MXPA99005484A - Methods for purifying viruses - Google Patents
Methods for purifying virusesInfo
- Publication number
- MXPA99005484A MXPA99005484A MXPA/A/1999/005484A MX9905484A MXPA99005484A MX PA99005484 A MXPA99005484 A MX PA99005484A MX 9905484 A MX9905484 A MX 9905484A MX PA99005484 A MXPA99005484 A MX PA99005484A
- Authority
- MX
- Mexico
- Prior art keywords
- virus
- column
- size exclusion
- chromatography
- regulator
- Prior art date
Links
- 241000700605 Viruses Species 0.000 title claims abstract description 60
- 239000011780 sodium chloride Substances 0.000 claims description 55
- 150000003839 salts Chemical class 0.000 claims description 30
- 238000001542 size-exclusion chromatography Methods 0.000 claims description 20
- 241000701161 unidentified adenovirus Species 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 11
- 238000005571 anion exchange chromatography Methods 0.000 claims description 9
- 238000005349 anion exchange Methods 0.000 claims description 8
- 239000012501 chromatography media Substances 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 5
- 239000012535 impurity Substances 0.000 abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 50
- 238000005267 amalgamation Methods 0.000 description 17
- 238000004587 chromatography analysis Methods 0.000 description 13
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L MgCl2 Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
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- CZMRCDWAGMRECN-UGDNZRGBSA-N D-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 11
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 11
- 239000005720 sucrose Substances 0.000 description 11
- 239000001488 sodium phosphate Substances 0.000 description 10
- 229910000162 sodium phosphate Inorganic materials 0.000 description 10
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 150000001450 anions Chemical class 0.000 description 8
- 210000004027 cells Anatomy 0.000 description 7
- 239000002609 media Substances 0.000 description 7
- 238000010828 elution Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- -1 dimethylaminoethyl groups Chemical group 0.000 description 5
- 238000011068 load Methods 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000012506 Sephacryl® Substances 0.000 description 3
- 239000012505 Superdex™ Substances 0.000 description 3
- 239000003957 anion exchange resin Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 125000004432 carbon atoms Chemical group C* 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 150000004676 glycans Polymers 0.000 description 2
- 239000006174 pH buffer Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 229960005486 vaccines Drugs 0.000 description 2
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 241000701372 Iridovirus Species 0.000 description 1
- UEEJHVSXFDXPFK-UHFFFAOYSA-N N-dimethylaminoethanol Chemical group CN(C)CCO UEEJHVSXFDXPFK-UHFFFAOYSA-N 0.000 description 1
- 101700080605 NUC1 Proteins 0.000 description 1
- 229920001850 Nucleic acid sequence Polymers 0.000 description 1
- 206010037844 Rash Diseases 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000002238 attenuated Effects 0.000 description 1
- 239000003637 basic solution Substances 0.000 description 1
- 239000012512 bulk drug substance Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing Effects 0.000 description 1
- 125000006264 diethylaminomethyl group Chemical group [H]C([H])([H])C([H])([H])N(C([H])([H])*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- SSVFMICWXDVRQN-UHFFFAOYSA-N ethanol;sodium Chemical compound [Na].CCO SSVFMICWXDVRQN-UHFFFAOYSA-N 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000002934 lysing Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
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- 238000006011 modification reaction Methods 0.000 description 1
- 230000003472 neutralizing Effects 0.000 description 1
- 101700006494 nucA Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
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- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 230000000644 propagated Effects 0.000 description 1
- 229910052904 quartz Inorganic materials 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
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Abstract
The invention provides methods for purifying a virus from impurities in an aqueous medium.
Description
METHODS FOR PURIFYING VIRUSES
BACKGROUND OF THE INVENTION
The cultivation and purification of viruses has become increasingly important for the development of gene and vaccine therapy. Huyghe et al. (Human Gene Therapy 6: 1403-1416 (1995)) disclose a comparison of several methods for the purification of recombinant adenoviruses, including anion exchange chromatography, size exclusion chromatography, affinity immobilized chromatography. zinc, ultracentrifugation, concluding that the preferred procedure for the purification of a recombinant adenovirus is the treatment with nuclease of one of a cell lysate, followed by filtration through membrane filters, followed by DEAE chromatography, followed by affinity chromatography of zinc. In view of the continually increasing need for purified viruses, for example for use as viral vectors for gene therapy, improved purification methods would be highly desired.
