U.S. patent application number 12/504073 was filed with the patent office on 2010-11-04 for purification of adenovirus and aav.
Invention is credited to Amy L. Erickson (Helgerson), Catherine E. O'Riordan, Alan E. Smith.
Application Number | 20100279385 12/504073 |
Document ID | / |
Family ID | 21703422 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279385 |
Kind Code |
A1 |
O'Riordan; Catherine E. ; et
al. |
November 4, 2010 |
PURIFICATION OF ADENOVIRUS AND AAV
Abstract
The present invention relates to the purification of large scale
quantities of active (infectious) adenovirus and AAV, especially
for use in therapeutic applications. In particular, the invention
provides improved methods for contacting such viruses with suitable
chromatographic materials in a fashion such that any damage to the
virus, particularly to surface components thereof, resulting from
contact with such chromatographic materials is minimized or
eliminated. The result is the ability to rapidly and efficiently
purify commercial level quantities of active (infectious) virus
suitable for use in therapeutic applications, e.g. gene
transfer/therapy procedures.
Inventors: |
O'Riordan; Catherine E.;
(Boston, MA) ; Erickson (Helgerson); Amy L.;
(Charlton, MA) ; Smith; Alan E.; (Dover,
MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Family ID: |
21703422 |
Appl. No.: |
12/504073 |
Filed: |
July 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179865 |
Jul 12, 2005 |
7579181 |
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12504073 |
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10470604 |
Mar 2, 2004 |
7015026 |
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11179865 |
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09604349 |
Jun 27, 2000 |
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10470604 |
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09011828 |
Jun 29, 1998 |
6143548 |
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PCT/US96/13872 |
Aug 30, 1996 |
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09604349 |
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60002967 |
Aug 30, 1995 |
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Current U.S.
Class: |
435/239 |
Current CPC
Class: |
Y10S 435/803 20130101;
C12N 2750/14143 20130101; C12N 2710/10351 20130101; C12N 2750/14151
20130101; A61P 43/00 20180101; C12N 15/86 20130101; C12N 7/00
20130101; C12N 2710/10343 20130101 |
Class at
Publication: |
435/239 |
International
Class: |
C12N 7/02 20060101
C12N007/02 |
Claims
1. A method of purifying active and infectious adenovirus
comprising the step of contacting said virus with a chromatographic
matrix material having binding groups with affinity for said virus
and wherein said binding groups are confined to pores in said
matrix sufficiently large to allow passage of said virus there
through in undamaged form.
2. A method of purifying active and infectious adenovirus
comprising the step of contacting said virus with a chromatographic
matrix material comprising pores and having binding groups with
affinity for said virus wherein said matrix further comprises
crosslinking or tentacles sufficient to substantially prevent the
virus from contacting said pores thereof.
3. A method of purifying adeno-associated virus from a sample also
containing adenovirus comprising the step of contacting said sample
with a chromatographic material capable of damaging adenovirus,
wherein said damaged adenovirus becomes non-infectious.
4. A method of purifying adeno-associated virus substantially in
conformity with the disclosure herein.
Description
[0001] The present application is a continuation of Ser. No.
10/470,604 filed on Mar. 2, 2004, which is a continuation of Ser.
No. 09/604,349 filed on Jun. 27, 2000 which is a continuation of
Ser. No. 09/011,828, filed Jun. 29, 1998, which is the U.S.
national stage filing under 35 U.S.C. Section 371 of the
application PCT/US96/13872, filed on Aug. 30, 1996, which is
related to and claims the benefit of the filing date of prior U.S.
Provisional Patent Application Ser. No. 60/002,967, filed Aug. 30,
1995, the text of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the purification of large
scale quantities of active (infectious) adenovirus and AAV,
especially for use in therapeutic applications. In particular, the
invention provides improved methods for 10 contacting such viruses
with suitable chromatographic materials in a fashion such that any
damage to the virus, particularly to surface components thereof,
resulting from contact with such chromatographic materials is
minimized or eliminated. The result is the ability to rapidly and
15 efficiently purify commercial level quantities of active
(infectious) virus suitable for use in therapeutic applications,
e.g. gene transfer/therapy procedures.
BACKGROUND OF THE INVENTION
[0003] Molecular therapy of disease often involves the
administration of nucleic acid to the cells of interest in order to
confer a therapeutic benefit. Most commonly, recombinant viruses
are engineered which take advantage of the natural infectivity of
viruses and their ability to transport heterologous nucleic acid
(transgene) to a cell. Widespread use of such recombinant viral
vectors depends on strategies for the design and production of such
viruses.
