization requires prolonged heating, which damages protein structure and may increase the risk of protein immunogenicity and inhibitor formation. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a process for preparing intravenous human immunoglobulin with low IgA / IgM impurity content, safer virus removal and lower side effects in clinical use.

[0009] The present invention provides a method for preparing intravenous human immunoglobulin using a sodium caprylate precipitation process, comprising the following steps:

[0010] 1) Plasma pooling and thawing: During the thawing process, the plasma temperature should not exceed 4°C, and the final plasma temperature should be controlled between 0 and 4°C. The completely thawed plasma should be centrifuged to collect the cryoprecipitate, which can be used to prepare human coagulation factor VIII. The decision to prepare human prothrombin complex (PC) will be based on market demand. If PC is to be prepared, gel adsorption separation is performed on the supernatant after removing the cryoprecipitate, and the adsorbate is used to purify and produce human prothrombin complex (PC).

[0011] 2) FI reaction: adjust the pH to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 8±1%, and control the reaction temperature at -5.0±1.0°C.

[0012] 3) FI+II+III separation: Adjust the pH of the FI reaction solution to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 15±5%, and control the reaction solution temperature at -5.0±1°C.

[0013] 4) FI+II+III filter press: Add filter aid, stir and filter, collect the FI+II+III precipitate and store below -30℃.

[0014] 5) Sodium Caprylate Precipitation Reaction: Dissolve the FI+II+III precipitate 15±5 times in water for injection at 2-8°C. Stir to dissolve. Adjust the pH of the solution to 4.0±0.5 and continue stirring. Add sodium caprylate directly to a final concentration of 15-35 mM. Adjust the pH to 5.0±0.5 with 0.5 M NaOH and allow the reaction to proceed with slow stirring.

[0015] 6) Filter press: The sodium octanoate precipitation reaction solution is filter press filtered through a pretreated plate-and-frame filter plate, and the supernatant is collected.

[0016] 7) Ultrafiltration and concentration: Concentrate the filtrate to 40% to 70% of the total volume, adjust the sample pH to 5.0 ± 0.5, and the conductivity to 1.4 ms / cm to 1.8 ms / cm.

[0017] 8) Fractogel EMD DEAE Chromatography: Load the sample onto a Fractogel EMD DEAE column equilibrated with 10-100 mmol / L acetate. After injection, wash the gel with 10-100 mmol / L acetate and collect the flow-through and washes. Adjust the pH of the flow-through washes to 6.0 ± 0.5, filter, and use in the next chromatography step.

[0018] 9) Fractogel EMD TMAE Chromatography: Load the filtered solution onto a Fractogel EMD TMAE column equilibrated with 10-100 mmol / L acetate. After injection, wash the gel with 10-100 mmol / L acetate, collecting the flow-through and washes.

[0019] 10) Ultrafiltration and Dialysis: The chromatography flow-through wash solution from step 9) was concentrated by ultrafiltration using an ultrafiltration membrane bag, dialyzed against 2% to 3% glycine, and the ultrafiltration concentrate was collected. The pH of the ultrafiltration solution was adjusted to 3.8 to 4.4, and the protein concentration was adjusted to 45 to 65 g / L.

[0020] 11) Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate is filtered through a 20 nm nanomembrane.

[0021] 12) Low pH incubation: After sterilization, the solution is incubated at a low pH with the incubation temperature controlled at 25±5°C for at least 21 days.

[0022] 13) Stock solution: After mixing with the low pH incubation liquid, ultrafiltration and concentration are performed using an ultrafiltration membrane package. The protein concentration reaches 95 g / L or above. The ultrafiltration concentrate is collected and the pH is adjusted to 4.4-5.2 to obtain the intravenous human immunoglobulin (10%) stock solution (Note: If the concentration is 5%, ultrafiltration and concentration are not required. The solution can be diluted and prepared directly after the low pH incubation and adjusted to pH 3.8-4.4 as required by the Chinese Pharmacopoeia. The protein concentration is >50 g / L).

[0023] 14) Semi-finished product: Determine whether to dilute and prepare the liquid based on the protein content test results of the original liquid and adjust the pH of the liquid to 4.4-5.2, which is the semi-finished product.

