CN103665100A - Method for extracting intravenous injection human immunoglobulin by low temperature ethanol - Google Patents
Method for extracting intravenous injection human immunoglobulin by low temperature ethanol Download PDFInfo
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Abstract
The invention discloses a method for extracting intravenous injection human immunoglobulin by low temperature ethanol. In the method, FI, FII+III, FIII and FII are sequentially separated; and when FII precipitates are subjected to ultrafiltration and purification, processes of ultrafiltration dealcoholization, Pasteur inactivation, primary precipitation separation, secondary precipitation separation and secondary precipitation ultrafiltration dealcoholization are sequentially adopted. According to the invention, in the filtering process, the temperature of a filter press is controlled in a low-temperature range; the proper electrical conductivity is adopted in the precipitation separation processes; yield of the immunoglobulin is improved; when the immunoglobulin is purified and refined, precipitation separation is carried out for twice; secondary clarification sterilization filtration is adopted in the process of carrying out precipitation separation for twice; and by a four-stage filtering technology, insoluble particles in a product are removed and clarity and yield of the product are improved.
Description
Technical field
The present invention relates to take human plasma and prepare the method for quiet note human normal immunoglobulin as raw material, relate in particular a kind of method that adopts cold ethanol method to extract quiet note human normal immunoglobulin.
Background technology
Cold ethanol method is one of preparation method of human plasma protein fraction, and the method is professor's EdwinJ.Cohn invention of 1940 medical colleges of Nian You Harvard University, is therefore called again " Kong Shi method ".Kong Shi method is used for Separation of Bovine serum albumin at first, is applied to subsequently human plasma separation.Cold ethanol method is to take pooled plasma as raw material, reduces step by step acidity (dropping to pH4.0 from pH7.0).Improve alcohol concn (being raised to 40% from 0), reduce temperature (dropping to-2 ℃ from 2 ℃) simultaneously, various albumen form substep with component (raw product) under different conditions is separated out from solution, and by centrifugal or filtering separation out.Affect protein precipitation reaction factor and mainly contain five, that is: pH value, temperature, protein concn, ionic strength and alcohol concn.Because processing parameter is selected not science of unreasonable and separation method, caused existing cold ethanol method to produce that human normal immunoglobulin yield is lower and purity is low.
The patent No. is that 201110030778.3 patent of invention discloses " a kind of method of producing quiet note human normal immunoglobulin ", the main technique of this invention is as follows: take human plasma as raw material, first from blood plasma, isolate components I+II+III, again from components I+II+III precipitate and separate components I+III, separated portion II again, use again purification by chromatography compositionⅱ, carry out afterwards inactivation of virus, after deactivation, prepare quiet note human normal immunoglobulin.The component separation cycle of the method is slightly long and purity is lower, and the yield that human normal immunoglobulin is produced need to improve.
Summary of the invention
The object of this invention is to provide a kind of method that cold ethanol extracts quiet note human normal immunoglobulin, is being that raw material is while preparing quiet note human normal immunoglobulin, to improve the yield of quiet note human normal immunoglobulin with human plasma.
Cold ethanol of the present invention extracts the method for quiet note human normal immunoglobulin, comprises the following steps:
Step 1, from human plasma separated F I, obtain F I supernatant liquor;
Step 2, from F I supernatant liquor, isolate F II+III precipitation;
Step 3, from F II+III precipitation separated F III precipitation, obtain F III supernatant liquor;
Step 4, from F III supernatant liquor, isolate F II precipitation;
Step 5, the ultrafiltration of F II precipitation, purifying, comprise the following steps:
1., ultrafiltration dealcoholysis: getting weight is that 8～10 times of above-mentioned F II precipitations, temperature are that 2～8 ℃ of waters for injection dissolve F II precipitation completely, adopts secondary clarification Sterile Filtration, regulator solution pH value to 4.30～4.70;
2., pasteurization: the solution after ultrafiltration dealcoholysis is regulated to protein content to 17～33g/l with water for injection, by 60～120g/l, add glycine, regulate pH value to 7.10～7.50, at the temperature of 55～65 ℃, heat and within 9～11 hours, carry out, after pasteurization, being cooled to 20～30 ℃;
3., primary sedimentation is separated: with water for injection, regulate the solution after pasteurization, making protein content in solution is 5.0～15.0g/l, regulator solution pH value to 6.70～7.20, control temperature to 0～2 ℃, regulate alcohol concn to volume percent 18～20%, control temperature to 2～6 ℃, the specific conductivity of regulator solution is 1.20～1.50ms/cm, stirring reaction 1～3 hour, in every kilogram of protein 0.08～0.12kg ratio, add respectively perlite and the diatomite of equivalent, continue reaction after 0.5～1.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control below pressure filter temperature to 5 ℃, filter to isolate primary sedimentation, obtain primary sedimentation supernatant liquor,
4., secondary sedimentation is separated: primary sedimentation supernatant liquor temperature is controlled to-1～-3 ℃, regulate pH value to 7.10～7.40, alcohol concn, to volume percent 24～26%, is controlled solution temperature to-6～-12 ℃, and the specific conductivity of regulator solution is 1.50-1.90ms/cm, stirring reaction 1～3 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-3 ℃, filter to isolate secondary sedimentation;
5., secondary sedimentation ultrafiltration dealcoholysis: the water for injection of getting weight and be 5～10 times of above-mentioned secondary sedimentations, temperature and be 2～8 ℃ dissolves secondary sedimentation completely, adopt secondary clarification Sterile Filtration, regulate pH value to 3.40～3.80 simultaneously, after ultrafiltration dealcoholysis, regulate again pH value to 3.8～4.4.
