CA2111998A1 - Process for the purification and concentration of rubella virus - Google Patents
Process for the purification and concentration of rubella virusInfo
- Publication number
- CA2111998A1 CA2111998A1 CA 2111998 CA2111998A CA2111998A1 CA 2111998 A1 CA2111998 A1 CA 2111998A1 CA 2111998 CA2111998 CA 2111998 CA 2111998 A CA2111998 A CA 2111998A CA 2111998 A1 CA2111998 A1 CA 2111998A1
- Authority
- CA
- Canada
- Prior art keywords
- virus
- concentration
- purification
- rubella
- erythrocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000008569 process Effects 0.000 title claims abstract description 19
- 241000710799 Rubella virus Species 0.000 title claims abstract description 12
- 238000000746 purification Methods 0.000 title claims abstract description 7
- 241000700605 Viruses Species 0.000 claims abstract description 18
- 239000002245 particle Substances 0.000 claims abstract description 13
- 230000002441 reversible effect Effects 0.000 claims abstract 2
- 210000003743 erythrocyte Anatomy 0.000 claims description 21
- 150000002500 ions Chemical class 0.000 claims description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000002738 chelating agent Substances 0.000 claims description 4
- 102000005962 receptors Human genes 0.000 claims description 4
- 108020003175 receptors Proteins 0.000 claims description 4
- 102000018265 Virus Receptors Human genes 0.000 claims description 3
- 108010066342 Virus Receptors Proteins 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 201000005404 rubella Diseases 0.000 description 6
- 241000726306 Irus Species 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 241000237074 Centris Species 0.000 description 4
- 239000008139 complexing agent Substances 0.000 description 4
- 239000000470 constituent Substances 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000272201 Columbiformes Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- TWXDDNPPQUTEOV-FVGYRXGTSA-N methamphetamine hydrochloride Chemical compound Cl.CN[C@@H](C)CC1=CC=CC=C1 TWXDDNPPQUTEOV-FVGYRXGTSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229940070376 protein Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36211—Rubivirus, e.g. rubella virus
- C12N2770/36251—Methods of production or purification of viral material
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Abstract of the disclosure Process for the purification and concentration of rubella virus The invention relates to a process for the purification and concentration of rubella virus for diagnostic and therapeutic purposes by reversible binding to natural or synthetic particles which carry receptors for the virus.
Description
'' 2 ~
sEHRINGwERKE AXTIENGESELLSCE~FT 92/~ 031 - Ma 940 Foreign text Process for the puxification and concentrakion of rubella virus The invention relates to a proce~ ~or the purification and concentration of rubella virus for diagno~tic and therapeutic purposes by rever~ible binding to natural or synthetic particles which carry receptor~ for the viru~.
Proce~ses have already been di~clo~ed for the i~olation of, for exa~ple, rubella antigen from th~ supernatant ~rom infected animal cells, ~uch as, for example, the i~olation by ultracentrifugation, ~hich tak~ place either as pelleting or a~ di~ferential centrifugation by mean8 of a density gradient (A. Paris-Hamelin et al., ~. Virol. Meth. 10, 1985, 355 - 361). Other processe~ use ultrafiltration through filter areas with different eparation properties or precipitation proce~e~ ~uch as, for example, those with polyethylene glycol (P~G) (D.S. Bowden et al., J. gen. VirolO 65 (1984), 933 -943)-It ha~ also been di~closed how to co~centrate and purify20 influenzaviru~ using erythrocyte~ (B. Giesendorf et al., ~iru~ Re earch 1, 1984, 655 - 667). Ths elimination takes place in thi~ case enzymatic~lly with the aid o~ neur-ami~ldase.
The known ~olutions for the purification of rubella antigen have, however, inter alia the disadvantage that the viru~ antigen obtained in this way can be contami-nated to a greater or le~er extent with cellular pro-teins and/or other cellular constituent~.
Furthermore, components o~ the ~etal calf serum (FCS~
used ~or the cell cultivation o~ten occur a~
co~tamination.
