CN101768206B - 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 virus vaccines, but bovine serum albumin is owing to deriving from animal and the pollution such as potential mad cow disease being arranged and the human serum albumin of being originated by human blood gradually substitutes.Yet same, blood source human serum albumin inevitably contains the danger of potential pathogenic infection, simultaneously blood source human serum albumin derive from human plasma and limited its output and batch between stability, quality is unstable.
WO98/06822 disclosed recombination human serum albumin (rHSA) for animal cell culture on February 19th, 1998, and wherein recombination human serum albumin is for the growth that promotes cell.WO99/12568 on March 18th, 1999 disclosed recombination human serum albumin and the stablizer of the compositions such as sugar, inorganic salt 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 in EP0612761, EP0570916 and EP0699687.Yet, these methods all many because of step, the cycle is long, cost is high becomes complicated, thereby is difficult to satisfy the economic requirement that industry is amplified.The own patent application CN1496993A of applicant and CN1854155A disclose and have prepared the method that production of vaccine is used rHSA.Compare with additive method, the impurity level decreases such as the substance that show color of the restructuring HSA feature that the method is purified and polysaccharide, but level of endotoxin is difficult to control, and need to carry out further optimization process, controls intracellular toxin, improves yield 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 efficiently industrialized rHSA.
The present invention is achieved through the following technical solutions:
At first have by the applicant gene engineering microzyme and the production method acquisition recombination human serum albumin fermented liquid that patent application technology (CN1854301 and CN1854306) builds by oneself, utilize the ceramic membrane in 0.5um aperture to filter, supernatant liquor after heating is through too high salt cation displacement chromatography, the solution of results carries out borate to be processed, and the solution of processing through tubular fibre carries out hydrophobic displacement chromatography and weak anionic displacement chromatography according to the technique in 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 removing method of borate precipitation is described as centrifugal removal in patent CN1854155A, but in actually operating, centrifugal rear feed clarification degree is low, power consumption is larger on producing, different from patent CN1854155A is, process operation by introducing tubular fibre, make fermented supernatant fluid the time need not add gac in heating, and the solution that borate is processed is without centrifugally operated, thereby makes operation easier.Introduce the advantage of tubular fibre: the tubular fibre in suitable aperture can the decrease endotoxin content, the feed clarification degree is good after filter, and saves gac and centrifugation to the absorption of albumen, 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 lower than 0.5EU/mL (product concentration is as 100mg/mL) take tachypleus amebocyte lysate.
Unless stated otherwise, in the application, the implication of the concentration of rHSA (%) is g/100mL, i.e. the content (g) of rHSA in every 100mL solution, and the content that is rHSA in every 100mL solution as the implication of rHSA10% is 10g.
Particularly, the present invention relates to:
1) method of purifying rHSA capable of being industrialized a kind of, 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 processing 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., be 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 lower than the 100ppm total protein, is preferably the 10ppm total protein; Polysaccharide is residual lower than 10ug/mg rHSA, is preferably lower than 1ug/mgrHSA; Endotoxin content is preferably 0.5EU/mL (10%rHSA) lower than 0.85EU/mL (10%rHSA).
5) the item 4) purposes of described rHSA in virus vaccines production cell VERO, MDCK or MRC-5 cell culture medium.
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 the cultural method of virus is rolling bottle technique, the instillation process of microcarrier suspension technique 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) the item 4) purposes of described rHSA in preparation Antirabic Vaccine preparation, 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 liquid drugs injection or freeze-dried.
The production of vaccine that the present invention obtains has following characteristics with rHSA: 1) protein concentration is 99%-100% monomer and dimer, preferably 100% monomer and dimer basically.2) host protein is residual lower than the 100ppm total protein, is preferably the 10ppm total protein.3) polysaccharide is residual lower than 10ug/mg rHSA, is preferably the rHSA lower than 1ug/mg.4) endotoxin content 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 maintain base that the rHA 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 rolling bottle technique, the instillation process of microcarrier suspension technique or the absorption of multi-polyester sheet.Simultaneously, the rHSA of the method preparation can also be used as the stablizer of virus vaccines finished product, can be freeze-dried, can be also 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 to carry out clarifying treatment to fermented liquid, get fermented supernatant fluid 80L, add 5mM Sodium octoate, 5mM EDTA, heating is 20 minutes under 70 ℃, and is cooling.