Step 6, step 5 gained secondary sedimentation solution is pressed to trimmed size preparation, make protein content be not less than 50g/l, add maltose, making maltose content is 90～110g/l, add Tween-80, make Tween-80 content be not more than 80 μ g/ml, regulate pH value to 3.8～4.4, realize after goods preparation, carry out Sterile Filtration packing.
Described step 1 is that the temperature of the human plasma after merging is controlled between 0.0～4.0 ℃, regulate protein concn to 40～65g/l, the specific conductivity of regulator solution is 12.0～14.0ms/cm, regulate pH value to 6.80～7.30, regulate alcohol concn to volume percent 7.0～10.0%, control temperature to-1.0～-3.0 ℃, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-1 ℃, filter to isolate F I precipitation, obtain F I supernatant liquor.
Described step 2 is, regulate F I supernatant liquor pH value to 5.70～6.30, alcohol concn to volume percent 18.0～22.0%, control temperature to-4.0～-6.0 ℃, according to every 16Kg protein, add the ratio of 0.5～1.5Kg and add respectively perlite and the diatomite of equivalent, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.7 μ m, control pressure filter temperature to below-3 ℃, filter to isolate F II+III precipitation.
Described step 3 is, getting weight is 8～10 times of above-mentioned F II+III precipitations, temperature is that the water for injection of 2～8 ℃ dissolves F II+III precipitation completely, regulate pH value to 4.60～5.00, after stirring reaction 1～2 hour, again the pH value of solution is adjusted to 5.00～5.40, carry out again stirring reaction 1～2 hour, regulator solution specific conductivity is 1.20～1.50ms/cm, regulate alcohol concn to volume percent 13～15%, react 1～3 hour, in every kilogram of F II+III precipitation, add the ratio of 0.07～0.1kg and add respectively perlite and the diatomite of equivalent, continue reaction 0.5～1.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.7 μ m, control pressure filter temperature to below-1 ℃, filter to isolate F III precipitation, get F III supernatant.
Described step 4 is, regulating F III supernatant liquor specific conductivity is that 6.50～8.50ms/cm, adjusting pH value to 7.00～7.40, alcohol concn are to volume percent 24～26%, control temperature to-6.0～-12.0 ℃, stirring reaction 1～3 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-3 ℃, filter to isolate F II precipitation.
Cold ethanol of the present invention extracts the method for quiet note human normal immunoglobulin, and separated F I, F II+III, F III, F II successively from human plasma make the refinement of extraction components precipitation lean, have shortened the separation cycle of component, are beneficial to the comprehensive utilization of human plasma.By controlling the temperature of pressure filter, avoided the intensification of goods pressure-filtering process to cause protein denaturation, simultaneously in precipitate and separate process using suitable specific conductivity, therefore improved the yield of immunoglobulin (Ig), the yield of immunoglobulin (Ig) can reach 5.2g/l above (yield of prior art immunoglobulin (Ig) is 4.8g/l).When refining to immunoglobulin purification, carried out precipitate and separate twice, twice precipitate and separate all adopted secondary clarification Sterile Filtration, by level Four, filters, and removed the insoluble particle in goods, improved the clarity of goods.The pH that carries out respectively solution before and after human normal immunoglobulin ultrafiltration controls, and reduces and removes the anticomplementary activity in human normal immunoglobulin, has improved the validity of human normal immunoglobulin.
