CN112375141A - Preparation method of subcutaneous injection human immunoglobulin - Google Patents
Preparation method of subcutaneous injection human immunoglobulin Download PDFInfo
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- CN112375141A CN112375141A CN202011369121.5A CN202011369121A CN112375141A CN 112375141 A CN112375141 A CN 112375141A CN 202011369121 A CN202011369121 A CN 202011369121A CN 112375141 A CN112375141 A CN 112375141A
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Abstract
According to the preparation method of the subcutaneous injection human immunoglobulin, phosphate is used for adjusting the ionic strength and pH in the FIII/FI + III separation step, selective dissolution is carried out at pH 4.6-4.9, and the precipitation of hybrid protein is carried out at pH 5.0-5.4, so that the anti-A and anti-B antibodies can be effectively removed, and the titer of the anti-A and anti-B antibodies in a product is reduced; the pH value selected in the second step of anion exchange chromatography is 6.5-7.5, and is higher than the isoelectric point of the anti-A anti-B antibody, the anti-A anti-B antibody carries negative charges, and is adsorbed by anion exchange gel to further remove the anti-A anti-B antibody, so that the risk of hemolytic reaction caused by subcutaneous injection of human immunoglobulin is reduced; the hybrid protein precipitation is carried out at the pH value of 5.0-5.4, so that the procoagulant active substances are reduced, and the risk of thromboembolism caused by subcutaneous injection of high-concentration human immunoglobulin is effectively reduced; the two-step anion exchange chromatography is adopted for further purification, so that the product purity can be improved, the contents of IgA and fibrinolytic enzyme are reduced, the incidence rate of adverse reactions is reduced, and the long-term stability of the product is improved.
Description
Technical Field
The invention relates to the technical field of preparation methods of immunoglobulin, in particular to a preparation method of subcutaneous injection human immunoglobulin.
Background
In 1952, human immunoglobulin is used for treating various immunodeficiency diseases for the first time, and the administration route is muscle or subcutaneous. 1980s third generation IVIG, characterized by intact biological activity with preservation of the natural structural integrity of IgG molecules, are marketed, with a protein content of typically 5%, and high concentrations of subcutaneously injected human immunoglobulin were developed internationally in 1990 s.
Human immunoglobulins have irreplaceably important roles in the treatment of various diseases, and the worldwide demand for human immunoglobulins is greatly increasing. Compared with intravenous injection human immunoglobulin, subcutaneous injection human immunoglobulin has considerable advantages, such as more stable IgG concentration in blood of a patient after injection, better tolerance, lower adverse reaction incidence rate, lower long-term use cost, and no need of close monitoring in the use process, so that the patient can use the immunoglobulin at home, the hospitalization time can be reduced, the life quality can be improved, and social resources can be saved.
Clinically, subcutaneous injection of human immunoglobulin is used for the prevention of Primary Immunodeficiency (PID), myeloma or chronic lymphocytic leukemia with secondary hypogammaglobulinemia and recurrent infections, Secondary Immunodeficiency (SID), Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Multifocal Motor Neuropathy (MMN), idiopathic inflammatory myopathy, including Polymyositis (PM), Dermatomyositis (DM), infection after organ transplantation surgery.
The existing technology for injecting the immunoglobulin under the skin with high concentration usually takes CHONFII + III/FI + II + III or KISTLER-NISTCHMANN precipitate A as raw material, adopts octanoic acid precipitation or PEG precipitation technology, and then prepares the immunoglobulin through chromatography and purification. The preparation processes abandon the FIII/FI + III separation step of the traditional low-temperature ethanol process, and FIII/FI + III precipitation is an important process step in the immunoglobulin separation and purification process and is a key step for removing homolectin to reduce hemolytic reaction, removing FXI/FXIa to reduce procoagulant activity of products, thereby improving product safety and reducing adverse reaction incidence, so that the products prepared by caprylic acid precipitation or PEG precipitation usually have higher anti-A and anti-B titer and FXIa activity, and the adverse reaction incidence such as hemolytic anemia, thromboembolism and the like is higher.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of subcutaneous injection human immunoglobulin with simple process and high safety.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a preparation method of subcutaneous injection human immunoglobulin comprises the following steps:
