A histamine human immunoglobulin preparation and a method for preparing the same

Abstract

The application provides a histamine human immunoglobulin preparation and a preparation method thereof, and the histamine human immunoglobulin preparation comprises human immunoglobulin, histamine phosphate, a protective agent, a pH buffer and polysorbate 80. The application also provides a method for preparing the histamine human immunoglobulin preparation, which comprises the steps of preparing a protein, preparing a product, and ultrafiltration and dialysis and sub-packaging. The application solves the problems of the existing histamine human immunoglobulin freeze-dried preparation, such as insecurity, high cost and complicated operation. The free histamine content of the product is as low as 0.01 μg / ml or below, which ensures the safety of the liquid preparation and reduces the occurrence of adverse reactions.

Description

Technical Field

[0001] This invention relates to the field of histamine human immunoglobulin preparation technology, and more particularly to a method for preparing a liquid histamine human immunoglobulin preparation. Background Technology

[0002] In the process of allergic reactions, allergic mediators play a direct role, with histamine being an important mediator of allergic active drugs. When the body comes into contact with a specific antigen, humoral or cellular immunity occurs, and histamine is released from cells such as mast cells. It then acts on receptors on the surface of target organs, causing tissue damage and lesions such as smooth muscle contraction, capillary dilation and changes in permeability, decreased blood pressure, and shock—a rapid-type hypersensitivity reaction. Normal human bodies contain a certain amount of histamine enzymes, which have a destructive effect on the histamine released during an allergic reaction. However, in some individuals who lack histamine enzymes, allergic symptoms may still occur.

[0003] Histamine-based human immunoglobulin is formulated from healthy human plasma through low-temperature ethanol protein separation or other approved separation methods to remove anti-complement activity. Appropriate amounts of histamine phosphate or histamine hydrochloride are then added, followed by virus removal and inactivation treatment. Sodium thiosulfate and glucose are added as excipients and protective agents. This product stimulates the body to produce anti-histamine antibodies, thereby eliminating the pathogenic effects of endogenous histamine. Clinically, it is mainly used for the prevention and treatment of allergic diseases such as bronchial asthma, allergic skin diseases, and urticaria.

[0004] Considering the stability of formulations, most histamine immunoglobulins on the market are currently lyophilized and contain sodium thiosulfate as a preservative. However, lyophilized formulations require reconstitution before use. Specifically, doctors or nurses must manually reconstitute the lyophilized formulation with sterile water using a syringe, and then inject the product. This process is cumbersome and increases the risk associated with using sterile products. Furthermore, the high levels of free histamine and sodium thiosulfate in these products can cause serious adverse reactions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a histamine human immunoglobulin preparation and its preparation method, which solves the problems of unsafe and inconvenient use of existing histamine human immunoglobulin lyophilized preparations.

[0006] According to an embodiment of the present invention, a histamine human immunoglobulin preparation is provided, the preparation being a liquid preparation comprising human immunoglobulin, histamine phosphate, a protective agent, a pH buffer, and polysorbate 80.

[0007] Preferably, the histamine phosphate can be replaced with histamine hydrochloride.

[0008] Preferably, the pH buffer is a hydrochloric acid solution or a sodium hydroxide solution, and the protectant is a glucose solution.

[0009] Preferably, the concentration of the human immunoglobulin is 60-100 g / L, the concentration of histamine phosphate or histamine hydrochloride is 0.75-2.5 μg / ml, the amount of histamine phosphate or histamine hydrochloride binding protein is 0.25-0.99 ug / ml, and the concentration of free histamine phosphate or histamine hydrochloride in the liquid preparation is less than 0.01 ug / ml; the concentration of polysorbate 80 is 40-60 g / L, and the concentration of glucose solution is 40-60 g / L.

[0010] The present invention also provides another embodiment, a method for preparing a histamine human immunoglobulin preparation, comprising the following steps:

[0011] Step 1: Prepare human immunoglobulin as a raw material;

[0012] Step 2, preparation of the product; take the human immunoglobulin prepared in Step 1, add histamine phosphate or histamine hydrochloride, add a protective agent, mix and stir, and finally adjust the pH value with a pH buffer.