[0024] 15) Finished product: The semi-finished product liquid in step 14) is sterilized, plugged, and capped to obtain the finished product of intravenous human immunoglobulin (10%).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention can significantly reduce the impurity content of IgA, IgM, and Alb in the product through sodium octanoate precipitation + two-step ion exchange chromatography, thereby reducing the occurrence of side effects during clinical use;

[0027] (2) The present invention uses sodium octanoate instead of octanoic acid for the precipitation reaction, mainly because the freezing point of octanoic acid is about 16°C. It is easy to solidify at low temperatures, which affects the addition effect. In addition, the solubility of octanoic acid is poor under low temperature conditions, and an oil-in-water pattern will form on the surface of the liquid. The effective concentration of octanoic acid will be reduced, which will cause batch differences. Sodium octanoate is easily soluble in water, and the effective concentration of sodium octanoate can be accurately controlled, resulting in good product uniformity.

[0028] (3) The present invention optimizes the formulation and does not add sugars as stabilizers. Instead, it uses glycine, a safer protective agent, which helps maintain the conformational stability of protein molecules in the liquid formulation and reduces their activity loss. At the same time, the use of amino acids as protective agents can reduce the patient's renal burden and enhance product stability. Diabetic patients can also receive large-dose infusions, expanding the product's user population.

[0029] (4) The present invention adopts a dual inactivation / removal of viruses process using low pH incubation + 20nm nano-membrane filtration. In addition, sodium octanoate precipitation and chromatography, as effective methods for separating plasma proteins, also have a certain effect in removing viruses. The S / D virus inactivation method reduces the complexity of the production process and the traditional pasteurization method requires long-term heating treatment, which has a certain destructive effect on the protein structure and may increase the immunogenicity of the protein and the risk of producing inhibitors. 20nm nano-membrane filtration can more effectively remove parvoviruses and ensure the virus safety of the product.

[0030] (5) Some domestic manufacturers use a two-step ion exchange chromatography + one-step anti-A and anti-B affinity gel chromatography process in their preparation process, which greatly increases production costs. The present invention uses a sodium octanoate precipitation + two-step ion exchange chromatography process to control the anti-A and anti-B quality indicators to be better than the quality standard requirements of the "Chinese Pharmacopoeia" and at the same time increase the product yield by about 10% to 20%, creating greater economic benefits for the enterprise and more social benefits for society;

[0031] (6) Sodium caprylate precipitation plus two-step ion exchange chromatography can be used to prepare intravenous human immunoglobulin with a concentration of 10%, which can reduce the volume of large-dose infusion in clinical use and facilitate the use of nurses;

[0032] (7) Sodium caprylate precipitation + two-step ion exchange chromatography can effectively remove the FXIa content in the product. FXIa in IVIG products is the main cause of thromboembolic complications. Therefore, the product prepared by the process of the present invention has a lower risk of thrombosis for patients with underlying diseases and patients who require large-dose infusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the process flow of the present invention for intravenous injection of human immunoglobulin

[0034] Figure 2 Circular dichroism detection result diagram of Example 1

[0035] Figure 3 Circular dichroism detection result diagram of Example 2

[0036] Figure 4 The circular dichroism detection result diagram of Example 3 DETAILED DESCRIPTION

[0037] Example 1

[0038] A method for preparing intravenous human immunoglobulin using a sodium caprylate precipitation process comprises the following steps:

[0039] 1) Plasma pooling and thawing: During the thawing process, the plasma temperature should not exceed 4°C, and the final plasma temperature should be controlled between 0 and 4°C. The completely thawed plasma should be centrifuged to collect the cryoprecipitate, which can be used to prepare human coagulation factor VIII. The decision to prepare human prothrombin complex (PC) will be based on market demand. If PC is to be prepared, gel adsorption separation is performed on the supernatant after removing the cryoprecipitate, and the adsorbate is used to purify and produce human prothrombin complex (PC).

[0040] 2) FI reaction: adjust the pH to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 8±1%, and control the reaction temperature at -5.0±1.0°C.

[0041] 3) FI+II+III separation: Adjust the pH of the FI reaction solution to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 15±5%, and control the reaction solution temperature at -5.0±1°C.

[0042] 4) FI+II+III filter press: Add filter aid, stir and filter, collect the FI+II+III precipitate and store below -30℃.