(1) FII + III/FI + II + III separation: adding an acetate buffer solution into the FI supernatant or the blood plasma, and adjusting the pH to 5.8-6.2; adding pre-cooled ethanol with the volume fraction of 95% until the volume of the ethanol reaches 18-22% of the volume of the mixed solution, simultaneously cooling the mixed solution to-6 to-4 ℃, precipitating and aging to obtain a first standing solution; centrifuging the first standing solution at-6 to-4 ℃ to obtain a centrifugally separated precipitate;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by using 10-15 times of water for injection at 0-5 ℃, adding a phosphate buffer solution to adjust the pH value to 4.6-4.9, reacting for 1 hour, adjusting the pH value to 5.0-5.4 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 0.6-1.6 ms/cm by using water for injection at 0-5 ℃, adding pre-cooled ethanol with the volume fraction of 95% until the volume of the ethanol reaches 10-12% of the volume of the mixed solution, and simultaneously reducing the temperature of the mixed solution to-5-3 ℃ for precipitation to obtain a precipitated mixed solution; centrifugally separating the precipitated mixed solution at the temperature of between 5 ℃ below zero and 3 ℃ below zero, and collecting the supernatant of FIII/FI + III;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 6-10 g/L, and performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, so as to obtain clear filtrate filtered by the filter plate;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 4.5-5.5% to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 0-24 ℃ injection water with the volume not less than 5 times that of the concentrated clear filtrate, wherein the transmembrane pressure is 0.1-0.3 Mpa, and thus obtaining a dialysis concentrated solution;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 0.9-1.1%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.28-0.32%, and inactivating the S/D virus for more than 6 hours at the temperature of 25 +/-1 ℃ to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 1.8-2.2%, adjusting the pH to 6.5-7.5, and adding PEG4000 until the mass fraction of PEG4000 is 10-16% to obtain a PEG precipitation reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with injection water at 2-8 ℃ in 4-6 times volume of the PEG centrifuged precipitate, and filtering with a deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarified filtrate to 5.0-5.4, balancing a chromatographic column with 5CV, 20-40 mM acetate buffer solution with pH value of 5.0-5.4, loading at a linear flow rate of 100-400 cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH value of the flow-through liquid to 6.5-7.5 by using 1mol/LNaOH, adjusting the conductivity of the flow-through liquid to 3.5-5.5 ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate balance liquid with 5CV, 25-45 mM and pH value of 6.5-7.5, loading the sample at a linear flow rate of 100-400 cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 3.8-4.4 by using 0.5MHCL, and filtering by using a degreasing filter element under the pressure of not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution by using water for injection at 0-24 ℃, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 3.8-4.4;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, and adding glycine until the concentration of glycine is 0.2-0.4 mol/L and the pH is 3.8-4.4 to obtain the subcutaneous injection human immunoglobulin.
Further, in the step (1), acetate buffer solution with pH of 4.0 is added into FI supernatant or blood plasma, and the pH is adjusted to 5.8-6.2; adding ethanol with the volume fraction of 95% at the temperature of below 15 ℃ below zero until the volume of the ethanol reaches 18-22% of the volume of the mixed solution, simultaneously cooling the mixed solution to 6 ℃ below zero to 4 ℃ below zero, precipitating and aging to obtain a first standing solution; centrifuging the first standing solution at-6 to-4 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the centrifuge speed was 15000 rpm.
Further, dissolving the centrifugally separated precipitate with 10-15 times of 0-5 ℃ injection water in the step (2), adding phosphate buffer solution to adjust the pH value to 4.6-4.9, after reacting for 1 hour, adjusting the pH value to 5.0-5.4 with the phosphate buffer solution, after reacting for 1 hour, adjusting the conductivity to 0.6-1.6 ms/cm with the 0-5 ℃ injection water, adding 95% ethanol with the volume fraction below-15 ℃ until the volume of the ethanol reaches 10-12% of the volume of the mixed solution, and simultaneously reducing the temperature of the mixed solution to-5-3 ℃ for precipitation to obtain a precipitated mixed solution; centrifugally separating the precipitated mixed solution at the temperature of between 5 ℃ below zero and 3 ℃ below zero, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the centrifuge speed was 15000 rpm.
Further, the filter aid in the step (3) is perlite.
Further, in the step (5), tween 80 is added to the dialysis concentrated solution until the mass fraction of tween 80 is 1%, tributyl phosphate is added until the mass fraction of tributyl phosphate is 0.3%, and the S/D virus is inactivated for more than 6 hours at 25 +/-1 ℃ to obtain the inactivated dialysis concentrated solution.
Further, in the step (6), the protein of the inactivated dialysis concentrate is diluted to a mass fraction of 2%, the pH is adjusted to 7, and PEG4000 is added to a mass fraction of 13% of PEG4000, to obtain a PEG precipitation reactant.
Further, the centrifugation process parameters in the step (7) are as follows: the temperature is 2-5 ℃, and the rotating speed of the centrifuge is 15000 rpm.
Further, in the step (9), the pH of the clarified filtrate of the PEG precipitation is adjusted to 5.2, the chromatographic column is equilibrated by 5CV, 20-40 mM and pH 5.2 acetate buffer solution, the sample is loaded at a linear flow rate of 200cm/h, and a 280nm ultraviolet absorption peak flow-through solution is collected for further chromatographic purification.