Table one: the main quality index of product
| Project | IgG monomer and dimer | Purity | PKA | ACA | The rate of recovery |
| Quiet note human normal immunoglobulin | ≥99.1% | ≥98.9% | ≤10IU/ml | ≤10% | ≥65% |
Accompanying drawing explanation
Fig. 1 is schema of the present invention.
Embodiment
In order further to explain technical scheme of the present invention, below in conjunction with accompanying drawing, the present invention will be described in detail.
Embodiment mono-
Referring to Fig. 1, this cold ethanol extracts the method for quiet note human normal immunoglobulin, take human plasma as raw material, adopts cold ethanol method and filter press technique to produce human normal immunoglobulin, and production operation pressure is less than 0.25MPa, comprises the following steps:
Step 1, from blood plasma separated F I:
The temperature of the human plasma after merging is controlled to 0.0 ℃, regulate protein concn to 40g/l, the specific conductivity of regulator solution is 12.0ms/cm, pH value to 6.80, alcohol concn is to volume percent 7.0%, control temperature to-1.0 ℃, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.9 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F I precipitation, obtain F I supernatant liquor.
Step 2, from F I supernatant liquor, isolate F II+III precipitation:
Regulate F I supernatant liquor pH value to 5.70, alcohol concn to volume percent 18.0%, control temperature to-4.0 ℃, after precipitation produces completely, according to every 16Kg protein, add the ratio of 0.5Kg and add respectively perlite and the diatomite (being each 0.25Kg of perlite and diatomite) of equivalent, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.7 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II+III precipitation.
Step 3, from F II+III precipitation separated F III precipitation:
Getting weight is 8 times of above-mentioned F II+III precipitations, temperature is that the water for injection of 2 ℃ dissolves F II+III precipitation completely, regulate pH value to 4.60, after stirring reaction 1 hour, again the pH value of solution is adjusted to 5.00, carry out again stirring reaction 1 hour, by water for injection regulator solution specific conductivity, the specific conductivity that makes solution is 1.20ms/cm, regulate alcohol concn to volume percent 13%, react 1 hour, after precipitation produces completely, in every kilogram of F II+III precipitation, add the ratio of 0.07kg and add respectively perlite and the diatomite (being each 0.035Kg of perlite and diatomite) of equivalent, continue reaction 0.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.7 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F III precipitation, get F III supernatant liquor.
Step 4, from F III supernatant liquor, isolate F II precipitation:
Regulate F III supernatant liquor specific conductivity, the specific conductivity that makes solution is 6.50ms/cm, adjusting pH value to 7.00, the concentration of ethanol is to volume percent 24%, control temperature to-6.0 ℃, stirring reaction 1 hour, after precipitation produces completely, carries out pressure filtration with pressure filter, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.9 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II precipitation.
Step 5, the ultrafiltration of F II precipitation, purifying, comprise the following steps:
1., ultrafiltration dealcoholysis: getting weight is that 8 times of above-mentioned F II precipitations, temperature are that 2 ℃ of waters for injection dissolve F II precipitation completely, adopts secondary clarification Sterile Filtration, regulates pH value to 4.30;
2., pasteurization: the solution after dealcoholysis is adjusted to protein content to 17g/l with water for injection, add protective material glycine by 60g/l, regulate pH value to 7.10, heat at the temperature of 55 ℃ and carry out for 9 hours, after pasteurization, being cooled to 20 ℃;
3., primary sedimentation is separated: with water for injection, regulate the solution after pasteurization, making protein content in solution is 5.0g/l, regulator solution pH to 6.70, control temperature to 0 ℃, regulate alcohol concn to volume percent 18%, control temperature to 2 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.20ms/cm, stirring reaction 1 hour, after precipitation produces completely, in every kilogram of protein 0.08kg ratio, add respectively perlite and the diatomite (being each 0.04Kg of perlite and diatomite) of equivalent, continue reaction after 0.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.9 μ m) deep layer filter plate, control below pressure filter temperature to 5 ℃, filter to isolate primary sedimentation, obtain primary sedimentation supernatant liquor,
4., secondary sedimentation is separated: primary sedimentation supernatant liquor temperature is controlled to-1 ℃, regulate pH value to 7.10, alcohol concn is to volume percent 24%, control solution temperature to-6 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.50ms/cm, stirring reaction 1 hour, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.9 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate secondary sedimentation;
5., secondary sedimentation ultrafiltration dealcoholysis: the water for injection of getting weight and be 5 times of above-mentioned secondary sedimentations, temperature and be 2 ℃ dissolves secondary sedimentation completely, adopt secondary clarification Sterile Filtration, regulate pH value to 3.40 simultaneously, carry out ultrafiltration dealcoholysis, after ultrafiltration dealcoholysis, regulate again pH value to 3.8.
