CN104193822B - Process for preparing rabies human immune globulin - Google Patents

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

A kind of rabies human immunoglobulin preparation technology

Technical field

The present invention relates to a kind of preparation technology of rabies human immunoglobulin, belong to field of biological pharmacy.

Background technology

Rabies are that a kind of pathogen is the elasticity virus of ribonucleic acid, are counted according to the World Health Organization, the whole nation three/ There is rubies epidemiology more than two countries and regions, and China and south east asia are in world rabies district occurred frequently, and its case is about The whole world more than 70%, rabies are accounted for once falling ill, there is no method to cure at present, the death rate is almost up to 100%.If providing in time enough Rabies human immunoglobulin, antibody reaches more than 0.5IU/ml in making its patients serum, and patient can be made effectively to be protected Shield.Therefore, it is necessary for treating preparing for rabic specific drug rabies human immunoglobulin, and just resist mad dog at present For the production technology of patient's immunoglobulin, single cold ethanol separating technology being used the product on domestic market more, point Many from step, the yield of immunoglobulin is low, causes domestic rabies immune globulin shortage.In the prior art such as《High-purity Rabies human immunoglobulin and its feedstock production process》CN200710114839.8, methods described is to include preparing for giving birth to Produce raw blood plasma of the antibody titer more than 10IU/ml, cold ethanol classification separation, the ion exchange of rabies human immunoglobulin Purification by chromatography, the treatment of incubated at low pH inactivation of viruses, cross filter virus, ultrafiltration, with liquid, degerming.The high-purity rabies of acquisition Human immunoglobulin(HIg), the rabies antibody containing high-titer, purity is more than 99% more than 98%, IgG monomers and dimer content, Rabies antibody potency is more than or equal to 150IU/ml, and protein content is less than 120g/L.Patient can be made to be effectively protected, and The consumption of product and the pain of patient and burden can be reduced.

It is effectively to extract separation acquisition rabies human to be immunized under reality of the immunoglobulin market still in short situation Globulin, particularly improves product yield, obtains safe and reliable product, and the preparation technology's of rabies human immunoglobulin enters One-step optimization will be significant.

The content of the invention

It is an object of the invention to provide a kind of high yield pulp1, the preparation technology of the rabies human immunoglobulin of high-purity.

The present invention is achieved like this, a kind of rabies human immunoglobulin preparation technology, and it includes low temperature second successively Alcohol filter press technique, chromatography, ultrafiltration, nanof iotaltration and washing, drying and pouring linkage layered water flood well technology extract separating immune globulin, Chromatography is a step ion exchange chromatography, and the supernatant of component III, then the supernatant of refined component III to obtaining first are refined before chromatography Liquid parameter is adjusted, and the protein concentration of the supernatant of refined component III is adjusted to 3~6%, pH to 6.4~6.6, and electrical conductivity exists It is 0.18~0.22s/m to be surveyed at T=19 DEG C, carries out purified by column chromatography after regulating with ion exchange column, and chromatography uses DEAE Sepharose FF fillers；It is 3.8~4.0 that chromatography is finished and adjust again pH.

Its concrete technology scheme is as follows：

（1）, by the centrifugation of the human plasma that it is qualified that quarantine quarantines, be centrifuged out liquid temperature control at 0 ~ 4 DEG C；Separate cryoprecipitate, Cryoprecipitate is produced for VIII factor, and its supernatant is delivered into Protein Separation retort, make the temperature of protein liquid 1~3 DEG C it Between, it is 6.70~7.20 to be slowly added to pH4.0 acetate buffer solutions regulation pH value；Plus less than -15 DEG C of 95% or 50% ethanol, make Ethanol final concentration is to 8%（V/V）, at -1~-3 DEG C, pH value is 6.80~7.20 to temperature control after adding ethanol；

（2）, by step（1）Prepared thing stirring more than 30min, be centrifuged, be centrifuged out liquid temperature control -1~-3 DEG C, component I precipitations and components I supernatant is centrifuged to obtain；Component I precipitations deposit in freezer or are directly used in fibrin original production；

