CN117756935A - Tetanus human immunoglobulin and preparation method thereof - Google Patents
Tetanus human immunoglobulin and preparation method thereof Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses tetanus human immunoglobulin and a preparation method thereof, comprising the following steps: s1, collecting plasma of a tetanus vaccine immune donor containing high-titer tetanus antibody; s2, separating plasma by a low-temperature ethanol protein separation method to obtain immunoglobulin; s3, removing impurities from the obtained immunoglobulin by an ion exchange chromatography to prepare high-purity immunoglobulin; s4, performing low-pH incubation and nano-membrane filtration on the high-purity immunoglobulin to remove or inactivate viruses, and preparing a specific immunoglobulin liquid preparation; the low pH incubation and 20nm membrane filtration can effectively inactivate and remove both lipid-enveloped viruses and non-lipid-enveloped viruses. Compared with the heating inactivation process, the virus inactivation process has milder inactivation conditions, not only effectively inactivates the virus, but also has smaller influence on the activity of the tetanus antibody. The 20nm membrane filtration can effectively remove human parvovirus B19 virus, and ensure the medication safety of people.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to tetanus human immunoglobulin and a preparation method thereof.
Background
The tetanus human immunoglobulin is prepared from high tetanus antibody titer plasma (more than or equal to 10 IU/ml) obtained by screening tetanus antibody titer of a plasma donor immunized by tetanus vaccine.
Tetanus (tetanus) is a specific infection in which clostridium tetani invades the human body through skin or mucosal wounds, grows and breeds in an anoxic environment, produces toxins, and causes muscle spasms. The main pathogenic substance is exotoxin, namely tetanus spasmodic toxin, which is a neurotoxin with extremely strong toxicity. The toxin has special affinity to the central nervous system, so that the toxin mainly attacks motor neurons in the nervous system, can prevent inhibitory synapse ends from releasing inhibitory neurotransmitters (glycine and gamma aminobutyric acid), and makes the excitation and inhibition of muscle activities disregulated, so that extensor and flexor simultaneously shrink strongly, and muscle spasms are caused, therefore, the disease is characterized in clinic by the tight jaw of teeth, paroxysmal spasms and spasms, the main affected muscle groups comprise the masseter muscle, dorsi-acanthus muscle, abdominal muscle, limb muscle and the like, and serious people can die due to asphyxia or respiratory failure. Tetanus latency is typically 7 to 8 days, as short as 24 hours or as long as several months. After injury, once the wound is infected with clostridium tetani, mortality rates are up to 40% +|! Currently, tetanus Antitoxin (TAT) and tetanus human immunoglobulin are used for treating the diseases, and Tetanus Antitoxin (TAT) is extracted from horse serum and is easy to cause human anaphylactic reaction, especially anaphylactic shock, seropathy and other adverse reactions, so that the use is gradually reduced. So, the passive immunization is mainly carried out by injecting tetanus human immunoglobulin at present, so the application of the medical market is wider.
There are a number of methods for extracting human immunoglobulins from plasma from the common cold, most of which involve at least two precipitation steps, one chromatography step and two viral removal or inactivation steps.
CN102178952a provides a process for chromatographic extraction of tetanus human immunoglobulin. The tetanus human immunoglobulin is only harvested in 420-500 ten thousand units per ton of blood plasma after coarse separation, one-step chromatographic purification and virus inactivation.
CN1660910 describes a stepwise precipitation method, in which plasma parameters are regulated in two steps and isolated by standing to obtain tetanus human immunoglobulin. The concentration of tetanus human immunoglobulin is below 18%, and the tetanus antibody titer is 110IU/ml.
CN114736294a provides a two-step method for virus inactivation of tetanus human immunoglobulin. The protein ultrafiltrate is diluted, and then is subjected to pasteurization and low pH incubation to inactivate and remove viruses. The natural structure of human immunoglobulin is easily changed by inactivating virus through Pasteur inactivation, so that polymer is produced, and adverse reaction is easily brought in the use process.