Step 6, step 5 gained secondary sedimentation solution is pressed to trimmed size preparation, make protein content be not less than 50g/l(as 50g/l), adding maltose to make maltose content is 90g/l, add Tween-80 to make Tween-80 content be not more than 80 μ g/ml(as 78 μ g/ml), regulate pH value to 3.8 to realize after goods preparation, carry out Sterile Filtration packing.
The inspection of goods censorship after packing, lamp, the goods after packing are placed on to incubate puts chamber, and goods are to incubate and put at least 24 days at 23 ℃ in pH3.8, temperature, put to incubate rear goods and by bottle, carry out outward appearance and visible foreign matters inspection, the qualified rear packing of lamp inspection, warehouse-in.
Embodiment bis-
Referring to Fig. 1, this cold ethanol extracts the method for quiet note human normal immunoglobulin, take human plasma as raw material, adopts cold ethanol method and filter press technique to produce human serum albumin, and production operation pressure is less than 0.25MPa, comprises the following steps:
Step 1, from blood plasma separated F I:
The temperature of the human plasma after merging is controlled to 2.0 ℃, regulate protein concn to 52.5g/l, the specific conductivity of regulator solution is 13.1ms/cm, regulates pH value to 7.05, alcohol concn is to volume percent 8.5%, control temperature to-2.0 ℃, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.6 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F I precipitation, get F I supernatant liquor.
Step 2, from F I supernatant liquor separated F II+III:
Regulate F I supernatant liquor pH value to 6.00, alcohol concn to volume percent 20.0%, control temperature to-5.0 ℃, after precipitation produces completely, according to every 16Kg protein, add the ratio of 1Kg and add respectively perlite and the diatomite (being each 0.5Kg of perlite and diatomite) of equivalent, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.4 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II+III precipitation.
Step 3, from F II+III precipitation separated F III precipitation:
Getting weight is 9 times of above-mentioned F II+III precipitations, temperature is that the water for injection of 5 ℃ dissolves F II+III precipitation completely, regulate pH value to 4.80, after stirring reaction 1.5 hours, again the pH value of solution is adjusted to 5.20, carry out again stirring reaction 1.5 hours, by water for injection regulator solution specific conductivity, the specific conductivity that makes solution is 1.35ms/cm, regulate alcohol concn to volume percent 14%, react 2 hours, after precipitation produces completely, in every kilogram of F II+III precipitation, add the ratio of 0.09kg and add respectively perlite and the diatomite (being each 0.045Kg of perlite and diatomite) of equivalent, continue reaction 1 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.4 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F III precipitation, get F III supernatant liquor.
Step 4, from F III supernatant liquor, isolate F II precipitation:
Regulate F III supernatant liquor specific conductivity, the specific conductivity that makes solution is 7.50ms/cm, the concentration that regulates pH value to 7.20, ethanol to volume very 25%, control extremely-9.0 ℃ of temperature, stirring reaction 2 hours, after precipitation produces completely, carries out pressure filtration with pressure filter, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.6 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II precipitation.