（3）, by step（2）Prepared thing component I supernatants add pH4.0 acetate buffer solutions, regulation pH value to 6.7~ 6.9；Plus less than -15 DEG C of 95% ethanol to concentration of alcohol is 20%（V/V）, temperature control is at -4~-6 DEG C；After adding ethanol PH should be 6.80~7.00；After stirring more than 120min, more than 60min is stood, be then turned on stirring, added by every liter of reaction solution 18g diatomite, carries out press filtration, and press filtration obtains the precipitation of compositionⅱ+III and the supernatant of compositionⅱ+III, and the supernatant of compositionⅱ+III is used for people Blood albumin is produced；

（4）, by step（3）Prepared thing compositionⅱ+III precipitation, add 10~15 times amount waters for injection, adjusted with NaCl Whole its Na ion concentration is 0.01mol/L；Stirring is opened, 3~5 DEG C are cooled to, the precipitation of compositionⅱ+III is poured into retort and is stirred Mix dissolving；PH4.0 acetate buffer solutions are slowly added in whipping process in reaction solution, adjustment pH is 5. 80~5.85, reaction Liquid temperature degree is 0~-1 DEG C, adds buffer solution, continues to stir at least 10min；Open 95% second below kind of refrigeration cycle, plus -15 DEG C Alcohol, makes the final concentration of alcohol of reaction solution be 19%；Reaction solution final temperature is controlled at -4~-6 DEG C, is added ethanol and is continued to stir 2h, stands more than 4h and is compressed filtering；First the compressed air below -4 DEG C of use slowly blows out the water in filter, starts retort Agitator, makes diatomite mix more than 10min with reaction solution and filters while stirring, and it is -4 that liquid temperature degree is kept out in filter process ~-6 DEG C, filter pressure is no more than 0.2Mpa, collects refined compositionⅱ+III and precipitates；

（5）, by step（4）The refined compositionⅱ+III of prepared thing precipitate, add 10~15 times of waters for injection of amount, use NaCl adjusts its Na ion concentration for 0.01mol/L；Stirring is opened, when being cooled to 2~4 DEG C, refined compositionⅱ+III precipitation is fallen Enter stirring and dissolving in retort；PH4.0 acetate buffer solutions are slowly added in whipping process in reaction solution, adjustment pH is 5.1 ~5.4, reacting liquid temperature is 0~-1 DEG C, adds buffer solution, continues to stir at least 10min；Open kind of refrigeration cycle, plus -15 DEG C with Under 95% ethanol, make the final concentration of alcohol of reaction solution be 17%；Reaction solution final temperature is controlled at -5~-7 DEG C, and pH is 5.2 ~5.4, add ethanol and continue to stir 2h, stand more than 4h and be compressed filtering；First the compressed air below -5 DEG C of use is slowly blown The water gone out in filter, starts reaction jar agitator, diatomite is mixed more than 10min with reaction solution and filters while stirring, in mistake Liquid temperature degree is kept out during filter for -5~-7 DEG C, the precipitation of component III and the supernatant of component III is obtained；

（6）By step（5）The prepared supernatant of thing component III metering after, start reaction jar agitator and kind of refrigeration cycle, control Temperature processed is -5~-7 DEG C, carries out in-depth filtration, filters out liquid temperature control at -5~-7 DEG C, with 1.0mol/L's after filtering HCl regulations pH is 4.0 or so；Protein concentrate concentration is dialysed to more than 5% with 5 times of volumes, 2~8 DEG C of waters for injection, dialysis Obtain final product the supernatant of refined component III；Protein concentration is adjusted to 3~6%, pH to 6.4~6.6 is adjusted with the NaOH of 0.5mol/L, so Afterwards plus 1mol/L phosphoric acid-NaOH buffer solutions regulation electrical conductivity is surveyed at T=19 DEG C be 0.18~0.22s/m, use ion after regulating Exchange column carries out purified by column chromatography, and chromatography uses DEAE Sepharose FF fillers；Chromatography is finished with the HCl of 1.0mol/L Regulation pH be 3.8~4.0, open ultrafilter be concentrated into more than 5%, dialysed with 2~8 DEG C of waters for injection, make ethanol content≤ 0.025%, protein liquid is then concentrated into more than 6%, the maltose of amount of calculation is added in protein liquid, with the HCl of 1.0mol/L Regulation pH value, makes maltose content 10 ± 1%, and pH is 3.9~4.3, protein content 5.5 ± 0.5%；

