CN101768206A - Method for purifying recombinant human serum albumin protein and application thereof - Google Patents
Method for purifying recombinant human serum albumin protein and application thereof Download PDFInfo
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Abstract
The present invention relates to a method for purifying recombinant human serum albumin (rHSA) protein. The method comprises the following steps: fermented liquid containing rHSA is processed by a ceramic membrane, supernatant liquid is orderly purified by high salt cation exchange chromatography, hydrophobic layer exchange chromatography and weak anion exchange chromatography, and purified rHSA is obtained. The present invention is characterized in that solution processed by high salt cation exchange chromatography is processed by borate and then filtered by hollow fibers. The rHSA obtained can be used for producing vaccines for humans against viruses with a cell culture method, particularly rabies vaccines.
Description
Technical field
The present invention has set forth a kind of purification process and application in cell culture method production human virus vaccines of recombination human serum albumin (rHSA) of pichia yeast expression.
Background technology
Bovine serum albumin or human serum albumin are that cell culture method is produced indispensable stablizer in the virus vaccines, but bovine serum albumin is owing to derive from animal and pollution such as potential mad cow disease is arranged and the human serum albumin of being originated by human blood gradually substitutes.Yet same, blood source human serum albumin contains the danger of potential pathogenic infection inevitably, simultaneously blood source human serum albumin derive from human plasma and limited its output and batch between stability, the quality instability.
WO98/068221998 disclosed recombination human serum albumin (rHSA) February 19 and has been used for animal cell culture, and wherein recombination human serum albumin is the growth that is used to promote cell.WO99/12568 on March 18th, 1999 disclosed the stablizer that recombination human serum albumin and compositions such as sugar, inorganic salt are used for varicella, zoster and measles, rubella, parotitis live virus.
The preparation method of rHSA is well known to those skilled in the art, and has all disclosed a method with the gene manipulation techniques recombination human serum albumin among EP0612761, EP0570916 and the EP0699687.Become complicated yet these methods are all many because of step, the cycle is long, cost is high, thereby be difficult to satisfy the economic requirement that industry is amplified.Own patent application CN1496993A of applicant and CN1854155A disclose and have prepared the method for production of vaccine with rHSA.Compare with additive method, impurity levels such as the substance that show color of the reorganization HSA feature that this method is purified and polysaccharide reduce significantly, but level of endotoxin is difficult to control, need carry out further optimization process, and control intracellular toxin, raising yield are to obtain purifying process capable of being industrialized.
Summary of the invention
The invention provides a kind of purification process and the purposes of rHSA in preparation virus vaccines substratum, particularly Rabies Vaccine substratum and Rabies Vaccine preparation that is fit to industrialized rHSA efficiently.
The present invention is achieved through the following technical solutions:
At first gene engineering microzyme and the production method that makes up by the own patent application technology (CN1854301 and CN1854306) of applicant obtains the recombination human serum albumin fermented liquid, utilize the ceramic membrane in 0.5um aperture to filter, supernatant liquor after the heating is through too high salt cation displacement chromatography, the solution of results carries out borate to be handled, and the solution of handling through tubular fibre carries out hydrophobic displacement chromatography and weak anionic displacement chromatography according to the technology among the CN1854155A.At last, the elution fraction currently known methods of weak anionic chromatography is made various finished products as ultrafiltration and concentration method, freeze-drying method etc.
The sedimentary removing method of borate is described as centrifugal removal in patent CN1854155A, but centrifugal back feed clarification degree is low in the actually operating, power consumption is bigger on producing, different with patent CN1854155A is, handle operation by introducing tubular fibre, make fermented supernatant fluid the time need not add gac, and the solution that borate is handled is without centrifugally operated, thereby makes operation easier in heating.Introduce the advantage of tubular fibre: the tubular fibre in suitable aperture can reduce endotoxin content significantly, filter back feed clarification degree is good, and saves gac and centrifugation to proteic absorption, and yield improves 2-10%.Wherein the tubular fibre aperture is molecular weight cut-off 100K-0.5um, is preferably 500K-0.2um.Level of endotoxin obtains stable control, detects with tachypleus amebocyte lysate to be lower than 0.5EU/mL (product concentration is 100mg/mL).