The object of the invention was therefore to find a -~ ' ' ' , 2111~.3~
sEHRINGwERKE AXTIENGESELLSCE~FT 92/~ 031 - Ma 940 Foreign text Process for the puxification and concentrakion of rubella virus The invention relates to a proce~ ~or the purification and concentration of rubella virus for diagno~tic and therapeutic purposes by rever~ible binding to natural or synthetic particles which carry receptor~ for the viru~.
Proce~ses have already been di~clo~ed for the i~olation of, for exa~ple, rubella antigen from th~ supernatant ~rom infected animal cells, ~uch as, for example, the i~olation by ultracentrifugation, ~hich tak~ place either as pelleting or a~ di~ferential centrifugation by mean8 of a density gradient (A. Paris-Hamelin et al., ~. Virol. Meth. 10, 1985, 355 - 361). Other processe~ use ultrafiltration through filter areas with different eparation properties or precipitation proce~e~ ~uch as, for example, those with polyethylene glycol (P~G) (D.S. Bowden et al., J. gen. VirolO 65 (1984), 933 -943)-It ha~ also been di~closed how to co~centrate and purify20 influenzaviru~ using erythrocyte~ (B. Giesendorf et al., ~iru~ Re earch 1, 1984, 655 - 667). Ths elimination takes place in thi~ case enzymatic~lly with the aid o~ neur-ami~ldase.
The known ~olutions for the purification of rubella antigen have, however, inter alia the disadvantage that the viru~ antigen obtained in this way can be contami-nated to a greater or le~er extent with cellular pro-teins and/or other cellular constituent~.
Furthermore, components o~ the ~etal calf serum (FCS~
used ~or the cell cultivation o~ten occur a~
co~tamination.
The object of the invention was therefore to find a -~ ' ' ' , 2111~.3~
- 2 ~
~imple process in which the yield is improved and the impurities are minimized.
It has been found, surprisingly, that human or ani~al erythrocytes (which differ markedly fro~ viruse~ in their density and their size) also under certain conditions reversibly bind rubella virus, the re~ulting complex of cell and virus can then be removed ea6ily by 8uitable proce~ses from the medium in which the virus has been repliaated, and then the virus can under the condition~
according to the in~ention be eliminated ~rom the erythrocytes and, where appropriate, further concen-tra~ed.
The process according to the invention can also be u~ed for the puri~;.cation and concentration of those virus constituents which reversibly bind to erythrocytes undsr the condition~ according to the invention.
The term viru~ for the purpose o~ this invention also includes these virus constituents.
The invention therefore relate~ to a proce B for the puri~ication and concentration o~ rubella virus and virus particle~ by re~arsible binding to natural or ~ynthetic particles which carry receptors for this ~irus, where the ~iru~ particles are iæolated ~rom the u~pension, and the ~irus i5 detached from the particles and subsequently isolated, where ~he binding of the ~irus takes place in the pre~ence of doubly charged ions, and the release takes place in thé pre~ence of a chelating agent ~or doubly charged ions.
A pre~erred proce~s in thi~ connection is one in which the doubly charged ions are Mg2+ and/or Ca2+.
The pre$erred proce~ is alRo one in which the chelating agent is EDTA.
~ . . . . . ~ . ~ . - - .. .
, ;: ,.
An advantageous process is one in which the particle i~
an erythrocyte.
For the purification and concentration in thi~ case, for example, the virus-containing supernatant from infected cells i8 adjusted to a p~ of 4 ~ 10, pre~erably 6 - 8, particularly preferably about 7. Methods suitable for thi~ are known to the skilled worker. The temperature during this i8 0C to 30C, preferably 0 - 10C~ very preferably about 4C.
The salt concentration in the solution which contains the viruse~ is moreo~er adjusted so that, at the time o~ the binding of the viruses to the erythrocyte~, it contain~
in respect of Mg2+ 4 x 10-4 to 0.4, pre~erably 10-3 to 10-2, particularly preferably 3-5 x 10-3 mol/l, and in re~pect of Cà2~ 3 x 10-4 to 0.4, preferably 10-3 to 10-2, partiau-larly preferably 2-4 x 10-3 mol/l.