（7）, by step（6）Prepared thing prepare after protein liquid in carrying out aseptic filtration in 6h；Product after aseptic filtration Incubated at low pH room is placed on, through 24 ± 1 DEG C of incubated at low pH 21 days；Incubate and remove virus filtration with DV50 filter cores after putting end；

（8）, by step（7）Prepared thing filtering after protein liquid, with 2~8 DEG C of 0.85%NaCl solution ultrafiltration, carry out five Times isometric washing, washing terminate after concentration, it is dilute match somebody with somebody, adjustment glycine is 26~30g/L, adjustment product pH value for 6.6~ 7.2, rabies antibody potency >=100IU/mL；

（9）, by step（8）Prepared thing dilute dispensed through 0.2 μm of degerming filter element filtering with protein liquid；Product after packing send Inspection, lamp inspection, pack, are put in storage after the assay was approved；

In addition to having and limiting, remaining is mass percent to the percentage.

The aqueous solution that the pH4.0 acetate buffer solutions are made up of sodium acetate and glacial acetic acid, 244.9mL is added in every liter of water Mass concentration is 99% glacial acetic acid and 65.6g sodium acetates.

Component I supernatants and its precipitation, the supernatant of compositionⅱ+III and its precipitation are the universal classification of art Method；It is also in the present invention the title by material after universal classification classification, does not imply that simple sequence number.

Cryoprecipitate is centrifuged by human plasma, the centrifuged supernatant after separation adds second by adjusting pH and temperature Alcohol is centrifuged to obtain component I supernatants and its precipitation again；

By adjusting pH and temperature to component I supernatants, press filtration obtains the supernatant of compositionⅱ+III and its sinks addition ethanol again Form sediment；

Component I precipitations mainly contain fine former, FV III, Ciq, Clr, Cls and fibronectin etc.；

Component I supernatants are mainly contained：Albumin, IgG, IgA, IgM, F II, VII, Ⅸ, Ⅹ, α, beta Globulin, covellite egg In vain, alpha1-antitrypsin, the complement component of IgM, AT- III, transferrins, haptoglobin etc.；

The precipitation of compositionⅱ+III mainly contains IgG, IgA, IgM, F II, VII, Ⅸ, Ⅹ, α, beta Globulin and CER etc.；

The supernatant of compositionⅱ+III is mainly contained：Albumin, α, beta Globulin, CER, alpha1-antitrypsin, IgM, The complement components of AT- III, transferrins, haptoglobin etc..

The positive effect of the present invention：

The present invention combines optimize technique, using cold ethanol press filtration extraction and separation technology and a step ion-exchange chromatography （Chromatogram）Technology is combined to extract to separate and obtains rabies human immunoglobulin, while removing disease using secondary Viral inactivation techniques Poison, can effectively improve product recovery rate, improve the purity and its security of product.Specially：

1st, using cold ethanol filter press technique, chromatography, ultrafiltration, the technique skill such as nanof iotaltration and washing, drying and pouring linkage packing Art, and through effective viral inactivation treatment, it is mature technology, stabilization, advanced.

2nd, clinical efficacy it is clear and definite, safe, can rapidly symptom management progress and epidemic situation development, while dosage it is small, Quiet third is reduced using so as to save rare blood plasma resource.

3rd, SIG derives from healthy human body, and its structure is safe without any transformation and modification, makees With clear and definite, the R＆D cycle is relatively short, and pharmacological effect effect is clear and definite, relatively easily goes out product, produces result.

Compared to the preparation technology of rabies human immunoglobulin compared to existing technology, innovative point is the present invention：

1st, by cold ethanol method below -15 DEG C of multistep, refine step by step, joined by adjusting temperature, PH, ionic strength etc. Number, makes whole concentration of alcohol obtain component I supernatants, the precipitation of compositionⅱ+III, refined compositionⅱ for 8%, 20%, 19%, 17% successively + III precipitation, the supernatant of component III, and carry out filter pressing it is standby go out purity more than 95.0%, IgG monomers and dimer total content exist More than 92.0% rabies human immunoglobulin.Remaining ingredient can be used for the production of other blood products simultaneously.