Unless stated otherwise, the implication of the concentration of rHSA (%) is g/100mL among the application, and the content (g) of rHSA in promptly every 100mL solution is that the content of rHSA in every 100mL solution is 10g as the implication of rHSA10%.
Particularly, the present invention relates to:
1) method of a kind of purifying rHSA capable of being industrialized, comprise that the fermented liquid that will contain rHSA is through the ceramic membrane processing, supernatant is successively through too high salt cation displacement chromatography, hydrophobic displacement chromatography and weak anionic displacement chromatography, obtain purifying rHSA, it is characterized in that: described solution through high salt cation displacement chromatography utilizes hollow fiber column to filter after handling through borate.
2) according to item 1) described method, wherein the aperture of tubular fibre is molecular weight cut-off 100K-0.5um, is preferably 500K-0.2um.
3) according to item 1) described method, wherein the damping fluid of weak anionic displacement chromatography use comprises sodium-acetate, sodium phosphate, sodium-chlor and Sodium octoate etc., is preferably Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC and Sodium octoate, concentration is 10mM-200mM, preferred 50-100mM.
4) the 1) rHSA of described method preparation, it possesses following characteristics: purity of protein is 99%-100% monomer and dimer, preferably 100% monomer and dimer basically; Host protein is residual to be lower than the 100ppm total protein, is preferably the 10ppm total protein; Polysaccharide is residual to be lower than 10ug/mg rHSA, is preferably to be lower than 1ug/mgrHSA; Endotoxin content is lower than 0.85EU/mL (10%rHSA), is preferably 0.5EU/mL (10%rHSA).
5) purposes of described rHSA in virus vaccines production cell VERO, MDCK or MRC-5 cell culture medium item 4).
6) according to item 5) described purposes, wherein the concentration of rHSA is 0.1%-10%, is preferably 0.1%-0.5%.
7) according to item 5)-6) described purposes, wherein Bing Du cultural method is a rolling bottle technology, the instillation process of microcarrier suspension technology or the absorption of multi-polyester sheet.
8) according to item 5)-6) arbitrary described purposes, wherein virus vaccines is the human Rabies Vaccine.
9) purposes of described rHSA in preparation Antirabic Vaccine preparation item 4), wherein the concentration of rHSA is 0.1%-10%, preferred 0.5%-5%.
10) according to item 9) described purposes, wherein said Rabies Vaccine is a liquid drugs injection or freeze-dried.
The production of vaccine that the present invention obtains has following feature with rHSA: 1) protein concentration is 99%-100% monomer and dimer, preferably 100% monomer and dimer basically.2) host protein is residual is lower than the 100ppm total protein, is preferably the 10ppm total protein.3) polysaccharide is residual is lower than 10ug/mg rHSA, is preferably to be lower than 1ug/mg rHSA.4) endotoxin content is lower than 0.85EU/mL (10%rHSA), is preferably 0.5EU/mL (10%rHSA).
The rHSA of the inventive method preparation can be used for the production that VERO, MDCK and MRC-5 cell carry out the production of virus vaccines, particularly Rabies Vaccine.Particularly, add 0.1%-10% in the DMEM substratum, the substratum of keeping that the recombinant human albumin that is preferably 0.1%-0.5% is received the liquid process as virus carries out virus receipts liquid.The virus culture method can be a rolling bottle technology, the instillation process of microcarrier suspension technology or the absorption of multi-polyester sheet.Simultaneously, the rHSA of this method preparation can also be used as the stablizer of virus vaccines finished product, can be freeze-dried, also can be aqua.
Embodiment
Following embodiment only understands the present invention better for those skilled in the art, should not be construed as limitation of the present invention.
The purifying of embodiment 1 recombination human serum albumin (rHSA)
Various damping fluids used in purge process see the following form:
| Title | Buffer formulation |
| Solution A | Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC of the 25mM of pH4.5 |
| Solution B | 50mM Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC of pH7.0,0.1M sodium-chlor, the Sodium octoate of 10mM |
| Solution C | 50mM Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC of pH6.0,0.1M sodium-chlor |
| Solution D | 50mM Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC of pH6.0,0.2M sodium-chlor |
A) clarification of fermented liquid and heat treated
Utilize the ceramic membrane of aperture 0.5um that fermented liquid is carried out clarifying treatment, get fermented supernatant fluid 80L, add 5mM Sodium octoate, 5mM EDTA, heated 20 minutes down, cooling at 70 ℃.