Further, in the step (10), the pH of the flow-through liquid is adjusted to 7 by using 1mol/L NaOH solution, the conductivity of the flow-through liquid is adjusted to 4ms/cm by using sodium chloride, a chromatographic column is balanced by using 5CV, 25-45 mM and pH 7 acetate equilibrium solution, the sample is loaded at a linear flow rate of 300cm/h, and the flow-through liquid with 280nm ultraviolet absorption peaks is collected for further treatment.
Further, in the step (11), the pH of the flow-through liquid collected in the step (10) is adjusted to 4.2 by 0.5MHCL, and the flow-through liquid is filtered by a degreasing filter element under the pressure of not more than 0.3MPa, so as to obtain a filtered clarified liquid.
The invention relates to a preparation method of subcutaneous injection human immunoglobulin, anti-A anti-B isolectin isoelectric point is about pH 6.3, have minimum solubility near isoelectric point, concentration and kind of salt used in the separation process have strong influence on the solubility of anti-A anti-B antibody, the solubility of anti-A anti-B antibody is reduced with the reduction of ionic strength in a certain range; under the same ionic strength condition, the phosphate can improve IgG solubility and reduce the solubility of the anti-A anti-B antibody, so that the ionic strength and pH are adjusted by using the phosphate in the FIII/FI + III separation step, selective dissolution is carried out at the pH value of 4.6-4.9, and the precipitation of hybrid protein is carried out at the pH value of 5.0-5.4, so that the anti-A anti-B antibody can be effectively removed, and the titer of the anti-A anti-B antibody in a product is reduced; the pH value selected in the second step of anion exchange chromatography is 6.5-7.5, and is higher than the isoelectric point of the anti-A anti-B antibody, the anti-A anti-B antibody carries negative charges, and is adsorbed by anion exchange gel to further remove the anti-A anti-B antibody, so that the risk of hemolytic reaction caused by subcutaneous injection of human immunoglobulin is reduced.
The invention relates to a preparation method of subcutaneous injection human immunoglobulin, FXI factor is activated due to various reasons in the separation process of plasma protein, and activated FXIa with procoagulant activity remained in a human immunoglobulin preparation is the root cause of thromboembolic events after products are used. The european union pharmacopoeia 8 th edition, officially effective 1/2014, requires that one or more steps be included in the production process of human immunoglobulin to demonstrate the removal of substances responsible for the production of thrombi. The step of separating FIII/FI + III in the low-temperature ethanol process is a key process for removing FXIa, and the key process parameter influencing the FXIa removal capacity is pH, so that the low-temperature ethanol FIII/FI + III separation step is adopted for separation and purification, and the pH is 5.0-5.4 for impurity protein precipitation, so that the procoagulant active substances are reduced, and the risk of thromboembolic events caused by subcutaneous injection of high-concentration human immunoglobulin is effectively reduced.
The invention relates to a preparation method of subcutaneous injection human immunoglobulin, which adopts a low-temperature ethanol FIII/FI + III separation process capable of effectively reducing anti-A anti-B antibodies and removing procoagulant activity, and adopts two-step anion exchange chromatography for further purification in the subsequent process, thereby improving the product purity, reducing the contents of IgA and fibrinolytic enzyme, reducing the incidence rate of adverse reactions and improving the long-term stability of the product.
The invention relates to a preparation method of subcutaneous injection human immunoglobulin, which adopts a three-step virus inactivation/removal process: the S/D treatment, the low pH incubation and the nano-membrane filtration can effectively inactivate/remove broad-spectrum viruses including enveloped viruses and non-enveloped viruses, and the B19 virus and prion can be effectively separated by the FIII/FI + III separation step, so that the subcutaneous injection human immunoglobulin prepared according to the method has good virus safety.
The FIII/FI + III production process removes B19 virus and prions.
Drawings
FIG. 1 is a process flow chart of a method for preparing human immunoglobulin for subcutaneous injection according to the present invention.
