CA2070903A1 - Process for purifying hepatitis a virus (hav), virus thus purified and vaccine compositions containing it - Google Patents
Process for purifying hepatitis a virus (hav), virus thus purified and vaccine compositions containing itInfo
- Publication number
- CA2070903A1 CA2070903A1 CA002070903A CA2070903A CA2070903A1 CA 2070903 A1 CA2070903 A1 CA 2070903A1 CA 002070903 A CA002070903 A CA 002070903A CA 2070903 A CA2070903 A CA 2070903A CA 2070903 A1 CA2070903 A1 CA 2070903A1
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- hav
- nacl
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Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- 241000709721 Hepatovirus A Species 0.000 title claims abstract description 24
- 241000700605 Viruses Species 0.000 title claims description 22
- 229960005486 vaccine Drugs 0.000 title claims description 6
- 239000000203 mixture Substances 0.000 title claims description 4
- 238000002523 gelfiltration Methods 0.000 claims abstract description 11
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 230000009089 cytolysis Effects 0.000 claims abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000000463 material Substances 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 14
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 12
- 229920004890 Triton X-100 Polymers 0.000 claims description 11
- 239000013504 Triton X-100 Substances 0.000 claims description 11
- 239000007983 Tris buffer Substances 0.000 claims description 9
- 239000003599 detergent Substances 0.000 claims description 9
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 229920002684 Sepharose Polymers 0.000 claims description 5
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 4
- 230000006037 cell lysis Effects 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 238000005571 anion exchange chromatography Methods 0.000 claims 1
- 238000004440 column chromatography Methods 0.000 claims 1
- 239000003480 eluent Substances 0.000 claims 1
- 208000005252 hepatitis A Diseases 0.000 claims 1
- 238000005342 ion exchange Methods 0.000 claims 1
- 229920002113 octoxynol Polymers 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 10
- 239000011368 organic material Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000012286 ELISA Assay Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 235000019766 L-Lysine Nutrition 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000003957 anion exchange resin Substances 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002510 pyrogen Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000709664 Picornaviridae Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
ABSTRACT
A process is described for the purification of the hepatitis A
virus, which allows to obtain with good yields a pure product, in which organic material collected by centrifugation after lysis of the culture cells is submitted to gel filtration and successively to ion exchange chromatography.
A process is described for the purification of the hepatitis A
virus, which allows to obtain with good yields a pure product, in which organic material collected by centrifugation after lysis of the culture cells is submitted to gel filtration and successively to ion exchange chromatography.
Description
PROCESS FOR PURIFYING HEPATITIS A VIRUS (HAV), VIRUS THUS
PURIFIED AND VACCINE COMPOSITIONS CONTAINING IT.
Invention Field A process is described for the purification of the heptatitis A
virus (HAV) in which the material from the culture cells, after cell lysis and centrifugation, is subuitted to gel filtration and the thus obtained eluate is submitted to ion exchange chromatography.
State of the Art The hepatitis A virus (HAV) is a hicosahedral morphology Picornavirus with 32 capsomeres on its surface, which presents four important structural VP1 polypeptides with a molecular weight MSW 30.000-33.000, VP2 24.000-27.000, VP3 21.000-23.000, VP4 7.000-14.000.
Said four proteins are the ones responsible for the antigenic virus power and are therefore the ones which the purification processes tend to put in evidence and to isolate in order to obtain, with a good degree of purity, an inactivated vaccine.
The purification processes seek to eliminate cellular contaminants and growth factors which are employed in the virus production process.
Various methods for the partial virus purification, both for vaccination and for virus characterization purposes, have been described. For example, through the virus sedimentation by 2Q~03 means of a 20% sucrose pad and successive centrifugation in a cesium chloride gradient [P.J. Provost et al. J. of Medical Virology 19, p.23-31 (1986)], through ammonium sulphate precipitation and sedimentation with a 20% sucrose pad and cesium chloride gradient ~Flehmig B. et al., J. of Medical Virology 22, p. 7-16 (1987)], with a lysis buffer, freezing, defrosting, sonication to set the virus free, ultrafiltration with tangential flow and purification in cesium chloride gradient [Flehmig et al., The Lancet, May 13, p.1039 (1989)~, through various clarification cycles and successive freon or chloroform extraction followed by gel filtration, ion exchange chromatography and purification in cesium chloride gradient ~S.A. Locarnini, Intervirology 10, 300-308 ~1978)].