2nd, prior art is such as《High-purity rabies human immunoglobulin and its feedstock production process》 CN200710114839.8 is carried out pure using Q-Sepharose FF and DEAE Sepharose FF two step ion exchange chromatographies methods Change.Before present invention optimization chromatography the refined Supernatant protein liquid of component III be concentration 3~6%, pH6.4~6.6, electrical conductivity 0.18~ 0.22 s/m（T=19℃）Etc. upper prop purifying again after response parameter, chromatography finish again with 1.0mol/L HCl adjust pH 3.8~ 4.0.One step ion exchange equally can effectively remove foreign protein, improve purity, and by ultrafiltration, concentration, addition maltose is protected The rabies human that the methods such as shield agent obtain the moderate purity 99.5% of the embodiment of the present invention 1, IgG monomers and dimer total content 98.8% is immunized Globulin liquid.The use of maltose, can protect immunoglobulin molecules functional characteristic not influenceed by temperature and time.Compared to Two step ion-exchanges, step chromatography and addition protective agent simple possible, save operation, reduce production cost.

3rd, the present invention obtains the various process parameters such as optimal column chromatography by experiment, and the technique can produce 200IU/ bottles Bottle/ton the blood plasma of rabies human immunoglobulin 17887, compared to the conventionally produced bottle/ton blood of immunoglobulin 15000 Slurry, the present invention can produce 2887 bottle/ton blood plasma more, and recovery rate can improve 19.2 ~ 25% compared with traditional handicraft.

The inventive method prepare product with《Chinese Pharmacopoeia》（Version in 2010, three）Described in the immune ball of rabies human Contrast such as following table of the albumen in Key Quality Indicator.

Brief description of the drawings

Fig. 1 is present invention process flow chart.

Fig. 2 is to prepare the molecular size distribution results scanning figure of sample 1 in the embodiment of the present invention 1.

Fig. 3 is to prepare the molecular size distribution results scanning figure of sample 2 in the embodiment of the present invention 1.

Fig. 4 is to prepare the purity result scanning figure of product in the embodiment of the present invention 1.

Specific embodiment

The present invention can be so that the invention will be further described, however, the scope of the present invention is simultaneously by the following examples It is not limited to following embodiments.

Embodiment 1：By taking 5000 liters of blood plasma as an example, specific preparation technology is as follows：

（1）, quarantine is quarantined qualified 5000 liters of human plasmas centrifugation, be centrifuged out liquid temperature control at 0 ~ 4 DEG C；Separate cold Precipitation, cryoprecipitate is produced for VIII factor, is taken its supernatant and is delivered to Protein Separation retort, and the temperature control of protein liquid is existed Between 1~3 DEG C, it is 6.70~7.20 to be slowly added to pH4.0 acetate buffer solutions regulation pH value；Plus less than -15 DEG C 95% or 50% ethanol, makes ethanol final concentration to 8%（V/V）, temperature control at -1~-3 DEG C, add pH value after ethanol should be 6.80~ 7.20；The aqueous solution that the pH4.0 acetate buffer solutions are made up of sodium acetate and glacial acetic acid, adds 244.9mL matter in every liter of water Amount concentration is 99% glacial acetic acid and 65.6g sodium acetates.

（2）, by step（1）Prepared thing stirring more than 30min, be centrifuged, be centrifuged out liquid temperature control -1~-3 DEG C, component I precipitations and components I supernatant is centrifuged to obtain；Component I precipitations deposit in freezer or are directly used in fibrin original production.

（3）, by step（2）Prepared thing component I supernatants add pH4.0 acetate buffer solutions in, adjust supernatant pH value To 6.7~6.9；Plus less than -15 DEG C of 95% ethanol to concentration of alcohol is 20%（V/V）, temperature control is at -4~-6 DEG C；Add PH should be 6.80~7.00 after ethanol；After stirring more than 120min, more than 60min is stood, open stirring, added by every liter of reaction solution Enter 18g diatomite, carry out press filtration, inlet hydraulic not more than 0.20Mpa, press filtration obtains the precipitation of compositionⅱ+III and compositionⅱ+III Supernatant, the supernatant of compositionⅱ+III is produced for human serum albumin.