[0004] Most attempts to use viral vectors for gene therapy have
relied on retrovirus vectors, chiefly because of their ability to
integrate into the cellular genome. However, the disadvantages of
retroviral vectors are becoming increasingly clear, including their
tropism for dividing cells only, the possibility of insertional
mutagenesis upon integration into the cell genome, decreased
expression of the transgene over time, rapid inactivation by serum
complement, and the possibility of generation of
replication-competent retroviruses (Jolly, D., Cancer Gene Therapy
1:51-64, 1994; Hodgson, C. P., Bio Technology 13:222-225,
1995).
[0005] Adenovirus is a nuclear DNA virus with a genome of about 36
kb, which has been well-characterized through studies in classical
genetics and molecular biology (Horwitz, M. S., "Adenoviridae and
Their Replication," in Virology, 2nd edition, Fields, B. N., et
al., eds., Raven Press, New York, 1990). Adenovirus-based vectors
offer several unique advantages for delivering a therapeutic
transgene to a cell, including, inter alia, tropism for both
dividing and non-dividing cells, minimal pathogenic potential,
ability to replicate to high titer for preparation of vector
stocks, and the potential to carry large inserts (Berkner, K. L.,
Curr. Top. Micro. Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene
Therapy 1:51-64, 1994).
[0006] Adeno-associated virus (AAV) is a single-stranded
non-pathogenic DNA virus which is capable of integrating into the
genome of an infected cell. This feature of the virus life cycle
has focused attention on the use of AAV as a gene therapy vehicle
(creating a recombinant adeno-associated vector, rAAV) to deliver a
gene of interest for gene therapy. The ability of AAV to insert a
therapeutic gene into the cell genome facilitates persistent
expression of the gene of interest and reduces the need for
repeated dosing of a gene therapy vector.
[0007] Current methods for the purification of adenovirus and
adeno-associated virus (AAV) involve the use of density gradient
centrifugation, which does not easily allow for large scale
production of virus stocks for therapeutic use. A further
limitation to widespread use of AAV vectors is the general lack of
any adequate purification methods which yield high titers of AAV,
while removing contaminating adenovirus required for the
propagation of AAV vector stocks.
[0008] Ion-exchange, affinity chromatography and gel filtration are
widely used column chromatography tools in protein purification.
Until recently, however, these methods have been inapplicable to
purification of adenoviruses. Such techniques have resulted in
damage to the viruses, thereby reducing their ability to bind and
infect a target cell. Provisional U.S. patent application Ser. No.
60/002,967, filed Aug. 30, 1995, set forth parameters for purifying
infectious adenovirus utilizing chromatographic fractionation
techniques as described more fully herein. Recent studies have
shown that column chromatography may be used in the purification of
recombinant adenovirus (Huyghe et al., Human Gene Therapy
6:1403-1416, 1995).
[0009] Column chromatography, using other systems such as so-called
"macroporous" resins, which comprise beads having pores therein,
the average diameter of which is approximately the same as the
diameter of adenovirus (diameter=about 80 nm, excluding the fibres
and about 140 nm with the fibre molecules), have not resulted in
the recovery of infectious adenovirus. The most likely reason for
this is that the passage of adenovirus through such resins shears
the fibres from the viral surface through intimate contact of the
virus with the pores in the beads. The adenovirus fibre molecules,
inter alia, are believed to be involved in the virus's ability to
bind to and infect target cells. Thus, damage or loss of the fibre
molecules (as well as other surface molecules) by such prior art
column methods results in the recovery of inactive (non-infectious)
virus.
[0010] As is well known in the art, AAV propagation requires the
use of helper virus, such as adenovirus. The requirement for helper
virus complicates purification of AAV. Current approaches to AAV
purification involve lysing of AAV infected cells using repeated
cycles of freeze-thawing followed by the use of density gradient
centrifugation to fractionate the cell lysate in order to obtain
infectious AAV, free of cellular contaminants and substantially
free of helper virus (such as adenovirus) required for AAV
propagation. (Flotte et al., Gene Therapy 2: 29-37, 1995; Chiorini
et al., Human Gene Therapy 6: 1531-1541, 1995; Fisher et al., J.
Virol. 70: 520-532, 1996). Standard purification techniques
generally result in very low yields (0.3-5%) of active (infectious)
virus. Moreover, because of the helper-virus requirement, it has
been difficult to obtain AAV that is totally free of the helper
(e.g. adenovirus).
SUMMARY OF THE INVENTION
[0011] The present invention is directed to methods for the
purification of commercially useful quantities of infectious
adenovirus and AAV, especially for therapeutic use.