Chlorides are preferably employed as compounds.
The proce 8 according to the invention can be carried out with in each case one type o~ ion~.
Both ion~ are preférably employed, and very pr~ferably in approximately the ~ame molar co~aentrations.
Erythrocyte able to bind rubellavirus are added to the solution described abo~e. Erythrocytes ~rom a human, ! guinea pig, pigeon, wether, ~heep, rabbit or hor e are used advantageously.
The concentration o~ th`e erythrocyte~ in~this case i~
0.04 - 4% by Yolume, preferably about 0.4% by ~olume.
The i~cubation is carried out ~or 1 min - 48 h, pre~er-ably 20 min - 4 h, ~ery preerably about 1 h.
A~ a~vantageou process i~ also one in which, for reason~
o~ economy of work, incubation i9 carried out 'lo~er-night".
: :' ' . ' ' .' ~ ' . ' -~. 211~ng The complex of virus and cell is removed by ~ui~able method~. Centrifugation and filtration proces~e~ known per se to the skilled worker are particularly advanta-geou~ for thi~.
To eliminate the virus, a complexing agent which com-plexe~ doubly charged cations i6 addedO Complexing agent~
of this type are known per se to tha skilled worker and pre~erably used, beside~ EDTA, are biodegradable complex-ing agent as disclosed, for example, in EP 0 488 168.
The complexing agent concentration required for the eli~ination i~ advantageously about 3 x 10-2 to 3 x 10'3 mol/l and very particularly ad~antageously about 7 x 10-3 mol/l. The elimination and elution advantageously take~
place at a pH of 6 - 12, particularly ad~antageously at a pH of about 9.
Since erythrocyte~ and virus di~er greatly in ~ize and density, they can subæequently easil.y be separated ~rom one another by suitable methods known per ~e to the skilled wor~er, and thi~ ~eparation advantageously takea place by low-speed centrifugation, in which ca~e ~he erythrocytes ediment and the virus remain~ in the ~upernatant. The op~imal centri~uga~ion conditions can easily be determined experi~entally where appropriate.
It i~ al~o ad~antageous to employ the process in a column - 25 proceR~ which includes the following ~tep~:
a3 preparation of a colum~ with ~uitable erythrocyte b) addition of the viru~-containing ~olution taking into account the required ionic conditions, whereupon the binding to the erythrocytes takes place, -c) washing of the colu~n and ~ub~equently d) elution o~ the viru. or of the ~iru~ constituents by . : ' ' . ' .".' : :.............. ' `
. i .
2 ~
addition of the elution bu~er.
After the disclosure o~ the process according to the invention it is easy for the skilled worker to optimiza a column process of this type.
Variations which are traceable to the pre~ent inventi~e principle are conceivable for the ~aid proce~s step Thus, for example, the cellular virus receptor can be used after isolation, for example ~rom the erythrocytes.
The virus receptor can in this case be coupled to sedi-mentable plastic particles or magnetizable carriers.
A viru~ receptor o~ this type can also be prepared in an alternative way, ~or example by genetic manipulation, The advantage o the present prccess i~ that the ~irus i8 concentrated and, at the same time, unwa~ted contaminan 8 are remo~ed with simple means.
The ~ollowing example is i~tended to illu~trate the i~vention but not to restrict it.
~xample:
Material required - 10 l of crude rubella viru~ ~uspen~ion - immobilize~ wether erythrocytes - 2 N HCl - 2 l PBS, pH 7.0 with O . 1 g/l MgCl2 ~ X 6 H2O and 0.132 g/l of CaCla x 2 H2O
- 0.2 1 PBS, pEI 7.5 - 0.5 g EDTA
Ad~orptio~
h ' . ':. ' -.