（4）, by step（3）Prepared thing compositionⅱ+III precipitate 467 kilograms, add 13 times amount waters for injection, use NaCl Its Na ion concentration is adjusted for 0.01mol/L；Stirring is opened, when being cooled to 3~5 DEG C, the precipitation of compositionⅱ+III retort is poured into Middle stirring and dissolving；PH4.0 acetate buffer solutions are slowly added in whipping process in reaction solution, adjustment pH is 5. 80~5.85, Reacting liquid temperature is 0~-1 DEG C, adds buffer solution, continues to stir at least 10min；Open below kind of refrigeration cycle, plus -15 DEG C 95% ethanol, makes the final concentration of alcohol of reaction solution be 19%；Reaction solution final temperature is controlled at -4~-6 DEG C, adds ethanol continuation Stirring 2h, stands more than 4h and is compressed filtering；First the compressed air below -4 DEG C of use slowly blows out the water in filter, starts anti- Jar agitator is answered, diatomite is mixed more than 10min with reaction solution and is filtered while stirring, liquid temperature degree is kept out in filter process It is -4~-6 DEG C, filter pressure is no more than 0.2Mpa, collects refined compositionⅱ+III and precipitate.

（5）, by step（4）The refined compositionⅱ+III of prepared thing precipitate 440 kilograms, add 13 times of waters for injection of amount, use NaCl adjusts its Na ion concentration for 0.01mol/L；Stirring is opened, when being cooled to 2~4 DEG C, refined compositionⅱ+III precipitation is fallen Enter stirring and dissolving in retort；PH4.0 acetate buffer solutions are slowly added in whipping process in reaction solution, adjustment pH is 5. 1 ~5.4, reacting liquid temperature is 0~-1 DEG C, adds buffer solution, continues to stir at least 10min；Open kind of refrigeration cycle, plus -15 DEG C with Under 95% ethanol, make the final concentration of alcohol of reaction solution be 17%；Reaction solution final temperature is controlled at -5~-7 DEG C, and pH is 5.2 ~5.4, add ethanol and continue to stir 2h, stand more than 4h and be compressed filtering；First the compressed air below -5 DEG C of use is slowly blown The water gone out in filter, starts reaction jar agitator, diatomite is mixed more than 10min with reaction solution and filters while stirring, in mistake It is -5~-7 DEG C that liquid temperature degree is kept out during filter；Obtain the precipitation of component III and the supernatant of component III；

（6）, by step（5）The prepared supernatant of thing component III metering after, start reaction jar agitator and kind of refrigeration cycle, control Temperature processed is -5~-7 DEG C, carries out in-depth filtration, filters out liquid temperature control at -5~-7 DEG C, with 1.0mol/L's after filtering HCl regulations pH is 4.0 or so；8563 kilograms of protein liquids are obtained after regulation PH, protein concentration more than 5% is concentrated into, volume after must concentrating 600 liters, dialysed with 5 times of volumes, 2~8 DEG C of waters for injection, dialysis obtains final product the supernatant of refined component III；Protein concentration is adjusted To 5.9%, pH to 6.53 is adjusted with the NaOH of 0.5mol/L, then add 1mol/L phosphoric acid-NaOH buffer solutions to adjust electrical conductivity in T=19 DEG C when to survey be 0.194s/m, carry out purified by column chromatography after regulating with ion exchange column, chromatography uses DEAE Sepharose FF fillers；It is 3.8~4.0 that chromatography is finished and adjusts pH with the HCl of 1.0mol/L, opens ultrafilter and is concentrated into more than 5%, with 2~8 DEG C water for injection is dialysed, and makes ethanol content≤0.025%, protein liquid then is concentrated into more than 6%, by the malt of amount of calculation Sugar is added in protein liquid, and with the HCl regulation pH value of 1.0mol/L, maltose content 9.7% in final protein liquid, pH is 4.10, egg Bai Hanliang 6.0%, 478.5 kilograms of solution weight.