Detailed Description
The following examples may help one skilled in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
A preparation method of subcutaneous injection human immunoglobulin requires the following steps:
1. the water for preparing acetate buffer solution, phosphate buffer solution and other main reagents is injection water;
2. the collection and quality requirements of raw material plasma used for preparing the FI supernatant fluid must meet the related requirements of human plasma for blood product production in pharmacopoeia of the people's republic of China;
3. production equipment facilities and environment meet GMP requirements;
4. FI supernatant refers to: removing fibrinogen supernatant
5. The centrifugation equipment used was: the high-capacity continuous flow centrifuge has the following model: GQ142
6. The main drugs used are all of pharmaceutical grade, and the manufacturer information is as follows
| Name of reagent | Manufacturer of the product |
| Glacial acetic acid | TAISHAN XINNING PHARMACEUTICAL Co.,Ltd. |
| Sodium acetate | TAISHAN XINNING PHARMACEUTICAL Co.,Ltd. |
| 95% ethanol | HUNAN XIANGYIKANG PHARMACEUTICAL Co.,Ltd. |
| Disodium hydrogen phosphate | Jiu Dian pharmaceutical Limited liability company of Hunan |
| Glycine | HUBEI PROVINCIAL BAFENG PHARMACEUTICAL & CHEMICAL SHARE Co.,Ltd. |
| PEG4000 | Hunan Er-Kang Pharmaceutical Co.,Ltd. |
| Phosphoric acid tributyl ester | Merck |
| Tween 80 | Hunan Er-Kang Pharmaceutical Co.,Ltd. |
| Hydrochloric acid | HUNAN ER-KANG PHARMACEUTICAL Co.,Ltd. |
Example 1
A preparation method of subcutaneous injection human immunoglobulin comprises the following steps:
(1) FII + III/FI + II + III separation: adding an acetate buffer solution with pH4.0 into the FI supernatant or the blood plasma, adjusting the pH to 5.8, and adding the acetate buffer solution with pH4.0 at a speed of 150 ml/min; adding 95% ethanol at a temperature below-15 deg.C until the volume of ethanol reaches 18% of the volume of the mixture, adding ethanol at a rate of 65L/h, gradually cooling the mixture to-4 deg.C while adding ethanol, stirring for reaction for 2 hr, standing for aging for 6 hr to obtain a first standing solution; centrifuging the first standing solution at-4 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the temperature is minus 4 ℃, and the rotating speed of a centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 1000-2000 ml/min;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by using 10 times of 0 ℃ water for injection, adding a phosphate buffer solution at a speed of 150ml/min to adjust the pH value to 4.6, reacting for 1 hour, adjusting the pH value to 5.0 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 0.6ms/cm by using 0 ℃ water for injection, adding 95% ethanol with the volume fraction of below-15 ℃ until the volume of the ethanol reaches 10% of the volume of the mixed solution, and reducing the temperature of the mixed solution to-3 ℃ for precipitation at the same time to obtain a precipitated mixed solution, wherein the volume of the mixed solution is 60L/h; centrifugally separating the precipitated mixed solution at the temperature of minus 3 ℃, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the temperature is minus 3 ℃, and the rotating speed of the centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 6g/L, mixing for more than 30 minutes, performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, and obtaining clear filtrate filtered by the filter plate; the filter aid is perlite Harbolite 1800;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 4.5 percent to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 0 deg.C water for injection with a volume not less than 5 times of the volume of the concentrated clear filtrate, and obtaining a dialysis concentrated solution with transmembrane pressure of 0.1 Mpa;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 0.9%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.28%, and inactivating the S/D virus at 25 +/-1 ℃ for more than 6h to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 1.8%, adjusting the pH to 6.5 by using 0.5mol/L sodium hydroxide, and adding PEG4000 until the mass fraction of PEG4000 is 10% to obtain a PEG precipitation reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate; the centrifugal process parameters are as follows: the temperature is 2 ℃, and the rotating speed of the centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with 4 times volume of 2 deg.C water for injection of PEG centrifuged precipitate, and filtering with a deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarifying filtrate to 5.0, and balancing the chromatographic column by using 5CV, 20-40 mM acetate buffer solution with the pH value of 5.0, namely balancing the chromatographic column by using 5 balancing solutions with the column volume concentration of 20-40 mM and the pH value of 5.0-5.4; sampling at a linear flow rate of 100cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH of the flow-through liquid to 6.5 by using 1mol/LNaOH, adjusting the conductivity of the flow-through liquid to 3.5ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate equilibrium liquid with 5CV, 25-45 mM and pH of 6.5, loading the sample at a linear flow rate of 100cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 3.8 by using 0.5MHCL, and filtering by using a degreasing filter element, wherein the pressure is not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution with 0 ℃ water for injection, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 3.8;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, adding glycine until the concentration of glycine is 0.2mol/L and the pH value is 3.8, and obtaining the subcutaneous injection human immunoglobulin.