BRIEF DESCRIPTION OF THE INVENTION
One aspect of the invention is a method for the purification of a virus preparation, comprising: a) subjecting the virus preparation to anion exchange chromatography, wherein the virus is eluted from anion exchange chromatographic medium; and b) subjecting the virus product of step a) to size exclusion chromatography, where the virus is eluted from a size exclusion chromatographic medium. The virus preparation can be a cell lysate, which can be filtered before step a). The virus can be a recombinant adenovirus, such as ACN53 (set forth in WO 95/11984). The anion exchange medium can comprise diethylaminomethyl groups on an interlaced base structure of agarose, cellulose, polyacrylamide or polystyrene, such as FRACTOGEL ™ -DEAD. The size exclusion means may comprise an entangled polysaccharide and may be a mixed body of interlaced agarose and dextran. An exemplary means of excluding size is Superdex-200. The medium can be washed extensively in the anion exchange chromatography medium prior to the application of the virus preparation. The size exclusion medium can be provided in a column prepared with a salt gradient that decreases in intensity
From the upper part of the column to the lower part, the upper part of the column having a pH regulator having an ionic strength substantially identical to that of the product of step a). A further aspect of the invention is a virus purified by the method of claim 1.
DESCRIPTION OF THE PREFERRED MODALITY
The present invention relates to the purification of a virus, which may have been produced, for example, by culture in a cell host and then released by lysis of the cells and separation of the cellular waste. The term "virus" includes wild type, mutant and recombinant viruses, especially adenoviral vectors for the expression of heterologous nucleic acid sequences. The embodiments of the invention form part of the general strategy of adsorption chromatography of a virus preparation followed by size exclusion chromatography. Typically, anion exchange chromatography on an anion exchange resin consisting of basic groups on fixed side chains with a macromolecular base structure is carried out. The basic groups are preferably substituted amino groups, in particular (lower dialkyl) -aminoalkyl groups wherein each lower alkyl group has from 1 to 4, preferably 2, carbon atoms, and each alkyl group has from 2 to 4, preferably 2, carbon atoms. The base structure may be composed of silica or an organic matrix, for example agarose, cellulose, polyacrylamide or entangled polystyrene, it is preferred to use in particular an anion exchange resin consisting of dimethylaminoethyl groups (DMAE groups) or especially diethylaminoethyl groups (groups DEAE) on an interlaced agarose base structure; Especially preferred DEAE type resins are those sold under the trade name "DEAE-Fractogel", for example "FRACTOGEL ™ EMD DEAE-650M" and "FRACTOGEL ™ AE." In some embodiments of the invention, the "structure of The base can be a solid support such as a globule.The anion exchange resin is preferably achieved extensively before loading the virus preparation to remove preservatives, such as sodium azide and ethanol, and other foreign materials, washing the column with about 5 to 10 column volumes of a basic solution, such as 50 mM NaOH / 1 M NaCl, followed by about 5 to 10 column volumes of a neutralizing solution, such as 50 mM HCl / NaCl a 1 M, followed by approximately 5 to 30 volumes of charge and / or elution pH regulators Optionally, the column is washed with a pH regulator of lower salt concentration the pH regulator charge and / or elution before washing with regula charge pH and / or elution. Typically, a virus preparation, such as a cell lysate, is loaded onto a chromatographic medium in a pH regulated solution at about 7.0-8.5, with a salt concentration of about 100-360 mM. The salt is typically NaCl. In some embodiments, other pH regulators are used, such as phosphate or Tris. Pollutants can be eluted by preferably washing the column with a pH regulator at a salt concentration of about 250-380 mM. The virus can then be eluted with a solution with a salt concentration of approximately 360-600 mM. The salt is typically NaCl. Typically, about 5 to 50, more preferably about 30, volumes of pH buffer are used to elute the virus. The fractions are gathered and analyzed for the presence of virus, measuring the fractions of peak A260 or A280 and of amalgamation; alternatively, the eluent containing the A260 or A2β peak, of a single fraction, can be combined. This single A260 or A2so fraction or those fractions amalgamated in the eluent containing the virus are referred to herein as "anion exchange amalgamation". In the step of size exclusion chromatography, the molecules are separated according to size in a bed