All the above mentioned processes employ, at least in one step, ultracentrifugation systems and high cost materials such as cesium chloricle, and therefore, although yielding excellent results on a small scale, are hardly suitable for an industrial production, in which process duration, costs and availability of sultable personnel have to be taken into consideration.
In the Europe~m Patent Application EP-A-302692 a process for the purification of hepatitis A virus is described, which employs sonication for the cell lysis, followed by extractlon with organic solvents and successive concentration, chromatography on anion exchange resins and, finally, gel filtration chromatography. Also this process, particularly in 207~903 view of the use of sonication and of organic phase extractions and concentrations, presents, on an industrial scale, certain operative difficulties.
Detailed description of the invention The process according to the present invention allows to obviate the mentioned drawbacks and is therefore a valid contribution to the purification on an industrial scale and the production of a purified HAV virus suitable for the use as a vaccine.
Diploid human cells MRC-5 designated by the World Health Organization as suitable for the production of vaccines for human use, cultivated and collected according to conventional techniques, were used.
The cells were infected with HAV at the 30th passage. After 21 days incubation, the cell substrate was washed with PBS-A to eliminate as much as possible the fetal bovine serum present in the culture medium and indispensable for the substrate.
The infected cells were taken up with trypsin EDTA following traditional methods and re-suspended in isotonic buPfer (Tris 10 mM, NaCl 10 mM, pH 7.5), this causing cell lysis and therefore setting the virus free, and frozen.
At the time of purification the material is defrosted and treated with 2Z Triton-X-100 for 30 min. at room temperature, stirring about every 5 min.; the material is then collected by centrifugation, so as to remove the cell fragments; this ~7~9~3 treatment allows the solubilization of membrane lipids with which the virus is strictly associated. The next step is gel filtration, employing gel filtration beds of both agarose and dextrane, e.g. Sepharose Cl-4B (Pharmacia) equilibrated in TNE
buffer (Tris 10 m~, NaCl 150 mM, EDTA 1 mM, pH 7.2-7.6), containing 0.1-0.2% Triton-X-100 or glycine buffer 0.1 M with 0.2% deoxycholate, pH 8.5. The eluated material is collected in 20 ml fractions which are tested for the presence of HAV by an ELISA assay. With this passage, yields of` 85-95% are obtained 10 with an approximate eight-fold purity increase (30-50 ug virus per mg of protein).
The eluate obtained in the preceding step is then submitted to ion exchange chromatography employing anion exchange resins, such as e.g. DEAE Sepharose CL-6B equilibrated in TNE
15 containing 0.1%-0.2~ Triton-X-100; in these conditions the virus is adsorbed on the bed while part of the contaminants are not retained.
After washing the column with TNE, to eLiminate the detergent, eluition is performed decreasing the pH and increasing the ion 20 strength. To this end a phosphate buffer may be employed with a continuous pH gradient from 7 . 4 to 4 and ionic strength from 0 to 0.3 M NaCl~ The yield in this second step is of the order of 50% with respect to the preceding step and the purity of the collected virus is increased 6 to 10 times (with an average 25 virus contents of 70~ on the total protain). The thus purified 2n7o~3 material is filtered on a membrane of 0.22 ~m porosity and inactivated with 1:2000 formalin at 35 C for 5 days under continuous stirring.
During the inactivation period, disaggregating treatments are performed, the 2nd day the material is sonicated at 50-60 W 1 sec/ml. The third day the material is filtered on a 0.22 ~m membrane and L-lysine.HCl 25 mM is added. After inactivation, the suspension is dialyzed against PBS A (1:100 v/v) for about 36 hrs, with an intermediate buffer substitution. After dialysis, the material is submitted to a sterilizing filtration and the product undergoes all the required controls: sterility, pyrogenicity, inactivation, antigenicity, pH, stability, residual formalin.
Experimental Section 15 MRC-5 cells at the 30th passage in rotating 850 cm3 bottles are infected with HAV (strain LSH/S ATCC VR 2266) at a 0.5 MOI.