[0012] The present invention avoids the problems associated with
prior art methods of purifying infectious virus and relies on
chromatographic fractionation techniques which provide for large
scale separation of infectious adenovirus that are useful in gene
transfer of therapeutic transgenes to a host. Thus, the present
invention provides improved methods for contacting therapeutically
useful viruses with suitable materials used in chromatographic
fractionation techniques in a fashion and under conditions such
that the viruses, especially surface components thereof believed
required for infectivity, are not damaged by such contact.
[0013] Especially in regard to adenovirus purification, the
invention encompasses several design considerations involved in
these improved methodologies. These design considerations are
related in that they accomplish a similar objective--minimizing or
eliminating damage to the virus by contact with various
chromatographic materials used in purification. In particular, the
approaches are intended to obviate the effect of the openings or
pores in such materials which are involved in the partitioning of
biological molecules.
[0014] In one aspect of the invention, a "batch"-type technique may
be used. In this aspect, the virus is mixed with a suitable
chromatographic material rather than subjected to a "flow-through"
procedure. It is believed that with this approach, the virus
particles are less likely to enter the pores in the beads and
suffer damage.
[0015] A second aspect of the invention involves using
chromatographic materials in which the pore size of the support
material is so small that the virus cannot enter the pores during
chromatographic fractionation (e.g. in a column or membrane). The
reduction in pore size of such chromatographic materials can be
accomplished, e.g. by increased cross-linking of the support
matrix. The reduction in pore size prevents the viruses from
entering the openings in the beads where they can be damaged.
[0016] Alternatively, chromatographic materials may be used which
contain structures, e.g. "tentacles," that prevent viruses from
getting close to the pores in the matrix material. Again, this
serves to prevent or minimize damage to the virus particles.
[0017] In a third aspect of the invention, chromatographic
materials may be used wherein the matrix of the materials contains
openings or pores that are very large in size, i.e. pores that have
a diameter significantly larger than the diameter of the adenovirus
particles. Thus, the virus can be partitioned through the pores in
such chromatographic materials without being damaged.
[0018] Based on these design considerations, the purification
methods of the present invention allow for the use of wide variety
of commercially available chromatographic materials known to be
useful in fractionating biological materials. Useful support
matrices of such materials can include, inter alia, polymeric
substances such as cellulose or silica gel type resins or membranes
or cross-linked polysaccharides (e.g. agarose) or other resins.
Also, the chromatographic materials can further comprise various
functional or active groups attached to the matrices that are
useful in separating biological molecules.
[0019] As such, the methods of the invention also exploit the use
of affinity groups bound to the support matrices with which the
viruses interact via various noncovalent mechanisms, and can
subsequently be removed therefrom. Preferred approaches presented
in the present invention exploit ion-exchange (especially
anion-exchange) type interactions useful in chromatographic
fractionation techniques. Other specific affinity groups can
involve, inter alia, the use of heparin and virus-specific
antibodies bound to the support matrix. Of consideration in the
choice of affinity groups in virus purification via the present
techniques is the avidity with which the virus interacts with the
chosen affinity group and ease of its removal without damaging
viral surface molecules involved in infectivity.
[0020] Commercial-scale production of relatively high molecular
weight virus species (e.g. adenovirus and AAV) at high yields of
active (infectious) virus is achieved with these methods.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1: Schematic diagram of Acti-Mod.RTM. Cartridge
resin.
[0022] FIG. 2: Chromatogram showing elution profile of adenovirus
from DEAE-MemSep.RTM. resin. Elution peak for adenovirus is
labelled.
[0023] FIG. 3: Chromatogram showing elution profile of adenovirus
from Superdex.RTM. 200 resin. Elution peak for adenovirus is
labelled.
[0024] FIG. 4: SDS-PAGE analysis of adenovirus purified by DEAE and
gel filtration chromatography (Lane A), compared to CsCl gradient
purified virus (Lane B).
[0025] FIG. 5: SDS-PAGE analysis of adenovirus purified by heparin,
DEAE and Superdex.RTM. chromatography (Lane B), compared to CsCl
gradient purified virus (Lane A).
[0026] FIG. 6: Densitometric analysis of adenovirus purified by
heparin, DEAE and Superdex.RTM. chromatography B, compared to CsCl
gradient purified virus A.
[0027] FIG. 7: Chromatogram showing elution profiles of AAV from
(A) Superdex.RTM. 200 resin and (B) DEAE-MemSep.RTM. resin.
[0028] FIG. 8: SDS-PAGE analysis of two fractions of AAV purified
by Superdex.RTM. and DEAE chromatography (Lanes A and B).
[0029] FIG. 9A: Ceramic hydroxyapatite chromatography of an
AAV/adenovirus-containing 293 cell lysate. Elution peaks for both
AAV and adenovirus are labelled.
