Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
1997-12-11
2001-07-17
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S235100, C210S660000
Reexamination Certificate
active
06261823
ABSTRACT:
BACKGROUND OF THE INVENTION
The cultivation and purification of viruses has become increasingly important for gene therapy and vaccine development. Huyghe et al. (
Human Gene Therapy
6: 1403-1416 (1995)) disclosed a comparison of several methods for purification of recombinant adenoviruses, including anion-exchange chromatography, size exclusion chromatography, immobilized zinc affinity chromatography, ultracentrifugation, concluding that the preferred process for purification of a recombinant adenovirus is nuclease treatment of a cell lysate, followed by filtration through membrane filters, followed by DEAE chromatography, followed by zinc affinity chromatography.
In view of the ever-increasing need for purified viruses, for example for use as viral vectors for gene therapy, improved methods of purification would be highly desired.
SUMMARY OF THE INVENTION
One aspect of the invention is a method for purification of a virus preparation comprising:
a) subjecting the virus preparation to anion-exchange chromatography, wherein the virus is eluted from an anion-exchange chromatographic medium; and
b) subjecting the virus product of step a to size exclusion chromatography, wherein 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 recombinant adenovirus, such as ACN53 (disclosed in WO 95/11984).
The anion exchange medium can comprise diethylaminoethyl groups on a cross-linked agarose, cellulose, polyacrylamide or polystyrene backbone, such as FRACTOGEL™-DEAE. The size-exclusion medium can comprise a cross-linked polysaccharide, and may be a composite of cross-linked agarose and dextran. An exemplary size exclusion medium is SUPERDEX-200. The anion exchange chromatographic medium can be extensively washed before application of the virus preparation.
The size-exclusion medium can be provided in a column prepared as a salt gradient decreasing in ionic strength from the top of the column towards the bottom, the top of the column having a buffer 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 EMBODIMENT
The present invention relates to the purification of a virus, which may for example have been produced by cultivation in a cellular host and then liberated by lysis of the cells and separation from cellular debris. The term “virus” includes wild type, mutant, and recombinant viruses, especially adenoviral vectors for expression of heterologous nucleic acid sequences.
The embodiments of the invention fall into the general strategy of adsorption chromatography of a virus preparation followed by size exclusion chromatography. Typically the anion exchange chromatography is carried out on an anion-exchange resin consisting of basic groups in side chains attached to a macromolecular backbone. The basic groups are preferably substituted amino groups, in particular diloweralkylaminoalkyl groups where each lower alkyl group has 1 to 4, preferably 2, carbon atoms, and each alkyl group has 2 to 4, preferably 2, carbon atoms. The backbone can be composed of silica or an organic matrix, for example cross-linked agarose, cellulose, polyacrylamide or polystyrene; it is particularly preferred to use an anion exchange resin consisting of dimethylaminoethyl groups (DMAE groups) or especially of diethylaminoethyl groups (DEAE groups) on a cross-linked agarose backbone; especially preferred resins of the DEAE type are those sold under the trade name DEAE-FRACTOGEL, e.g., “FRACTOGEL™EMD DEAE-650M”, and “FRACTOGEL™AE”. In some embodiments of the invention, the “backbone” can be a solid support such as a bead.
The anion-exchange-resin is preferably washed extensively before loading the virus preparation to remove preservatives such as sodium azide and ethanol, and other extraneous materials, by 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/1 M NaCl, followed by about 5 to 30 volumes of loading and/or elution buffers. Optionally, the column is washed with a buffer of lower salt concentration than the loading and/or elution buffer before washing with loading and/or elution buffer.
Typically, a preparation of virus such as a cell lysate is loaded onto the chromatographic medium in a buffered solution of about pH 7.0-8.5, with a salt concentration of about 100-360 mM. The salt is typically NaCl. In some embodiments other buffers such as phosphate or Tris are used. Contaminants can be preferentially eluted by washing the column with a buffer at a salt concentration of about 250-380 mM. The virus can then be eluted by a solution with a salt concentration of about 360-600 mM. The salt is typically NaCl. Typically, about 5 to 50, more preferably about 30 volumes of buffer are used to elute the virus. Fractions may be collected and assayed for the presence of virus by measuring the A
260
or A
280
and pooling peak fractions; alternatively, the eluant containing the A
260
or A
280
peak may be collected in a single fraction. This single A
260
or A
280
fraction or pooled fractions in the eluant containing the virus are termed “anion-exchange pool” herein.
In the size-exclusion chromatography step, molecules are separated according to size in a bed packed with an inert porous medium, especially an inert gel medium, which is preferably a composite of cross-linked polysaccharides, e.g., cross-linked agarose and dextran in the form of spherical beads. Molecules larger than the largest pores in the swollen gel beads do not enter the gel beads and therefore move through the chromatographic bed fastest. Smaller molecules, which enter the gel beads to varying extent depending on their size and shape, are retarded in their passage through the bed. Molecules are thus generally eluted in the order of decreasing molecular size. Viruses, because of their large size, generally elute in the void volume. For example, adenoviruses have a diameter of approximately 80 nm. Media appropriate for size-exclusion chromatography of adenoviruses include but are not limited to such resins as G6000PWXL (TosoHaas); SB-806 (Alltech); SEPHACRYL S400 HR, SEPHACRYL S-500 HR, SEPHACRYL S-1000 SF, SEPHADEX G-200, SEPHAROSE CL-2B; SUPERDEX 200 prep grade, SUPEROSE 6 prep grade (Pharmacia); TSK 6000PWXL (Bodman), and ULTRAHYDROGEL 2000 (Waters).
“Size-exclusion” chromatography as used herein is intended to include gel filtration chromatography. A particularly preferred size-exclusion medium is that sold under the trade name “SUPERDEX 200”; see the Pharmacia Catalog, 1996, pages 338-339, code no. 17-1043-01 (bulk), or 17-1069-01 or 17-1071-01 (pre-packed columns). Since a group separation of virus from impurities of lower molecular weight is achieved, the loading volume of starting materials from the anion-exchange pool can be relatively large, e.g., up to 20%, more preferably 15%, of the bed volume.
Exemplary materials for the practice of the anion-exchange and size exclusion chromatographic steps of the invention are provided in Table I. Exemplary variables and controls are provided in Tables II and III.
TABLE I
Exemplary Materials Used In Anion-Exchange and Size Exclusion
Chromatography
Purification Step
Procedure
Solution Used
DEAE-FRACTOGEL
Salt
4M NaCl
Adjustment
Equilibration
265 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer A)
50 mM NaOH, 1M NaCl
100 mM HCl, 1M NaCl
Wash 1
265 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer A)
Wash 2
265 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer A)
600 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer B)
Elution
265 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer A)
600 mM NaCl, 2 mM MgCl
2
, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (
Bondoc, Jr. Laureano L.
Tang John Chu-Tay
Vellekamp Gary
Gould James M.
Lankford , Jr. Leon B.
Schering Corporation
Schram David B.
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