B) high salt cation chromatographic separation and concentrated
Above-mentioned treated sample with the high salt cation CST-II of sample FF post on the 150cm/h flow velocity (GE company makes, and uses the solution A balance in advance for XK50/60, Vt=300ml), is used the solution B wash-out, get CST-II BD component.Concentrated film bag (production of MILLIPORE company) with molecular weight cut-off 300K is concentrated into about 100mg/ml.
C) borate is handled and the tubular fibre processing
Add sodium tetraborate and calcium chloride in concentrating component, making ultimate density is 50-100mM, spends the night.Hollow Fiber Ultrafiltration post (GE company) with molecular weight cut-off 500K separates.
D) heat treated
Handle adding Sodium octoate and halfcystine in the solution of back to tubular fibre, to each 5mM of ultimate density and 10mM, in 60 ℃ of heating 60 minutes, cooling rapidly.
E) hydrophobic chromatography separates and concentrates
Heating back sample enters Phenyl SFF post (GE company uses the solution C balance for XK50/60, Vt=500ml) through 0.45 and the membrane filtration of 0.22um.Collect stream and wear part.
F) weak anionic chromatographic separation
Above-mentioned stream is worn component enter Aminobutyl SFF post (GE company makes, and uses the solution C balance for XK50/60, Vt=500ml), carry out wash-out, get the Amino-BD component with solution D.
G) the concentrated liquid that changes
Ultra-filtration membrane with molecular weight cut-off 30K concentrates, and changes liquid with the Sodium phosphate dibasic-sodium dihydrogen phosphate that contains Sodium octoate then, is finally contained the finished product 517g of rHSA 10%.
H) evaluation of product
The present invention respectively goes on foot rHSA concentration, productive rate, sugared content and purity and sees the following form:
Wherein, measure protein concentration, calculate total protein concentration, respectively go on foot yield thereby calculate with the Bradford method; Sugar content is measured with the phenolsulfuric acid method; Purity utilizes the SDS-PAGE electrophoretic method to analyze in conjunction with the HPLC method; Level of endotoxin utilizes limulus reagent test to detect.
The application of embodiment 2 rHSA in Rabies Vaccine rolling bottle culture process
A) cell preparation:
With Vero cell (deriving from U.S. ATCC CRL-1586) recover, the rolling bottle amplification cultivation, the double-deck rolling bottle of the 3.5L that goes down to posterity.37 ℃ of culture condition, CO
25% (v/v).
B) virus inoculation:
When the Vero cell cultures becomes individual layer, be 0.025～0.125 inoculation CTN strain seed culture of viruses (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) with MOI.After the inoculation, use the interpolation production of vaccine instead and keep liquid with the DMEM substratum of rHSA 0.35%.34 ℃ of culture temperature.
C) virus is received liquid:
Use instead to keep after the liquid to receive in per three days and change liquid once, receive and continue to renew aquatic foods behind the liquid and keep liquid, receive liquid continuously three times.Adopt Monoclonal Antibody double-antibody sandwich elisa method and mouse titration experiments to measure the G protein content and the virus titer of three receipts liquid respectively, determination data sees the following form:
| Receive liquid batch | ??Gp(IU/ml) | Virus titer (LD50/ml) |
| 0.35%rHSA receives liquid 1 | ??10.8±5.4 | ??4.9±0.9 |
| 0.35%rHSA receives liquid 2 | ??13.3±4.7 | ??6.1±0.7 |
| 0.35%rHSA receives liquid 3 | ??12.9±4.1 | ??5.4±0.8 |
D) purifying of Rabies Vaccine virus results liquid:
Carry out ultrafiltration and concentration behind the virus results liquid clarification filtration: use the hollow fiber column of molecular weight cut-off 750K, virus results liquid is carried out ultrafiltration and concentration.In the ultra-filtration process, virion obtains highly concentrated, and small-molecule substance in the nutrient solution such as albumin, major part such as phenol red are being removed in seeing through liquid.