[0030] FIG. 9B: DEAE-MemSep.RTM. chromatography of an
hydroxyapatite AAV-containing eluate. Elution peaks for both AAV
and adenovirus are labelled.
[0031] FIG. 9C: Cellufine.RTM. sulfate Chromatography of an
AAV-containing DEAE eluate. The eluted AAV peak from the resin is
labelled.
[0032] FIG. 10: Coomassie-stained SDS-PAGE analysis of the AAV
containing fractions recovered from various columns: lane 1:
hydroxyapatite load; lane 2: hydroxyapatite eluate; lane 3: DEAE
eluate; lane 4: Cellufine.RTM. sulfate eluate; and lane 5:
Cellufine.RTM. sulfate eluate (100 .mu.l). Control protein
standards (in kD) are shown in the left column.
[0033] FIG. 11: SDS-PAGE analysis of AAV purified by ceramic
hydroxyapatite, DEAE and Cellufine.RTM. sulfate chromatography.
[0034] FIG. 12: Coumassie-stained protein gel of AAV purified by
ceramic hydroxyapatite, DEAE and Cellufine.RTM. sulfate
chromatography (Lane 1), compared to CsCl gradient purified AAV
(Lanes 2-4).
[0035] FIG. 13: Immunoblot of AAV purified by ceramic
hydroxyapatite, DEAE and Cellufine.RTM. sulfate chromatography
(Lane AAV), using an anti-Rep antibody, compared to Rep
controls.
[0036] FIG. 14: Schematic diagram of pTRlacZ.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention is directed to chromatographic
fractionation methods adaptable for large scale for the
purification of active (infectious) adenovirus and adeno-associated
virus (AAV), especially for use in therapeutic applications, such
as gene transfer, including gene therapy. The chromatography
methods of the invention are intended to replace the current
non-scaleable method of density-gradient ultracentrifugation of
virus purification and allow for production of active (infectious)
viruses on an industrial scale. The design strategies for
purification of the two viruses are interrelated. As
aforementioned, propagation of AAV requires the presence of
helper-virus components, most commonly provided by adenovirus. The
purification of AAV has been problematic because of adenoviral
contamination.
[0038] The chromatographic fractionation techniques of the
invention offer several advantages over prior virus purification
procedures based solely upon centrifugation: the methods are rapid;
the protocols are efficient, permitting separation of milligram
quantities of virus in a single run; virus integrity is not
compromised; and high yields of infectious virus are obtained.
Purification of Adenovirus
[0039] As aforementioned, prior attempts to adapt conventional
chromatographic procedures and materials, such as those routinely
used in the purification of proteins or other viruses, to the
purification of adenovirus, have been unsuccessful. It is believed
that in applying such procedures, insufficient care has been taken
to protect the adenovirus from damage, and in particular, to
macromolecular components thereof necessary for infectivity.
Without being limited as to theory, it is recognized that
particular adenoviral proteins, including most particularly the
protein species known as "fibre" are involved in the binding of
adenovirus to target cells during infection. Although numerous
copies of such proteins may be found on each adenovirus, viral
infectivity is relatively low for most cell types, and thus damage
to even a small portion of, for example, the fiber molecules or
other viral macromolecules can substantially prevent the
establishment of successful infection. Maintaining high infectivity
is therefore of considerable importance with respect to the
commercial scale production of adenoviral vectors intended for
therapeutic use, such as for gene therapy.
[0040] Accordingly, there are provided a wide variety of
chromatography methods to take properly into account the fragile
nature of adenovirus particles. As presently disclosed, a wide
variety of conventional chromatographic materials including matrix
or support materials, and the active (binding) groups routinely
coupled thereto, are useful in the practice of the invention.
[0041] The methods described here in permit retrieval of purified
adenoviral particles at high concentration in aqueous media without
damage to adenoviral components. Similarly, the methods are suited
to the preparation of milligram quantities of virus.
[0042] Adenovirus is isolated from virus-infected cells, for
example, 293 cells. Cells may be infected at high multiplicity of
infection (MOI) in order to optimize yield. Any method suitable for
recovering virus from infected cells may be utilized. Preferred
techniques for the recovery of virus from infected cells include
freeze-thawing and the use of a microfluidiser. However, in order
to make purification of viruses a scaleable process it is
preferable to use procedures which lyse the virus infected cells
without repeated freeze thawing and to remove cellular debris from
the cell lysate without centrifugation. Optimal conditions for
lysis of virus infected 293 cells for release of active virus may
be achieved using a pressure cell, e.g. a Microfluidiser pressure
cell (Microfluidics, Newton, Mass.). Ultrafiltration of the lysate
using, for example, a Minitan system (Millipore, Bedford, Mass.),
which comprises a High Resolution Tangential Flow System, can be
used to further concentrate the virus fraction prior to
chromatographic fraction techniques.