After a 20 day incubation period, the cellular substrate is washed 3 times with serum-free medium maintaining the last washing overnight. The following day the cells are removed with tripsine-EDTA following traditional methods, and suspended again in isotonic buffer (Tris 10 mM, NaCl mM, pH 7.5) 1 ml for each 100 cm2 cell culture and Prozen.
60 ml of the frozen suspension, deriving from approximately 5.700 cm2 culture are defrosted and treated with a non ionic 25 detergent (2% Triton-X-100) for 20 to 30 minutes at room 2~7~9~3 temperature under moderate stirring every 5-10 minutes.
The sample is centrifuged at 400 g for 10 minutes while cooling to remove cellular fragments. The supernatant is purified through gel filtration on a agarose resin (Sepharose CL4B
Pharmacia)column 5 x 90 cm (K 50/100 column, Pharmacia) equilibrated with Tris lO mM, NaCl 150 mM, EDTA 1 mM buffer, pH
7.4, containing 0.1% Triton-X-100 at a 75 ml/h flow rate. The eluted material is collected in 20 ml fractions which are tested for the presence of HAV by a ELISA assay. The HAV
containing fractions are collected, obtaining approximately 400 ml. This material is further purified by ion exchange chromatography seeding about 200 ml, at a flow rate of 100 ml/h on a anionic resin (Sepharose CL6B Pharmacia) column 5 x 5 (column XK 50/30 Pharmacia) which had previously been equilibrated in Tris 10 mM, NaCl 150 mM, EDTA 1 mM, pH 7.4 buffer containing 0.1% Triton-X-100. Under such conditions the virus is adsorbed on the matrix. The matrix is washed with Triton-X-100 free buffer to remove the detergent and the virus is eluted at a flow of approximately 160 ml/h, applying a continuous pH gradient and ionic strength, starting from pH 7.4 and NaCl 0 mM to pH 4 and NaCl 0.3 M. The eluted material is collected in fractions of about 10 ml and the fractions found positive for the presence of HAV at a ELISA assay are put together.
A virus of 70X purity is thus obtained. The thus purified 207Q~
material is filtered on 0.22 ~m porous membrane and inactivated with formalin 1.2000 at 35 C for 5 days under continuous stirring. During the inactivation period, disaggregation treatments are performed: on the 2nd day the material is sonicated at 50-60 W per 1 second/l ml; on the 3rd day it is filtered on a 0.22 ~m membrane and L-lysine.HCl 25 mM is added.
After inactivation, the suspension is dialyzed against PBS-A
(KCl 2.7 mM, KH2P04 1.5 mM, NaCl 137 mM, NaH2P04 ~.1 mM, pH
7.4) in a 1:100 v/v ratio for 36 hours with an intermediate buffer substitution. After dialysis, the material undergoes a sterilizing filtration and is then submitted to the usual controls for sterility, pyrogenicity, inactivation, antigenicity, pH, stability and residual formalin.
PURIFIED AND VACCINE COMPOSITIONS CONTAINING IT.
Invention Field A process is described for the purification of the heptatitis A
virus (HAV) in which the material from the culture cells, after cell lysis and centrifugation, is subuitted to gel filtration and the thus obtained eluate is submitted to ion exchange chromatography.
State of the Art The hepatitis A virus (HAV) is a hicosahedral morphology Picornavirus with 32 capsomeres on its surface, which presents four important structural VP1 polypeptides with a molecular weight MSW 30.000-33.000, VP2 24.000-27.000, VP3 21.000-23.000, VP4 7.000-14.000.
Said four proteins are the ones responsible for the antigenic virus power and are therefore the ones which the purification processes tend to put in evidence and to isolate in order to obtain, with a good degree of purity, an inactivated vaccine.
The purification processes seek to eliminate cellular contaminants and growth factors which are employed in the virus production process.
