U.S. patent application number 09/872134 was filed with the patent office on 2001-11-01 for methods for purifying viruses.
Invention is credited to Bondoc, Laureano L. JR., Tang, John Chu-Tay, Vellekamp, Gary.
Application Number | 20010036657 09/872134 |
Document ID | / |
Family ID | 26709386 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036657 |
Kind Code |
A1 |
Tang, John Chu-Tay ; et
al. |
November 1, 2001 |
Methods for purifying viruses
Abstract
The invention provides methods for purifying a virus from
impurities in an aqueous medium.
Inventors: |
Tang, John Chu-Tay;
(Livingston, NJ) ; Vellekamp, Gary; (Glen Ridge,
NJ) ; Bondoc, Laureano L. JR.; (Piscataway,
NJ) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
26709386 |
Appl. No.: |
09/872134 |
Filed: |
June 1, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09872134 |
Jun 1, 2001 |
|
|
|
08989227 |
Dec 11, 1997 |
|
|
|
6261823 |
|
|
|
|
60033176 |
Dec 13, 1996 |
|
|
|
Current U.S.
Class: |
435/239 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 7/00 20130101; C12N 2710/10351 20130101 |
Class at
Publication: |
435/239 |
International
Class: |
C12N 007/02 |
Claims
What is claimed is:
1. 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.
2. The method of claim 1, wherein the virus preparation is a cell
lysate.
3. The method of claim 2, wherein the cell lysate is filtered
before step a.
4. The method of claim 1, wherein the virus is a recombinant
adenovirus.
5. The method of claim 1, wherein the anion-exchange medium is
FRACTOGEL.TM.-DEAE.
6. The method of claim 1, wherein the size exclusion medium is
Superdex-200.
7. The method of claim 1, wherein the size-exclusion medium is
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.
8. The method of claim 1 wherein the anion exchange medium
comprises diethylaminoethyl groups on a cross-linked agarose,
cellulose, polyacrylamide or polystyrene backbone.
9. The method of claim 1, wherein the size-exclusion medium
comprises a cross-linked polysaccharide.
10. The method of claim 9, wherein the cross-linked polysaccharide
is a composite of cross-linked agarose and dextran.
17. The method of claim 1 wherein the virus is ACN53.
18. The method of claim 1, wherein the anion exchange
chromatographic medium is extensively washed before application of
the virus preparation.
19. A virus purified by the method of claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] 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.
[0002] 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
[0003] One aspect of the invention is a method for purification of
a virus preparation comprising:
[0004] a) subjecting the virus preparation to anion-exchange
chromatography, wherein the virus is eluted from an anion-exchange
chromatographic medium; and
[0005] 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).
[0006] The anion exchange medium can comprise diethylaminoethyl
groups on a cross-linked agarose, cellulose, polyacrylamide or
polystyrene backbone, such as FRACTOGEL.TM.-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.
[0007] 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.
[0008] A further aspect of the invention is a virus purified by the
method of claim 1.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0009] 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.
[0010] 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.TM. EMD DEAE-650M", and "FRACTOGEL.TM. AE. In some
embodiments of the invention, the "backbone" can be a solid support
such as a bead.
[0011] 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.
[0012] 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.sub.260 or A.sub.280 and pooling peak fractions; alternatively,
the eluant containing the A.sub.260 or A.sub.280 peak may be
collected in a single fraction. This single A.sub.260 or A.sub.280
fraction or pooled fractions in the eluant containing the virus are
termed "anion-exchange pool" herein.
[0013] 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 S-400 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).
[0014] "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.
[0015] 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.
1TABLE I Exemplary Materials Used In Anion-Exchange and Size
Exclusion Chromatography Purification Step Procedure Solution Used
DEAE-Fractogel Salt Adjustment 4 M NaCl Equilibration 265 mM NaCl,
2 mM MgCl.sub.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.sub.2, 2% (w/v) sucrose, 50 mM sodium phosphate at
pH 7.5 (Buffer A) Wash 2 265 mM NaCl, 2 mM MgCl.sub.2, 2% (w/v)
sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer A) 600 mM NaCl, 2
mM MgCl.sub.2, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5
(Buffer B) Elution 265 mM NaCl, 2 mM MgCl.sub.2, 2% (w/v) sucrose,
50 mM sodium phosphate at pH 7.5 (Buffer A) 600 mM NaCl, 2 mM
MgCl.sub.2, 2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5
(Buffer B) Superdex 200 Equilibration 130 mM NaCl, 2 mM MgCl.sub.2,
2% (w/v) sucrose, 50 mM sodium phosphate at pH 7.5 (Buffer C)
Elution 130 mM NaCl, 2 mM MgCl.sub.2, 2% (w/v) sucrose, 50 mM
sodium phosphate at pH 7.5 (Buffer C)
[0016]
2TABLE II In-process Control and Operating Variables for the
Anion-Exchange Chromatography Purification Procedure Variable
Recommended Value All procedures Temperature 4-12.degree. C.