[0043] The adenovirus-containing lysate so obtained may then be
subjected to the chromatographic fractionation techniques of the
invention. In regard to adenovirus purification, the invention
encompasses several design considerations involved in these
improved methodologies. These design considerations are related in
that they accomplish a similar objective--minimizing or eliminating
damage to the virus by contact with various chromatographic
materials used in purification. In particular, the approaches are
intended to obviate the effect of the openings or pores in such
materials which are involved in the partitioning of biological
molecules.
[0044] In one aspect of the invention, a "batch"-type technique may
be used. In this aspect, the virus is mixed with a suitable
chromatographic material rather than subjected to a "flow-through"
procedure. It is believed that with this approach, the virus
particles are less likely to enter the pores in the beads where
they can become damaged.
[0045] A second aspect of the invention involves using
chromatographic materials in which the pore size of the support
material is smaller than that of the virus particles so that the
virus cannot enter the pores during chromatographic fractionation
(e.g., in a column or membrane). The reduction in pore size of such
chromatographic materials can be accomplished, e.g. by increased
cross-linking of the support matrix. The reduction in pore size
prevents the viruses from entering the openings in the beads where
they can be damaged.
[0046] Alternatively, chromatographic materials may be used which
contain structures, e.g. "tentacles," that prevent viruses from
getting close to the pores in the matrix material. Again, this
serves to prevent or minimize damage to the virus particles.
[0047] In a third aspect of the invention, chromatographic
materials may be used wherein the matrix of the materials contains
openings or pores that are very large in size, i.e., pores that
have a diameter significantly larger than the diameter of the
adenovirus particles. Thus, the virus can be partitioned through
the pores in such chromatographic materials without being
damaged.
[0048] Based on these design considerations, the purification
methods of the present invention allow for the use of wide variety
of commercially available chromatographic materials known to be
useful in fractionating biological materials. Useful support
matrices of such materials can include, inter alia, polymeric
substances such as cellulose or silica gel type resins or membranes
or cross-linked polysaccharides (e.g. agarose) or other resins.
Also, the chromatographic materials can further comprise various
functional or active groups attached to the matrices that are
useful in separating biological molecules.
[0049] As such, the methods of the invention also exploit the use
of affinity groups bound to the support matrices with which the
viruses interact via various non-covalent mechanisms, and can
subsequently be removed therefrom. Preferred approaches presented
in the present invention exploit ion-exchange (especially
anion-exchange) type interactions useful in chromatographic
fractionation techniques. Other specific affinity groups can
involve, inter alia, the use of heparin and virus-specific
antibodies bound to the support matrix. Of consideration in the
choice of affinity groups in virus purification via the present
techniques is the avidity with which the virus interacts with the
chosen affinity group and ease of its removal without damaging
viral surface molecules involved in infectivity.
[0050] The following types of chromatographic materials are
suitable for the batch-type chromatographic methods discussed
above.
[0051] The teachings of the present disclosure also make possible
the use of other commercially available chromatographic materials,
if only in batch form. Although not a preferred embodiment of the
invention, once it is understood that adenovirus can be damaged by
contacting such chromatographic materials, purification procedures
can be redesigned to minimize damage to the virus. To the extent
that many polymer materials contain pores which can damage any
contacting adenovirus, such damage can be limited by minimizing,
for example, column pressure, thereby limiting entry of adenovirus
into matrix pores. In the simplest example thereof, the
purification step is simply conducted in batch form. Polymer
materials useful according to this aspect of the invention include
the products Heparin Sepharose High Performance, of Pharmacia;
macroporous hydroxyapatite such as Macro-Prep Ceramic
Hydroxyapatite, Bio-Rad, Richmond, Calif.; and cellufine sulfate
from Amicon.
[0052] Chromatography materials comprise polymers having sufficient
matrix crosslinking such that interaction of adenovirus with any
pore spaces thereof is minimized, wherein also are present any of a
number of binding groups (including, for example, ion exchange
groups or heparins) having affinity for the adenovirus.
Representative heparinized polymers useful in the practice of the
invention include those having about 6% more of crosslinking, such
as Heparin Superflow Plus.RTM. (or Sterogene), a 6% crosslinked
heparin agarose. Such an agarose matrix has an exclusion limit of
about 6 million daltons which is expected to be much lower than
that of a 4% crosslinked product, such as Heparin Agarose (Sigma
Chemical Co.). In one experiment, recovery of adenovrius in
infective form was substantially improved when the 6% rather than
the 4% product was used. Additional polymers containing heparin
groups that are useful in the practice of the invention include
Heparin Sepharose.RTM. CL6B (Pharmacia).