Example 2
A preparation method of subcutaneous injection human immunoglobulin comprises the following steps:
(1) FII + III/FI + II + III separation: adding an acetate buffer solution with pH4.0 into the FI supernatant or the blood plasma, adjusting the pH to 6.2, and adding the acetate buffer solution with pH4.0 at a speed of 150 ml/min; adding 95% ethanol at a temperature below-15 deg.C until the volume of ethanol reaches 22% of the volume of the mixture, adding ethanol at a rate of 65L/h, gradually cooling the mixture to-6 deg.C while adding ethanol, stirring for reaction for 2 hr, standing for aging for 6 hr to obtain a first standing solution; centrifuging the first standing solution at-6 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 1000-2000 ml/min;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by 15 times of 5 ℃ injection water, adding a phosphate buffer solution at a speed of 150ml/min to adjust the pH to 4.9, reacting for 1 hour, adjusting the pH to 5.4 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 1.6ms/cm by using 5 ℃ injection water, adding 95% ethanol with the volume fraction of below-15 ℃ until the volume of the ethanol reaches 12% of the volume of the mixed solution, and reducing the temperature of the mixed solution to-5 ℃ for precipitation at the same time to obtain a precipitated mixed solution, wherein the volume of the mixed solution is 60L/h; centrifugally separating the precipitated mixed solution at the temperature of-5 ℃, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 10g/L, mixing for more than 30 minutes, performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, and obtaining clear filtrate filtered by the filter plate; the filter aid is perlite Harbolite 1800;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 5.5 percent to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 24 deg.C water for injection with a volume not less than 5 times that of the concentrated clear filtrate under transmembrane pressure of 0.3Mpa to obtain dialysis concentrated solution;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 1.1%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.32%, and inactivating the S/D virus for more than 6h at the temperature of 25 +/-1 ℃ to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 2.2%, adjusting the pH to 7.5 by using 0.5mol/L sodium hydroxide, and adding PEG4000 until the mass fraction of PEG4000 is 16% to obtain a PEG precipitation reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate; the centrifugal process parameters are as follows: the temperature is 5 ℃, and the rotating speed of the centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with 6 times volume of 8 deg.C water for injection, and filtering with deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarified filtrate to 5.4, balancing a chromatographic column by using 5CV, 20-40 mM acetate buffer solution with the pH value of 5.4, loading the sample at a linear flow rate of 400cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH of the flow-through liquid to 7.5 by using 1mol/LNaOH, adjusting the conductivity of the flow-through liquid to 5.5ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate equilibrium liquid with 5CV, 25-45 mM and pH of 7.5, loading the sample at a linear flow rate of 400cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 4.4 by using 0.5MHCL, and filtering by using a degreasing filter element, wherein the pressure is not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution with 24 ℃ water for injection, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 4.4;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, adding glycine until the concentration of glycine is 0.4mol/L and the pH value is 4.4, and obtaining the subcutaneous injection human immunoglobulin.
Example 3
A preparation method of subcutaneous injection human immunoglobulin comprises the following steps:
(1) FII + III/FI + II + III separation: adding a pH4.0 acetate buffer solution into the FI supernatant or the blood plasma, adjusting the pH to 6, and adding the pH4.0 acetate buffer solution at a speed of 150 ml/min; adding 95% ethanol at a temperature below-15 deg.C until the volume of ethanol reaches 20% of the volume of the mixture, adding ethanol at a rate of 65L/h, gradually cooling the mixture to-5 deg.C while adding ethanol, stirring for reaction for 2 hr, standing for aging for 6 hr to obtain a first standing solution; centrifuging the first standing solution at-5 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 1000-2000 ml/min;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by using 12 times of water for injection at 2 ℃, adding a phosphate buffer solution at a speed of 150ml/min to adjust the pH value to 4.7, reacting for 1 hour, adjusting the pH value to 5.2 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 1.2ms/cm by using water for injection at 2 ℃, adding ethanol with the volume fraction of 95% at the temperature of below-15 ℃ until the volume of the ethanol reaches 11% of the volume of the mixed solution, and reducing the temperature of the mixed solution to-4 ℃ for precipitation at the same time to obtain a precipitated mixed solution, wherein the volume of the mixed solution is 60L/h; centrifugally separating the precipitated mixed solution at the temperature of-4 ℃, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 8g/L, mixing for more than 30 minutes, performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, and obtaining clear filtrate filtered by the filter plate; the filter aid is perlite Harbolite 1800;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 5 percent to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 5 deg.C water for injection with a volume not less than 5 times that of the concentrated clear filtrate under transmembrane pressure of 0.2Mpa to obtain dialysis concentrated solution;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 1%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.3%, and inactivating the S/D virus for more than 6h at the temperature of 25 +/-1 ℃ to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 2%, adjusting the pH to 7 by using 0.5mol/L sodium hydroxide, and adding PEG4000 until the mass fraction of PEG4000 is 13% to obtain a PEG precipitate reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate; the centrifugal process parameters are as follows: the temperature is 3 ℃, and the rotating speed of the centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with 5 times volume of 5 deg.C water for injection, and filtering with deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarified filtrate to 5.2, balancing a chromatographic column by using an acetate buffer solution with 5CV, 20-40 mM and pH value of 5.2, loading the sample at a linear flow rate of 200cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH value of the flow-through liquid to 7 by using 1mol/LNaOH, adjusting the conductivity of the flow-through liquid to 4ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate equilibrium liquid with 5CV, 25-45 mM and pH value of 7, loading the sample at a linear flow rate of 300cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 4.2 by using 0.5MHCL, and filtering by using a degreasing filter element, wherein the pressure is not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution by using water for injection at the temperature of 5 ℃, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 3.8-4.4;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, adding glycine until the concentration of glycine is 0.3mol/L and the pH value is 3.9, and obtaining the subcutaneous injection human immunoglobulin.