Equilibration Flow rate <5 cm/min. 1) NaOH/NaCl Volume 5 column
volumes pH of effluent 12.0 2) HCl/NaCl Volume 6 column volumes pH
of effluent 8.0 3) Buffer A Volume 10 column volumes pH of effluent
equivalent to Buffer A_0.2 pH Load Flow rate <5 cm/min
Conductivity of the feed 20-30 mS Wash 1: Buffer A Flow rate <5
cm/min Volume 4 column volumes Wash 2: Buffer A/ Flow rate <5
cm/min Buffer B Volume 8 column volumes Elution: Buffer A/ Flow
rate <5 cm/min Buffer B Volume 30 column volumes Fraction
Selection A.sub.280 >>background
[0017]
3TABLE III In-Process Control and Operating Variables for the
Size-Exclusion Chromatography Purification Procedure Variable
Recommended Value All procedures Temperature 4-12.degree. C.
Equilibration: Buffer C Flow rate <1 cm/min Volume 1 column
volume pH of Effluent equivalent to Buffer C_0.2 pH Load Flow Rate
<1 cm/min Volume 0.2 column volumes Concentration 30
A.sub.280/mL Elution: Buffer C: Flow rate <1 cm/min Volume 1
column volume Fraction Selection A.sub.280 >>background
[0018] In an embodiment of the invention, the virus is loaded from
the anion-exchange pool onto a size-exclusion column. In some
embodiments, the column is prepared with a salt gradient decreasing
in ionic strength from the top towards the bottom of the column.
After loading, the virus moves down through the salt gradient
(since the virus is preferentially not adsorbed by the resin) and
the gentle change in ionic strength avoids damage to the virus.
After overtaking the salt gradient, the virus is eluted in a
low-salt (e.g. 0-200 mM NaCl) buffer. Such low salt buffers
include, but are not limited to, formulations for long term storage
or administration to patients.
[0019] In some embodiments, glycerol is added to the
chromatographic buffers, such as elution buffer, or to the pooled
fractions containing virus. Typically the 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
process. In further embodiments, other excipients, such as about
2-16% sucrose, may be used in place of the glycerol.
[0020] In a preferred embodiment, the size exclusion chromatography
column is equilibrated with buffer at low salt concentration, e.g.,
about 100 to 150 mM, especially about 130 mM NaCl. Just prior to
loading the feed, a salt gradient is loaded, equivalent to a
moderate fraction of the bed volume, e.g. 10 to 20%, preferably
about 15%, from low salt concentration (about 130 mM NaCl) to the
higher salt concentration of the feed (e.g., 400 to 450 mM NaCl,
especially about 420 nM NaCl).
[0021] A simple test is performed to determine the quality of the
DEAE-Fractogel pool and consequently whether a salt gradient should
be used. This test depends on the constancy of the
A.sub.320/A.sub.260 ratio of the DEAE-Fractogel pool diluted with
an appropriate pH 7.5 buffer over a period of a few minutes (e.g.,
5 minutes). An appropriate buffer consists of 50 mM sodium
phosphate, pH 7.5, 2 mM MgCl.sub.2, 2% sucrose, no NaCl. If the
A.sub.320/A.sub.260 ratio remains substantially constant over a
5-minute period (e.g., if it increases by no more than 0.04), then
that sample is suitable for either isocratic or salt-gradient
size-exclusion chromatography. If the ratio of A.sub.320/A.sub.260
increases more than about 0.04 during that period, then that
DEAE-Fractogel pool is preferably performed on salt-gradient
size-exclusion chromatography to improve the yield. Table IV
provides exemplary materials and protocols for the use of salt
gradient size exclusion chromatography.