CN102603891a provides a process for preparing tetanus human immunoglobulin by double virus inactivation, and after the product is separated and purified by a low-temperature ethanol protein separation method, the virus is removed by low-pH incubation inactivated virus and DV50 nano-film. In general, a two-step process is adopted for removing or inactivating viruses in the industry, a low pH incubation method is not suitable for inactivating non-lipid-coated viruses, a 50nm membrane filtration method is generally adopted for nanofiltration membrane filtration, and the effect of removing human parvovirus B19 viruses is poor.
CN102584993a provides a preparation process of anti-tetanus immune plasma, the immune program adopts 1-1 scheme and 0, 42 inoculation program, respectively, on day 1 (0), 43 days 1/time of injection, respectively, but the above-mentioned method has the defects of low immunogenicity, high efficiency and low cost of plasma, only collecting single person plasma with tetanus antibody titer above 12IU, low pulp yield and low comprehensive utilization rate of plasma, so that it is necessary to raise the effectiveness of immune program and raise the pulp yield of tetanus antibody plasma.
Disclosure of Invention
The invention aims to provide tetanus human immunoglobulin and a preparation method thereof, which are used for solving the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions: s1, collecting plasma of a tetanus vaccine immune donor containing high-titer tetanus antibody;
s2, separating plasma by a low-temperature ethanol protein separation method to obtain immunoglobulin;
s3, removing impurities from the obtained immunoglobulin by an ion exchange chromatography to prepare high-purity immunoglobulin;
s4, performing low-pH incubation and nano-membrane filtration on the high-purity immunoglobulin to remove or inactivate viruses, and preparing a specific immunoglobulin liquid preparation.
Preferably, the physical condition of the tetanus vaccine immune donor is healthy, and the immunization program adopts a 2-1-1 scheme and 0, 28 and 84 inoculation programs, namely, on day 1 (0) (1 injection for each of the left and right arms and 2 injections for each time), and on day 29, namely, 1 injection for each of 28 days and 85 days and 84 days; boosting based on the plasma titer test results, it was determined whether boosting was needed or not, with 1 injection at 56 day intervals. The immunization method gives plasma fighters immunity to self-produce tetanus antibody through tetanus toxin, and collects plasma with the antibody titer of more than 5 IU/ml.
Preferably, the tetanus antibody titer after mixing the plasma collected in S1 should not be less than 10IU/ml.
Preferably, the thickness of the nano film in the S3 is 20nm.
Preferably, the specific method of S2 is as follows:
s21, melting the plasma by using circulating water at 30-35 ℃, centrifuging and separating the melted plasma to obtain cryoprecipitate, controlling the temperature of the liquid outlet at 0-4 ℃, and using the cryoprecipitate for producing human blood coagulation factor VIII;
s22, heating the plasma to 10-20 ℃ for gel adsorption after centrifuging the S21 preparation,
adsorbates are used for human prothrombin complex production;
s23, diluting the plasma protein content of the S22 preparation by using 0.85% sodium chloride solution to be within 4.5-5.5%, regulating the plasma pH value to be 6.80-7.00 by using a pH4.0 acetic acid-sodium acetate buffer solution, adding ethanol to the final concentration of the ethanol to be 8%, stirring, centrifuging to obtain a component I precipitate and a component I supernatant, wherein the component I precipitate is directly used for the production or freezing storage of human fibrinogen;
s24, adding acetic acid-sodium acetate buffer solution into supernatant of the component I of the product obtained in the S23 to adjust the pH value to 6.80-6.85, adding ethanol until the concentration of the ethanol is 20%, and adding ethanol until the pH value is 6.80-7.00; stirring, standing, starting stirring, adding diatomite, carrying out filter pressing, controlling the temperature of supernatant liquid in the filter pressing process to be between-4 and-6 ℃, and carrying out filter pressing to obtain component II+III sediment and component II+III supernatant liquid, wherein the component II+III supernatant liquid is used for producing human serum albumin;