':, ' ;'' "',' ' :
2 ~
10 1 of crude rubella virus 6u~pension were cooled to 4C
and adjusted to pH 7 with 2 N HCl. 160 ml of erythrocyte ~uæpension (25% by volume) were added; this correspond~
to a final concentration of 0.4% by volume. The mixture was ætirred with a stirrer at 4C ~or l - 16 h. The suspension was centrifuged in 1 l centrifuge cups in a Cryofuge 8000 (Heraeus, Germany) at 4C and 2700 rpm for 1 0 ~rli~ .
After the centrifugation, the erythrocyte pellet-was resu~pended in 1000 ml of PBS, pH 7Ø A second centri~ugation wa~ carried out undex the abovementioned conditions, and the ~upernatant was decanted off.
Preparation of the elution buffer 0-25% EDTA (ClOHl~N2O8Na2 2H2O; SERVA, Order No. 11280) was added to P~S p~ 7.5 without Mg2~/Ca2~ and completely dis~ol~ed. The ~olutio~ was ~ub~equently titrated to pH 9 with 2 N NaOH and cooled to 4C in a cold room.
The elution buffer i8 prepared fre~hly each time where poesible.
The erythrocyte pellet obtained by the aboveme~tioned proce~ wa cautiou~ly u~pended in 1~100 of the volume o~ the crude viru~ suspension u~ed (10 1 ~ 100 ml) of elution buffer and gently ~tirred at 4C for 15 min.
The su~pen~ion waæ centri~uged in a Cryofuge 8000 at 4C
and 3000 rpm for 10 min. The supernatant contained the large~t part of the ~irus employed and wa~ called "eluate 1". Eluate 1 was centri~uged once more to clarify ~rom remaining erythrocyte~: 4C, 3000 rpm, 10 min.
- The erythrocyte pellet was eluted once more under identical conditions~ this re~ulted in "eluate 2".
The eluates can be pooled or further processed ,: .
:.
- 7 ~ 3 separately.
The purificatlon was checked inter alia by employi~g an immunoblot quantification. This entailed the proteins being fractionated in a reducing SDS polyacrylamide gel (Laem~li, U.K., Nature 227 ~1970), pages 680 - 685), th~
proteins being transferred to nitrocellulo~e (sowen~ B .
et al., Nucleic Acids Re~. 8 (1980~, page~ 1 - 20) and the protein band being detected by the following detec-tion system which i~ know~ per se to the skilled worker, employing rubella i~munoglobulin, biotinylated anti-human immu~oglobulin and peroxida~e-labeled strepta~idin.
The quantification of an immunoblot of this tXpe using a ~canner i~ depicted in Figures 1 and 2, where 1, ~2 and C identify structural protein of rubella virus. PEGAg (Fig. 1) i~ material processed according to the prior art (Bowden et al. (1984)) and EryAg (Fig. 2) i~ matsrial purified by the process according to the invention.
A 2D-lD video den~itometer (Biotech/F:ischer Software) wa~
employed for the sca~ning (peak hsight: 5, i~tegration 20 resolution: 100%, peak width: 2, graph length: 15).
.
. , . .:
~ 5i3 De~criptio~ of the igure~
Figure l: Immunoblot quantification of a rubella antigen obtained by PEG precipitation.
Figure 2~ Immunoblot quantification of a rubella antigen i~olated by the process according to the inven-tio~.
El, E2, C: Structural proteins of rubella virus .
-.
:' ' ' ' '~- :
~imple process in which the yield is improved and the impurities are minimized.
It has been found, surprisingly, that human or ani~al erythrocytes (which differ markedly fro~ viruse~ in their density and their size) also under certain conditions reversibly bind rubella virus, the re~ulting complex of cell and virus can then be removed ea6ily by 8uitable proce~ses from the medium in which the virus has been repliaated, and then the virus can under the condition~
according to the in~ention be eliminated ~rom the erythrocytes and, where appropriate, further concen-tra~ed.
The process according to the invention can also be u~ed for the puri~;.cation and concentration of those virus constituents which reversibly bind to erythrocytes undsr the condition~ according to the invention.
The term viru~ for the purpose o~ this invention also includes these virus constituents.