4TABLE IV Exemplary materials and protocols for the use of salt
gradient size exclusion chromatography Step Procedure Typical Range
Preferred values Step (I) Equilibrate the 100 to 150 mM 130 mM NaCl
column with the NaCl low salt buffer Step (ii) Generate salt
(100-150) to 130 to 450 mM gradient at top (400-500) NaCl; of
column mM NaCl; 10 to 15% bed volume 20% bed volume Step (iii) Load
the DEAE- 10 to 20% bed 15% bed volume pool onto the volume column.
Step (iv) Elute the adenovirus 400 to 500 mM 450 mM NaCl with
high-salt NaCl solution Step (v) Complete the 400 to 500 mM 450 mM
NaCl elution of the NaCl Virus.
[0022] The purification method of the present invention is suitable
for scaling-up (or scaling down) and for large-scale containment.
Suitable procedures and guidelines well-known in the art can be
used and followed to control the virus and prevent biohazardous
situations: see, e.g., "Biosafety in Microbiological and Biomedical
Laboratories", 3rd Edition, edited by Richman and McKinney, U.S.
Department of Health and Human Services, published by the Center
for Disease Control and the National Institute of Health,
Washington, D.C., U.S. Government Printing Office, May 1993.
[0023] The methods of the instant invention are amenable to a wide
range of viruses, including but not limited to adenoviruses, pox
viruses, iridoviruses, herpes viruses, papovaviruses,
paramyxoviruses, orthomyxoviruses, retroviruses, and rotaviruses.
The viruses are preferably recombinant viruses, but can include
clinical isolates, attenuated vaccine strains, and so on.
[0024] Thus, for example, an exemplary recombinant adenovirus is
that can be purified by the method of the invention is ACN53, which
is disclosed in PCT patent application no. WO 95/11984.
[0025] In the first step, the ACN53 adenoviral vector 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. The concentrate is
frozen and stored at about -20_C until use. Frozen concentrate is
thawed, clarified by filtration through a 0.45_m filter, the
conductivity of the preparation adjusted to about 250-360 mM NaCl,
and subjected to DEAE chromatography. Solutions used in the
chromatography are listed in Table 1.
[0026] In the second step, the ACN53 adenoviral vector is purified
by size-exclusion chromatography on a Superdex-200 column. Selected
fractions containing virus are identified by A.sub.260 or A.sub.280
and pooled. The pooled fractions constitute the purified bulk ACN53
adenoviral vector which is then filtered sterile through a 0.2_m
filter and stored at about -20_C.
[0027] The virus in the DEAE-Fractogel pool may be unstable due to
the presence of a high concentration of salt (about 420 mM NaCl).
It is preferably processed immediately or stored at 4-12_C for not
more than about 24 hours.
[0028] Fractions of the elution profile showing the ACN53
adenoviral vector peak as determined by A.sub.260 or A.sub.280 are
pooled for further processing. The size-exclusion pool is filtered
through a 0.2_m filter. This filtrate, the final purified bulk
ACN53 adenoviral vector, is then transferred into sterile plastic
bottles (e.g. Teflon) and stored at about -20.degree. C. The
in-process controls for this step are listed in Table III.
[0029] The increase in purity of the ACN53 adenoviral vector at
each step of the purification method can be followed by Resource Q
HPLC (see Huyghe et al., Human Gene Therapy, Vol. 6 (November
1995), pp. 1403-1416 at p. 1405). The quality of the virus in the
first and second chromatographic pools is also monitored by
spectroscopic methods. The characteristic ratio of
A.sub.260/A.sub.280 is 1.23-1.31:1 for final purified virus. The
light scattering which results from the high molecular weight of
the virus is derived from the ratio of A.sub.320/A.sub.260 nm and
is also used to monitor the chromatographic pools. Purified, free
virus particles display a light scattering ratio of about
0.22-0.30:1.
[0030] The following Examples serve to illustrate the present
invention. The selected vectors and hosts and other materials, the
concentration of reagents, the temperatures, and the values of
other variables are only to exemplify how the present invention may
be carried out, and are not to be considered limitations
thereof.