s25, adding 10-15 times of injection water into the sediment of the component II+III prepared in the S24, and adding sodium chloride to adjust the concentration of sodium ions to 0.01mol/L; stirring is started, and stirring and dissolution are carried out when the temperature is reduced to 2-4 ℃. After the precipitate is completely dissolved, regulating the pH to 5.10-5.30 by using an acetic acid-sodium acetate buffer solution with the pH of 4.0, cooling to 0-minus 1 ℃, continuously stirring for at least 10 minutes, adding 95% ethanol to ensure that the final ethanol concentration of the reaction solution is 17%, and controlling the final temperature to minus 5-minus 7 ℃ and the pH to 5.20-5.30; continuously stirring for 2 hours, standing for more than 4 hours, and performing filter pressing to obtain a component III precipitate and a component III supernatant;
s26, performing deep filtration on supernatant of the component III obtained in the step S25, controlling the temperature to be-5 to-7 ℃, adjusting the pH of the deep filtrate to be 3.90-4.10 by using 1mol/L HCl after filtration, performing ultrafiltration concentration to a protein concentration of more than 5%, and performing dialysis dealcoholization by using water for injection; the concentration of the protein is regulated to 3 to 6 percent, the pH value is 6.40 to 6.60, and the conductivity is 1750 to 2050us/cm; purifying the protein solution with adjusted conductivity and pH value by column chromatography, wherein the chromatography uses weak anion exchange gel filler, and collecting the flow-through solution;
s27, adding 1.0mol/L hydrochloric acid solution into the logistics threading liquid prepared in the S26 to adjust the pH value to 3.8-4.0, ultrafiltering and concentrating to more than 5% of protein concentration, dialyzing with water for injection, concentrating to obtain primary ultrafiltrate, and adding 10+/-1% of maltose to dilute to obtain primary diluted liquid;
s28, sterilizing and filtering the dilute protein solution prepared in the step S27 by using a 0.2 mu m sterilizing filter element; placing the sterilized and filtered product in a low pH incubation chamber, and incubating at 24+ -1deg.C for 21 days; after the low pH incubation is finished, performing virus removal filtration by a 20nm nano membrane;
s29, concentrating, dialyzing and diluting the protein solution obtained by filtering the preparation of S28 by using 0.85% sodium chloride solution, and adding 28g glycine per liter according to the final volume to adjust the tetanus antibody titer to be more than or equal to 100IU/ml.
Preferably, the percentage in S24 is the mass percentage except the limit.
Preferably, the acetic acid-sodium acetate buffer in S23, S24 and S25 comprises 108.9g/L sodium acetate and 255.6g/L glacial acetic acid, and the acetic acid-sodium acetate buffer in S26 comprises 40g/L NaOH and 85.9g/L phosphoric acid.
It should be noted that, in the present invention, the component I supernatant and the precipitate thereof, and the component ii+iii supernatant and the precipitate thereof are all general classification methods in the technical field; in the present invention, the names of the classified substances according to the general classification method are not simply numbers.
Separating human plasma by centrifugation and cold precipitation, regulating pH and temperature of separated centrifugal supernatant, adding ethanol, and centrifuging to obtain component I supernatant and precipitate thereof;
adjusting the pH and temperature of the component I supernatant, adding ethanol, and then performing pressure filtration to obtain a component II+III supernatant and a precipitate thereof;
the component I precipitate mainly contains fibrinogen, FVIII, ciq, clr, cls and fibronectin;
the component I supernatant mainly contains: albumin, igG, igA, igM, F II, VII, IX, X, alpha, beta globulin, ceruloplasmin, alpha 1-antitrypsin, igM, AT-III complement component, transferrin, haptoglobin;
the component II+III precipitate mainly contains IgG, igA, igM, F II, VII, IX, X, alpha, beta globulin and ceruloplasmin;
the supernatant of component II+III mainly contains: albumin, alpha, beta globulin, ceruloplasmin, alpha 1-antitrypsin, igM, AT-III complement components, transferrin, haptoglobin.