B) high salt cation chromatographic separation and concentrated
Above-mentioned treated sample with the high salt cation CST-II of 150cm/h flow velocity loading FF post (GE company makes, and uses in advance the solution A balance for XK50/60, Vt=300ml), is used the solution B wash-out, get CST-II BD component.Be concentrated into the 100mg/ml left and right with the concentrated film bag of molecular weight cut-off 300K (MILLIPORE company produce).
C) borate is processed and the tubular fibre processing
Add sodium tetraborate and calcium chloride in concentrated 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
Add Sodium octoate and halfcystine in solution after processing to tubular fibre, to each 5mM of ultimate density and 10mM, 60 ℃ of heating 60 minutes, cooling rapidly.
E) hydrophobic chromatography separates and is concentrated
After heating sample is through 0.45 and the membrane filtration of 0.22um, enters Phenyl SFF post (GE company uses the solution C balance for XK50/60, Vt=500ml).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 with solution D, get the Amino-BD component.
G) the concentrated liquid that changes
Ultra-filtration membrane with molecular weight cut-off 30K is concentrated, then changes liquid with the Sodium phosphate dibasic-sodium dihydrogen phosphate that contains Sodium octoate, obtains finally containing 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 with the Bradford method, calculate total protein concentration, respectively go on foot yield thereby calculate; 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 2rHSA in Rabies Vaccine spinner culture technique
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, take MOI as 0.025～0.125 inoculation CTN strain seed culture of viruses (Nat'l Pharmaceutical ﹠ Biological Products Control Institute).After inoculation, use instead and add the DMEM substratum maintenance medium that production of vaccine is used rHSA 0.35%.34 ℃ of culture temperature.
C) virus is received liquid:
After using maintenance medium instead, every three days receipts are changed liquid once, continue to renew bright maintenance medium after receipts liquid, receive continuously liquid three times.Adopt respectively monoclonal antibody to prepare G protein content and the virus titer of double antibody sandwich ELISA and three receipts liquid of mouse titration experiments mensuration, 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 harvest liquid:
Carry out ultrafiltration and concentration after the virus harvest liquid clarification filtration: use the hollow fiber column of molecular weight cut-off 750K, virus harvest liquid is carried out ultrafiltration and concentration.In ultra-filtration process, virion is highly concentrated, and the small-molecule substance in nutrient solution such as albumin, the major part such as phenol red are being removed in seeing through liquid.
4 ℃ of deactivations of beta-propiolactone (1: 4000) 24 hours, 37 ℃ are hydrolyzed 2 hours;
Column chromatography purification: use Sepharose 4 Fast Flow chromatography columns, press column volume 10% loading, collect the 1st peak.After chromatography purification, the low molecular weight protein molecule in virus liquid (being mainly the albumin in nutrient solution) is removed, and in viral peak, protein content reduces greatly.
From chromatography column collect the peak extract measure the protein content sample after, as protective material, its final concentration is 1%, adds simultaneously Thiomersalate to cook sanitas, Thiomersalate final concentration≤0.08mg/ml to add immediately human serum albumin (Hengyang, Hunan Province the Southern Mountain pharmaceutical factory).Prepare according to protein content and antigenic content, packing becomes liquid preparation.
The preparation finished product is through check, indices all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC), be mainly: it is 4.6IU/ dosage that the NIH method is measured vaccine potency, particularly, utilizes Elisa test kit method the yeast cell host protein to be detected residual less than 5ng/ dosage.
The application of embodiment 3rHSA in Rabies Vaccine NBS bioreactor culture technique
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 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 production of vaccine with the DMEM substratum of rHSA 0.35%, take MOI as 0.025～0.125 inoculation CTN strain seed culture of viruses in the time of 4～5 days.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 6.5lgLD50.
D) purifying of Rabies Vaccine virus harvest liquid:
Purification process is carried out according to purification process in embodiment 2.
The preparation finished product is through check, and indices all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Be mainly: it is 4.6IU/ dosage that the NIH method is measured vaccine potency, particularly, utilizes Elisa test kit method the yeast cell host protein to be detected residual less than 5ng/ dosage.
The application of embodiment 4rHSA in Rabies Vaccine microcarrier bioreactor culture technique
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 under pH value 7.4 conditions, culturing cell is 5, and cell concn reaches 1.2 * 10
7Virus inoculation during cells/ml.