charged with an inert porous medium, especially an inert gel medium, which is preferably a mixed body of cross-linked polysaccharides, for example , agarose and dextran crosslinked in the form of spherical globules. Molecules larger than the larger pores in the dilated gel cells do not enter the gel globules and therefore move through the chromatographic bed in the fastest manner. Smaller molecules, which enter the gel globules to varying degrees depending on their size and shape, are retarded as they pass through the bed. All molecules are thus eluted, usually in the order of decreasing molecular size. Viruses, because of their large size, usually elute in the empty volume. For example, the adenoviruses have a diameter of about 80 nm. Suitable means for size exclusion chromatography of adenoviruses include, but are not limited to, resins such as G600PWXL (TosoHass); SB-806 (Altech);
Sephacryl S-400 HR, Sephacryl S-500 HR, Sephacryl S-1000 SF, Sephadex
G-200, Sepharose CL-2B; Superdex 200 prep grade, Superóse 6 prep grade (Pharmacia); TSK 6000PWXL (Bodman), and Ultrahydrogel 2000 (Waters). The "size exclusion" chromatography as used herein is intended to include gel filtration chromatography. A particularly preferred means of exclusion is the one sold under the factory name "Superdex 200"; see Pharmacia Catalog, 1996, p. 338-339, code no. 17-1043-01 (in bulk) or 17-1069-01 or 17-1071-01 (previously loaded columns). Since a separation of virus groups from lower molecular weight impurities is achieved, the loading volume of the anion exchange amalgamation starting materials can be relatively large, for example up to 20%, more preferably 15%. %, of the bed volume. In Table I exemplary materials are provided for the practice of the chromatographic steps of anion exchange and size exclusion of the invention. In tables II and III exemplary variables and controls are provided.
TABLE 1 EXEMPLARY MATERIALS USED IN CHROMATOGRAPHY OF
EXCHANGE OF ANIONS AND SIZE EXCLUSION
TABLE II VARIABLES OF CONTROL AND OPERATION IN PROCESS FOR THE
CHROMATOGRAPHY OF EXCHANGE OF ANIONS
TABLE III VARIABLES OF CONTROL AND OPERATION IN PROCESS FOR THE
SIZE EXCLUSION CHROMATOGRAPHY
In one embodiment of the invention, the anion exchange amalgamation virus is loaded onto a size exclusion column. In some embodiments, the column is prepared with a salt gradient that decreases the ionic intensity from the top to the bottom of the column. After loading, the virus moves downstream of the salt gradient (since the virus is not preferentially absorbed by the resin) and the slight change in ionic strength prevents damage to the virus. After reaching the salt gradient, the virus is eluted in a low salt pH regulator (for example NaCl at 0-200 mM). Such low salt pH regulators include, but are not limited to, formulations for long-term storage or administration to patients. In some embodiments, glycerol is added to chromatographic pH regulators, such as elution pH regulator, or amalgamated fractions containing viruses. Typically, glycerol is present in a final concentration of 5-20%, more typically 10%. Thus, in some embodiments, glycerol is present in all solutions throughout the procedure. In other embodiments, other excipients, such as sucrose at about 2-16%, may be used in place of glycerol. In a preferred embodiment, the size exclusion chromatography column is equilibrated with a pH regulator at a low salt concentration, for example, NaCl at about 100 and up to 150 mM, especially at about 130 mM. Shortly before loading the feed, a salt gradient is loaded, equivalent to a moderate fraction of the bed volume, for example to 10 and up to 20%, preferably to about 15%, of the low salt concentration (NaCl a about 130 mM) at the highest concentration of feed salt (for example, NaCl at 400 and up to 450 mM, especially at approximately 420 mM). A simple test is carried out to determine the quality of the DEAE-Fractogel amalgamation and therefore if a salt gradient should be used. This test depends on the consistency of the 320/260 ratio of DEAE-Fractogel amalgamation with a pH regulator appropriate at 7.5 over a period of a few minutes (eg, 5 minutes). A suitable pH regulator consists of 50 mM sodium phosphate, pH 7.5, 2 mM MgCl 2, 2% sucrose ai, without NaCl. If the ratio of 320 / A260 remains substantially constant in a period of 5 minutes
(for example, if it grows at no more than 0.04), then that sample is suitable for either isocratic or size exclusion chromatography with salt gradient. If the ratio of A32o / A26o increases by more than about 0.04 during that period, the amalgamation of
DEAE-Fractogel preferably on size exclusion chromatography with salt gradient to improve the yield. Table IV provides exemplary materials and protocols for the use of size exclusion chromatography with salt gradient.