[0053] Once the advantages of carefully controlled crosslinking are
understood, it is apparent that those skilled in the art could
substitute any number of polymer materials composed of any number
of recognized matrix materials and binding groups such that the
adenovirus is not damaged by contact therewith.
[0054] As to the second design consideration provided,
representative chromatographic materials contain functional groups
that interact effectively with adenovirus but which have a design
that minimizes access of the virus to any pore spaces thereof (such
as about 0.1 micron), which can damage the virus, and in
particular, the fiber protein thereof.
[0055] Likewise, so-called tentacled polymers containing core
regions to which are attached polymerized chains of varying
lengths, and to which may be attached further functional groups;
for example, Fractogel Tentacle Ion 25 Exchange Media available
from E. Merck, Wakefield, R.I. These chromatographic materials are
described as having an insoluble matrix copolymerized from
oligoethyleneglycol, glycidylmethacrylate, and
penta-erythrold-imethacrylate, to which are grafted polymerized
chains of acrylamide derivatives, ending in ion exchanging groups
such as DEAE, or quaternary aminoethyl, quaternary ammonimum, DMAE,
TMAE, and the like, or, for example, S0.sub.3- or carboxyl. It is
within the practice of the invention to utilize similar "tentacled"
materials.
[0056] Preferred, chromatographic materials regarding the third
design comprise matrices having pores of at least about 0.1 .mu.m
(the size of an adenovirus), but preferably at least about 1
.mu.m.
[0057] Such materials can be cellulose membrane or silica membrane
cartridges characterized by high substrate specificity for target
protomers, negligible binding of non-specific proteins, and a pore
size (.about.1.2 .mu.m) which is sufficient for purification of the
largest known spheroidal viruses. The cartridge design, which
consists of a stack of low-binding cellulose or silica membrane
filters, is suited to high flow rates, while the large pore size
(1.2 .mu.m) of the cartridges eliminates the diffusion associated
with beaded gel resins packed in columns.
[0058] For example, the ACTI-MOD.RTM. (American International,
Natick, Mass.) cartridges consist of sheets of microporous
silica/PVC. These silica sheets have a large surface area with
numerous uniform pores (See FIG. 1, panel A). The pores are lined
with silica to which different active side chains may be attached,
e.g. DEAE or heparin structural units. During chromatography, the
movement of virus through the highly porous silica/PVC sheets of
ACTI-MOD.RTM. (or through the openings in similarly effective
MemSep.RTM. (Millipore, Bedford, Mass.) cartridges) permits direct
contact of the virions with the activated silica surface (FIG. 1,
panel B). Such contact permits appropriate partitioning while, at
the same time, avoiding adverse interaction of the virions with
pores in the beads of the resin that are approximately the same
size, or slightly smaller, than the size of the encapsulated
virions themselves that occurs with the use of traditional beaded
gel-type resins. Such adverse interaction may involve damage to
virion surface components, including, for example, fibre protein,
thereby reducing the ability of the virion to successfully infect
target cells.
[0059] Additional polymer products which are useful in the practice
of the invention are those containing pores which are large enough
not to damage the virion and include, inter alia, spiral
preparative chromatography modules, such as the CycloSep.TM. module
(American International Chemical Inc., Natick, Mass.). The
CycloSep.TM. spiral purification column has a matrix comprising a
microporous plastic sheet (MPS.RTM.), with an integral rib design
available in various spacing configurations. The matrix is wound
into a spiral and is coated with silica. The resultant hydrophilic
surface can be derivatized with various affinity ligands such as
heparin, or those used in ion-exchange chromatography, such as DEAE
and carboxymethyl.
[0060] Anion-exchange chromatography may be performed utilizing
various functional moieties known in the art for anion-exchange
techniques, including, but not limited to, DEAE, (diethyl
aminoethyl), QAE (quaternary aminoethyl), and Q (quaternary
ammonium). These functional moieties may be attached to any
suitable resin useful in the present invention, including the
cellulose and silica resins described herein. For example, DEAE may
be attached to various resins, including cellulose resins, in
columns such as DEAE-MemSep.RTM. (Millipore, Bedford, Mass.)
Sartobind.TM. membrane absorbers (Sartorius, Edgewood, N.J.) and
silica resins such as ACTI-MOD.TM., (American International
Chemical, Natick, Mass.).
[0061] Cation-exchange chromatography also may be used for
adenovirus purification, including, but not limited to, the use of
such columns as SP MemSep.RTM. (Millipore, Bedford, Mass.), CM
MemSep.RTM. (Millipore, Bedford, Mass.), Fractogel.RTM.