Various methods for the partial virus purification, both for vaccination and for virus characterization purposes, have been described. For example, through the virus sedimentation by 2Q~03 means of a 20% sucrose pad and successive centrifugation in a cesium chloride gradient [P.J. Provost et al. J. of Medical Virology 19, p.23-31 (1986)], through ammonium sulphate precipitation and sedimentation with a 20% sucrose pad and cesium chloride gradient ~Flehmig B. et al., J. of Medical Virology 22, p. 7-16 (1987)], with a lysis buffer, freezing, defrosting, sonication to set the virus free, ultrafiltration with tangential flow and purification in cesium chloride gradient [Flehmig et al., The Lancet, May 13, p.1039 (1989)~, through various clarification cycles and successive freon or chloroform extraction followed by gel filtration, ion exchange chromatography and purification in cesium chloride gradient ~S.A. Locarnini, Intervirology 10, 300-308 ~1978)].
All the above mentioned processes employ, at least in one step, ultracentrifugation systems and high cost materials such as cesium chloricle, and therefore, although yielding excellent results on a small scale, are hardly suitable for an industrial production, in which process duration, costs and availability of sultable personnel have to be taken into consideration.
In the Europe~m Patent Application EP-A-302692 a process for the purification of hepatitis A virus is described, which employs sonication for the cell lysis, followed by extractlon with organic solvents and successive concentration, chromatography on anion exchange resins and, finally, gel filtration chromatography. Also this process, particularly in 207~903 view of the use of sonication and of organic phase extractions and concentrations, presents, on an industrial scale, certain operative difficulties.
Detailed description of the invention The process according to the present invention allows to obviate the mentioned drawbacks and is therefore a valid contribution to the purification on an industrial scale and the production of a purified HAV virus suitable for the use as a vaccine.
Diploid human cells MRC-5 designated by the World Health Organization as suitable for the production of vaccines for human use, cultivated and collected according to conventional techniques, were used.
The cells were infected with HAV at the 30th passage. After 21 days incubation, the cell substrate was washed with PBS-A to eliminate as much as possible the fetal bovine serum present in the culture medium and indispensable for the substrate.
The infected cells were taken up with trypsin EDTA following traditional methods and re-suspended in isotonic buPfer (Tris 10 mM, NaCl 10 mM, pH 7.5), this causing cell lysis and therefore setting the virus free, and frozen.
At the time of purification the material is defrosted and treated with 2Z Triton-X-100 for 30 min. at room temperature, stirring about every 5 min.; the material is then collected by centrifugation, so as to remove the cell fragments; this ~7~9~3 treatment allows the solubilization of membrane lipids with which the virus is strictly associated. The next step is gel filtration, employing gel filtration beds of both agarose and dextrane, e.g. Sepharose Cl-4B (Pharmacia) equilibrated in TNE
buffer (Tris 10 m~, NaCl 150 mM, EDTA 1 mM, pH 7.2-7.6), containing 0.1-0.2% Triton-X-100 or glycine buffer 0.1 M with 0.2% deoxycholate, pH 8.5. The eluated material is collected in 20 ml fractions which are tested for the presence of HAV by an ELISA assay. With this passage, yields of` 85-95% are obtained 10 with an approximate eight-fold purity increase (30-50 ug virus per mg of protein).
The eluate obtained in the preceding step is then submitted to ion exchange chromatography employing anion exchange resins, such as e.g. DEAE Sepharose CL-6B equilibrated in TNE
15 containing 0.1%-0.2~ Triton-X-100; in these conditions the virus is adsorbed on the bed while part of the contaminants are not retained.
After washing the column with TNE, to eLiminate the detergent, eluition is performed decreasing the pH and increasing the ion 20 strength. To this end a phosphate buffer may be employed with a continuous pH gradient from 7 . 4 to 4 and ionic strength from 0 to 0.3 M NaCl~ The yield in this second step is of the order of 50% with respect to the preceding step and the purity of the collected virus is increased 6 to 10 times (with an average 25 virus contents of 70~ on the total protain). The thus purified 2n7o~3 material is filtered on a membrane of 0.22 ~m porosity and inactivated with 1:2000 formalin at 35 C for 5 days under continuous stirring.
During the inactivation period, disaggregating treatments are performed, the 2nd day the material is sonicated at 50-60 W 1 sec/ml. The third day the material is filtered on a 0.22 ~m membrane and L-lysine.HCl 25 mM is added. After inactivation, the suspension is dialyzed against PBS A (1:100 v/v) for about 36 hrs, with an intermediate buffer substitution. After dialysis, the material is submitted to a sterilizing filtration and the product undergoes all the required controls: sterility, pyrogenicity, inactivation, antigenicity, pH, stability, residual formalin.