The invention therefore relate~ to a proce B for the puri~ication and concentration o~ rubella virus and virus particle~ by re~arsible binding to natural or ~ynthetic particles which carry receptors for this ~irus, where the ~iru~ particles are iæolated ~rom the u~pension, and the ~irus i5 detached from the particles and subsequently isolated, where ~he binding of the ~irus takes place in the pre~ence of doubly charged ions, and the release takes place in thé pre~ence of a chelating agent ~or doubly charged ions.
A pre~erred proce~s in thi~ connection is one in which the doubly charged ions are Mg2+ and/or Ca2+.
The pre$erred proce~ is alRo one in which the chelating agent is EDTA.
~ . . . . . ~ . ~ . - - .. .
, ;: ,.
An advantageous process is one in which the particle i~
an erythrocyte.
For the purification and concentration in thi~ case, for example, the virus-containing supernatant from infected cells i8 adjusted to a p~ of 4 ~ 10, pre~erably 6 - 8, particularly preferably about 7. Methods suitable for thi~ are known to the skilled worker. The temperature during this i8 0C to 30C, preferably 0 - 10C~ very preferably about 4C.
The salt concentration in the solution which contains the viruse~ is moreo~er adjusted so that, at the time o~ the binding of the viruses to the erythrocyte~, it contain~
in respect of Mg2+ 4 x 10-4 to 0.4, pre~erably 10-3 to 10-2, particularly preferably 3-5 x 10-3 mol/l, and in re~pect of Cà2~ 3 x 10-4 to 0.4, preferably 10-3 to 10-2, partiau-larly preferably 2-4 x 10-3 mol/l.
Chlorides are preferably employed as compounds.
The proce 8 according to the invention can be carried out with in each case one type o~ ion~.
Both ion~ are preférably employed, and very pr~ferably in approximately the ~ame molar co~aentrations.
Erythrocyte able to bind rubellavirus are added to the solution described abo~e. Erythrocytes ~rom a human, ! guinea pig, pigeon, wether, ~heep, rabbit or hor e are used advantageously.
The concentration o~ th`e erythrocyte~ in~this case i~
0.04 - 4% by Yolume, preferably about 0.4% by ~olume.
The i~cubation is carried out ~or 1 min - 48 h, pre~er-ably 20 min - 4 h, ~ery preerably about 1 h.
A~ a~vantageou process i~ also one in which, for reason~
o~ economy of work, incubation i9 carried out 'lo~er-night".
: :' ' . ' ' .' ~ ' . ' -~. 211~ng The complex of virus and cell is removed by ~ui~able method~. Centrifugation and filtration proces~e~ known per se to the skilled worker are particularly advanta-geou~ for thi~.
To eliminate the virus, a complexing agent which com-plexe~ doubly charged cations i6 addedO Complexing agent~
of this type are known per se to tha skilled worker and pre~erably used, beside~ EDTA, are biodegradable complex-ing agent as disclosed, for example, in EP 0 488 168.
The complexing agent concentration required for the eli~ination i~ advantageously about 3 x 10-2 to 3 x 10'3 mol/l and very particularly ad~antageously about 7 x 10-3 mol/l. The elimination and elution advantageously take~
place at a pH of 6 - 12, particularly ad~antageously at a pH of about 9.
Since erythrocyte~ and virus di~er greatly in ~ize and density, they can subæequently easil.y be separated ~rom one another by suitable methods known per ~e to the skilled wor~er, and thi~ ~eparation advantageously takea place by low-speed centrifugation, in which ca~e ~he erythrocytes ediment and the virus remain~ in the ~upernatant. The op~imal centri~uga~ion conditions can easily be determined experi~entally where appropriate.
It i~ al~o ad~antageous to employ the process in a column - 25 proceR~ which includes the following ~tep~:
a3 preparation of a colum~ with ~uitable erythrocyte b) addition of the viru~-containing ~olution taking into account the required ionic conditions, whereupon the binding to the erythrocytes takes place, -c) washing of the colu~n and ~ub~equently d) elution o~ the viru. or of the ~iru~ constituents by . : ' ' . ' .".' : :.............. ' `
. i .