4 ℃ of deactivations of beta-propiolactone (1: 4000) 24 hours, 37 ℃ of hydrolysis 2 hours;
Column chromatography purification: use Sepharose 4Fast Flow chromatography column, press sample on the column volume 10%, collect the 1st peak.Behind chromatography purification, the low molecular weight protein molecule in the viral liquid (mainly being the albumin in the nutrient solution) is removed, and protein content reduces greatly in the viral peak.
Behind chromatography column collection peak extraction mensuration protein content sample, add human serum albumin (Hengyang, Hunan Southern Mountain pharmaceutical factory) immediately as protective material, its final concentration is 1%, adds Thiomersalate simultaneously and cooks sanitas, Thiomersalate final concentration≤0.08mg/ml.Prepare according to protein content and antigenic content, packing becomes liquid preparation.
The preparation finished product is through check, every index all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC), mainly be: it is 4.6IU/ dosage that the NIH method is measured vaccine potency, and particularly, it is residual less than 5ng/ dosage to utilize Elisa test kit method to detect the yeast cell host protein.
The application of embodiment 3 rHSA in Rabies Vaccine NBS reactor culture process
A) cell preparation:
With the Vero cell recover, the rolling bottle amplification cultivation, with 4 * 10
5The density of cells/ml is inoculated in the NBS5L bio-reactor, device 160g polyester chips in the reactor ester sheet basket, working volume 2.5L, 37 ℃ of culture condition, dissolved oxygen 45%, stirring velocity 70rpm, pH value 7.4.
B) virus inoculation:
The Vero cell cultures was used instead and is added the DMEM substratum of production of vaccine with rHSA 0.35% in the time of 4～5 days, was 0.025～0.125 inoculation CTN strain seed culture of viruses with MOI.34 ℃ of culture temperature, dissolved oxygen 45%, stirring velocity 70rpm, pH value 7.4.
C) virus is received liquid:
Continous pouring was received liquid 20 days, received liquid measure 24L, and hybrid virus is received the liquid virus titer and all reaches 6.51gLD50.
D) purifying of Rabies Vaccine virus results liquid:
Purification process is carried out according to purification process among the embodiment 2.
The preparation finished product is through check, and every index all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Mainly be: it is 4.6IU/ dosage that the NIH method is measured vaccine potency, and particularly, it is residual less than 5ng/ dosage to utilize Elisa test kit method to detect the yeast cell host protein.
The application of embodiment 4 rHSA in Rabies Vaccine microcarrier reactor culture process
A) cell preparation:
With the Vero cell recover, the rolling bottle amplification cultivation, with 5 * 10
5The density of cells/ml is inoculated in the microcarrier reactor of working volume 5L, microcarrier consumption 125 grams.
Retort is 37 ℃ of temperature, dissolved oxygen 45%, and culturing cell is 5 under pH value 7.4 conditions, and cell concn reaches 1.2 * 10
7Virus inoculation during cells/ml.
B) virus inoculation:
With MOI is 0.025～0.125 inoculation CTN strain seed culture of viruses.With the DMEM substratum that contains rHSA 0.35%, 34 ℃ of culture temperature, dissolved oxygen 45%, pH value 7.4 was cultivated 72 hours.
C) virus is received liquid
Virus culture begins perfusion and receives liquid after 72 hours.
In the 5th, 9,13 day, and mix the sampling of results liquid behind the virus infection.Carry out the detection of microscopy, titration of virus and glycoprotein.Glycoprotein content converges rate less than 1IU/ml or cell and is lower than 20%, stops to receive liquid.
Receive liquid altogether 14 days, and gathered in the crops the long-pending 58L of viral liquid.5th, 9,13 days and mixing receipts liquid detection data such as following table:
| RHSA receives the liquid sample | Glycoprotein content IU/ml | Titre lgLD50 |
| The 5th day | ??3.3 | ??6.8 |
| The 9th day | ??2.6 | ??7.2 |
| The 13rd day | ??2.0 | ??6.4 |
| Biased sample | ??4.6 | ??6.8 |
D) purifying of Rabies Vaccine virus results liquid:
Purification process is carried out according to purification process among the embodiment 2.
