CN104193822A - Process for preparing rabies human immune globulin - Google Patents
Process for preparing rabies human immune globulin Download PDFInfo
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- CN104193822A CN104193822A CN201410454232.4A CN201410454232A CN104193822A CN 104193822 A CN104193822 A CN 104193822A CN 201410454232 A CN201410454232 A CN 201410454232A CN 104193822 A CN104193822 A CN 104193822A
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Abstract
The invention discloses a process for preparing rabies human immune globulin. According to the process, immune globulin is extracted and separated by using a low-temperature ethanol filter press technique, a chromatography method, an ultrafiltration method, a nano filtering method, a secondary ultrafiltration method and a washing, baking and filling linked sub-packaging process technique. The process is characterized in that a one-step ion exchange chromatography method is adopted as the chromatography method, the component III supernate is firstly refined before chromatography, then the premasters of the refined component III supernate are adjusted so that the protein concentration of the refined component III supernate is 3-6%, the pH value of the refined component III supernate is 6.4-6.6, and the conductivity is 0.18-0.22s/m when the temperature is 19 DEG C, after the adjustment, column chromatography purification is performed by using an ion exchange column, DEAESepharoseFF packing is adopted in chromatography, and the pH value is adjusted to be 3.8-4.0 after chromatography is completed. Compared with a conventional process, the process has the advantages that the yield is increased by 19.2-25%, small dosage is needed, and the use of human immune globulin is reduced, so that the scarce plasma resource is saved.
Description
Technical field
The preparation technology who the present invention relates to a kind of rabies human immunoglobulin, belongs to field of biological pharmacy.
Background technology
Rabies are that a kind of pathogenic agent is the elasticity virus of Yeast Nucleic Acid, add up according to the World Health Organization, there is rubies epidemiology more than 2/3rds countries and regions, the whole nation, China and south east asia are in rabies district occurred frequently, the world, its case accounts for the whole world more than 70%, once rabies morbidity, the method that there is no is at present cured, and mortality ratio almost reaches 100%.If enough rabies human immunoglobulins are provided in time, make in its patients serum more than antibody reaches 0.5IU/ml, can to make patient be effectively protected.Therefore, preparation for the rabic specifics rabies human immunoglobulin for the treatment of is necessary, and with regard to the production technology of current rabies human normal immunoglobulin, the single cold ethanol separating technology of the many employings of product on domestic market, separating step is many, the yield of immunoglobulin (Ig) is low, causes domestic rabies immune globulin shortage.In prior art, as " high-purity rabies human immunoglobulin and feedstock production process thereof " CN200710114839.8, described method is to comprise the raw blood plasma, cold ethanol fractional separation, ion exchange chromatography purifying, the processing of incubated at low pH inactivation of viruses, filtering virus, ultrafiltration, dosing, the degerming excessively that are greater than 10IU/ml for the preparation of the antibody titer of producing rabies human immunoglobulin.The high-purity rabies human immunoglobulin obtaining, the rabies antibody that contains high-titer, purity exceedes 98%, IgG monomer and dimer content is greater than 99%, and rabies antibody is tired and is more than or equal to 150IU/ml, and protein content is less than 120g/L.Can make patient be effectively protected, and can reduce the consumption of product and patient's painful and burden.
In immunoglobulin (Ig) market still under the reality in short situation, obtain rabies human immunoglobulin for effectively extracting to separate, particularly carry high product yield, obtain safe and reliable product, the preparation technology's of rabies human immunoglobulin further optimization is by significant.
Summary of the invention
The object of the present invention is to provide the preparation technology of a kind of high yield, highly purified rabies human immunoglobulin.
The present invention is achieved like this, a kind of rabies human immunoglobulin preparation technology, it comprises cold ethanol filter press technique successively, chromatography, ultrafiltration process, nanofiltration method and washing, drying and pouring linkage packing Technology are extracted separating immune globulin, chromatography is a step ion exchange chromatography, before chromatography, first refine component III supernatant liquor, again the refining component III supernatant liquor parameter obtaining is adjusted, the protein concentration of refining component III supernatant liquor is adjusted to 3～6%, pH to 6.4～6.6, it is 0.18～0.22s/m that specific conductivity is surveyed in the time of T=19 DEG C, regulate and carry out upper column chromatography purification with ion exchange column afterwards, chromatography uses DEAE Sepharose FF filler, the complete pH that regulates again of chromatography is 3.8～4.0.