TABLE IV EXEMPLARY MATERIALS AND PROTOCOLS FOR THE USE OF THE
SIZE EXCLUSION CHROMATOGRAPHY WITH GRADIENT OF
SALT
The purification method of the present invention is suitable for scaling increase (or reduction) or large scale containment. Appropriate procedures and guidelines well known in the art for controlling the virus and avoiding biologically hazardous situations can be used and followed: see, for example, "Biosafety in Microbiological and Biomedical Laboratories," 3rd Edition, edited by Richman and McKinney, Department of Health and Humanitarian Services of the US: A. published by the Center for Disease Control and the National Institute of Health, Washington, DC, US Government Press, May 1993. The methods of the present invention are suitable for a wide variety of viruses, including, but not limited to, the same, adenoviruses, pustular eruption virus, iridovirus, herpes virus, papovavirus, paramyxovirus, otomyxovirus, retrovirus and rotavirus. The viruses are preferably recombinant viruses, but may include clinical isolates, attenuated vaccine strains, etc. Thus, for example, an exemplary recombinant adenovirus that can be purified by the method of the invention is ACN53, which is set forth in PCT patent application No. WO 95/11984. In the first step, the adenoviral vector ACN53 is purified by anion exchange chromatography on a DEAE column. For this, the virus is typically propagated in 293 kidney cells, harvested and subjected to concentration and ultrafiltration. Freeze and store the concentrate at approximately -20 ° C until use. The frozen concentrate is thawed, clarified by filtration through a 0.45 μm filter, the conductivity of the preparation is adjusted to approximately 250-360 mM NaCl and subjected to DEAE chromatography. The solutions used in the chromatography are listed in Table I. In the second step, the adenoviral vector ACN53 is purified by size exclusion chromatography on a Superdex-200 column.
Selected reactions containing viruses are identified by A26o or A28o and amalgamated. The amalgamated fractions constitute the purified massive ACN53 adenoviral vector which is then sterile filtered back from a 0.2 μm filter and stored at about -20 ° C. The virus in the amalgamation of DEAE-Fractogel can be unstable due to the presence of a high concentration of salt
(approximately NaCl at 420 mM). It is preferably treated immediately or stored at 4.12 ° C for no more than about 24 hours. The elution profile fractions that exhibit the peak of the adenoviral vector ACN53 determined by A260 or A280 are amalgamated for further treatment. The size exclusion amalgamation was filtered through a 0.2 μm filter. This filtrate, the final purified purified ACN53 adenoviral vector, is then transferred to sterile plastic bottles (for example Teflon) and stored at about -20 ° C. The controls in process for this step are listed in Table III. The purity increase of the adenoviral vector can be followed
ACN53 at each step of the purification method by Resource Q HPLC (see Huyghe et al., Human Genae Therapy, Vol. 6 (November 1995), pages 1403-1416 on page 1405). The quality of the virus is also monitored in the first and second chromatographic amalgamations, by spectroscopic methods. The characteristic ratio of A260 / A280 is 1.23-1.31: 1 for the final purified virus. The diffusion of light resulting from the high molecular weight of the virus is derived from the A320 / 360 nm ratio and is also used to monitor the amalgamate chromatographies. The purified free virus particles exhibit a light diffusion ratio of approximately
0. 22-0.30: 1. The following examples serve to illustrate the present invention. The vectors and hosts and other selected materials, the concentration of the reagents, the temperatures and the values of other variables are only to exemplify how the present invention can be carried out and are not to be considered limitations thereof.