Experimental Section 15 MRC-5 cells at the 30th passage in rotating 850 cm3 bottles are infected with HAV (strain LSH/S ATCC VR 2266) at a 0.5 MOI.
After a 20 day incubation period, the cellular substrate is washed 3 times with serum-free medium maintaining the last washing overnight. The following day the cells are removed with tripsine-EDTA following traditional methods, and suspended again in isotonic buffer (Tris 10 mM, NaCl mM, pH 7.5) 1 ml for each 100 cm2 cell culture and Prozen.
60 ml of the frozen suspension, deriving from approximately 5.700 cm2 culture are defrosted and treated with a non ionic 25 detergent (2% Triton-X-100) for 20 to 30 minutes at room 2~7~9~3 temperature under moderate stirring every 5-10 minutes.
The sample is centrifuged at 400 g for 10 minutes while cooling to remove cellular fragments. The supernatant is purified through gel filtration on a agarose resin (Sepharose CL4B
Pharmacia)column 5 x 90 cm (K 50/100 column, Pharmacia) equilibrated with Tris lO mM, NaCl 150 mM, EDTA 1 mM buffer, pH
7.4, containing 0.1% Triton-X-100 at a 75 ml/h flow rate. The eluted material is collected in 20 ml fractions which are tested for the presence of HAV by a ELISA assay. The HAV
containing fractions are collected, obtaining approximately 400 ml. This material is further purified by ion exchange chromatography seeding about 200 ml, at a flow rate of 100 ml/h on a anionic resin (Sepharose CL6B Pharmacia) column 5 x 5 (column XK 50/30 Pharmacia) which had previously been equilibrated in Tris 10 mM, NaCl 150 mM, EDTA 1 mM, pH 7.4 buffer containing 0.1% Triton-X-100. Under such conditions the virus is adsorbed on the matrix. The matrix is washed with Triton-X-100 free buffer to remove the detergent and the virus is eluted at a flow of approximately 160 ml/h, applying a continuous pH gradient and ionic strength, starting from pH 7.4 and NaCl 0 mM to pH 4 and NaCl 0.3 M. The eluted material is collected in fractions of about 10 ml and the fractions found positive for the presence of HAV at a ELISA assay are put together.
A virus of 70X purity is thus obtained. The thus purified 207Q~
material is filtered on 0.22 ~m porous membrane and inactivated with formalin 1.2000 at 35 C for 5 days under continuous stirring. During the inactivation period, disaggregation treatments are performed: on the 2nd day the material is sonicated at 50-60 W per 1 second/l ml; on the 3rd day it is filtered on a 0.22 ~m membrane and L-lysine.HCl 25 mM is added.
After inactivation, the suspension is dialyzed against PBS-A
(KCl 2.7 mM, KH2P04 1.5 mM, NaCl 137 mM, NaH2P04 ~.1 mM, pH
7.4) in a 1:100 v/v ratio for 36 hours with an intermediate buffer substitution. After dialysis, the material undergoes a sterilizing filtration and is then submitted to the usual controls for sterility, pyrogenicity, inactivation, antigenicity, pH, stability and residual formalin.
Claims (18)
1. Process for purifying the hepatitis A virus (HAV) in which the material from the culture is submitted, after cell lysis and centrifugation, to gel filtration, and the thus obtained eluate is submitted to ion exchange column chromatography.
2. Process according to claim 1 in which the gel filtration is performed with agarose or dextrane matrices.
3. Process according to claim 2 in which the gel filtration matrix employed is Sepharose CL-4B.
4. Process according to claim 1 in which the gel filtration matrix is equilibrated with a pH 7.2-7.6 detergent containing buffer.
5. Process according to claim 4 in which the detergent containing buffer consists of Tris 10 mM, NaCl 150 mM, EDTA 1 mM, pH 7.4.