Compared with the prior art, the invention has the beneficial effects that: the invention combines the optimized process, adopts the low-temperature ethanol filter-pressing extraction separation technology and the ion exchange chromatography (chromatographic) technology to combine the extraction separation to obtain the tetanus human immunoglobulin, and performs ultrafiltration and concentration. And meanwhile, the two-step virus inactivation technology is adopted to remove viruses, so that the product yield can be effectively improved, and the purity and the safety of the product can be improved. The method comprises the following steps:
according to the invention, the Capto DEAE ion exchange chromatography is utilized to purify tetanus human immunoglobulin, and the optimal technological parameters are determined through the research on the PH, the flow rate and the consumption of balance liquid in the chromatography process, the PH, the ionic strength and the protein content parameters of the sample liquid, and the liquid medicine can remove foreign proteins to the greatest extent, improve the purity and maintain the activity of the immunoglobulin through the sample loading, washing and elution processes, wherein the biological activity of an FC section reaches 120%, the molecular size distribution of the chromatography liquid reaches 100%, and the purity is more than 99%. The product is purified by ion exchange chromatography, so that the contents of immunoglobulin A (IgA), PKA and ACA are reduced, and the risk of anaphylactic reaction is reduced; reduces the content of immunoglobulin M (IgM), improves nanofiltration flux and enhances the effect of virus inactivation.
2, the invention adopts advanced two-step virus inactivation/removal steps, low PH incubation and 20nm membrane filtration, and can effectively inactivate and remove both lipid-enveloped viruses and non-lipid-enveloped viruses. Compared with the heating inactivation process, the virus inactivation process has milder inactivation conditions, not only effectively inactivates the virus, but also has smaller influence on the activity of the tetanus antibody. The 20nm membrane filtration can effectively remove human parvovirus B19 virus, and ensure the medication safety of people.
And 3, separating by adopting a two-step precipitation method to obtain a component III supernatant, preparing, press-filtering and separating the component I supernatant to obtain a component II+III precipitate, and directly performing press-filtering on the component II+III precipitate in the second step without refining to obtain the component III precipitate and the component III supernatant. The precipitation refining of components II and III is reduced, the loss of protein in the low-temperature ethanol process can be reduced, and the recovery rate of the product is improved.
4, the invention adopts the 2-1-1 scheme and the immunization programs of 0, 28 and 84, can obtain the tetanus antibody plasma with high titer, and can reach the standard production feed mixing titer of more than 10IU of Chinese pharmacopoeia by only collecting the plasma with tetanus antibody titer of more than 5 IU/ml. The industry usually collects the plasma with the antibody titer of more than 8IU/ml, and can reach the production and feeding mixed plasma titer of more than 10 IU. The method improves blood plasma utilization rate, and further improves product yield.
5, the purity of the tetanus human immunoglobulin prepared by the invention can reach more than 99 percent (the requirements of Chinese pharmacopoeia are more than 90 percent and European pharmacopoeia are more than or equal to 95 percent); the molecular size distribution can reach more than 97 percent (the requirement of the Chinese pharmacopoeia is more than 90 percent); the protein concentration is lower and the protein content is controlled between 95 and 120g/L under the same tetanus antibody titer; can produce a unit product with higher antibody titer, and the tetanus antibody titer reaches 140IU/ml; finally, the impurity content of the residual amount of IgA, igG, fibrinogen, plasminogen, ceruloplasmin, C3, albumin and aluminum in the finished product is very tiny, and the safety of the clinical use of the product can be further ensured.
6, the invention obtains various technological parameters of optimal column chromatography through experiments, and the process can produce 100 IU/bottle of tetanus human immunoglobulin 20000 bottles/ton of plasma, and the yield of tetanus human immunoglobulin per ton of plasma is about 600 ten thousand units to 700 ten thousand units. Compared with 15000 bottles/ton of plasma of immunoglobulin produced by the traditional process, the invention can produce 5000 bottles/ton of plasma, and the yield can be improved by 25-33% compared with the traditional process.
Drawings
FIG. 1 is a flow chart of the preparation of human immunoglobulin for tetanus according to the invention.
FIG. 2 is a scan of the molecular size distribution of the product prepared in example 1 of the present invention.