2 ~
addition of the elution bu~er.
After the disclosure o~ the process according to the invention it is easy for the skilled worker to optimiza a column process of this type.
Variations which are traceable to the pre~ent inventi~e principle are conceivable for the ~aid proce~s step Thus, for example, the cellular virus receptor can be used after isolation, for example ~rom the erythrocytes.
The virus receptor can in this case be coupled to sedi-mentable plastic particles or magnetizable carriers.
A viru~ receptor o~ this type can also be prepared in an alternative way, ~or example by genetic manipulation, The advantage o the present prccess i~ that the ~irus i8 concentrated and, at the same time, unwa~ted contaminan 8 are remo~ed with simple means.
The ~ollowing example is i~tended to illu~trate the i~vention but not to restrict it.
~xample:
Material required - 10 l of crude rubella viru~ ~uspen~ion - immobilize~ wether erythrocytes - 2 N HCl - 2 l PBS, pH 7.0 with O . 1 g/l MgCl2 ~ X 6 H2O and 0.132 g/l of CaCla x 2 H2O
- 0.2 1 PBS, pEI 7.5 - 0.5 g EDTA
Ad~orptio~
h ' . ':. ' -.
':, ' ;'' "',' ' :
2 ~
10 1 of crude rubella virus 6u~pension were cooled to 4C
and adjusted to pH 7 with 2 N HCl. 160 ml of erythrocyte ~uæpension (25% by volume) were added; this correspond~
to a final concentration of 0.4% by volume. The mixture was ætirred with a stirrer at 4C ~or l - 16 h. The suspension was centrifuged in 1 l centrifuge cups in a Cryofuge 8000 (Heraeus, Germany) at 4C and 2700 rpm for 1 0 ~rli~ .
After the centrifugation, the erythrocyte pellet-was resu~pended in 1000 ml of PBS, pH 7Ø A second centri~ugation wa~ carried out undex the abovementioned conditions, and the ~upernatant was decanted off.
Preparation of the elution buffer 0-25% EDTA (ClOHl~N2O8Na2 2H2O; SERVA, Order No. 11280) was added to P~S p~ 7.5 without Mg2~/Ca2~ and completely dis~ol~ed. The ~olutio~ was ~ub~equently titrated to pH 9 with 2 N NaOH and cooled to 4C in a cold room.
The elution buffer i8 prepared fre~hly each time where poesible.
The erythrocyte pellet obtained by the aboveme~tioned proce~ wa cautiou~ly u~pended in 1~100 of the volume o~ the crude viru~ suspension u~ed (10 1 ~ 100 ml) of elution buffer and gently ~tirred at 4C for 15 min.
The su~pen~ion waæ centri~uged in a Cryofuge 8000 at 4C
and 3000 rpm for 10 min. The supernatant contained the large~t part of the ~irus employed and wa~ called "eluate 1". Eluate 1 was centri~uged once more to clarify ~rom remaining erythrocyte~: 4C, 3000 rpm, 10 min.
- The erythrocyte pellet was eluted once more under identical conditions~ this re~ulted in "eluate 2".
The eluates can be pooled or further processed ,: .
:.
- 7 ~ 3 separately.
The purificatlon was checked inter alia by employi~g an immunoblot quantification. This entailed the proteins being fractionated in a reducing SDS polyacrylamide gel (Laem~li, U.K., Nature 227 ~1970), pages 680 - 685), th~
proteins being transferred to nitrocellulo~e (sowen~ B .
et al., Nucleic Acids Re~. 8 (1980~, page~ 1 - 20) and the protein band being detected by the following detec-tion system which i~ know~ per se to the skilled worker, employing rubella i~munoglobulin, biotinylated anti-human immu~oglobulin and peroxida~e-labeled strepta~idin.
The quantification of an immunoblot of this tXpe using a ~canner i~ depicted in Figures 1 and 2, where 1, ~2 and C identify structural protein of rubella virus. PEGAg (Fig. 1) i~ material processed according to the prior art (Bowden et al. (1984)) and EryAg (Fig. 2) i~ matsrial purified by the process according to the invention.