Step 5, the ultrafiltration of F II precipitation, purifying, comprise the following steps:
1., ultrafiltration dealcoholysis: getting weight is that 9 times of above-mentioned F II precipitations, temperature are that 5 ℃ of waters for injection dissolve F II precipitation completely, adopts secondary clarification Sterile Filtration, regulates pH value to 4.50;
2., pasteurization: the solution after dealcoholysis is adjusted to protein content to 25g/l with water for injection, add protective material glycine by 80g/l, regulate pH value to 7.30, heat at the temperature of 60 ℃ and carry out for 10 hours, after pasteurization, being cooled to 25 ℃;
3., primary sedimentation is separated: with water for injection, regulate the solution after pasteurization, making protein content in solution is 10.0g/l, regulator solution pH to 6.95, control temperature to 1 ℃, regulate alcohol concn to volume percent 19%, control temperature to 4 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.35ms/cm, stirring reaction 2 hours, after precipitation produces completely, the perlite and the diatomite (being each 0.5Kg of perlite and diatomite) that in every kilogram of protein, add 0.10kg ratio, continue reaction after 1 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.6 μ m) deep layer filter plate, control below pressure filter temperature to 5 ℃, filter to isolate primary sedimentation, obtain primary sedimentation supernatant,
4., secondary sedimentation is separated: primary sedimentation supernatant liquor temperature is controlled to-2 ℃, regulated pH to 7.25, alcohol concn is to volume percent 25%, control solution temperature to-8 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.70ms/cm, stirring reaction 2 hours, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.6 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate secondary sedimentation;
5., secondary sedimentation ultrafiltration dealcoholysis: the water for injection of getting weight and be 7.5 times of above-mentioned secondary sedimentations, temperature and be 5 ℃ dissolves secondary sedimentation completely, adopt secondary clarification Sterile Filtration, regulate pH to 3.60 simultaneously, carry out ultrafiltration dealcoholysis, after ultrafiltration dealcoholysis, regulate again pH value to 4.1.
Step 6, will be after the ultrafiltration of step 5 gained secondary sedimentation solution by trimmed size preparation protein content, be not less than 50g/l(as 60g/l), adding maltose to make maltose content is 100g/l, add Tween-80, make Tween-80 content be not more than 80 μ g/ml(as 70 μ g/ml), regulate pH value to 4.1 to realize after goods preparation, carry out Sterile Filtration packing.
The inspection of goods censorship after packing, lamp, the goods after packing are placed on to incubate puts chamber, and goods are to incubate and put at least 24 days at 24 ℃ in pH4.1, temperature, put to incubate rear goods and by bottle, carry out outward appearance and visible foreign matters inspection, the qualified rear packing of lamp inspection, warehouse-in.
Embodiment tri-
Referring to Fig. 1, this cold ethanol extracts the method for quiet note human normal immunoglobulin, take human plasma as raw material, adopts cold ethanol method and filter press technique to produce human serum albumin, and production operation pressure is less than 0.25MPa, comprises the following steps:
Step 1, from blood plasma separated F I:
The temperature of the human plasma after merging is controlled to 4.0 ℃, regulate protein concn to 65g/l, the specific conductivity of regulator solution is 14.0ms/cm, regulates pH value to 7.30, alcohol concn is to volume percent 10.0%, control temperature to-3.0 ℃, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.3 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F I precipitation, get F I supernatant liquor.
Step 2, from F I supernatant liquor, isolate F II+III precipitation:
Regulate F I supernatant liquor pH value to 6.30, alcohol concn to volume percent 22.0%, control temperature to-6.0 ℃, after precipitation produces completely, according to every 16Kg protein, add the ratio of 1.5Kg and add respectively perlite and the diatomite of equivalent, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.2 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II+III precipitation.
Step 3, from F II+III precipitation separated F III precipitation:
Getting weight is 10 times of above-mentioned F II+III precipitations, temperature is that the water for injection of 8 ℃ dissolves F II+III precipitation completely, regulate pH value to 5.00, after stirring reaction 2 hours, again the pH value of solution is adjusted to 5.40, carry out again stirring reaction 2 hours, by sodium-chlor regulator solution specific conductivity, the specific conductivity that makes solution is 1.50ms/cm, regulate alcohol concn to volume percent 15%, react 3 hours, after precipitation produces completely, in every thousand can F II+ratio that III precipitation is added 0.1kg adds respectively perlite and the diatomite (being each 0.05Kg of perlite and diatomite) of equivalent, continue reaction 1.5 hours, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.7 μ m(as 0.2 μ m) deep layer filter plate, control pressure filter temperature to below-1 ℃, filter to isolate F III precipitation, obtain F III supernatant liquor.
Step 4, from F III supernatant liquor, isolate F II precipitation:
Regulate F III supernatant liquor specific conductivity, the specific conductivity that makes solution is 8.50ms/cm, regulates pH value to 7.40, and the concentration of ethanol is to volume percent 26%, control temperature to-12.0 ℃, stirring reaction 3 hours, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.3 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate F II precipitation, obtain F II precipitation.