EXPERIMENTAL EXAMPLES
[0031] A. Small Scale Purification of Adenovirus
[0032] (1) Anion-Exchange Chromatography (DEAE-Fractogel)
[0033] A DEAE-EMD Fractogel 650M column (E. Merck), 5.times.18 cm.,
was pre-equilibrated with 5 bed volumes (B.V.) of 0.5 M NaOH/1 M
NaCl followed by 6 B.V. of 0.1 M HCl/1 M NaCl, and then by 20 B.V.
of Buffer A (265 mM NaCl, 2 mM MgCl.sub.2, 2% (w/v) sucrose, 50 mM
sodium phosphate 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_membrane, and adjusted with a small volume of 4 M NaCl to a
conductivity equal to that of Buffer A. The feed was loaded onto
the column at a linear flow rate of 1 cm/min. The column was washed
with 4 B.V. of Buffer A. The column was then washed with 8 B.V. of
94% Buffer A/6% Buffer B (identical to Buffer A except that NaCl
was 600 mM). The column was eluted with 30 B.V. of a linear
gradient from 94% Buffer A/6% Buffer B to 100% Buffer B. Fractions
containing substantial virus were pooled to form the feed ("DEAE
pool") for the following column.
[0034] (2) Isocratic Size-Exclusion Chromatography
(Superdex-200)
[0035] Size exclusion chromatography was performed on a
Superdex-200 column (Pharmacia), 5.times.73 cm, pre-equilibrated
with 0.5 B.V. 0.5 M NaOH, 1 B.V. of H.sub.2O, and 2 B.V. of Buffer
C (130 mM NaCl, 2 mM MgCl.sub.2, 2% (w/v) sucrose, 50 mM sodium
phosphate at pH 7.5) at a linear flow rate of 0.6 cm/min. The feed
consisting of 220 ml of DEAE pool was loaded onto the column. ACN53
was eluted with Buffer C at a linear flow rate of 0.6 cm/min.
Fractions with substantial virus were pooled, passed through a
0.2_microfilter and stored. The virus concentrate can be stored at
low temperature, e.g., at 0-10_C, preferably at about 4_C, or if
the volume is small, e.g., less than about 50 ml, frozen at
-80_C.
[0036] (3) Salt-Gradient Size-Exclusion Chromatography
[0037] (a). Salt Dilution Test
[0038] The DEAE-Fractogel pool (0.4 ml) was mixed with of a buffer
consisting of 50 mM sodium phosphate, pH 7.5, 2 mM MgCl.sub.2, 2%
sucrose, no NaCl (0.8 ml) and immediately placed in a quartz
cuvette and measured for absorbance at 260 and 320 nm on a UV
spectrometer equipped with a photodiode array. Without removal of
the sample from the cuvette, the reading was repeated at 1-2 minute
intervals over a 5-minute period. If the ratio of
A.sub.320/A.sub.260 was substantially constant during that period,
then that DEAE-Fractogel pool was suitable for either isocratic or
salt-gradient size-exclusion chromatography. If the ratio of
A.sub.320/A.sub.260 increased more than about 4% during that
period, then that DEAE-Fractogel pool required salt-gradient
size-exclusion chromatography to improve the yield.
[0039] (b) Salt-Gradient Size-Exclusion Chromatography
[0040] A salt-gradient chromatography was performed on a
Superdex-200 column, 2.6 cm.times.60 cm, pre-equilibrated with 0.5
B.V. 0.5 M NaOH, 1 B.V. of H.sub.2O, and 2 B.V. of Buffer C (20 mM
sodium phosphate, pH 8.0, 130 mM NaCl, 2mM MgCl.sub.2, 2% sucrose).
Immediately prior to loading the DEAE pool, a linear gradient from
100% Buffer C to 100% Buffer D (20 mM sodium phosphate, pH 8.0, 420
mM NaCl, 2 mM MgCl.sub.2, 2% sucrose) of 0.15 B.V. (48 ml) was
applied to the Superdex-200 column. The feed consisting of 20 ml of
a DEAE pool that had failed the above test was then loaded onto the
column and eluted with Buffer D at a linear flow rate of 0.6
cm/min. Fractions with substantial virus eluting in or near the
void volume were pooled, passed through a sterilizing filter and
stored at -80_C. The step yield was 60% and the A.sub.320/A.sub.260
ratio was 0.24:1.