Its concrete technology scheme is as follows:
(1), by quarantining quarantine, qualified human plasma is centrifugal, centrifugal go out liquid temp be controlled at 0 ~ 4 DEG C; Separate cryoprecipitate, cryoprecipitate is produced for the VIII factor, and its supernatant liquor is delivered to albumen sepn retort, makes the temperature of protein liquid between 1～3 DEG C, and slowly adding pH4.0 acetate buffer solution to regulate pH value is 6.70～7.20; Add-15 DEG C of 95% or 50% following ethanol, make ethanol final concentration to 8%(V/V), temperature is controlled at-1～-3 DEG C, and adding pH value after ethanol is 6.80～7.20;
(2), the thing that makes of step (1) is stirred more than 30min, carry out centrifugal, centrifugal go out liquid temp be controlled at-1～-3 DEG C, centrifugal component I precipitation and components I supernatant liquor; Component I precipitation is deposited in freezer or is directly used in scleroproein original production;
(3), the thing component I supernatant liquor that makes of step (2) is added in pH4.0 acetate buffer solution to adjusting pH value to 6.7～6.9; Adding-15 DEG C of following 95% ethanol to alcohol concn is 20%(V/V), temperature is controlled at-4～-6 DEG C; After adding ethanol, pH should be 6.80～7.00; After stirring more than 120min, more than standing 60min, then open and stir, add 18g diatomite by every liter of reaction solution, carry out press filtration, press filtration obtains compositionⅱ+III precipitation and compositionⅱ+III supernatant liquor, and compositionⅱ+III supernatant liquor is for human serum albumin production;
(4), by step (3) make thing compositionⅱ+III precipitation, add the water for injection of 10～15 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, be cooled to 3～5 DEG C, pour compositionⅱ+III precipitation in retort stirring and dissolving; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5. 80～5.85, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 19%; Reaction solution outlet temperature is controlled at-4～-6 DEG C, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-4 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-4～-6 DEG C, filter pressure is no more than 0.2Mpa, collects refining compositionⅱ+III precipitation;
(5), by step (4) make the refining compositionⅱ of thing+III precipitation, add the water for injection of 10～15 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, while being cooled to 2～4 DEG C, will refining compositionⅱ+III precipitation and pour stirring and dissolving in retort into; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5.1～5.4, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 17%; Reaction solution outlet temperature is controlled at-5～-7 DEG C, and pH is 5.2～5.4, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-5 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-5～-7 DEG C, obtains component III precipitation and component III supernatant liquor;
(6) making after the metering of thing component III supernatant liquor step (5), start retort agitator and refrigeration cycle, controlling temperature is-5～-7 DEG C, carries out Depth Filtration, filter out liquid temp and be controlled at-5～-7 DEG C, after filtering, regulating pH with the HCl of 1.0mol/L is 4.0 left and right; More than protein concentrate concentration to 5%, dialyse with 2～8 DEG C of waters for injection of 5 times of volumes, dialyse and obtain and refine component III supernatant liquor; Protein concentration is adjusted to 3～6%, with NaOH tune pH to 6.4～6.6 of 0.5mol/L, then adding 1mol/L phosphoric acid-NaOH damping fluid regulates specific conductivity in the time of T=19 DEG C, to survey is 0.18～0.22s/m, regulate and carry out upper column chromatography purification with ion exchange column afterwards, chromatography uses DEAE Sepharose FF filler; It is 3.8～4.0 that the complete HCl with 1.0mol/L of chromatography regulates pH, opening ultrafilter is concentrated into more than 5%, dialyse with 2～8 DEG C of waters for injection, make ethanol content≤0.025%, then protein liquid is concentrated into more than 6%, the maltose of calculated amount is added in protein liquid, by the HCl adjusting pH value of 1.0mol/L, make maltose content 10 ± 1%, pH is 3.9～4.3, protein content 5.5 ± 0.5%;
(7), the protein liquid making after thing preparation of step (6) is carried out to Sterile Filtration in 6h; After Sterile Filtration, goods were placed on incubated at low pH chamber, through 24 ± 1 DEG C of incubated at low pHs 21 days; Incubate to put and finish the rear DV50 of using filter core except virus filtration;
(8), by protein liquid after the making thing and filter of step (7), with the 0.85%NaCl solution ultrafiltration of 2～8 DEG C, carry out five times of equal-volumes and wash, washing finishes rear concentrated, rare joining, adjustment glycine is 26～30g/L, and adjusting goods pH value is 6.6～7.2, tire >=100IU/mL of rabies antibody;
(9), step (8) made to the rare protein liquid of joining of thing through 0.2 μ m degerming filter element filtering packing; Goods censorship after packing, lamp inspection, pack after the assay was approved, put in storage;
Described percentage ratio is apart from outside limiting, and all the other are mass percent.