Comparative example 1
A preparation method of subcutaneous injection human immunoglobulin comprises the following steps:
(1) FII + III/FI + II + III separation: adding a pH4.0 acetate buffer solution into the FI supernatant or the blood plasma, adjusting the pH to 6, and adding the pH4.0 acetate buffer solution at a speed of 150 ml/min; adding ethanol with volume fraction of 95% at a temperature below-15 deg.C until the volume of ethanol reaches 20% of the volume of the mixed solution, adding ethanol at a rate of 65L/h, cooling the mixed solution to-5 deg.C while adding ethanol, stirring for reaction for 2 hr, standing, and aging for 6 hr to obtain a first standing solution; centrifuging the first standing solution at-5 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 1000-2000 ml/min;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by using 12 times of water for injection at 2 ℃, adding a phosphate buffer solution at a speed of 150ml/min to adjust the pH value to 4.7, reacting for 1 hour, adjusting the pH value to 4.9 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 1.2ms/cm by using water for injection at 2 ℃, adding ethanol with the volume fraction of 95% at the temperature of below-15 ℃ until the volume of the ethanol reaches 11% of the volume of the mixed solution, and reducing the temperature of the mixed solution to-4 ℃ for precipitation at the same time to obtain a precipitated mixed solution, wherein the volume of the mixed solution is 60L/h; centrifugally separating the precipitated mixed solution at the temperature of-4 ℃, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the rotating speed of the centrifuge is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 8g/L, mixing for more than 30 minutes, performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, and obtaining clear filtrate filtered by the filter plate; the filter aid is perlite Harbolite 1800;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 5 percent to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 5 deg.C water for injection with a volume not less than 5 times that of the concentrated clear filtrate under transmembrane pressure of 0.2Mpa to obtain dialysis concentrated solution;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 1%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.3%, and inactivating the S/D virus for more than 6h at the temperature of 25 +/-1 ℃ to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 2%, adjusting the pH to 7 by using 0.5mol/L sodium hydroxide, and adding PEG4000 until the mass fraction of PEG4000 is 13% to obtain a PEG precipitate reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate; the centrifugal process parameters are as follows: the temperature is 3 ℃, and the rotating speed of the centrifugal machine is 15000 rpm; the outflow speed of the supernatant is 500-1000 ml/min;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with 5 times volume of 5 deg.C water for injection, and filtering with deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarified filtrate to 5.2, balancing a chromatographic column by using an acetate buffer solution with 5CV, 20-40 mM and pH value of 5.2, loading the sample at a linear flow rate of 200cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH value of the flow-through liquid to 6.3, adjusting the conductivity of the flow-through liquid to 4ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate equilibrium liquid with 5CV, 25-45 mM and pH value of 7, loading at a linear flow rate of 300cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 4.2 by using 0.5MHCL, and filtering by using a degreasing filter element, wherein the pressure is not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution by using water for injection at the temperature of 5 ℃, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 3.8-4.4;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, adding glycine until the concentration of glycine is 0.3mol/L and the pH value is 3.9, and obtaining the subcutaneous injection human immunoglobulin.
The following table 1 shows the detection results of the FIII/FI + III separation to reduce the content of the anti-A and anti-B antibodies in examples 1-3 and comparative example 1, respectively;
the detection method of the anti-A comprises the following steps: indirect anti-human globulin method
The detection method of the anti-B comprises the following steps: indirect anti-human globulin method
TABLE 1
The data in Table 1 above show that the method of the invention is effective in removing anti-A anti-B antibodies.
The following table 2 shows the results of detection of FXIa removal by FIII/FI + III separation in examples 1-3 and comparative example 1;
the detection method of the FXIa content comprises the following steps: chromogenic substrate method
TABLE 2
The data in Table 2 above show that the method of the invention is effective in removing procoagulant actives;
table 3 below shows the results of accelerated stability tests at 25. + -. 2 ℃ for the products prepared in examples 1-3;
TABLE 3
The data in table 3 above show that the high concentration subcutaneous human immunoglobulin prepared by the method has good stability.