SO.sub.3.sup.- (EM Separation Technology, Gibbstown, N.J.) and
Macroprep S.RTM. (BioRad, Melville, N.Y.), as well as heparin-based
resins. Heparin ACTI-MOD.RTM. Cartridge (American International
Chemical Inc., Natick, Mass.), and POROS.RTM. Perfusion
chromatography media (Boehringer Mannheim) represent additional
examples of this embodiment of the invention.
[0062] Other affinity ligands which may be used to purify
adenovirus include anti-adenovirus antibodies attached to suitable
resins as provided herein as well as others known to those skilled
in the art.
[0063] Preferably, purification of adenovirus includes the
following steps: pressure lysis of 293 cells infected with
adenovirus in the presence of a detergent such as Tween-80 and
recovery of virus in the lysate; clarification of lysate by passage
through a 3 .mu.m glass fiber filter and an 0.8 .mu.m cellulose
acetate filter; heparin chromatography using an ACTI-MOD.RTM.
silica cartridge where adenovirus is recovered in the flow-through
fraction; and DEAE-ion exchange chromatography using a MemSep.RTM.
cartridge. Bound adenovirus is eluted from the DEAE-resin using
500-700 mM NaCl in a suitable buffer; followed by gel filtration
(size exclusion) chromatography of the DEAE-eluate, where
adenovirus is recovered in the void volume of the column
eluate.
[0064] Gel filtration chromatography using, for example, resins
such as Superdex.RTM. (Pharmacia, Piscataway, N.J.) to purify can
be used to recover active adenovirus away from any contamination
process. Such resins have a very small pore size (exclusion limit 2
million daltons) which results in the adenovirus being completely
excluded from the beads of the resin and eluting in the void volume
of the column. The partitioning functions in a similar fashion to a
protein de-salting column.
[0065] Adenovirus purification may be determined by assaying for
viral proteins, using, for example, Western blotting or SDS-PAGE
analysis. The identification of adenoviral DNA may be used as an
indicator of virus recovery, using, for example, slot blot
analysis, Southern blotting, or restriction enzyme analysis of
viral DNA. Purification is evidenced by the predominance of viral
proteins or nucleic acid in an assayed sample.
[0066] The identification of adenovirus particles may also be used
to assay virus purification, using, for example, the
spectrophotometric absorbance at 260 nm of a purified fraction, or
the observance of virus particle by, for example, electron
microscopy.
[0067] The recovery of infectious adenovirus may be determined by
infection of a suitable host cell line (e.g., 293 cells) with a
chromatographed sample. Infectious adenovirus may be dentified and
titrated by plaque assays. Alternatively, infected cells may be
stained for the abundant adenoviral hexon protein. Such staining
may be performed by fixing the cells with acetone: methanol seven
days after infection, and staining with a polyclonal FITC-labeled
anti-hexon antibody (Chemicon, Temecula, Calif.). The 10 activity
of a purified fraction may be determined by the comparison of
infectivity before and after chromatography.
[0068] The use of other columns in the methods of the invention is
within the scope of the invention directed to purification of
adenovirus.
[0069] Since, as discussed above, AAV propagation requires helper
adenovirus, which can purify with and contaminate AAV preparations,
AAV purification takes advantage of chromatographic materials which
damage adenovirus (e.g., through contact with the pores of such
materials) in the purification of AAV. Thus, AAV purification uses
chromatographic materials that are not preferred for adenovirus
purification. For example, such materials can include the so-called
"macroporous" resins discussed above, whose pore size is
approximately that of adenovirus. In general, it is advantageous to
select matrices with pore sizes that would damage adenovirus
leading to its inactivation during purification.
[0070] The present invention also relates to methods for the
separation of AAV from adenoviral and cellular proteins in the cell
lysate, using column chromatography. The advantage of column
chromatography over current non-scaleable methods of
density-gradient ultracentrifugation is that large quantities of
AAV can be produced. Another advantage of using column
chromatography for the purification of AAV is that it effectively
removes contaminating adenovirus which is used routinely as a
helper virus in the production of AAV.
Purification of AAV
[0071] The initial lysis of AAV-infected cells can be accomplished
via methods that chemically or enzymatically treat rather than
physically manipulate the cells in order to release infectious
virions. Such methods include the use of nucleases (such as
benzonase.RTM.; DNAse) to enzymati-tally degrade host cell,
non-encapsulated or incomplete adenoviral DNA. Proteases, such as
trypsin, can be used to enzymatically degrade host cell, adenoviral
or free AAV proteins. Detergents, surfactants and other chemical
agents known in the art can also be used alone or in conjunction
with enzymatic treatment.