The aqueous solution that described pH4.0 acetate buffer solution is made up of sodium acetate and glacial acetic acid, in every premium on currency, adding 244.9mL mass concentration is 99% glacial acetic acid and 65.6g sodium acetate.
Component I supernatant liquor and precipitation thereof, the universal classification method of technical field under compositionⅱ+III supernatant liquor and precipitation thereof are; Also be by the title of material after universal classification classification in the present invention, do not refer to simple sequence number.
By human plasma, through centrifugation cryoprecipitate, the centrifuged supernatant after separation, by regulating pH and temperature, is added ethanol centrifugal component I supernatant liquor and the precipitation thereof of obtaining again;
By component I supernatant liquor is regulated to pH and temperature, interpolation ethanol again press filtration obtains compositionⅱ+III supernatant liquor and precipitation thereof;
That component I precipitation mainly contains is fine former, FV III, Ciq, Clr, Cls and Fiberonectin etc.;
Component I supernatant liquor mainly contains: albumin, IgG, IgA, IgM, F II, VII, IX, X, α, beta Globulin, copper-protein, alpha1-antitrypsin, IgM, AT-III complement component, Transferrins,iron complexes, haptoglobin etc.;
Compositionⅱ+III precipitation mainly contains IgG, IgA, IgM, F II, VII, IX, X, α, beta Globulin and copper-protein etc.;
Compositionⅱ+III supernatant liquor mainly contains: albumin, α, beta Globulin, copper-protein, alpha1-antitrypsin, IgM, AT-III complement component, Transferrins,iron complexes, haptoglobin etc.
Positively effect of the present invention:
The present invention combines Optimization Technology, adopt cold ethanol press filtration extraction and separation technology and step ion exchange chromatography (chromatogram) technology to obtain rabies human immunoglobulin in conjunction with extracting to separate, adopt secondary virus inactivation technology to remove virus simultaneously, can effectively improve product recovery rate, improve purity and the security thereof of product.Be specially:
1, adopt cold ethanol filter press technique, chromatography, ultrafiltration process, the Technologies such as nanofiltration method and washing, drying and pouring linkage packing, and through effective viral inactivation treatment, Technology maturation, stable, advanced.
2, clinical efficacy clear and definite, safe, can control rapidly the development of disease progression and epidemic situation, use and save rare blood plasma resource thereby using dosage is little, reduce quiet third simultaneously.
3, specific immunoglobulin derives from healthy human body, and its structure is without any transformation and modification, safe, and effect is clear and definite, and the R&D cycle is relatively short, and pharmacological effect effect is clear and definite, relatively easily goes out product, produces result.
The present invention is than the preparation technology of rabies human immunoglobulin compared to existing technology, and innovative point is:
1, by multistep-15 DEG C following cold ethanol method, step by step refining, by regulating the parameters such as temperature, PH, ionic strength, making successively whole alcohol concn is 8%, 20%, 19%, 17% to obtain component I supernatant liquor, compositionⅱ+III precipitation, refining compositionⅱ+III precipitation, component III supernatant liquor, and carry out filter pressing standby go out purity more than 95.0%, IgG monomer and the rabies human immunoglobulin of dimer total content more than 92.0%.Remaining ingredient can be used for the production of other blood productss simultaneously.