FIG. 3 is a scan of the purity results of the product prepared in example 1 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-3, the present invention provides a technical solution:
example 1: the preparation process comprises the following steps:
laminar flow in class D environment after 1.2 tons of qualified human tetanus antibody plasma were collected during quarantine period
And (5) carrying out bag breaking under protection. Mixing the mixed materials into a slurry melting tank, carrying out interlayer circulating slurry melting by using circulating water at 30-35 ℃, controlling the temperature of plasma between 0-4 ℃, centrifuging and cryoprecipitating the melted plasma, controlling the temperature of liquid outlet between 0-4 ℃, and using the cryoprecipitate for producing human blood coagulation factor VIII;
centrifuging the product obtained in the step (1), heating the plasma to 10-20 ℃ for gel adsorption,
adsorbates are used for human prothrombin complex production;
(3) Diluting the plasma protein content of the product obtained in the step (2) to be within 4.5-5.5% by using 0.85% sodium chloride solution with the temperature of 0-4 ℃, regulating the pH value of the plasma to be 6.80-7.00 by using acetic acid-sodium acetate buffer solution with the pH value of 4.0, adding ethanol with the temperature of-15 ℃ or colder until the final concentration of the ethanol is 8%, stirring for 30-90 minutes, centrifuging to obtain component I precipitate and component I supernatant, wherein the component I precipitate is directly used for the production or freezing storage of human fibrinogen;
(4) Adding acetic acid-sodium acetate buffer with pH of 4.0 into the supernatant liquid of the component I obtained in the step (3) to adjust the pH value to 6.80-6.85, adding 95% ethanol with the temperature of minus 15 ℃ or colder until the ethanol concentration is 20%, and adding the ethanol until the pH value is 6.80-7.00; after stirring for 2 hours, standing for 1-17 hours, starting stirring, adding 1.8% diatomite, performing filter pressing, controlling the temperature of supernatant in the filter pressing process to be-4 to-6 ℃, and performing filter pressing to obtain component II+III precipitate and component II+III supernatant, wherein the component II+III supernatant is used for producing human serum albumin;
(5) Adding 13 times of injection water into 109 kg of component II+III sediment prepared in the step (4), and adding sodium chloride to adjust the sodium ion concentration to 0.01mol/L; stirring is started, and stirring and dissolution are carried out when the temperature is reduced to 2-4 ℃. After the precipitate is completely dissolved, regulating the pH to 5.10-5.30 by using a pH4.0 acetic acid-sodium acetate buffer solution, cooling to 0-1 ℃, continuously stirring for at least 10 minutes, adding 95% ethanol at-15 ℃ or colder to ensure that the final ethanol concentration of the reaction solution is 17%, and controlling the final temperature to be-5-7 ℃ and the pH to be 5.20-5.30; continuously stirring for 2 hours after adding ethanol, standing for more than 4 hours, performing compression filtration, starting a reaction tank stirrer, uniformly mixing diatomite with the reaction liquid for more than 10 minutes, stirring and filtering, keeping the liquid outlet temperature at-5 to-7 ℃ in the filtering process, keeping the filtering pressure at not more than 0.2Mpa, and performing filter pressing to obtain a component III precipitate and a component III supernatant;
(6) Carrying out deep filtration on the supernatant of the component III obtained in the step (5), controlling the temperature to be-5 to-7 ℃, regulating the pH value of the deep filtrate to be 3.90-4.10 by using 1mol/L HCl after filtration, obtaining 1987 kg protein liquid after regulating the pH value, carrying out ultrafiltration concentration to more than 5% of protein concentration, obtaining 100L of concentrated volume, and carrying out dialysis dealcoholization by using 5 times of 2-8 ℃ of water for injection; adjusting the protein concentration of the dialyzate to 3-6% by using water for injection, adjusting the pH to 6.40-6.60 by using 0.5mol/L NaOH, and adjusting the conductivity of the dialyzate to 1750-2050 us/cm by using 1mol/L phosphoric acid-NaOH buffer solution; purifying the protein solution with adjusted conductivity and pH value by column chromatography using Capto DEAE (weak anion exchange gel) filler, and collecting flow-through solution;