As can be seen from the detection data in tables 1-3, the preparation method of the subcutaneous injection human immunoglobulin disclosed by the invention has the advantages that the ionic strength and pH are adjusted by using phosphate in the FIII/FI + III separation step, selective dissolution is carried out at pH 4.6-4.9, and the precipitation of hybrid protein is carried out at pH 5.0-5.4, so that the anti-A and anti-B antibodies can be effectively removed, and the titer of the anti-A and anti-B antibodies in a product is reduced; the pH value selected in the second step of anion exchange chromatography is 6.5-7.5, and is higher than the isoelectric point of the anti-A anti-B antibody, the anti-A anti-B antibody carries negative charges, and is adsorbed by anion exchange gel to further remove the anti-A anti-B antibody, so that the risk of hemolytic reaction caused by subcutaneous injection of human immunoglobulin is reduced; low-temperature ethanol FIII/FI + III separation steps are adopted for separation and purification, and hybrid protein precipitation is carried out at pH 5.0-5.4, so that procoagulant active substances are reduced, and the risk of thromboembolism caused by subcutaneous injection of high-concentration human immunoglobulin is effectively reduced; the low-temperature ethanol FIII/FI + III separation process capable of effectively reducing the anti-A and anti-B antibodies and removing the procoagulant activity is adopted, and two-step anion exchange chromatography is adopted in the subsequent process for further purification, so that the product purity can be improved, the contents of IgA and fibrinolytic enzyme are reduced, the incidence rate of adverse reactions is reduced, and the long-term stability of the product is improved.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A preparation method of subcutaneous injection human immunoglobulin is characterized by comprising the following steps:
(1) FII + III/FI + II + III separation: adding an acetate buffer solution into the FI supernatant or the blood plasma, and adjusting the pH to 5.8-6.2; adding pre-cooled ethanol with the volume fraction of 95% until the volume of the ethanol reaches 18-22% of the volume of the mixed solution, simultaneously cooling the mixed solution to-6 to-4 ℃, precipitating and aging to obtain a first standing solution; centrifuging the first standing solution at-6 to-4 ℃ to obtain a centrifugally separated precipitate;
(2) FIII/FI + III separation: dissolving the centrifugally separated precipitate by using 10-15 times of water for injection at 0-5 ℃, adding a phosphate buffer solution to adjust the pH value to 4.6-4.9, reacting for 1 hour, adjusting the pH value to 5.0-5.4 by using the phosphate buffer solution, reacting for 1 hour, adjusting the conductivity to 0.6-1.6 ms/cm by using water for injection at 0-5 ℃, adding pre-cooled ethanol with the volume fraction of 95% until the volume of the ethanol reaches 10-12% of the volume of the mixed solution, and simultaneously reducing the temperature of the mixed solution to-5-3 ℃ for precipitation to obtain a precipitated mixed solution; centrifugally separating the precipitated mixed solution at the temperature of between 5 ℃ below zero and 3 ℃ below zero, and collecting the supernatant of FIII/FI + III;
(3) clarifying and filtering: adding a filter aid into the FIII/FI + III supernatant according to the proportion of 6-10 g/L, and performing deep filtration by using a degreasing filter plate, wherein the filtration pressure is not more than 0.3Mpa, so as to obtain clear filtrate filtered by the filter plate;
(4) ultrafiltration concentration dialysis: concentrating the clear filtrate filtered by the filter plate by using a 50KD ultrafiltration membrane until the protein content is 4.5-5.5% to obtain concentrated clear filtrate; dialyzing the concentrated clear filtrate with 0-24 ℃ injection water with the volume not less than 5 times that of the concentrated clear filtrate, wherein the transmembrane pressure is 0.1-0.3 Mpa, and thus obtaining a dialysis concentrated solution;
(5) S/D virus inactivation: adding Tween 80 into the dialysis concentrated solution until the mass fraction of Tween 80 is 0.9-1.1%, adding tributyl phosphate until the mass fraction of tributyl phosphate is 0.28-0.32%, and inactivating the S/D virus for more than 6 hours at the temperature of 25 +/-1 ℃ to obtain an inactivated dialysis concentrated solution;
(6) PEG precipitation: diluting the inactivated dialysis concentrated solution until the mass fraction of protein is 1.8-2.2%, adjusting the pH to 6.5-7.5, and adding PEG4000 until the mass fraction of PEG4000 is 10-16% to obtain a PEG precipitation reactant;
(7) centrifugal separation: centrifuging the PEG precipitation reactant to obtain PEG centrifuged precipitate;
(8) dissolving and filtering the precipitate: dissolving the PEG centrifuged precipitate with injection water at 2-8 ℃ in 4-6 times volume of the PEG centrifuged precipitate, and filtering with a deep filter plate to obtain PEG precipitate clarified filtrate;
(9) first step anion exchange chromatography: adjusting the pH value of the PEG precipitation clarified filtrate to 5.0-5.4, balancing a chromatographic column with 5CV, 20-40 mM acetate buffer solution with pH value of 5.0-5.4, loading at a linear flow rate of 100-400 cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further chromatographic purification;
(10) the second step of anion exchange chromatography: adjusting the pH value of the flow-through liquid to 6.5-7.5 by using 1mol/LNaOH, adjusting the conductivity of the flow-through liquid to 3.5-5.5 ms/cm by using sodium chloride, balancing a chromatographic column by using an acetate balance liquid with 5CV, 25-45 mM and pH value of 6.5-7.5, loading the sample at a linear flow rate of 100-400 cm/h, and collecting 280nm ultraviolet absorption peak flow-through liquid for further treatment;
(11) degreasing and filtering: adjusting the pH value of the flow-through liquid collected in the step (10) to 3.8-4.4 by using 0.5MHCL, and filtering by using a degreasing filter element under the pressure of not more than 0.3Mpa to obtain filtered clarified liquid;
(12) and (3) ultrafiltration preparation: concentrating the filtered clarified solution with a 50KD ultrafiltration membrane to obtain a concentrated solution; dialyzing the concentrated solution by using water for injection at 0-24 ℃, wherein the using amount of the water for injection is not less than 8 times of the volume of the concentrated solution, and concentrating the product after dialysis to prepare a stock solution with the mass fraction of protein being 5% and the pH value being 3.8-4.4;
(13) low pH viral inactivation: continuously incubating the stock solution after sterilization and filtration for more than 21 days at 24 ℃ to inactivate residual lipid-enveloped viruses to obtain virus inactivation stock solution;
(14) virus removal and filtration by a nano membrane: performing virus removal filtration on the virus inactivation stock solution by using a nano membrane of 20 nanometers to obtain virus removal filtration stock solution;
(15) concentrating and preparing: concentrating the virus-removing filtration stock solution by using a 100Kd ultrafiltration membrane to obtain a concentrated stock solution; preparing according to 200g/L protein content, and adding glycine until the concentration of glycine is 0.2-0.4 mol/L and the pH is 3.8-4.4 to obtain the subcutaneous injection human immunoglobulin.