B) virus inoculation:
Take MOI as 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 harvest liquid sampling after virus infection.Carry out the detection of microscopy, titration of virus and glycoprotein.Glycoprotein Content converges rate lower than 20% less than 1IU/ml or cell, stops receiving liquid.
Received altogether liquid 14 days, results virus liquid volume 58L.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 harvest liquid:
Purification process is carried out according to purification process in embodiment 2.
The preparation finished product is through check, and indices all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Be mainly: it is 4.7IU/ dosage that the NIH method is measured vaccine potency, particularly, utilizes Elisa test kit method the yeast cell host protein to be detected residual less than 5ng/ dosage.
The application of embodiment 5rHSA in human diploid cell spinner culture production Rabies Vaccine technique
A) cell preparation:
Human diploid cell line MRC-5 is 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:
After inoculation 3～5 days, when the MRC-5 Growth of Cells when cultivating into individual layer, inoculation CTN strain seed culture of viruses, inoculating MOI is 0.025～0.125.After connecing poison, use the DMEM substratum maintenance medium of adding rHSA 0.35% instead.34 ℃ of culture temperature.
C) virus is received liquid:
Connect rear 3 days of poison, begin to receive liquid, continue to renew bright maintenance medium after receipts liquid, receive continuously liquid three times.Adopt respectively monoclonal antibody to prepare G protein content and the virus titer of double antibody sandwich ELISA and three receipts liquid of mouse titration experiments mensuration, 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 harvest liquid:
Purification process is carried out according to purification process in embodiment 2.
The preparation finished product is through check, and indices all meets 2005 editions pharmacopeia requirements of the People's Republic of China (PRC).Be mainly: it is 4.5IU/ dosage that the NIH method is measured vaccine potency, particularly, utilizes Elisa test kit method the yeast cell host protein to be detected residual less than 5ng/ dosage.
Embodiment 6rHSA uses 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, add respectively the additives such as amino acid, vegetable protein hydrolyzate, recombination human serum albumin, ester class in DMEM (Hyclone company produce) substratum, 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, result shows that the alternative blood of rHSA source albumin is used for VERO cell non-serum culture medium, both 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, maintain.
C) change every other day liquid.
Experimental result: kept altogether 42 days, and kept when finishing adherence 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 MDCK serum-free maintain, both with contain 5% blood serum medium without 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, maintain.
3) change every other day liquid.
Experimental result: kept altogether 30 days, and kept when finishing adherence rate: SFMI and be 83.5%, SFMII is 85.0%.Show that the alternative human serum albumin of rHSA is used for MRC-5 cell non-serum maintain.
Embodiment 7 use recombination human serum albumins are made the stablizer of Rabies Vaccine
Aqueous injection: rabies virus particle after chromatography purification receive liquid extract measure the protein concentration sample after, add immediately final concentration be 1% rHSA as protective material, add simultaneously Thiomersalate to 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 result is 3.8IU/ml.
Freeze-dried: after chromatography purification, virion is received liquid according to protein content and antigenic content preparation work in-process, adds the 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 result is that 3.7IU/ props up.
Claims (4)
1. the method for a purifying rHSA capable of being industrialized, is characterized in that, utilizes ceramic membrane to carry out clarifying treatment to the fermented liquid that contains rHSA, with the supernatant liquor heating, need not add gac during heating; Supernatant liquor after heating is through too high salt cation displacement chromatography, and the solution of results utilizes hollow fiber column to filter after borate is processed, and the solution after filtration passes through hydrophobic displacement chromatography and weak anionic displacement chromatography successively, obtains the rHSA of purifying.
2. method according to claim 1, wherein the molecular weight cut-off of hollow fiber column is 500K.
3. method according to claim 1 and 2, wherein the aperture of ceramic membrane is 0.5 μ m.
4. method according to claim 1 and 2, wherein the damping fluid that uses of weak anionic displacement chromatography is the 50mM Sodium phosphate dibasic-sodium dihydrogen phosphate that contains 0.2M sodium-chlor.
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|---|---|---|---|---|
| WO2016108211A1 (en) * | 2015-01-01 | 2016-07-07 | Navya Biologicals Pvt Ltd | Novel method for efficient purification of human serum albumin |
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| CN101768206A (en) | 2010-07-07 |
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