[0013] Step 3: Ultrafiltration dialysis and dispensing; add an equal volume of dialysis solution for at least 1 times the amount of dialysis solution, then add polysorbate 80, and then dispense.

[0014] Preferably, the concentration of human immunoglobulin added in step two is 120-150 g / L, the concentration of histamine phosphate or histamine hydrochloride is 1 g / L, and the protective agent is a glucose solution of 300 g / L.

[0015] Preferably, the stirring time in step two is at least 30 minutes.

[0016] Preferably, the dialysate in step three is a glucose solution of 40–60 g / L.

[0017] Preferably, the concentration of polysorbate 80 added in step three is 10 g / L, and its final concentration is 40-60 ug / ml.

[0018] Preferably, the pH buffer added in step two is a 0.5M hydrochloric acid solution or a 1M sodium hydroxide solution, and the adjusted pH value is 4.50 to 6.50.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This liquid formulation is stable and highly effective, and easy to use. In clinical applications, it combines the functions of both anti-allergy and human immunoglobulin, thus expanding the product's applicability.

[0021] 2. Compared with histamine human immunoglobulin lyophilized preparations, the present invention is prepared in the form of a liquid preparation, which is more convenient to use and does not require reconstitution, while reducing the risk of bacterial contamination and making storage more convenient.

[0022] 3. The liquid formulation of the present invention does not contain sodium thiosulfate as a preservative and has a low free histamine content, as low as 0.01 μg / ml, which ensures the safety of the liquid formulation and reduces the occurrence of adverse reactions. Detailed Implementation

[0023] Example 1: Preparation of liquid formulation

[0024] (1) Preparation of human immunoglobulins

[0025] The immunoglobulin stock solution was separated from healthy human plasma using a low-temperature ethanol precipitation method. Specifically, qualified plasma from healthy individuals was subjected to a stepwise separation process using low-temperature ethanol precipitation: precipitation of component I, component II+III, component III, and component II. Component II precipitation underwent pasteurization, primary precipitation, secondary precipitation, TMAE chromatography, and ultrafiltration concentration to obtain immunoglobulin IgG stock solution. Precipitation was achieved by adjusting pH, conductivity, ethanol concentration, temperature, and protein concentration. Glycine was used as a protective agent for pasteurization, and the temperature was maintained at 60℃ for 10 hours. The TMAE chromatography parameters were: pH 5.0–6.00, protein concentration 10–50 g / L, and conductivity not exceeding 2.00 mS / cm. The ultrafiltration membrane had a pore size of 10–50 kDa.

[0026] There are various methods for producing human immunoglobulins, including low-temperature ethanol precipitation, ammonium sulfate precipitation, PEG precipitation, and chromatography. However, these methods are not limited to those listed above; high-purity human immunoglobulins can be obtained using existing techniques.

[0027] (2) Product preparation

[0028] Product 1:

[0029] 1. Take the 120 g / L human immunoglobulin prepared above, add 1 g / L histamine phosphate, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 60 g / L, histamine phosphate concentration 0.75 ug / ml, glucose concentration 40 g / L.

[0030] 2. Use 40 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 5.50 with 0.5 M hydrochloric acid solution, add 10 g / L polysorbate 80 to achieve a final concentration of 40 μg / ml, stir well, and dispense the product.

[0031] Product 2:

[0032] 1. Take the 120 g / L human immunoglobulin prepared above, add 1 g / L histamine hydrochloride, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 60 g / L, histamine phosphate concentration 1.625 ug / ml, glucose concentration 40 g / L.

[0033] 2. Use 40 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 5.50 with 0.5 M hydrochloric acid solution, add 10 g / L of polysorbate 80 to achieve a final concentration of 50 μg / ml, stir well, and dispense the product.

[0034] Product 3:

[0035] 1. Take the 120 g / L human immunoglobulin prepared above, add 1 g / L histamine hydrochloride, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 60 g / L, histamine phosphate concentration 2.5 ug / ml, glucose concentration 50 g / L.

[0036] 2. Use 50 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 4.50 with 0.5 M hydrochloric acid solution, add 10 g / L of polysorbate 80 to achieve a final concentration of 60 μg / ml, stir well, and dispense the product.