（7）, by step（6）Prepared thing prepare after 478.5 kilograms of protein liquid in carrying out aseptic filtration in 6h；Degerming mistake Product was placed on incubated at low pH room after filter, through 24 DEG C of ± 1 DEG C of incubated at low pH 21 days；Incubate and remove viral mistake with DV50 filter cores after putting end Filter；

（8）, by step（7）Prepared thing filtering after protein liquid, with 2~8 DEG C of 0.85%NaCl solution ultrafiltration, carry out five Times isometric washing, washing terminate rear protein concentration to 14.4% carry out it is dilute match somebody with somebody, adjustment glycine is 27g/L, adjustment product pH value It is 6..8, rabies antibody potency >=100IU/mL.

（9）, by step（8）Prepared thing it is dilute with protein liquid 185.4 kilograms carry out protein filtering, mistake through 0.2 μm of filter core 183.2 kilograms of albumen liquid measure is obtained after filter；Packing, packing loading amount is every bottle of 2mL, and packing quantity is 89438 bottles；Product after packing Censorship, lamp inspection, pack, are put in storage after the assay was approved.

In addition to having and limiting, remaining is mass percent to the percentage.

The verification result of the product prepared through the present embodiment is referring to table 1 below~table 4.

As can be seen from the table, the rabies human immunoglobulin purity that embodiment is prepared is more than 95.0% （99.5%）, IgG monomers and dimer total content be more than 92.0%（98.8%）.

The molecular size distribution results scanning figure for preparing product is shown in Fig. 2 and Fig. 3.

The purity result scanning figure for preparing product is shown in Fig. 4.

Table 1：Heat stabilization test

Table 2：Molecular size measure of spread

Table 3：Protein content determination

Table 4：Purity testing

Claims (1)

1. a kind of rabies human immunoglobulin preparation technology, it include successively cold ethanol filter press technique, chromatography, ultrafiltration, Nanof iotaltration and washing, drying and pouring linkage layered water flood well technology extract separating immune globulin, it is characterised in that preparation technology is as follows：

（1）The preparation of the precipitation of compositionⅱ+III

By the human plasma centrifugation that it is qualified that quarantine quarantines, liquid temperature control is centrifuged out at 0 ~ 4 DEG C；Cryoprecipitate is separated, cryoprecipitate is used In the production of VIII factor, its supernatant is delivered to Protein Separation retort, makes the temperature of protein liquid between 1~3 DEG C, slowly added It is 6.70~7.20 to enter pH4.0 acetate buffer solutions regulation pH value；Plus less than -15 DEG C of 95% or 50% ethanol, make ethanol dense eventually Spend to 8%（V/V）, at -1~-3 DEG C, pH value is 6.80~7.20 to temperature control after adding ethanol；Be obtained thing stirring 30min with On, it is centrifuged, liquid temperature control is centrifuged out at -1~-3 DEG C, component I precipitations and components I supernatant is centrifuged to obtain；Component I is precipitated Deposit in freezer or be directly used in fibrin original production；Component I supernatants are added in pH4.0 acetate buffer solutions, adjust pH value To 6.7~6.9；Plus less than -15 DEG C of 95% ethanol to concentration of alcohol is 20%（V/V）, temperature control is at -4~-6 DEG C；Add PH should be 6.80~7.00 after ethanol；After stirring more than 120min, more than 60min is stood, stirring is then turned on, by every liter of reaction solution 18g diatomite is added, press filtration is carried out, press filtration obtains the precipitation of compositionⅱ+III and the supernatant of compositionⅱ+III, and the supernatant of compositionⅱ+III is used In human serum albumin production；

（2）The preparation of the refined precipitation of compositionⅱ+III

Compositionⅱ+III is precipitated, and adds 10~15 times of waters for injection of amount, and it is 0.01mol/L to adjust its Na ion concentration with NaCl； Stirring is opened, 3~5 DEG C are cooled to, the precipitation of compositionⅱ+III is poured into stirring and dissolving in retort；Slowly add in whipping process Enter pH4.0 acetate buffer solutions in reaction solution, adjustment pH is 5. 80~5.85, and reacting liquid temperature is 0~-1 DEG C, adds buffering Liquid, continues to stir at least 10min；95% ethanol below kind of refrigeration cycle, plus -15 DEG C is opened, makes the concentration of alcohol that reaction solution is final It is 19%；Reaction solution final temperature is controlled at -4~-6 DEG C, is added ethanol and is continued to stir 2h, is stood more than 4h and is compressed filtering； First the compressed air below -4 DEG C of use slowly blows out the water in filter, starts reaction jar agitator, diatomite is mixed with reaction solution Even more than 10min is filtered while stirring, keeps out liquid temperature degree for -4~-6 DEG C in filter process, and filter pressure is no more than 0.2Mpa, collects refined compositionⅱ+III and precipitates；