The preparation finished product is through check, and every index all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Mainly be: it is 4.7IU/ dosage that the NIH method is measured vaccine potency, and particularly, it is residual less than 5ng/ dosage to utilize Elisa test kit method to detect the yeast cell host protein.
Embodiment 5 rHSA cultivate the application of producing in the Rabies Vaccine technology at the human diploid cell rolling bottle
A) cell preparation:
Human diploid cell is MRC-5, from U.S. ATCC CCL-171.Clone is through recovery, rolling bottle amplification cultivation, and double-deck rolling bottle goes down to posterity.37 ℃ of culture condition, CO
25%.
B) virus inoculation:
Inoculation back 3～5 days, when the MRC-5 cell grows into when cultivating into individual layer, inoculation CTN strain seed culture of viruses, inoculation MOI is 0.025～0.125.After connecing poison, use the DMEM substratum that adds rHSA 0.35% instead and keep liquid.34 ℃ of culture temperature.
C) virus is received liquid:
Connect back 3 days of poison, begin to receive liquid, continue to renew aquatic foods behind the receipts liquid and keep liquid, receive liquid continuously three times.Adopt Monoclonal Antibody double-antibody sandwich elisa method and mouse titration experiments to measure the G protein content and the virus titer of three receipts liquid respectively, determination data sees the following form:
| Receive liquid batch | Average Gp (IU/ml) | Average virus titer (LD50/ml) |
| 0.35%rHSA receives liquid | ??5.8±1.2 | ??4.5±0.5 |
D) purifying of Rabies Vaccine virus results liquid:
Purification process is carried out according to purification process among the embodiment 2.
The preparation finished product is through check, and every index all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Mainly be: it is 4.5IU/ dosage that the NIH method is measured vaccine potency, and particularly, it is residual less than 5ng/ dosage to utilize Elisa test kit method to detect the yeast cell host protein.
Embodiment 6 rHSA use in VERO, MDCK and MRC-5 cell non-serum culture medium
According to VERO, MDCK (ATCC CCL-34), MRC-5 cell cultures Nutrition and Metabolism situation, in DMEM (production of Hyclone company) substratum, add additives such as amino acid, vegetable protein hydrolyzate, recombination human serum albumin, ester class respectively, make VERO, MDCK, MRC-5 cell serum free medium.
Main improvement is as follows:
| Amino acid | ??mg/L |
| ??Asp | ??175 |
| ??Ser | ??155.1 |
| ??Glu | ??209.7 |
| ??Gly | ??117.3 |
| ??His | ??336.1 |
| ??Thr | ??150.9 |
| ??Arg | ??411.4 |
| ??Ala | ??126.2 |
| ??Pro | ??145.7 |
| ??Cys | ??109.3 |
| ??Tyr | ??129.2 |
| ??Val | ??144.7 |
| ??Met | ??48.2 |
| ??Lys | ??240.3 |
| ??Ile | ??182.4 |
| ??Leu | ??209.7 |
| ??Phe | ??123.3 |
Adopt above-mentioned formulated to become two kinds of serum free mediums:
SFMI: add 1g/l rHSA
SFMII: add the 1g/l human serum albumin.
Experimental program 1: above-mentioned two kinds of substratum are used for the VERO passage to be cultivated, and compares with the DMEM substratum that contains 10% (v/v) serum.
Experimental result: 50 generations of continuous passage, cell state no significant difference in above-mentioned three kinds of substratum, cytoactive all＞99%, the cell colony doubling time is SFMI 23 hours, SFMII 23.5 hours, contrast culture liquid is 22 hours, the result shows that the alternative blood of rHSA source albumin is used for the VERO cell non-serum culture medium, the two performance no significant difference.
Experimental program 2: adopt above-mentioned SFMI, SFMII substratum to carry out mdck cell and keep parallel check experiment, compare with the DMEM nutrient solution that contains 5% (v/v) serum.
A) 6 2L rolling bottles of mdck cell inoculation.
B) after cell covers with individual layer, change and state three kinds of substratum, each 2 bottles, keep cultivation.
C) change liquid every other day.