[0043] 5) Sodium Caprylate Precipitation: Dissolve the FI+II+III precipitate 15-fold in water for injection at 2-8°C. Stir to dissolve. Adjust the pH of the solution to 4.0 and continue stirring. Add sodium caprylate directly to a final concentration of 15 mM. Adjust the pH to 5.0 with 0.5 M NaOH and allow the reaction to proceed with slow stirring.

[0044] 6) Filter press: The sodium octanoate precipitation reaction solution is filter press filtered through a pretreated plate-and-frame filter plate, and the supernatant is collected.

[0045] 7) Ultrafiltration and concentration: The filtrate was concentrated to 40% of the total volume, and the pH of the sample was adjusted to 5.0 and the conductivity to 1.7 ms / cm.

[0046] 8) Fractogel EMD DEAE Chromatography: Load the sample onto a Fractogel EMD DEAE column equilibrated with 20 mmol / L acetate. After injection, wash the gel with 20 mmol / L acetate and collect the flow-through and washes. Adjust the pH of the flow-through washes to 5.5, filter, and use in the next chromatography step.

[0047] 9) Fractogel EMD TMAE Chromatography: The filtered solution was loaded onto a Fractogel EMD TMAE column equilibrated with 20 mmol / L acetate. After injection, the gel was washed with 20 mmol / L acetate, and the flow-through and washes were collected.

[0048] 10) Ultrafiltration and dialysis: The chromatographic flow-through washing solution in step 9) was concentrated by ultrafiltration using an ultrafiltration membrane bag, dialyzed against 3% glycine, and the ultrafiltration concentrate was collected. The pH of the ultrafiltration solution was adjusted to 4.1, and the protein concentration was adjusted to 50 g / L.

[0049] 11) Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate is filtered through a 20 nm nanomembrane.

[0050] 12) Low pH incubation: After sterilization, the feed solution is incubated at a low pH with the incubation temperature controlled at 25±5°C for 21 days.

[0051] 13) Stock solution: After mixing with the low pH incubation liquid, ultrafiltration and concentration are performed using an ultrafiltration membrane package. The protein concentration reaches 95 g / L or above. The ultrafiltration concentrate is collected and the pH is adjusted to 4.8 to obtain the intravenous human immunoglobulin (10%) stock solution (Note: If the concentration is 5%, ultrafiltration and concentration are not required. The solution can be directly diluted and prepared after the low pH incubation and adjusted to pH 3.8-4.4 as required by the Chinese Pharmacopoeia. The protein concentration is >50 g / L).

[0052] 14) Semi-finished product: Determine whether to dilute and prepare the liquid based on the protein content test results of the original liquid and adjust the pH of the liquid to 4.8, which is the semi-finished product.

[0053] 15) Finished product: The semi-finished product liquid in step 14) is sterilized, plugged, and capped to obtain the finished product of intravenous human immunoglobulin (10%).

[0054] Activity index test results:

[0055] Table 1 Circular dichroism spectrum of intravenous human immunoglobulin product (Example 1, scanning wavelength 250-190 nm)

[0056]

[0057] Table 2 Key Quality Indicators (Example 1)

[0058] Detection indicators Test results 2020 edition of the Chinese Pharmacopoeia purity(%) 99.1 ≥95 IgA (ug / ml) 18 / Molecular size distribution (%) 99.2 ≥95

[0059] Example 2

[0060] A method for preparing intravenous human immunoglobulin using a sodium caprylate precipitation process comprises the following steps:

[0061] 1) Plasma pooling and thawing: During the thawing process, the plasma temperature should not exceed 4°C, and the final plasma temperature should be controlled between 0 and 4°C. The completely thawed plasma should be centrifuged to collect the cryoprecipitate, which can be used to prepare human coagulation factor VIII. The decision to prepare human prothrombin complex (PC) will be based on market demand. If PC is to be prepared, gel adsorption separation is performed on the supernatant after removing the cryoprecipitate, and the adsorbate is used to purify and produce human prothrombin complex (PC).

[0062] 2) FI reaction: adjust the pH to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 8±1%, and control the reaction temperature at -5.0±1.0°C.

[0063] 3) FI+II+III separation: Adjust the pH of the FI reaction solution to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 15±5%, and control the reaction solution temperature at -5.0±1°C.

[0064] 4) FI+II+III filter press: Add filter aid, stir and filter, collect the FI+II+III precipitate and store below -30℃.