2. The method for preparing human immunoglobulin for subcutaneous injection according to claim 1, wherein in the step (1), acetate buffer with pH4.0 is added to FI supernatant or plasma to adjust pH to 5.8-6.2; adding ethanol with the volume fraction of 95% at the temperature of below 15 ℃ below zero until the volume of the ethanol reaches 18-22% of the volume of the mixed solution, simultaneously cooling the mixed solution to 6 ℃ below zero to 4 ℃ below zero, precipitating and aging to obtain a first standing solution; centrifuging the first standing solution at-6 to-4 ℃ to obtain a centrifugally separated precipitate; the centrifugal process parameters are as follows: the centrifuge speed was 15000 rpm.
3. The method according to claim 1, wherein the centrifuged precipitate is dissolved in 10-15 times of 0-5 ℃ water for injection in the step (2), a phosphate buffer is added to adjust the pH value to 4.6-4.9, the pH value is adjusted to 5.0-5.4 by the phosphate buffer after 1 hour of reaction, the conductivity is adjusted to 0.6-1.6 ms/cm by 0-5 ℃ water for injection after 1 hour of reaction, 95% ethanol is added until the volume of the ethanol reaches 10-12% of the volume of the ethanol at a temperature below-15 ℃, and the mixture is precipitated by reducing the temperature of the mixture to-5-3 ℃ to obtain a precipitated mixture; centrifugally separating the precipitated mixed solution at the temperature of between 5 ℃ below zero and 3 ℃ below zero, and collecting the supernatant of FIII/FI + III; the centrifugal process parameters are as follows: the centrifuge speed was 15000 rpm.
4. The method for preparing human immunoglobulin for subcutaneous injection according to claim 1, wherein the filter aid in step (3) is perlite.
5. The method according to claim 1, wherein tween 80 is added to the dialysis concentrate in step (5) until the mass fraction of tween 80 is 1%, and tributyl phosphate is added until the mass fraction of tributyl phosphate is 0.3%, and S/D virus is inactivated for more than 6 hours at 25 ± 1 ℃ to obtain the inactivated dialysis concentrate.
6. The method according to claim 1, wherein the inactivated dialysis concentrate is diluted to have a protein content of 2% by mass in the step (6), the pH is adjusted to 7, and PEG4000 is added to have a PEG4000 content of 13% by mass, thereby obtaining a PEG precipitate.
7. The method for preparing human immunoglobulin for subcutaneous injection according to claim 1, wherein the centrifugation process parameters in step (7) are: the temperature is 2-5 ℃, and the rotating speed of the centrifuge is 15000 rpm.
8. The method according to claim 1, wherein the pH of the clarified PEG precipitate filtrate in step (9) is adjusted to 5.2, the column is equilibrated with 5CV of 20-40 mM acetate buffer solution having pH of 5.2, and the sample is applied at a linear flow rate of 200cm/h, and the flow-through solution of 280nm UV absorption peak is collected for further chromatographic purification.
9. The method according to claim 1, wherein in step (10), the pH of the flow-through solution is adjusted to 7 with 1mol/L NaOH solution, the conductivity of the flow-through solution is adjusted to 4ms/cm with sodium chloride, the chromatographic column is equilibrated with 5CV, 25-45 mM and pH 7 acetate equilibrium solution, the sample is loaded at a linear flow rate of 300cm/h, and the flow-through solution with 280nm UV absorption peak is collected for further processing.
10. The method according to claim 1, wherein the pH of the flow-through solution collected in step (10) is adjusted to 4.2 with 0.5MHCL in step (11), and the solution is filtered through a filter element with degreasing activity, and the pressure is not higher than 0.3MPa, so as to obtain a clear filtered solution.
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