EXPERIMENTAL EXAMPLES
A. Small scale purification of adenovirus (1) Anion exchange chromatography The DEAE-EMD Fractogel column was previously equilibrated
650M (E. Merck), 5 x 18 cm, with 5 bed volumes (V.L.) of NaOH at 0.5M / NaCl at 1 M, followed by 6 V.L. from 0.1 MHCI to 1 M / NaCl to 1M, and then by 20 V.L. of pH A regulator (NaCl at 265 mM, MgCl2 at 2 mM, sucrose
2% (w / w), sodium phosphate at 50 mM, at pH 7.5) at a linear flow rate of 2 cm / min. The feed for this column was derived from 2 liters of frozen crude virus solution, which was thawed, microfiltered through a 0.45 μm membrane and adjusted with a small volume of
NaCl at 4 M at a conductivity equal to that of the pH regulator A. The feed was loaded onto the column at a linear flow rate of 1 cm / min. The column was washed with 4 V.L. of pH regulator A. The column was then washed with 8 V.L. of regulator at 94% pH A / regulator at 6% pH B (identical to pH A regulator except that NaCl was 600 mM). The column was eluted with 30 V.L. from a linear gradient of the regulator to 94% of pH A / regulator at
6% of pH B to the buffer at 100% of pH B. The fractions containing substantial virus were mismanaged to form the feed ("amalgamation of
DEAE ") for the next column.
(2) Size-exclusion Socratic Chromatography (Superdex-200) Size exclusion chromatography was performed on a Superdex-200 (Pharmacia) column, 5 x 73 cm, broadly equilibrated with 0.5 V.L. of NaOH at 0.5 M, 1 V.L. of H2O and 2 V.L. of pH C regulator (130 mM NaCl, 2 mM MgCl 2, 2% sucrose (w / v), 50 mM sodium phosphate, at pH 7.5) at a linear flow rate of 0.6 cm / min. The feeding consisting of 220 ml of DEAE amalgamation was loaded onto the column. ACN53 was eluted with a pH C regulator at a linear flow rate of 0.6 cm / min. Fractions were amalgamated with substantial virus, passed through a 0.2 μ microfilter and stored. The virus concentrate can be stored at low temperature, for example, 0-10 ° C, preferably at about 4 ° C, or if the volume is small, for example, less than about 50 ml, freeze at -80 ° C.
(3) Size exclusion chromatography with gradient and salt (a) Salt dilution test The amalgamation of DEAE-Fractogel (0.4 ml) was mixed with a pH buffer consisting of 50 mM sodium phosphate, pH 7.5, MgCl2 at 2 mM, 2% sucrose (w / v), without NaCl (0.8 ml) and placed immediately in a quartz specimen and measured in terms of absorbance at 260 and 320 nm on a UV spectrometer equipped with a series of photodiodes. Without removing the sample from the specimen, the reading was repeated at 1-2 minute intervals for a period of 5 minutes. If the A320 / A260 ratio was sufficiently constant during that period, then amalgamation of DEAE-Fractogel was suitable for isocratic size exclusion chromatography or with salt gradient. If the ratio of A320 / A260 increased more than about 4% during that period, then the amalgamation of DEAE-Fractogel required that size exclusion chromatography with salt gradient will improve the yield.
(b) Size Excluding Chromatography with Salt Gradient Salt gradient chromatography was performed on a Superdex-200 2.6 cm x 60 cm column, was actually balanced with 0.5 or V.L. of NaOH at 0.5 M, 1 V.L. of H20, and 2 V.L. of the pH C regulator (20 mM sodium phosphate, pH 8.0, 130 mM NaCl, 2 mM MgCl 2, 2% sucrose). Immediately before loading the DEAE smear, a linear gradient of the 100% regulator of pH C was applied to the buffer at 100% pH D (20 mM sodium phosphate, pH 8.0, 420 mM NaCla, 2 mM MgCl 2, sucrose
2%) of 0.15 or V.L. (48 ml) to a column of Superdex-200. The feed consisting of 20 ml of a DEAE amalgamation that had been missing in the previous test was then loaded onto the column and eluted with pH D regulator at a linear flow rate of 0.6 cm / min. The fractions were amalgamated with substantial virus eluting in or near the empty volume, passed through a sterilization filter and stored at room temperature.
-80 ° C. The yield in this step was 60% and the A320 / A260 ratio was 0.24: 1.