[0037] Product 4:

[0038] 1. Take the 150 g / L human immunoglobulin prepared above, add 1 g / L histamine phosphate, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 100 g / L, histamine phosphate concentration 0.75 ug / ml, glucose concentration 50 g / L.

[0039] 2. Use 50 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 5.00 with 1 M sodium hydroxide solution, add 10 g / L polysorbate 80 to achieve a final concentration of 40 μg / ml, stir well, and dispense the product.

[0040] Product 5:

[0041] 1. Take the 150 g / L human immunoglobulin prepared above, add 1 g / L histamine phosphate, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 100 g / L, histamine phosphate concentration 1.625 ug / ml, glucose concentration 60 g / L.

[0042] 2. Use 40 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 6.00 with 1 M sodium hydroxide solution, add 10 g / L polysorbate 80 to achieve a final concentration of 50 μg / ml, stir well, and dispense the product.

[0043] Product 6:

[0044] 1. Take the 120 g / L human immunoglobulin prepared above, add 1 g / L histamine phosphate, then add 300 g / L glucose solution, mix and stir for at least 30 minutes, and prepare according to the following formula: human immunoglobulin final concentration 60 g / L, histamine phosphate concentration 2.5 ug / ml, glucose concentration 60 g / L.

[0045] 2. Use 40 g / L glucose solution as the dialysate, dialyzing an equal volume of 1. Then, use a 50 kDa membrane-coated ultrafiltration membrane to dialyze an equal volume of 2 times the volume of water for injection, maintaining the dialysis pressure within the range of 1–1.2 Pa. Adjust the pH to 6.50 with 1 M sodium hydroxide solution, add 10 g / L polysorbate 80 to achieve a final concentration of 60 μg / ml, stir well, and dispense the product.

[0046] Example 2: Histamine determination of products 1-6

[0047] (1) Histamine detection

[0048] Products 1-6 were tested. Two volumes of water for injection were dialyzed using a 50KD membrane-coated ultrafiltration machine. The dialysate was collected, and the free histamine content in the dialyzed products and dialysate was determined. The free histamine content and histamine-binding protein levels were determined according to the 2020 edition of the Chinese Pharmacopoeia, Volume IV, "General Chapter 3125 Determination of Free Phosphate Histamine in Human Immunoglobulins," as shown in Table 1.

[0049] Table 1. Determination of free histamine content and histamine-binding protein content in products 1-6

[0050]

[0051] Remark:

[0052] 1. The free histamine detection method measures the content of histamine in the product that is not bound to the product; the content of bound free histamine cannot be detected.

[0053] 2. The total amount of histamine added to the predialysis preparation = the total amount of histamine in the dialysate + the total amount of free histamine in the postdialysis preparation + the total amount of bound histamine in the postdialysis preparation.

[0054] As can be seen from Table 1, the free histamine content of products 1-6 is low, all less than 0.175 μg / ml. In addition, the amount of histamine-binding protein in the products of this embodiment is much greater than the free histamine content, which ensures the safety and reliability of the product while ensuring the effectiveness of the formulation.

[0055] (2) Comparative Experiment

[0056] This embodiment compares the lyophilized formulations of lyophilizers A and B, as shown in Table 2.

[0057] Table 2. Determination of free histamine content and histamine-binding protein content in lyophilized formulations.

[0058]

[0059] Note: Add 0.1625 ug / ml of histamine phosphate or histamine hydrochloride to 6 g / L of protein.

[0060] The comparison revealed that the ratio of free histamine content to histamine-bound protein in the lyophilized formulations in Table 2 was approximately 1:1. The amount of histamine-bound protein reflects the effectiveness of the formulation, while the free histamine content must be less than 0.175 μg / ml.

[0061] In addition, the amount of histamine-binding protein in the above liquid products 1-6 is much greater than that in the two lyophilized preparations, while the content of free histamine is much less than that in the two lyophilized preparations, which means that this liquid preparation is safer, more reliable and more effective.

[0062] Example 3: Animal efficacy experiment

[0063] Products 1-6 prepared in Example 1 were all used in animal efficacy experiments, which mainly included animal immunization tests and shock tests.