Step 5, the ultrafiltration of F II precipitation, purifying, comprise the following steps:
1., ultrafiltration dealcoholysis: getting weight is that 10 times of above-mentioned F II precipitations, temperature are that 8 ℃ of waters for injection dissolve F II precipitation completely, adopts secondary clarification Sterile Filtration, regulates pH value to 4.70;
2., pasteurization: the solution after dealcoholysis is adjusted to protein content to 33g/l with water for injection, add protective material glycine by 120g/l, regulate pH value to 7.50, heat at the temperature of 65 ℃ and carry out for 11 hours, after pasteurization, being cooled to 30 ℃;
3., primary sedimentation is separated: with water for injection, regulate the solution after pasteurization, making protein content in solution is 15.0g/l, regulator solution pH to 7.20, control temperature to 2 ℃, regulate alcohol concn to volume percent 20%, control temperature to 6 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.50ms/cm, stirring reaction 3 hours, after precipitation produces completely, in every kilogram of protein, add the ratio of 0.12kg to add respectively perlite and the diatomite (being each 0.06Kg of perlite and diatomite) of equivalent, continue reaction after 1.5 hours, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.3 μ m) deep layer filter plate, control below pressure filter temperature to 5 ℃, filter to isolate primary sedimentation, obtain primary sedimentation supernatant,
4., secondary sedimentation is separated: primary sedimentation supernatant liquor temperature is controlled to-3 ℃, regulate pH value to 7.40, alcohol concn is to volume percent 26%, control solution temperature to-12 ℃, regulator solution specific conductivity, the specific conductivity that makes solution is 1.90ms/cm, stirring reaction 3 hours, after precipitation produces completely, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than 0.9 μ m(as 0.3 μ m) deep layer filter plate, control pressure filter temperature to below-3 ℃, filter to isolate secondary sedimentation;
5., secondary sedimentation ultrafiltration dealcoholysis: the water for injection of getting weight and be 10 times of above-mentioned secondary sedimentations, temperature and be 8 ℃ dissolves secondary sedimentation completely, adopt secondary clarification Sterile Filtration, regulate pH value to 3.80 simultaneously, carry out ultrafiltration dealcoholysis, after ultrafiltration dealcoholysis, regulate again pH value to 4.4.
Step 6, step 5 gained secondary sedimentation solution is pressed to trimmed size preparation, make protein content be not less than 50g/l(as 70g/l), add maltose, making maltose content is 110g/l, add Tween-80 to make Tween-80 content be not more than 80 μ g/ml(as 60 μ g/ml), pH regulator to 4.4 is realized after goods preparation, carries out Sterile Filtration packing.
The inspection of goods censorship after packing, lamp, the goods after packing are placed on to incubate puts chamber, and goods are 4.4 at pH, and temperature is to incubate and put at least 24 days at 25 ℃, puts to incubate rear goods and by bottle, carry out outward appearance and visible foreign matters inspection, the qualified rear packing of lamp inspection, warehouse-in.
Claims (5)
1. cold ethanol extracts a method for quiet note human normal immunoglobulin, it is characterized in that: comprise the following steps:
Step 1, from human plasma separated F I, obtain F I supernatant liquor;
Step 2, from F I supernatant liquor, isolate F II+III precipitation;
Step 3, from F II+III precipitation separated F III precipitation, obtain F III supernatant liquor;
Step 4, from F III supernatant liquor, isolate F II precipitation;
Step 5, the ultrafiltration of F II precipitation, purifying, comprise the following steps:
1., ultrafiltration dealcoholysis: getting weight is that 8～10 times of above-mentioned F II precipitations, temperature are that 2～8 ℃ of waters for injection will
F II precipitation is dissolved completely, adopts secondary clarification Sterile Filtration, regulator solution pH value to 4.30～4.70;
2., pasteurization: the solution after ultrafiltration dealcoholysis is regulated to protein content to 17～33g/l with water for injection, by 60～120g/l, add glycine, regulate pH value to 7.10～7.50, at the temperature of 55～65 ℃, heat and within 9～11 hours, carry out, after pasteurization, being cooled to 20～30 ℃;
3., primary sedimentation is separated: with water for injection, regulate the solution after pasteurization, making protein content in solution is 5.0～15.0g/l, regulator solution pH value to 6.70～7.20, control temperature to 0～2 ℃, regulate alcohol concn to volume percent 18～20%, control temperature to 2～6 ℃, the specific conductivity of regulator solution is 1.20～1.50ms/cm, stirring reaction 1～3 hour, in every kilogram of protein 0.08～0.12kg ratio, add respectively perlite and the diatomite of equivalent, continue reaction after 0.5～1.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control below pressure filter temperature to 5 ℃, filter to isolate primary sedimentation, obtain primary sedimentation supernatant liquor,
4., secondary sedimentation is separated: primary sedimentation supernatant liquor temperature is controlled to-1～-3 ℃, regulate pH value to 7.10～7.40, alcohol concn, to volume percent 24～26%, is controlled solution temperature to-6～-12 ℃, and the specific conductivity of regulator solution is 1.50～1.90ms/cm, stirring reaction 1～3 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-3 ℃, filter to isolate secondary sedimentation;
5., secondary sedimentation ultrafiltration dealcoholysis: the water for injection of getting weight and be 5～10 times of above-mentioned secondary sedimentations, temperature and be 2～8 ℃ dissolves secondary sedimentation completely, adopt secondary clarification Sterile Filtration, regulate pH value to 3.40～3.80 simultaneously, after ultrafiltration dealcoholysis, regulate again pH value to 3.8～4.4.