(7) Adding 1.0mol/L hydrochloric acid solution into the logistics threading liquid prepared in the step (6) to adjust the pH value to 3.8-4.0, ultrafiltering and concentrating to more than 5% of protein concentration, dialyzing with 5 times of 2-8 ℃ water for injection to ensure that the ethanol content is less than or equal to 0.025%, concentrating the protein liquid to more than 6%, adding calculated amount of maltose into the protein liquid, adjusting the pH value with 1.0mol/L HCl, and finally adjusting the pH value to 10.2% of maltose content, the pH value to 4.10, the protein content to 5.9% of tetanus antibody titer to 65IU and the solution weight to 83.0 kg;
(8) Performing degerming filtration on 83.0 kg of the dilute protein solution prepared in the step (7) within 6 h; placing the sterilized and filtered product in a low pH incubation chamber, and incubating at 24+ -1deg.C for 21 days; after the low pH incubation is finished, performing virus removal filtration by a 20nm nano membrane;
(9) Concentrating and dialyzing the protein solution obtained in the step (8) by using a solution of 0.85% sodium chloride with the volume of 5 times of 2-8 ℃, concentrating the protein to 16.7% after washing, diluting, adding 27g glycine per liter according to the final volume, and adjusting the tetanus antibody titer to be more than or equal to 100IU/ml, wherein the glycine content is 26-30 g/L, pH and the value is 6.6-7.2;
(10) Filtering and subpackaging the diluted protein liquid prepared in the step (9) through a 0.2 mu m degerming filter element; inspecting the products after sub-packaging, inspecting by a lamp, packaging and warehousing after the inspection is qualified;
the invention is compared with the key quality index of tetanus human immunoglobulin recorded in Chinese pharmacopoeia (2010 edition, three parts) in the following table.
Key quality index comparison table for tetanus human immunoglobulin
The key quality index results of the tetanus human immunoglobulin of the invention are compared with those of other manufacturers, and the table is shown below.
The results of the assays for the products prepared in this example are shown in tables 1-3 below.
As can be seen from the data in the table, the purity of the tetanus human immunoglobulin prepared in the examples was 95.0% or more (99.6%), and the total content of IgG monomers and dimers was 92.0% or more (97.4%).
The scan of the molecular size distribution results of the prepared product is shown in figure 2.
A scan of the purity results of the prepared product is shown in FIG. 3.
Table 1: determination of molecular size distribution
Table 2: protein content determination
Table 3: purity determination
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for preparing tetanus human immunoglobulin, which is characterized by comprising the following steps: the method comprises the following steps:
s1, collecting plasma of a tetanus vaccine immune donor containing high-titer tetanus antibody;
s2, separating plasma by a low-temperature ethanol protein separation method to obtain immunoglobulin;
s3, removing impurities from the obtained immunoglobulin by an ion exchange chromatography to prepare high-purity immunoglobulin;
s4, performing low-pH incubation and nano-membrane filtration on the high-purity immunoglobulin to remove or inactivate viruses.
2. A process for the preparation of human immunoglobulin for tetanus according to claim 1, characterized in that: the plasma is obtained by immunizing a plasma donor with a tetanus vaccine, and collecting healthy human plasma containing high-titer tetanus antibodies.
3. A process for the preparation of human immunoglobulin for tetanus according to claim 1, characterized in that: the tetanus antibody titer after mixing the plasma collected in S1 should not be lower than 10IU/ml.
4. A process for the preparation of human immunoglobulin for tetanus according to claim 1, characterized in that: the thickness of the nano film in the S3 is 20nm.