Experimental result: kept altogether 42 days, and kept when finishing cell attachment rate: SFMI and be 72.7%, SFMII is 70.5%, contrast is 78.6%.Show that the alternative human serum albumin of rHSA is used for the MDCK serum-free and keeps cultivation, the two with contain 5% blood serum medium and do not have significant difference.
Experimental program 3: adopt above-mentioned SFMI, SFMII substratum to carry out the MRC-5 cell and keep parallel control and test.
1) 4 2L rolling bottles of MRC-5 cell inoculation.
2) after cell covers with individual layer, change and state two kinds of substratum, each 2 bottles, keep cultivation.
3) change liquid every other day.
Experimental result: kept altogether 30 days, and kept when finishing cell attachment rate: SFMI and be 83.5%, SFMII is 85.0%.Show that the alternative human serum albumin of rHSA is used for the MRC-5 cell non-serum and keeps cultivation.
Embodiment 7 usefulness recombination human serum albumins are made the stablizer of Rabies Vaccine
Aqueous injection: rabies virus particle behind the chromatography purification receive liquid extract measure the protein concentration sample after, add final concentration immediately and be 1% rHSA as protective material, add Thiomersalate simultaneously and cook sanitas, final concentration＜0.08mg/ml.According to protein content and antigenic content preparation work in-process, add rHSA (final concentration 1%) as stablizer, packing becomes liquid preparation.Mensuration is tired and is 4.8IU/ml.Sample is carried out heat stability test: after 37 ℃ of 4 weeks of placement, mensuration is tired, and the result is 3.8IU/ml.
Freeze-dried: virion is received liquid according to protein content and antigenic content preparation work in-process behind the chromatography purification, adds stablizers such as rHSA (final concentration 2%), maltose, packing, and freeze-drying becomes freeze-dried preparation.Mensuration is tired and is propped up for 4.6IU/.Sample is carried out heat stability test: after 37 ℃ of 4 weeks of placement, mensuration is tired, and the result props up for 3.7IU/.
Claims (10)
1. the method for a purifying rHSA capable of being industrialized, comprise that the fermented liquid that will contain rHSA is through the ceramic membrane processing, supernatant is successively through too high salt cation displacement chromatography, hydrophobic displacement chromatography and weak anionic displacement chromatography, obtain purifying rHSA, it is characterized in that: described solution through high salt cation displacement chromatography utilizes hollow fiber column to filter after handling through borate.
2. method according to claim 1, wherein the aperture of tubular fibre is molecular weight cut-off 100K-0.5um, is preferably 500K-0.2um.
3. method according to claim 1, wherein the damping fluid of weak anionic displacement chromatography use comprises sodium-acetate, sodium phosphate, sodium-chlor and Sodium octoate etc., be preferably Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC and Sodium octoate, concentration is 10mM-200mM, preferred 50-100mM.
4. the rHSA of the described method of claim 1 preparation, it possesses following characteristics: purity of protein is 99%-100% monomer and dimer, preferably 100% monomer and dimer basically; Host protein is residual to be lower than the 100ppm total protein, is preferably the 10ppm total protein; Polysaccharide is residual to be lower than 10ug/mg rHSA, is preferably to be lower than 1ug/mg rHSA; Endotoxin content is lower than 0.85EU/mL (10%rHSA), is preferably 0.5EU/mL (10%rHSA).
5. the described rHSA of claim 4 produces purposes in cell VERO, MDCK or the MRC-5 cell culture medium at virus vaccines.
6. purposes according to claim 5, wherein the concentration of rHSA is 0.1%-10%, is preferably 0.1%-0.5%.
7. according to the arbitrary described purposes of claim 5-6, wherein Bing Du cultural method is a rolling bottle technology, the instillation process of microcarrier suspension technology or the absorption of multi-polyester sheet.
8. according to the arbitrary described purposes of claim 5-6, wherein virus vaccines is the human Rabies Vaccine.
9. the purposes of the described rHSA of claim 4 in preparation Antirabic Vaccine preparation, wherein the concentration of rHSA is 0.1%-10%, preferred 0.5%-5%.
10. purposes according to claim 9, wherein said Rabies Vaccine are liquid drugs injections or freeze-dried.
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