2, prior art is carried out purifying as " high-purity rabies human immunoglobulin and feedstock production process thereof " CN200710114839.8 adopts Q-Sepharose FF and DEAE Sepharose FF bis-step ion exchange chromatographies.It is concentration 3～6%, pH6.4～6.6, specific conductivity 0.18～0.22 s/m(T=19 DEG C that the present invention optimizes refining component III supernatant liquor protein liquid before chromatography) etc. go up again column purification after reaction parameter, chromatography is complete again with 1.0mol/L HCl adjusting pH 3.8～4.0.Foreign protein can be effectively removed in one step ion-exchange equally; improve purity; and by ultrafiltration, concentrated, the methods such as interpolation maltose protective material obtain the rabies human immunoglobulin liquid of the embodiment of the present invention 1 moderate purity 99.5%, IgG monomer and dimer total content 98.8%.The use of maltose, can protect immunoglobulin molecules functional performance not to be subject to the impact of temperature and time.Than two step ion exchange methods, a step chromatography and interpolation protective material simple possible, save operation, reduces production costs.
3, the present invention obtains the various process parameters such as optimum column chromatography by test, this technique can be produced 17887 bottles of/ton of blood plasma of rabies human immunoglobulin of 200IU/ bottle, 15000 bottles of/ton of blood plasma of immunoglobulin (Ig) of producing than traditional technology, the present invention can produce 2887 bottles of/ton of blood plasma more, and recovery rate can improve 19.2 ~ 25% compared with traditional technology.
The rabies human immunoglobulin of describing in product prepared by the inventive method and " Chinese Pharmacopoeia " (version in 2010, three) in the contrast of Key Quality Indicator as following table.
Brief description of the drawings
Fig. 1 is process flow sheet of the present invention.
Fig. 2 is the molecular size distribution result scintigram of preparing sample 1 in the embodiment of the present invention 1.
Fig. 3 is the molecular size distribution result scintigram of preparing sample 2 in the embodiment of the present invention 1.
Fig. 4 is the purity result scintigram of preparing product in the embodiment of the present invention 1.
Embodiment
The present invention is by the following examples can the invention will be further described, but scope of the present invention is not limited to following embodiment.
Embodiment 1: taking 5000 liters of blood plasma as example, concrete preparation technology is as follows:
(1), by quarantining quarantine, 5000 liters of qualified human plasmas are centrifugal, centrifugal go out liquid temp be controlled at 0 ~ 4 DEG C; Separate cryoprecipitate, cryoprecipitate is produced for the VIII factor, gets its supernatant liquor and is delivered to albumen sepn retort, and the temperature of protein liquid is controlled between 1～3 DEG C, and slowly adding pH4.0 acetate buffer solution to regulate pH value is 6.70～7.20; Add-15 DEG C of 95% or 50% following ethanol, make ethanol final concentration to 8%(V/V), temperature is controlled at-1～-3 DEG C, add ethanol after pH value should be 6.80～7.20; The aqueous solution that described pH4.0 acetate buffer solution is made up of sodium acetate and glacial acetic acid, in every premium on currency, adding 244.9mL mass concentration is 99% glacial acetic acid and 65.6g sodium acetate.
(2), the thing that makes of step (1) is stirred more than 30min, carry out centrifugal, centrifugal go out liquid temp be controlled at-1～-3 DEG C, centrifugal component I precipitation and components I supernatant liquor; Component I precipitation is deposited in freezer or is directly used in scleroproein original production.
(3), the thing component I supernatant liquor that makes of step (2) is added in pH4.0 acetate buffer solution to adjusting supernatant liquor pH value to 6.7～6.9; Adding-15 DEG C of following 95% ethanol to alcohol concn is 20%(V/V), temperature is controlled at-4～-6 DEG C; After adding ethanol, pH should be 6.80～7.00; After stirring more than 120min, more than standing 60min, open and stir, add 18g diatomite by every liter of reaction solution, carry out press filtration, the highest 0.20Mpa that is no more than of inlet hydraulic, press filtration obtains compositionⅱ+III precipitation and compositionⅱ+III supernatant liquor, and compositionⅱ+III supernatant liquor is produced for human serum albumin.