B. Large Scale Purification Adenovirus (1) Anion exchange chromatography The frozen concentrate was thawed in a flask from the fermentation and recovery step and filtered through the 0.45 μm Durapore hydrophilic membrane in a Millipore 10"Opticap capsule. The filtrate was collected in a closed tank To minimize losses, the filter cartridge was washed with approximately 1.5 I of the pH regulator J-1 (50 mM sodium phosphate, pH 7.5, 265 mM sodium chloride, Magenium chloride at 2 mM, 2% sucrose (w / v)) was supplemented with 5.4% (w / w) of solution J-3 (sodium chloride at 4M) The salt concentration of filtrate was adjusted by adding 5.4% (w / v) of J-3 solution (4M sodium chloride) This feed solution was then applied to a Fractogel EMD DEAE-650 M column (7 cm diameter, bed height 14.8 cm) , 570 ml of bed volume) previously equilibrated with pH regulator J-1 (sodium phosphate
50mM, pH 7.5, sodium chloride at 265mM, magenation chloride at 2mM, sucrose at 2% (w / v)). The adenovirus binds to the exchange resin of
Ones, while most of the impurities media of the host cells pass through the column of the consumed charge. A column was initially developed with 4 volumes of 94% buffer at pH J-1 and 6% buffer at pH J-2 (50 mM sodium phosphate, pH 7.5, 600 mM sodium chloride, magnesium chloride at 2 mM, sucrose at 2% (w / v)) to remove additional impurities. The virus was eluted from the column with a linear gradient of 30 volumes of pH regulator J-2 at 6% to 7%. The adenavirus peak of the present invention determined by A280 was pooled and mixed for further treatment. The process control parameters in process for the anion exchange chromatography step are summarized in Table V.
TABLE V CONTROL AND OPERATION PARAMETERS IN PROCESS FOR THE
CHROMATOGRAPHY OF EXCHANGE OF ANIONS
(2) Size exclusion chromatography The amalgamation of DEAE was immediately applied to a size exclusion column of Superdex-200 (14 cm diameter, bed height 77 cm, bed volume 11.9) pre-equilibrated with regulator pH K-1 (sodium phosphate at 20 mM, pH 8.0, sodium chloride at 100 mM, magnesium chloride at 2 mM, sucrose at 2% (w / v)). The column was eluted with pH regulator K-1. The adenovirus peak of the elution profile determined by A2β- was pooled and amalgamated. The chromatography step achieved an exchange of pH regulator and separation of low molecular weight impurities from the adenovirus product. The parameters of controls and in-process operation of the size exclusion chromatography step are summarized in Table VI.
TABLE VI
CONTROL AND OPERATION PARAMETERS IN PROCESS FOR THE
SIZE EXCLUSION CHROMATOGRAPHY
(3) Final filtration of 0.2 μm The amalgamation of Superdex 200 was filtered through a Durapore hydrophilic membrane of 0.2 μm at 2 and up to 15 ° C. This step was carried out under sterile conditions in a biosafety cabinet. Since several filtering devices were used, the individual filtrates were amalgamated and aliquots were then introduced into autoclaved containers. The bulk drug substance containers were frozen in solution in a dry ice / ethanol bath and stored at about -20 ° C. All publications and patent applications cited in this document are incorporated by reference., to the same degree as if it were specifically and individually indicated that each individual manufacturing or patent application is incorporated by reference. The modifications and variations of this invention will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and the invention is not to be construed as limited by the same
Claims (8)
1. - A method for the purification of a virus preparation comprising: a) subjecting the virus preparation to anion exchange chromatography, wherein the virus is eluted from anion exchange chromatographic medium; and b) subjecting the virus product of step a) to size exclusion chromatography, where the virus is eluted from a size exclusion chromatographic medium.
2. The method according to claim 1, further characterized in that the virus preparation is a cell lysate.
3. The method according to claim 2, further characterized in that the cell lysate is filtered before step a).
4. The method according to claim 1, further characterized in that the virus is a recombinant adenovirus.
5. The method according to claim 1, further characterized in that the size exclusion means is provided in a column prepared as a salt gradient that decreases the ionic strength of the upper part of the bottom column, having the upper part of the column a regular pH having an ionic intensity substantially identical to that of the product of step a).
6. - The method according to claim 1, further characterized in that the virus is ACN53.
7. The method according to claim 1, further characterized in that it is extensively washed in anion exchange chromatographic medium before application and virus preparation.
8. A virus purified by the method of claim 1.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/766,835 | 1996-12-13 |
Publications (1)
Publication Number | Publication Date |
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MXPA99005484A true MXPA99005484A (en) | 2000-01-21 |
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