A 2D-lD video den~itometer (Biotech/F:ischer Software) wa~
employed for the sca~ning (peak hsight: 5, i~tegration 20 resolution: 100%, peak width: 2, graph length: 15).
.
. , . .:
~ 5i3 De~criptio~ of the igure~
Figure l: Immunoblot quantification of a rubella antigen obtained by PEG precipitation.
Figure 2~ Immunoblot quantification of a rubella antigen i~olated by the process according to the inven-tio~.
El, E2, C: Structural proteins of rubella virus .
-.
:' ' ' ' '~- :
Claims (5)
1. A process for the purification and concentration of rubella virus by reversible binding to natural or synthetic particles which carry receptors for this virus, where the virus is isolated from a suspension, detached from the particles and subsequently isolated, wherein the binding of the virus takes place in the presence of doubly charged ions, and the release takes place in the presence of a chelating agent for doubly charged ions.
2. The process as claimed in claim 1, wherein the doubly charged ions are Mg2+ and/or Ca2+.
3. The process as claimed in claim 1 or 2, wherein the chelating agent is EDTA.
4. The process as claimed in claim 1, wherein the particle is an erythrocyte.
5. The process as claimed in claim 1, wherein the particle is a synthetic particle to which an isolated virus receptor is bound.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4243491.2 | 1992-12-22 | ||
| DE19924243491 DE4243491A1 (en) | 1992-12-22 | 1992-12-22 | Process for the purification and enrichment of Rubella virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2111998A1 true CA2111998A1 (en) | 1994-06-23 |
Family
ID=6476127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2111998 Abandoned CA2111998A1 (en) | 1992-12-22 | 1993-12-21 | Process for the purification and concentration of rubella virus |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0603615A3 (en) |
| JP (1) | JPH06217767A (en) |
| AU (1) | AU678404B2 (en) |
| CA (1) | CA2111998A1 (en) |
| DE (1) | DE4243491A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1010981A1 (en) * | 1998-12-18 | 2000-06-21 | Diagor GmbH | Conjugates to detect viruses and their use |
| WO2001079456A2 (en) * | 2000-04-18 | 2001-10-25 | Cancer Research Technology Limited | Materials and methods relating to increasing viral titre |
| JP2006204201A (en) * | 2005-01-28 | 2006-08-10 | Nippon Sekijiyuujishiya | Method for separation/concentration and recovery/sample preparation of virus |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3316153A (en) * | 1965-03-29 | 1967-04-25 | Lilly Co Eli | Virus purification |
| FR2242463B1 (en) * | 1973-09-04 | 1976-06-18 | Bellon Labor Sa Roger | |
| AU7104194A (en) * | 1993-06-11 | 1995-01-03 | Peter Palese | High yielding influenza viruses |
| US5447859A (en) * | 1993-07-16 | 1995-09-05 | Viagene | Method for the purification or removal of retroviruses using sulfated cellulose |
| EP0714306B1 (en) * | 1993-08-06 | 1998-03-25 | Connaught Laboratories Limited | Inactivated respiratory syncytial viral vaccines |
-
1992
- 1992-12-22 DE DE19924243491 patent/DE4243491A1/en not_active Withdrawn
-
1993
- 1993-12-04 EP EP93119575A patent/EP0603615A3/en not_active Withdrawn
- 1993-12-17 AU AU52509/93A patent/AU678404B2/en not_active Ceased
- 1993-12-21 CA CA 2111998 patent/CA2111998A1/en not_active Abandoned
- 1993-12-21 JP JP32073593A patent/JPH06217767A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| DE4243491A1 (en) | 1994-06-23 |
| AU5250993A (en) | 1994-07-07 |
| AU678404B2 (en) | 1997-05-29 |
| JPH06217767A (en) | 1994-08-09 |
| EP0603615A2 (en) | 1994-06-29 |
| EP0603615A3 (en) | 1995-04-19 |
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