(4), by step (3) make 467 kilograms of thing compositionⅱ+III precipitations, add the water for injection of 13 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, while being cooled to 3～5 DEG C, pour compositionⅱ+III precipitation in retort stirring and dissolving; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5. 80～5.85, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 19%; Reaction solution outlet temperature is controlled at-4～-6 DEG C, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-4 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-4～-6 DEG C, filter pressure is no more than 0.2Mpa, collects refining compositionⅱ+III precipitation.
(5), by step (4) make 440 kilograms of the refining compositionⅱ of thing+III precipitations, add the water for injection of 13 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, while being cooled to 2～4 DEG C, will refining compositionⅱ+III precipitation and pour stirring and dissolving in retort into; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5. 1～5.4, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 17%; Reaction solution outlet temperature is controlled at-5～-7 DEG C, and pH is 5.2～5.4, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-5 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-5～-7 DEG C; Obtain component III precipitation and component III supernatant liquor;
(6), making after the metering of thing component III supernatant liquor step (5), start retort agitator and refrigeration cycle, controlling temperature is-5～-7 DEG C, carries out Depth Filtration, filter out liquid temp and be controlled at-5～-7 DEG C, after filtering, regulating pH with the HCl of 1.0mol/L is 4.0 left and right; After regulating PH, obtain 8563 kilograms of protein liquids, be concentrated into protein concentration more than 5%, after must concentrating, 600 liters of volumes, dialyse with 2～8 DEG C of waters for injection of 5 times of volumes, dialyse and obtain and refine component III supernatant liquor; Protein concentration is adjusted to 5.9%, with the NaOH tune pH to 6.53 of 0.5mol/L, then add 1mol/L phosphoric acid-NaOH damping fluid and regulate specific conductivity in the time of T=19 DEG C, to survey as 0.194s/m, regulate and carry out upper column chromatography purification, chromatography use DEAE Sepharose FF filler with ion exchange column afterwards; It is 3.8～4.0 that the complete HCl with 1.0mol/L of chromatography regulates pH, opening ultrafilter is concentrated into more than 5%, dialyse with 2～8 DEG C of waters for injection, make ethanol content≤0.025%, then protein liquid is concentrated into more than 6%, the maltose of calculated amount is added in protein liquid, with the HCl adjusting pH value of 1.0mol/L, maltose content 9.7% in final protein liquid, pH is 4.10, protein content 6.0%, 478.5 kilograms of solution weights.
(7), 478.5 kilograms of the protein liquids after thing preparation that make of step (6) are carried out to Sterile Filtration in 6h; After Sterile Filtration, goods were placed on incubated at low pH chamber, through 24 DEG C ± 1 DEG C incubated at low pH 21 days; Incubate to put and finish the rear DV50 of using filter core except virus filtration;
(8), the thing that makes of step (7) is filtered to rear protein liquid, with the 0.85%NaCl solution ultrafiltration of 2～8 DEG C, carry out five times of equal-volume washings, washing finishes rear albumen and is concentrated into 14.4% and carries out rare joining, adjustment glycine is 27g/L, adjusting goods pH value is 6..8, tire >=100IU/mL of rabies antibody.
(9), rare 185.4 kilograms of the protein liquids of joining of thing that make of step (8) are carried out to protein filtration through 0.2 μ m filter core, after filtration 183.2 kilograms of albumen liquid measures; Packing, packing loading amount is every bottle of 2mL, packing quantity is 89438 bottles; Goods censorship after packing, lamp inspection, pack after the assay was approved, put in storage.
Described percentage ratio is apart from outside limiting, and all the other are mass percent.
The verification result of the product of preparing through the present embodiment sees table 1～table 4.
As can be seen from the table, the rabies human immunoglobulin purity that embodiment prepares is (99.5%), IgG monomer and dimer total content more than 92.0% (98.8%) more than 95.0%.
Prepare the molecular size distribution result scintigram of product and see Fig. 2 and Fig. 3.