6. Process according to claim 5 in which said buffer contains an amount of 0.1-0.2% of Triton-X-100 as detergent.
7. Process according to claim 1 in which the ion exchange chromatography is performed on DEAE Sepharose CL-6B.
8. Process according to claim 7 in which DEAE Sepharose CL-6B
is equilibrated with a pH 7.2-7.6 detergent containing buffer.
is equilibrated with a pH 7.2-7.6 detergent containing buffer.
9. Process according to claim 8 in which the detergent containing buffer consists of Tris 10 mM, NaCl 150 mM, EDTA 1 mM, pH 7-4-
10. Process according to claim 9, in which said buffer contains an amount of 0.1-0.2% of Triton-X 100 as detergent.
11. Process according to claim 1, in which the ion exchange chromatography column is eluted decreasing the pH and increasing the ionic strength.
12. Process according to claim 11, in which a phosphate buffer with continuous pH gradient from 7.4 to 4 and ionic strength from 0 to 0.3 M NaCl is employed as eluent.
13. Process according to claim 11 in which the virus contained in the eluate is successively inactivated with 1:2000 formalin.
14. Process for purifying the hepatitis A virus (HAV) comprising the following steps:
a) MRC-5 cells infected by strain LSH/S (ATCC VR 2266) HAV
virus are cultivated and collected;
b) the cells are submitted to lysis with 2% Triton-X-100 and centrifuged;
c) the collected material is submitted to gel filtration on Sepharose CL-4B equilibrated with pH 7.4 Tris 10 mM, NaCl 150 mM, EDTA 1 mM, containing 0.1% of Triton-X-100;
d) the eluate from (c) is submitted to anion exchange chromatography on DEAE Sepharose CL-6B equilibrated with pH 7.4 Tris 10 mM, NaCl 150 mM, EDTA 1 mM, containing 0.1% of Triton-X-100;
e) the HAV virus adsorbed on the column in (d) is eluted in a continuous pH gradient employing phosphate buffer with a continuous gradient from 7.4 to 4 and a ionic strength gradient from 0 to 0.3 M NaCl f) the purified virus obtained in (e) is inactivated in 1:2000 formalin.
a) MRC-5 cells infected by strain LSH/S (ATCC VR 2266) HAV
virus are cultivated and collected;
b) the cells are submitted to lysis with 2% Triton-X-100 and centrifuged;
c) the collected material is submitted to gel filtration on Sepharose CL-4B equilibrated with pH 7.4 Tris 10 mM, NaCl 150 mM, EDTA 1 mM, containing 0.1% of Triton-X-100;
d) the eluate from (c) is submitted to anion exchange chromatography on DEAE Sepharose CL-6B equilibrated with pH 7.4 Tris 10 mM, NaCl 150 mM, EDTA 1 mM, containing 0.1% of Triton-X-100;
e) the HAV virus adsorbed on the column in (d) is eluted in a continuous pH gradient employing phosphate buffer with a continuous gradient from 7.4 to 4 and a ionic strength gradient from 0 to 0.3 M NaCl f) the purified virus obtained in (e) is inactivated in 1:2000 formalin.
15. Hepatitis A virus (HAV) strain LSH/S deposited as ATCC VR
2226.
2226.
16. HAV virus purified and inactivated, obtained according to the process claimed in claim 14.
17. The use of the inactivated virus according to claim 16 for the preparation of vaccine compositions against hepatitis A.
18. Vaccine compositions containing the virus according to claim 16.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITMI91A001662 | 1991-06-18 | ||
| ITSI911662 | 1991-06-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2070903A1 true CA2070903A1 (en) | 1992-12-19 |
Family
ID=11407671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002070903A Abandoned CA2070903A1 (en) | 1991-06-18 | 1992-06-10 | Process for purifying hepatitis a virus (hav), virus thus purified and vaccine compositions containing it |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2070903A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5602023A (en) * | 1992-03-24 | 1997-02-11 | Csatary; Laszlo K. | Pharmaceutical product containing live, stabilized virus for the therapy of viral and malignant diseases and process for preparing the same |
-
1992
- 1992-06-10 CA CA002070903A patent/CA2070903A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5602023A (en) * | 1992-03-24 | 1997-02-11 | Csatary; Laszlo K. | Pharmaceutical product containing live, stabilized virus for the therapy of viral and malignant diseases and process for preparing the same |
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