（3）The preparation of the refined supernatant of component III, chromatography

Refined compositionⅱ+III is precipitated, and adds 10~15 times of waters for injection of amount, and adjusting its Na ion concentration with NaCl is 0.01mol/L；Stirring is opened, when being cooled to 2~4 DEG C, refined compositionⅱ+III precipitation stirring and dissolving in retort is poured into； PH4.0 acetate buffer solutions being slowly added in whipping process in reaction solution, adjustment pH is 5.1~5.4, reacting liquid temperature is 0~- 1 DEG C, buffer solution is added, continue to stir at least 10min；95% ethanol below kind of refrigeration cycle, plus -15 DEG C is opened, makes reaction solution most Whole concentration of alcohol is 17%；At -5~-7 DEG C, pH is 5.2~5.4 to the control of reaction solution final temperature, adds ethanol and continues to stir 2h, stands more than 4h and is compressed filtering；First the compressed air below -5 DEG C of use slowly blows out the water in filter, starts retort Agitator, makes diatomite mix more than 10min with reaction solution and filters while stirring, and it is -5 that liquid temperature degree is kept out in filter process ~-7 DEG C, obtain the precipitation of component III and the supernatant of component III；After the metering of the supernatant of component III, start reaction jar agitator and refrigeration is followed Ring, controls temperature for -5~-7 DEG C, carries out in-depth filtration, filters out liquid temperature control at -5~-7 DEG C, and 1.0mol/ is used after filtering The HCl regulations pH of L is 4.0 or so；Protein concentrate concentration is dialysed, thoroughly to more than 5% with 5 times of volumes, 2~8 DEG C of waters for injection Analysis obtains final product the supernatant of refined component III；

Protein concentration is adjusted to 3~6%, pH to 6.4~6.6 is adjusted with the NaOH of 0.5mol/L, then plus 1mol/L phosphoric acid- It is 0.18~0.22s/m that NaOH buffer solutions regulation electrical conductivity is surveyed at T=19 DEG C, and upper prop is carried out with ion exchange column after regulating Chromatographic purifying, chromatography uses DEAE Sepharose FF fillers；Chromatography finish with the HCl of 1.0mol/L adjust pH be 3.8~ 4.0, open ultrafilter and be concentrated into more than 5%, dialysed with 2~8 DEG C of waters for injection, make ethanol content≤0.025%, then will Protein liquid is concentrated into more than 6%, and the maltose of amount of calculation is added in protein liquid, and pH value is adjusted with the HCl of 1.0mol/L, makes wheat Bud sugared content 10 ± 1%, pH is 3.9~4.3, protein content 5.5 ± 0.5%；

（4）Inactivation of virus, removal

Protein liquid is in carrying out aseptic filtration in 6h；Product is placed on incubated at low pH room after aseptic filtration, through 24 ± 1 DEG C of incubated at low pH 21 days；Incubate and remove virus filtration with DV50 filter cores after putting end；

（5）Ultrafiltration, it is dilute match somebody with somebody, dispense, lamp inspection, packaging, storage

Protein liquid after filtering, with 2~8 DEG C of 0.85%NaCl solution ultrafiltration, carries out five times of isometric washings, and washing terminates rear dense Contracting, it is dilute match somebody with somebody, adjustment glycine is 26~30g/L, and adjustment product pH value is 6.6~7.2, rabies antibody potency >=100IU/ mL；

It is dilute to be dispensed through 0.2 μm of degerming filter element filtering with protein liquid；Product censorship after packing, lamp inspection, pack, enter after the assay was approved Storehouse；

The aqueous solution that the acetate buffer solutions of the pH 4.0 are made up of sodium acetate and glacial acetic acid, adds 244.9mL matter in every liter of water Amount concentration is 99% glacial acetic acid and 65.6g sodium acetates；

In addition to having and limiting, remaining is mass percent to the percentage.