[0065] 5) Sodium Caprylate Precipitation: Dissolve the FI+II+III precipitate 20 times in water for injection at 2-8°C. Stir to dissolve. Adjust the pH of the solution to 4.0 and continue stirring. Add sodium caprylate directly to a final concentration of 25 mM. Adjust the pH to 5.0 with 0.5 M NaOH and allow the reaction to proceed with slow stirring.

[0066] 6) Filter press: The sodium octanoate precipitation reaction solution is filter press filtered through a pretreated plate-and-frame filter plate, and the supernatant is collected.

[0067] 7) Ultrafiltration and concentration: The filtrate was concentrated to 40% of the total volume, and the pH of the sample was adjusted to 5.0 and the conductivity to 1.7 ms / cm.

[0068] 8) Fractogel EMD DEAE Chromatography: Load the sample onto a Fractogel EMD DEAE column equilibrated with 20 mmol / L acetate. After injection, wash the gel with 20 mmol / L acetate and collect the flow-through and washes. Adjust the pH of the flow-through washes to 5.5, filter, and use in the next chromatography step.

[0069] 9) Fractogel EMD TMAE Chromatography: The filtered solution was loaded onto a Fractogel EMD TMAE column equilibrated with 20 mmol / L acetate. After injection, the gel was washed with 20 mmol / L acetate, and the flow-through and washes were collected.

[0070] 10) Ultrafiltration and dialysis: The chromatographic flow-through washing solution in step 9) was concentrated by ultrafiltration using an ultrafiltration membrane bag, dialyzed against 3% glycine, and the ultrafiltration concentrate was collected. The pH of the ultrafiltration solution was adjusted to 4.1, and the protein concentration was adjusted to 50 g / L.

[0071] 11) Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate is filtered through a 20 nm nanomembrane.

[0072] 12) Low pH incubation: After sterilization, the feed solution is incubated at a low pH with the incubation temperature controlled at 25±5°C for 21 days.

[0073] 13) Stock solution: After mixing with the low pH incubation liquid, ultrafiltration and concentration are performed using an ultrafiltration membrane package. The protein concentration reaches 95 g / L or above. The ultrafiltration concentrate is collected and the pH is adjusted to 4.8 to obtain the intravenous human immunoglobulin (10%) stock solution (Note: If the concentration is 5%, ultrafiltration and concentration are not required. The solution can be directly diluted and prepared after the low pH incubation and adjusted to pH 3.8-4.4 as required by the Chinese Pharmacopoeia. The protein concentration is >50 g / L).

[0074] 14) Semi-finished product: Determine whether to dilute and prepare the liquid based on the protein content test results of the original liquid and adjust the pH of the liquid to 4.8, which is the semi-finished product.

[0075] Activity index test results:

[0076] Table 3 Circular dichroism spectrum of intravenous human immunoglobulin product (Example 2, scanning wavelength 250-190 nm)

[0077]

[0078] Table 4 Key Quality Indicators (Example 2)

[0079] Detection indicators Test results 2020 edition of the Chinese Pharmacopoeia purity(%) 99.5 ≥95 IgA (ug / ml) 12 / Molecular size distribution (%) 99.3 ≥95

[0080] Example 3

[0081] A method for preparing intravenous human immunoglobulin using a sodium caprylate precipitation process comprises the following steps:

[0082] 1) Plasma pooling and thawing: During the thawing process, the plasma temperature should not exceed 4°C, and the final plasma temperature should be controlled between 0 and 4°C. The completely thawed plasma should be centrifuged to collect the cryoprecipitate, which can be used to prepare human coagulation factor VIII. The decision to prepare human prothrombin complex (PC) will be based on market demand. If PC is to be prepared, gel adsorption separation is performed on the supernatant after removing the cryoprecipitate, and the adsorbate is used to purify and produce human prothrombin complex (PC).

[0083] 2) FI reaction: adjust the pH to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 8±1%, and control the reaction temperature at -5.0±1.0°C.

[0084] 3) FI+II+III separation: Adjust the pH of the FI reaction solution to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 15±5%, and control the reaction solution temperature at -5.0±1°C.

[0085] 4) FI+II+III filter press: Add filter aid, stir and filter, collect the FI+II+III precipitate and store below -30℃.