Step 6, step 5 gained secondary sedimentation solution is pressed to trimmed size preparation, make protein content be not less than 50g/l, add maltose, making maltose content is 90～110g/l, add Tween-80, make Tween-80 content be not more than 80 μ g/ml, regulate pH value to 3.8～4.4, realize after goods preparation, carry out Sterile Filtration packing.
2. cold ethanol extracts the method for quiet note human normal immunoglobulin according to claim 1, it is characterized in that: described step 1 is that the temperature of the human plasma after merging is controlled between 0.0～4.0 ℃, regulate protein concn to 40～65g/l, the specific conductivity of regulator solution is 12.0～14.0ms/cm, regulate pH value to 6.80～7.30, regulate alcohol concn to volume percent 7.0～10.0%, control temperature to-1.0～-3.0 ℃, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-1 ℃, filter to isolate F I precipitation, obtain F I supernatant liquor.
3. cold ethanol extracts the method for quiet note human normal immunoglobulin according to claim 1, it is characterized in that: described step 2 is, regulate F I supernatant liquor pH value to 5.70～6.30, alcohol concn to volume percent 18.0～22.0%, control temperature to-4.0～-6.0 ℃, according to every 16Kg protein, add the ratio of 0.5～1.5Kg and add respectively perlite and the diatomite of equivalent, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.7 μ m, control pressure filter temperature to below-3 ℃, filter to isolate
F II+III precipitation.
4. cold ethanol extracts the method for quiet note human normal immunoglobulin according to claim 1, it is characterized in that: described step 3 is, getting weight is 8～10 times of above-mentioned F II+III precipitations, temperature is that the water for injection of 2～8 ℃ dissolves F II+III precipitation completely, regulate pH value to 4.60～5.00, after stirring reaction 1～2 hour, again the pH value of solution is adjusted to 5.00～5.40, carry out again stirring reaction 1～2 hour, regulator solution specific conductivity is 1.20～1.50ms/cm, regulate alcohol concn to volume percent 13～15%, react 1～3 hour, in every kilogram of F II+III precipitation, add the ratio of 0.07～0.1kg and add respectively perlite and the diatomite of equivalent, continue reaction 0.5～1.5 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.7 μ m, control pressure filter temperature to below-1 ℃, filter to isolate F III precipitation, get F III supernatant.