[0064] 1. Animal immunization experiments

[0065] Double amplification of the immune system:

[0066] Commercially available lyophilized formulation group: The commercially available lyophilized histamine human immunoglobulin product was reconstituted with water for injection, mixed with 1 ml of Freund's complete adjuvant, and emulsified until it became a white viscous substance. Six New Zealand rabbits were randomly selected and injected subcutaneously with 1 ml per rabbit once a week for a total of 5 weeks.

[0067] Liquid formulation group: Take 1 ml of each of the products 1-6 prepared in Example 1, mix and emulsify with 1 ml of Freund's complete adjuvant until it becomes white and viscous, and randomly select 6 New Zealand rabbits corresponding to different products. 1 ml / rabbit is injected subcutaneously once a week for a total of 5 times.

[0068] One week after immunization, blood was collected from each group of New Zealand rabbits to separate serum for a double amplification immunoassay to determine whether the immunization produced the corresponding antibodies. Specific steps: Weigh 1.5g of agarose, add 100ml of purified water, and heat to boiling until completely dissolved to obtain a 1.5% agarose solution. Immediately transfer 6ml of the solution onto a glass plate using a pipette, and after cooling, punch wells. Take 0.1ml of immunoglobulin and add 0.9ml of 0.9% sodium chloride solution to dilute the sample to a 0.5% protein concentration. Take the prepared agar plate, add 20μl of diluted immunoglobulin to each well, and add 20μl of rabbit serum to each well in turn. Place the prepared agar plate horizontally in a sealed humidified chamber and incubate at 37℃ for 24 hours, observing the precipitation line.

[0069] The presence of a clear precipitation line indicates that the corresponding antibodies have been produced by the immune system. This confirms the effectiveness of products 1-6.

[0070] Final results: Clear precipitation lines were observed in both commercially available lyophilized histamine immunoglobulin products and preparations 1-6, indicating that New Zealand rabbits developed corresponding antibodies in response to both products. Both preparations 1-6 and the commercially available lyophilized formulations were effective.

[0071] 2. Shock experiment

[0072] In this embodiment, New Zealand rabbits were selected for the shock experiment. The specific steps are as follows:

[0073] (1) Immunization preparation:

[0074] New Zealand rabbits were divided into three groups: a commercially available freeze-dried formulation group, a liquid formulation group, and a histamine phosphate group. Six New Zealand rabbits were randomly selected from each group for immunization.

[0075] Commercially available lyophilized formulation: The commercially available lyophilized histamine human immunoglobulin product was reconstituted with water for injection, mixed with 2.5 ml of Freund's complete adjuvant, and emulsified until a white, viscous consistency was achieved. 2.5 ml per rabbit was subcutaneously injected into New Zealand rabbits, once every two weeks, for a total of three times.

[0076] Liquid formulation group: Take 2.5 ml of each product and mix it with 2.5 ml of Freund's complete adjuvant until it becomes a white, viscous substance. Administer 2.5 ml subcutaneously to New Zealand rabbits. Provided that products 1-6 are effective, conduct a shock experiment. Randomly select 6 New Zealand rabbits to receive injections of different products, once every two weeks, for a total of three times.

[0077] Histamine phosphate group: Weigh 1.07g of histamine phosphate and dissolve it in 20ml of water for injection, mix well to obtain the histamine phosphate stock solution; transfer 9.5ml of the stock solution to 500ml of 0.9% sodium chloride solution, mix well to obtain a 1.0g / L histamine phosphate sodium chloride solution. Transfer 2.5ml of the 1.0g / L histamine phosphate sodium chloride solution and mix it with 2.5ml of Freund's complete adjuvant, emulsify until it becomes a white viscous consistency. Administer 2.5ml subcutaneously per animal, once every two weeks, for a total of three times. The experiment was conducted 7-8 weeks post-immunization.

[0078] (2) Observation after injection:

[0079] After the three immunization groups were completed, shock experiments were performed on New Zealand rabbits in the commercially available lyophilized formulation group, the liquid formulation group, and the histamine phosphate group, respectively. 1.07 g of histamine phosphate was weighed and dissolved in 20 ml of water for injection, and mixed thoroughly to obtain the histamine phosphate stock solution. 9.5 ml of the stock solution was transferred to 500 ml of 0.9% sodium chloride solution and mixed thoroughly to obtain a 1.0 g / L histamine phosphate sodium chloride solution. Each New Zealand rabbit was intravenously injected with the 1.0 g / L histamine phosphate sodium chloride solution at a concentration of 480 μg / kg body weight.