[0086] 5) Sodium Caprylate Precipitation: Dissolve the FI+II+III precipitate 20 times in water for injection at 2-8°C. Stir to dissolve. Adjust the pH of the solution to 4.2 and continue stirring. Add sodium caprylate directly to a final concentration of 35 mM. Adjust the pH to 5.0 with 0.5 M NaOH and allow the reaction to proceed with slow stirring.

[0087] 6) Filter press: The sodium octanoate precipitation reaction solution is filter press filtered through a pretreated plate-and-frame filter plate, and the supernatant is collected.

[0088] 7) Ultrafiltration and concentration: The filtrate was concentrated to 40% of the total volume, and the pH of the sample was adjusted to 5.0 and the conductivity to 1.7 ms / cm.

[0089] 8) Fractogel EMD DEAE Chromatography: Load the sample onto a Fractogel EMD DEAE column equilibrated with 20 mmol / L acetate. After injection, wash the gel with 20 mmol / L acetate and collect the flow-through and washes. Adjust the pH of the flow-through washes to 5.5, filter, and use in the next chromatography step.

[0090] 9) Fractogel EMD TMAE Chromatography: The filtered solution was loaded onto a Fractogel EMD TMAE column equilibrated with 20 mmol / L acetate. After injection, the gel was washed with 20 mmol / L acetate, and the flow-through and washes were collected.

[0091] 10) Ultrafiltration and dialysis: The chromatographic flow-through washing solution in step 9) was concentrated by ultrafiltration using an ultrafiltration membrane bag, dialyzed against 3% glycine, and the ultrafiltration concentrate was collected. The pH of the ultrafiltration solution was adjusted to 4.1, and the protein concentration was adjusted to 50 g / L.

[0092] 11) Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate is filtered through a 20 nm nanomembrane.

[0093] 12) Low pH incubation: After sterilization, the feed solution is incubated at a low pH with the incubation temperature controlled at 25±5°C for 21 days.

[0094] 13) Stock solution: After mixing with the low pH incubation liquid, ultrafiltration and concentration are performed using an ultrafiltration membrane package. The protein concentration reaches 95 g / L or above. The ultrafiltration concentrate is collected and the pH is adjusted to 4.8 to obtain the intravenous human immunoglobulin (10%) stock solution (Note: If the concentration is 5%, ultrafiltration and concentration are not required. The solution can be directly diluted and prepared after the low pH incubation and adjusted to pH 3.8-4.4 as required by the Chinese Pharmacopoeia. The protein concentration is >50 g / L).

[0095] 14) Semi-finished product: Determine whether to dilute and prepare the liquid based on the protein content test results of the original liquid and adjust the pH of the liquid to 4.8, which is the semi-finished product.

[0096] Activity index test results:

[0097] Table 5 Circular dichroism spectrum of intravenous human immunoglobulin product (Example 3, scanning wavelength 250-190nm)

[0098]

[0099] Table 6 Key Quality Indicators (Example 3)

[0100] Detection indicators Test results 2020 edition of the Chinese Pharmacopoeia purity(%) 99.7 ≥95 IgA (ug / ml) 7 / Molecular size distribution (%) 99.8 ≥95

[0101] As can be seen from the above table, above-mentioned 3 implementation cases, difference is precipitation dissolution multiple, pH adjustment range and sodium octanoate addition concentration, as can be seen from the result, dissolution multiple is 15~20 times, pH is 4.0~4.2, sodium octanoate addition concentration can prepare qualified product in the range of 15~35mmol, its product secondary structure activity index tends to be consistent, shows that this process is feasible, possesses reproducibility, in addition the sodium octanoate addition of different concentrations can make a difference to the purity of product, molecular size distribution, IgA, the sodium octanoate addition is larger, its precipitation miscellaneous protein is more, product quality index is higher, but simultaneously accompanying yield will receive certain impact, therefore need comprehensive consideration selection. Adopt sodium octanoate precipitation+2 step ion exchange chromatography process can remove a large amount of miscellaneous proteins such as IgA, IgM, Alb, reduce the generation of clinical side effects and the present invention uses 20nm virus removal nanofiltration membrane, can remove smaller potential virus, product quality safety is guaranteed.

[0102] The above specific implementations are only a part of the examples and do not include all limitations on the scope of the present invention. Simple modifications or replacements made by technicians in this professional and technical field based on the present invention are deemed to be within the scope of protection of the present invention.