Prepare the purity result scintigram of product and see Fig. 4.
table 1: heat stability test
table 2: molecular size distribution is measured
table 3: protein content determination
table 4: purity testing
Claims (6)
1. a rabies human immunoglobulin preparation technology, it comprises cold ethanol filter press technique successively, chromatography, ultrafiltration process, nanofiltration method and washing, drying and pouring linkage packing Technology are extracted separating immune globulin, it is characterized in that, chromatography is a step ion exchange chromatography, before chromatography, first refine component III supernatant liquor, again the refining component III supernatant liquor parameter obtaining is adjusted, the protein concentration of refining component III supernatant liquor is adjusted to 3～6%, pH to 6.4～6.6, it is 0.18～0.22s/m that specific conductivity is surveyed in the time of T=19 DEG C, regulate and carry out upper column chromatography purification with ion exchange column afterwards, chromatography uses DEAE Sepharose FF filler, the complete pH that regulates again of chromatography is 3.8～4.0.
2. a kind of rabies human immunoglobulin preparation technology according to claim 1, it is characterized in that adjusting pH to 6.4～6.6 with the NaOH of 0.5mol/L, then add 1mol/L phosphoric acid-NaOH damping fluid and regulate specific conductivity in the time of T=19 DEG C, to survey be 0.18～0.22s/m, the complete adjusting of the HCl with 1.0mol/L of chromatography pH is 3.8～4.0.
3. a kind of rabies human immunoglobulin preparation technology according to claim 1 and 2, it is characterized in that after the metering of component III supernatant liquor, start retort agitator and refrigeration cycle, controlling temperature is-5～-7 DEG C, carry out Depth Filtration, filter out liquid temp and be controlled at-5～-7 DEG C, after filtering, regulating pH with the HCl of 1.0mol/L is 4.0 left and right; More than protein concentrate concentration to 5%, dialyse with 2～8 DEG C of waters for injection of 5 times of volumes, dialyse and obtain and refine component III supernatant liquor.
4. a kind of rabies human immunoglobulin preparation technology according to claim 1 and 2, it is characterized in that it is 3.8～4.0 that the complete HCl with 1.0mol/L of chromatography regulates pH, opening ultrafilter is concentrated into more than 5%, dialyse with 2～8 DEG C of waters for injection, make ethanol content≤0.025%, then protein liquid is concentrated into more than 6%, the maltose of calculated amount is added in protein liquid, by the HCl adjusting pH value of 1.0mol/L, make maltose content 10 ± 1%, pH is 3.9～4.3, protein content 5.5 ± 0.5%; Enter again follow-up Sterile Filtration.
5. a kind of rabies human immunoglobulin preparation technology according to claim 1 and 2, is characterized in that preparation technology is as follows:
(1), by quarantining quarantine, qualified human plasma is centrifugal, centrifugal go out liquid temp be controlled at 0 ~ 4 DEG C; Separate cryoprecipitate, cryoprecipitate is produced for the VIII factor, and its supernatant liquor is delivered to albumen sepn retort, makes the temperature of protein liquid between 1～3 DEG C, and slowly adding pH4.0 acetate buffer solution to regulate pH value is 6.70～7.20; Add-15 DEG C of 95% or 50% following ethanol, make ethanol final concentration to 8%(V/V), temperature is controlled at-1～-3 DEG C, and adding pH value after ethanol is 6.80～7.20;
(2), the thing that makes of step (1) is stirred more than 30min, carry out centrifugal, centrifugal go out liquid temp be controlled at-1～-3 DEG C, centrifugal component I precipitation and components I supernatant liquor; Component I precipitation is deposited in freezer or is directly used in scleroproein original production;
(3), the thing component I supernatant liquor that makes of step (2) is added in pH4.0 acetate buffer solution to adjusting pH value to 6.7～6.9; Adding-15 DEG C of following 95% ethanol to alcohol concn is 20%(V/V), temperature is controlled at-4～-6 DEG C; After adding ethanol, pH should be 6.80～7.00; After stirring more than 120min, more than standing 60min, then open and stir, add 18g diatomite by every liter of reaction solution, carry out press filtration, press filtration obtains compositionⅱ+III precipitation and compositionⅱ+III supernatant liquor, and compositionⅱ+III supernatant liquor is for human serum albumin production;