[0041] B. Large-Scale Purification of Adenovirus
[0042] (1) Anion Exchange Chromatography
[0043] The frozen vial concentrate from the fermentation and
recovery step was thawed and filtered through a 0.45_m hydrophilic
Durapore membrane in a Millipore 10" Opticap capsule. The filtrate
was collected in a closed tank. To minimize losses, the filter
cartridge was washed with about 1.5 L of buffer J-1 (50 mM sodium
phosphate pH 7.5, 265 mM sodium chloride, 2 mM magnesium chloride,
2% (w/v) sucrose) supplemented with 5.4% (w/w) of solution J-3 (4 M
sodium chloride). The salt concentration of the filtrate was
adjusted by adding 5.4% (w/v) of solution J-3 (4 M sodium
chloride). This feed solution was then applied to a Fractogel EMD
DEAE-650 M column (7 cm diameter, 14.8 cm bed height, 570 ml bed
volume) pre-equilibrated with buffer J-1 (50 mM sodium phosphate pH
7.5, 265 mM sodium chloride, 2 mM magnesium chloride, 2% (w/v)
sucrose). The Adenovirus binds to the anion exchange resin, whereas
the majority of media and host cell impurities pass through the
column in the spent charge. The column was initially washed with 4
bed volumes of buffer J-1 followed by a second isocratic wash of 8
bed volumes of 94% buffer J-1 and 6% buffer J-2 (50 mM sodium
phosphate pH 7.5, 600 mM sodium chloride, 2 mM magnesium chloride,
2% (w/v) sucrose) to remove additional impurities. The virus was
eluted from the column with a 30 bed volume linear gradient from 6%
to 100% buffer J-2. The adenovirus peak of the elution profile as
determined by A.sub.280 was collected and pooled for further
processing. The in-process controls and operating parameters for
the anion exchange chromatography step are summarized in Table
V.
5TABLE V In-Process Control and Operating Parameters for the
Anion-Exchange Chromatography Column size: 7 cm diameter, 14.8 cm
bed height, 570 ml bed volume Purification Procedure Parameter
Value All Procedures Temperature 3 to 9_C. Equilibration Flow Rate
4 cm/min Volume 22 Column Volumes pH of Effluent Equal to Buffer
J-1 (7.4) Load Flow Rate 1 cm/min Conductivity of the Feed 28.2 mS
Wash 1 Flow Rate 2 cm/min Volume 4 Column Volumes Wash 2 Flow Rate
2 cm/min Volume 8 Column Volumes Elution Flow Rate 2 cm/min Volume
30 Column Volumes Fraction Selection A.sub.280 Peak area
[0044] (2) Size Exclusion Chromatography
[0045] The DEAE-Pool was applied immediately to a Superdex-200 size
exclusion column (14 cm diameter, 77 cm bed height, 11.9 L bed
volume) pre-equilibrated with buffer K-1 (20 mM sodium phosphate pH
8.0, 100 mM sodium chloride, 2 mM magnesium chloride, 2% (w/v)
sucrose). The column was eluted with buffer K-1. The adenovirus
peak of the elution profile as determined by A.sub.280 was
collected and pooled. This chromatography step achieved a buffer
exchange and separation of low molecular weight impurities from the
adenovirus product. The in-process controls and operating
parameters for the size exclusion chromatography step are
summarized in Table VI.
6TABLE VI In-Process Control and Operating Parameters for the Size
Exclusion Chromatography Column size: 14 cm diameter, 77 cm bed
height, 11.9 L bed volume Purification Procedure Parameter Value
All Procedures Temperature 3 to 9_C. Equilibration Flow Rate 0.43
cm/min Volume 2.3 Column Volume Load Flow Rate 0.43 cm/min Volume
_0.09 Column Volumes Concentration 2.7 A.sub.280/mL Elution Flow
Rate 0.43 cm.min Volume 1.5 Column Volume Fraction Selection
A.sub.280 Peak area
[0046] (3) Final 0.2_m Filtration
[0047] The Superdex 200-pool was filtered through a 0.2_m
hydrophilic Durapore membrane (Stericup, Millipore) at 2 to 15_C.
This step was carried out under sterile conditions in a biosafety
cabinet. Because several filtration devices were used, the
individual filtrates were pooled and then aliquoted into autoclaved
containers. The containers of bulk drug substance in solution were
frozen in a dry ice/ethanol bath and stored at about -20 C.
[0048] All publications and patent applications cited herein are
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
[0049] 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 thereby.
* * * * *