[0080] Observe and record the shock symptoms of New Zealand rabbits in each group after injection, observe the occurrence of anaphylactic shock reactions, and record the symptoms, severity, and activity status throughout the process. Six New Zealand rabbits were randomly selected from each group. Shock symptoms were mainly assessed in terms of respiration, limb weakness, lying down, tremors, urinary and fecal incontinence, shock, death, and recovery. As shown in Table 3.

[0081] Table 3 Levels of Shock Symptoms

[0082]

[0083]

[0084] Symptom grades "Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ, Ⅵ" are assigned 1, 2, 3, 4, 5, and 6 points respectively; the shock coefficient = (score of each symptom grade * sum of the number of each symptom) / number. The larger the shock coefficient, the worse the immune effect of the immunization injection product.

[0085] The criteria for judging the symptoms of shock in New Zealand rabbits are shown in Table 4.

[0086] Table 4. Criteria for Judging Shock Symptoms in New Zealand Rabbits

[0087] Ⅰ No response Ⅱ Breathing quickens, gradually recovering Ⅲ Rapid breathing, weakness in limbs, gradually recovering Ⅳ Rapid breathing, weakness in the limbs, lying down, and then gradually recovering. Ⅴ Rapid breathing, weakness in the limbs, lying down, trembling, incontinence, then gradually recovering. Ⅵ death from shock

[0088] As shown in the table above, the shock coefficient of the liquid histamine globulin products (products 1-6) produced by this patent is 3.33, while the shock coefficient of commercially available histamine globulin products is 3.67. The control group, rabbits directly injected with histamine, achieved a shock coefficient of 5.5. This demonstrates that the liquid formulation produced by this patent can prevent or reduce allergic reactions in immunized animals, and its immunization effect is better than that of commercially available products.

[0089] Example 4: Stability Test

[0090] Products 1-6 were stored at room temperature, with 40 samples in each group, for a total of 240 samples. The samples were stored for 0 months, 1 month, 2 months, 3 months and 6 months respectively, and the following stability tests were performed.

[0091] The results relied primarily on thermal stability experiments, abnormal toxicity tests, and kallikrein activator (PKA), as shown in Table 5.

[0092] Table 5. Results of Stability Indicators 1

[0093] Month 0 qualified qualified Not detected Compliant Month 1 qualified qualified Not detected Compliant 2nd month qualified qualified Not detected Compliant 3rd month qualified qualified Not detected Compliant 6th month qualified qualified Not detected Compliant

[0094] (1) Thermal stability test: Products 1-6 were placed in a water bath at 57.0±0.5℃ for 4 hours and then visually inspected for gelation or flocculent matter using a visible foreign matter inspection device. Ten samples of each of products 1-6 were taken at different time periods for testing, and all samples passed the test.

[0095] (2) Abnormal toxicity tests were conducted in accordance with the General Chapter 1141 of Part IV of the 2020 Chinese Pharmacopoeia to evaluate the safety of the product. Ten samples of each of the products 1-6 were taken at different time periods for abnormal toxicity tests, and all of them passed the tests.

[0096] (3) PKA was tested according to the 2020 edition of the Chinese Pharmacopoeia, Volume IV, General Chapter 3409. The kallikrein activator assay is used to determine the content of kallikrein activator in drugs. Ten samples each from products 1-6 at different time periods were taken for the kallikrein activator (PKA) assay. No kallikrein activator was detected in any of the products from any time period, indicating extremely low levels.

[0097] (4) All products were subjected to sterility testing and passed the test at each time period.

[0098] In addition, stability testing also relies on pH value, molecular size distribution (%), free histamine content, shock test, and guinea pig protection test.

[0099] A total of 240 products, from product 1 to product 6, were selected from the same batch. The following experiments were conducted on the products after the initial storage period of 0 months, 1 month, 2 months, 3 months, and 6 months, as shown in Table 6.