5. A process for the preparation of human immunoglobulin for tetanus according to claim 1, characterized in that: the specific method of the S2 is as follows:
s21, melting the plasma by using circulating water at 30-35 ℃, centrifuging and separating the melted plasma to obtain cryoprecipitate, controlling the temperature of the liquid outlet at 0-4 ℃, and using the cryoprecipitate for producing human blood coagulation factor VIII;
s22, heating the plasma to 10-20 ℃ for gel adsorption after centrifuging the S21 preparation,
adsorbates are used for human prothrombin complex production;
s23, diluting the plasma protein content of the S22 preparation by using 0.85% sodium chloride solution to be within 4.5-5.5%, regulating the plasma pH value to be 6.80-7.00 by using a pH4.0 acetic acid-sodium acetate buffer solution, adding ethanol to the final concentration of the ethanol to be 8%, stirring, centrifuging to obtain a component I precipitate and a component I supernatant, wherein the component I precipitate is directly used for the production or freezing storage of human fibrinogen;
s24, adding acetic acid-sodium acetate buffer solution into supernatant of the component I of the product obtained in the S23 to adjust the pH value to 6.80-6.85, adding ethanol until the concentration of the ethanol is 20%, and adding ethanol until the pH value is 6.80-7.00; stirring, standing, starting stirring, adding diatomite, carrying out filter pressing, controlling the temperature of supernatant liquid in the filter pressing process to be between-4 and-6 ℃, and carrying out filter pressing to obtain component II+III sediment and component II+III supernatant liquid, wherein the component II+III supernatant liquid is used for producing human serum albumin;
s25, adding 10-15 times of injection water into the sediment of the component II+III prepared in the S24, and adding sodium chloride to adjust the concentration of sodium ions to 0.01mol/L; stirring is started, and stirring and dissolution are carried out when the temperature is reduced to 2-4 ℃. After the precipitate is completely dissolved, regulating the pH to 5.10-5.30 by using an acetic acid-sodium acetate buffer solution with the pH of 4.0, cooling to 0-minus 1 ℃, continuously stirring for at least 10 minutes, adding 95% ethanol to ensure that the final ethanol concentration of the reaction solution is 17%, and controlling the final temperature to minus 5-minus 7 ℃ and the pH to 5.20-5.30; continuously stirring for 2 hours, standing for more than 4 hours, and performing filter pressing to obtain a component III precipitate and a component III supernatant;
s26, performing deep filtration on supernatant of the component III obtained in the step S25, controlling the temperature to be-5 to-7 ℃, adjusting the pH of the deep filtrate to be 3.90-4.10 by using 1mol/L HCl after filtration, performing ultrafiltration concentration to a protein concentration of more than 5%, and performing dialysis dealcoholization by using water for injection; the concentration of the protein is regulated to 3 to 6 percent, the pH value is 6.40 to 6.60, and the conductivity is 1750 to 2050us/cm; purifying the protein solution with adjusted conductivity and pH value by column chromatography, wherein the chromatography uses weak anion exchange gel filler, and collecting the flow-through solution;
s27, adding 1.0mol/L hydrochloric acid solution into the logistics threading liquid prepared in the S26 to adjust the pH value to 3.8-4.0, ultrafiltering and concentrating to more than 5% of protein concentration, dialyzing with water for injection, concentrating to obtain primary ultrafiltrate, and adding 10+/-1% of maltose to dilute to obtain primary diluted liquid;
s28, sterilizing and filtering the dilute protein solution prepared in the step S27 by using a 0.2 mu m sterilizing filter element; placing the sterilized and filtered product in a low pH incubation chamber, and incubating at 24+ -1deg.C for 21 days; after the low pH incubation is finished, performing virus removal filtration by a 20nm nano membrane;
s29, concentrating, dialyzing and diluting the protein solution obtained by filtering the preparation of S28 by using 0.85% sodium chloride solution, and adding 28g glycine per liter according to the final volume to adjust the tetanus antibody titer to be more than or equal to 100IU/ml.
6. The method for preparing tetanus human immunoglobulin according to claim 5, wherein the method comprises the steps of: the percentages in the S24 are mass percentages except for the limitation.
7. The method for preparing tetanus human immunoglobulin according to claim 5, wherein the method comprises the steps of: the acetic acid-sodium acetate buffer in S23, S24 and S25 comprises 108.9g/L sodium acetate and 255.6g/L glacial acetic acid, and the acetic acid-sodium acetate buffer in S26 comprises 40g/L NaOH and 85.9g/L phosphoric acid.
8. The method for preparing tetanus human immunoglobulin according to claim 5, wherein the method comprises the steps of: the specific method of the S4 is as follows:
s41, filtering and split charging the diluted protein solution prepared in the step S29 through a 0.2 mu m sterilization filter element;
s42, delivering and inspecting the packaged products and lamp inspection;
s43, packaging and warehousing after the inspection is qualified.
9. A tetanus human immunoglobulin prepared by the method of any one of claims 1-8.
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