(4), by step (3) make thing compositionⅱ+III precipitation, add the water for injection of 10～15 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, be cooled to 3～5 DEG C, pour compositionⅱ+III precipitation in retort stirring and dissolving; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5. 80～5.85, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 19%; Reaction solution outlet temperature is controlled at-4～-6 DEG C, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-4 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-4～-6 DEG C, filter pressure is no more than 0.2Mpa, collects refining compositionⅱ+III precipitation;
(5), by step (4) make the refining compositionⅱ of thing+III precipitation, add the water for injection of 10～15 times of amounts, adjusting its Na ion concentration with NaCl is 0.01mol/L; Open and stir, while being cooled to 2～4 DEG C, will refining compositionⅱ+III precipitation and pour stirring and dissolving in retort into; In whipping process, slowly add pH4.0 acetate buffer solution in reaction solution, adjusting pH is 5.1～5.4, and reacting liquid temperature is 0～-1 DEG C, adds damping fluid, continues to stir at least 10min; Open refrigeration cycle, add-15 DEG C of 95% following ethanol, making the final alcohol concn of reaction solution is 17%; Reaction solution outlet temperature is controlled at-5～-7 DEG C, and pH is 5.2～5.4, adds ethanol and continues to stir 2h, and standing 4h compresses filtration above; First slowly blow out the water in filter with-5 DEG C of following pressurized air, start retort agitator, make diatomite and reaction solution mix 10min and filter while stirring above, in filtration procedure, keeping liquid temp is-5～-7 DEG C, obtains component III precipitation and component III supernatant liquor;
(6) making after the metering of thing component III supernatant liquor step (5), start retort agitator and refrigeration cycle, controlling temperature is-5～-7 DEG C, carries out Depth Filtration, filter out liquid temp and be controlled at-5～-7 DEG C, after filtering, regulating pH with the HCl of 1.0mol/L is 4.0 left and right; More than protein concentrate concentration to 5%, dialyse with 2～8 DEG C of waters for injection of 5 times of volumes, dialyse and obtain and refine component III supernatant liquor; Protein concentration is adjusted to 3～6%, with NaOH tune pH to 6.4～6.6 of 0.5mol/L, then adding 1mol/L phosphoric acid-NaOH damping fluid regulates specific conductivity in the time of T=19 DEG C, to survey is 0.18～0.22s/m, regulate and carry out upper column chromatography purification with ion exchange column afterwards, chromatography uses DEAE Sepharose FF filler; It is 3.8～4.0 that the complete HCl with 1.0mol/L of chromatography regulates pH, opening ultrafilter is concentrated into more than 5%, dialyse with 2～8 DEG C of waters for injection, make ethanol content≤0.025%, then protein liquid is concentrated into more than 6%, the maltose of calculated amount is added in protein liquid, by the HCl adjusting pH value of 1.0mol/L, make maltose content 10 ± 1%, pH is 3.9～4.3, protein content 5.5 ± 0.5%;
(7), the protein liquid making after thing preparation of step (6) is carried out to Sterile Filtration in 6h; After Sterile Filtration, goods were placed on incubated at low pH chamber, through 24 ± 1 DEG C of incubated at low pHs 21 days; Incubate to put and finish the rear DV50 of using filter core except virus filtration;
(8), by protein liquid after the making thing and filter of step (7), with the 0.85%NaCl solution ultrafiltration of 2～8 DEG C, carry out five times of equal-volumes and wash, washing finishes rear concentrated, rare joining, adjustment glycine is 26～30g/L, and adjusting goods pH value is 6.6～7.2, tire >=100IU/mL of rabies antibody;
(9), step (8) made to the rare protein liquid of joining of thing through 0.2 μ m degerming filter element filtering packing; Goods censorship after packing, lamp inspection, pack after the assay was approved, put in storage;
Described percentage ratio is apart from outside limiting, and all the other are mass percent.
6. a kind of rabies human immunoglobulin preparation technology according to claim 5, it is characterized in that: the aqueous solution that described pH 4.0 acetate buffer solutions are made up of sodium acetate and glacial acetic acid, in every premium on currency, adding 244.9mL mass concentration is 99% glacial acetic acid and 65.6g sodium acetate.
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