[0100] Table 6. Results of Stability Indicators 2

[0101]

[0102] (1) pH values ​​of all products were tested, with pH values ​​determined according to General Chapter 0631 of the 2020 edition of the Chinese Pharmacopoeia, Volume IV. This experiment used the acidity meter method, which measures the pH value of the solution using the potentiometric method, and has high accuracy and sensitivity. The acceptable standard for this experiment was 4.5 to 5.5, and all products from all time periods fell within this range.

[0103] (2) Molecular size distribution was determined for all products, referring to Chapter 3122 of the 2020 edition of the Chinese Pharmacopoeia, Volume IV, using gel permeation chromatography. The principle is to pass substances through a gel chromatography column at a certain flow rate, and then determine their percentages based on molecular size by passing them sequentially through a detector. The standard was that the sum of IgG monomer and dimer content should not be less than 95.0%, and all products from all time periods met this standard.

[0104] (3) The free histamine content of all products was tested according to the General Chapter 3125 of the 2020 edition of the Chinese Pharmacopoeia, Volume IV. The method used was fluorescence method, which involved measuring the fluorescence intensity in the sample using a fluorophotometer to determine the free histamine content. The acceptable standard for this experiment was no higher than 0.175 ug / ml. The free histamine content in this product was low, and even after a period of time, the free histamine content remained low.

[0105] (4) Guinea pig protection experiment and shock experiment

[0106] One sample of each of products 1 to 6 was randomly selected for shock and immune tests. The steps of the shock test in Example 3 were repeated, and the shock coefficients for the first month, second month, third month, fourth month, and fifth month were 3.50, 3.50, 3.67, 3.50, and 3.50, respectively.

[0107] Healthy guinea pigs were randomly divided into several groups. Rabbit serum, prepared before the shock experiment, was used, with three guinea pigs used for each rabbit's serum. A lethal dose of histamine was mixed with an equal volume of rabbit serum and incubated at 37°C for 1 hour. The mixture was then intravenously injected into the guinea pigs. Experimental symptoms were observed and recorded. Symptom judgment criteria:

[0108] After injection, observe for any signs of anaphylactic shock and record the symptoms, severity, time required for recovery, and overall activity level. Shock symptoms should primarily be assessed for excitement, coughing, difficulty breathing, lying down, tremors, incontinence, death from shock, and recovery to normal.

[0109] The criteria for judging the symptoms of shock in guinea pigs are shown in Table 7.

[0110] Table 7. Criteria for Judging Symptoms of Shock in Guinea Pigs

[0111] Ⅰ No response Ⅱ Excitement, coughing, gradually recovered Ⅲ Excitement, coughing, and difficulty breathing gradually subsided. Ⅳ Excitement, coughing, difficulty breathing, lying down, gradually recovering Ⅴ Excitement, coughing, difficulty breathing, lying down, tremorous movements, urinary and fecal incontinence, gradually recovering Ⅵ Death from shock

[0112] The final shock coefficients were 3.28, 3.50, 3.61, 3.61, and 3.61, respectively.

[0113] Final conclusion: After testing the above-mentioned multiple indicators, there was no significant difference between the product at the beginning and before and after a period of time, indicating that the product has high stability.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a histamine human immunoglobulin preparation, characterized in that: Includes the following steps: Step 1: Prepare human immunoglobulin as a raw material; Step 2, preparation of the product; take the human immunoglobulin prepared in Step 1, add histamine phosphate or histamine hydrochloride, then add 300g / L glucose solution, mix and stir well; Step 3: Ultrafiltration dialysis and dispensing; add an equal volume of dialysis solution and dialyze at least 1 times, then adjust the pH value to 4.50-6.50 with a pH buffer, add polysorbate 80, and then dispense; the dialysis solution added in step 3 is a glucose solution of 40-60 g / L.

2. The method as described in claim 1, characterized in that: In step two, the concentration of human immunoglobulin added is 120–150 g / L, and the concentration of histamine phosphate or histamine hydrochloride is 1 g / L.

3. The method as described in claim 1, characterized in that: The stirring time in step two should be at least 30 minutes.

4. The method as described in claim 1, characterized in that: The concentration of polysorbate 80 added in step three is 10 g / L, and its final concentration is 40-60 ug / ml.

5. The method as described in claim 1, characterized in that: The pH buffer added in step three is a 0.5M hydrochloric acid solution or a 1M sodium hydroxide solution.

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