5. cold ethanol extracts the method for quiet note human normal immunoglobulin according to claim 1, it is characterized in that: described step 4 is, regulating F III supernatant liquor specific conductivity is that 6.50～8.50ms/cm, adjusting pH value to 7.00～7.40, alcohol concn are to volume percent 24～26%, control temperature to-6.0～-12.0 ℃, stirring reaction 1～3 hour, with pressure filter, carry out pressure filtration, during pressure filtration, select aperture to be no more than the deep layer filter plate of 0.9 μ m, control pressure filter temperature to below-3 ℃, filter to isolate F II precipitation.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104558163A (en) * | 2014-12-18 | 2015-04-29 | 华兰生物工程重庆有限公司 | Method for purifying human immune globulin from component III precipitate |
| WO2016088135A1 (en) | 2014-12-02 | 2016-06-09 | Hemarus Therapeutics Ltd. | A process for increased yield of immunoglobulin from human plasma |
| CN106699835A (en) * | 2017-01-12 | 2017-05-24 | 苏州美极医疗科技有限公司 | Full-automatic plasma processing equipment and method |
| CN108003236A (en) * | 2017-11-06 | 2018-05-08 | 山东泰邦生物制品有限公司 | A kind of human immunoglobulin(HIg) F II is precipitated and filter-pressing process |
| CN110054685A (en) * | 2019-04-26 | 2019-07-26 | 兰州兰生血液制品有限公司 | A kind of production method of intravenous human immunoglobulin(HIg) (pH4) cold ethanol component III |
| CN111110876A (en) * | 2020-01-20 | 2020-05-08 | 华兰生物工程重庆有限公司 | Specific human immunoglobulin virus inactivation process |
| CN111499736A (en) * | 2020-04-28 | 2020-08-07 | 国药集团武汉血液制品有限公司 | Preparation method of intravenous injection COVID-19 human immunoglobulin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101249265B (en) * | 2008-04-11 | 2010-10-27 | 三九集团湛江开发区双林药业有限公司 | Intravenous injection employ persons hepatitis B immune globulin and method of preparing the same |
| EP2270044A1 (en) * | 1998-06-09 | 2011-01-05 | CSL Behring AG | Liquid immunoglobulin G (lgG) product |
| CN101972479A (en) * | 2010-11-08 | 2011-02-16 | 江西博雅生物制药股份有限公司 | Preparation process of intravenous injection human immunoglobulin |
| CN102552906A (en) * | 2012-01-11 | 2012-07-11 | 西安回天血液制品有限责任公司 | Productive technology of intravenous injection human immunoglobulin |
| CN102584934A (en) * | 2012-03-19 | 2012-07-18 | 江西博雅生物制药股份有限公司 | Process for preparing human immunoglobulin for intravenous injection |
-
2014
- 2014-01-03 CN CN201410003241.1A patent/CN103665100A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2270044A1 (en) * | 1998-06-09 | 2011-01-05 | CSL Behring AG | Liquid immunoglobulin G (lgG) product |
| CN101249265B (en) * | 2008-04-11 | 2010-10-27 | 三九集团湛江开发区双林药业有限公司 | Intravenous injection employ persons hepatitis B immune globulin and method of preparing the same |
| CN101972479A (en) * | 2010-11-08 | 2011-02-16 | 江西博雅生物制药股份有限公司 | Preparation process of intravenous injection human immunoglobulin |
| CN102552906A (en) * | 2012-01-11 | 2012-07-11 | 西安回天血液制品有限责任公司 | Productive technology of intravenous injection human immunoglobulin |
| CN102584934A (en) * | 2012-03-19 | 2012-07-18 | 江西博雅生物制药股份有限公司 | Process for preparing human immunoglobulin for intravenous injection |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016088135A1 (en) | 2014-12-02 | 2016-06-09 | Hemarus Therapeutics Ltd. | A process for increased yield of immunoglobulin from human plasma |
| US9556253B2 (en) | 2014-12-02 | 2017-01-31 | Hemarus Therapeutics Limited | Process for increased yield of immunoglobulin from human plasma |
| CN104558163A (en) * | 2014-12-18 | 2015-04-29 | 华兰生物工程重庆有限公司 | Method for purifying human immune globulin from component III precipitate |
| CN106699835A (en) * | 2017-01-12 | 2017-05-24 | 苏州美极医疗科技有限公司 | Full-automatic plasma processing equipment and method |
| CN106699835B (en) * | 2017-01-12 | 2020-03-20 | 苏州美极医疗科技有限公司 | Full-automatic plasma treatment equipment and treatment method |
| CN108003236A (en) * | 2017-11-06 | 2018-05-08 | 山东泰邦生物制品有限公司 | A kind of human immunoglobulin(HIg) F II is precipitated and filter-pressing process |
| CN110054685A (en) * | 2019-04-26 | 2019-07-26 | 兰州兰生血液制品有限公司 | A kind of production method of intravenous human immunoglobulin(HIg) (pH4) cold ethanol component III |
| CN111110876A (en) * | 2020-01-20 | 2020-05-08 | 华兰生物工程重庆有限公司 | Specific human immunoglobulin virus inactivation process |
| CN111499736A (en) * | 2020-04-28 | 2020-08-07 | 国药集团武汉血液制品有限公司 | Preparation method of intravenous injection COVID-19 human immunoglobulin |
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