Claims

1. The present invention relates to a method for preparing intravenous human immunoglobulin using a sodium caprylate precipitation process, characterized in that: Using mixed human plasma as the starting material, intravenous human immunoglobulin is prepared from FI+II+III, which specifically includes the following steps: 1) Plasma pooling and thawing: During the thawing process, the plasma temperature should not exceed 4°C, and the final plasma temperature should be controlled between 0 and 4°C. The completely thawed plasma should be centrifuged to collect the cryoprecipitate, which can be used to prepare human coagulation factor VIII. The decision to prepare human prothrombin complex (PC) will be based on market demand. If PC is to be prepared, gel adsorption separation is performed on the supernatant after removing the cryoprecipitate, and the adsorbate is used to purify and produce human prothrombin complex (PC). 2) FI reaction: adjust the pH to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 8±1%, and control the reaction temperature at -5.0±1.0°C. 3) FI+II+III separation: Adjust the pH of the FI reaction solution to 7.0±0.5, add 95% ethanol until the ethanol concentration of the reaction solution is 15±5%, and control the reaction solution temperature at -5.0±1°C. 4) FI+II+III filter press: Add filter aid, stir and filter, collect the FI+II+III precipitate and store below -30℃. 5) Sodium Caprylate Precipitation Reaction: Dissolve the FI+II+III precipitate 15±5 times in water for injection at 2-8°C. Stir to dissolve. Adjust the pH of the solution to 4.0±0.5 and continue stirring. Add sodium caprylate directly to a final concentration of 15-35 mM. Adjust the pH to 5.0±0.5 with 0.5 M NaOH and allow the reaction to proceed with slow stirring. 6) Filter press: The sodium octanoate precipitation reaction solution is filter press filtered through a pretreated plate-and-frame filter plate, and the supernatant is collected. 7) Ultrafiltration and concentration: Concentrate the filtrate to 40% to 70% of the total volume, adjust the sample pH to 5.0 ± 0.5, and the conductivity to 1.4 ms / cm to 1.8 ms / cm. 8) Fractogel EMD DEAE Chromatography: Load the sample onto a Fractogel EMD DEAE column equilibrated with 10-100 mmol / L acetate. After injection, wash the gel with 10-100 mmol / L acetate and collect the flow-through and washes. Adjust the pH of the flow-through washes to 6.0 ± 0.5, filter, and use in the next chromatography step. 9) Fractogel EMD TMAE Chromatography: Load the filtered solution onto a Fractogel EMD TMAE column equilibrated with 10-100 mmol / L acetate. After injection, wash the gel with 10-100 mmol / L acetate, collecting the flow-through and washes. 10) Ultrafiltration and Dialysis: The chromatography flow-through wash solution from step 9) was concentrated by ultrafiltration using an ultrafiltration membrane bag, dialyzed against 2% to 3% glycine, and the ultrafiltration concentrate was collected. The pH of the ultrafiltration solution was adjusted to 3.8 to 4.4, and the protein concentration was adjusted to 45 to 65 g / L. 11) Nanomembrane filtration: After pre-filtration, the ultrafiltration dialysate is filtered through a 20 nm nanomembrane. 12) Low pH incubation: After sterilization, the solution is incubated at a low pH with the incubation temperature controlled at 25±5°C for at least 21 days. 13) Stock solution: After mixing with the low pH incubation liquid, ultrafiltration and concentration are performed using an ultrafiltration membrane package. The protein concentration reaches 95 g / L or above. The ultrafiltration concentrate is collected and the pH is adjusted to 4.4-5.2 to obtain the intravenous human immunoglobulin (10%) stock solution (Note: If the concentration is 5%, ultrafiltration and concentration are not required. The solution can be diluted and prepared directly after the low pH incubation and adjusted to pH 3.8-4.4 as required by the Chinese Pharmacopoeia. The protein concentration is >50 g / L). 14) Semi-finished product: Determine whether to dilute and prepare the liquid based on the protein content test results of the original liquid and adjust the pH of the liquid to 4.4-5.2, which is the semi-finished product. 15) Finished product: The semi-finished product liquid in step 14) is sterilized, plugged, and capped to obtain the finished product of intravenous human immunoglobulin (10%).

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