[0072] AAV-infected cells can be further lysed by application to a
pressure cell, such as a Microfluidiser pressure cell, wherein the
intensified pumping system employs an accelerated suction stroke
and a long slow pressure stroke to create a pressure profile of
briefly interrupted constant pressure. This pressure is then used
to lyse the AAV-infected cells, while gently retaining maximal
activity of the AAV. A further advantage of the use of such
pressure techniques for lysis is that such methods can be applied
to scaled up cell culture conditions, including propagating cells
on microcarriers. Alternatively, a French Pressure Cell (Baxter,
Deerfield, Ill.), Manton Goulin Homgeniser (Baxter, Deerfield,
Ill.) or Dynomill can be used. The resulting lysate can then be
clarified by filtration through glass fiber filters or cellulose
acetate filters. Alternatives include Millexe Durapore (Millipore,
Bedford, Mass.) and Gelman Science Tuffryne filters (Gelman
Science, Ann Arbor, Mich.). Filter sizes which may be used include
0.8 .mu.m and 0.45 .mu.m. If vacuum filtration is not used, glass
wool also may be employed to clarify the lysate.
[0073] Suitable column chromatography methods to fractionate
infected cell lysates for large-scale purification of AAV include
the use of sulfated resins, such as Sterogene-S (Sulfated Hi Flow)
(Sterogene, Carlsbad, Calif.), Spherilose-S (Isco, Lincoln, Nebr.),
Cellufine.RTM. sulfate (Amicon, Beverly, Mass.). Such sulfated
resins are capable of removing contaminating adenovirus from AAV
using elution buffers containing about 400-500 mM, preferably about
475 mM NaCl.
[0074] DEAE containing resins used in AAV purification, include,
but are not limited to, Puresyn DEAE (Puresyn, Malvern, Pa.), EM
Merck Tentacle DEAE (Merk, Whitehouse Station, N.J.), Sterogene
Superflow Plus DEAE (Sterogene, Carlsbad, Calif.), macroporous DEAE
resins (Biorad, Melville, N.Y.), DEAE ACTI-MOD.RTM. (American
International, Natick, Mass.), DEAE MemSep.RTM. (Millipore,
Bedford, Mass.), all of which are capable of removing contaminating
adenovirus.
[0075] Em Merck Tentacle DEAE is an ion exchange media consisting
of a matrix copolymerized from oligoethyleneglycol,
glycidymethacrylate and pentaerythroid to which are grafted
polymerized chains of acrylamide derivatives approximately 15-50
units in length. Sterogene Superflow Plus.RTM. DEAE consists of a
6% cross-linked agarose to which is attached DEAE reactive groups.
Macroporous DEAE resins are rigid hydrophilic supports with pore
sizes of 80-100 nm, where the DEAE reactive groups are attached to
the hydrophilic support. DEAE Acti-Mod.RTM. cartridge consists of
sheets of microporous silica/PVC. These silica sheets have a large
surface area with numerous uniform pores. The pores are lined with
silica to which may be attached active side chains such as DEAE.
This type of macroporous structure has pores of about 12,000 .ANG.,
or 1.2 .mu.m, in width. In the DEAE MemSep.RTM. resin, the DEAE
groups are covalently linked to the polymer matrix cellulose. A
suitable elution buffer for the recovery of AAV from such resins
comprises 200 mM NaCl in a phosphate buffer, pH 7.5.
[0076] Purification of AAV also utilizes hydroxyapatite resins,
including the ceramic hydroxyapatite resins from Biorad (Melville,
N.Y.). The recovery of AAV from such hydroxyapatite resins utilizes
elution with a 100-135 mM phosphate buffer (pH 6.4, 10-400 mM
phosphate gradient. The column is first washed with 30 mM phosphate
and AAV elutes around 135 mM phosphate).
[0077] In a particular aspect of the invention, chromatography on
cellulose or silica membrane resins is employed in conjunction with
the use of macroporous resins in for effective large-scale
purification of AAV. Examples of macroporous resins include the
BioRad macroporous series (Melville, N.Y.) or the DEAE-Thruput (6%
cross-linked agarose) (Sterogene, Carlsbad, Calif.). Silica or
cellulose membrane resins include the DEAE-MemSep.TM. 1010 HP
(Millipore), the ACTI-MOD.RTM. cartridge, or the CycloSep.TM.
(American Chemical International) spiral purification column.
Previous studies with macroporous resins showed them not to be very
useful for the purification of adenovirus because the 80 nm pore
size of the beads excludes and damages adenovirus particles having
a diameter of 140 nm. However, this size limitation is an advantage
for AAV purification, because these resins can remove contaminating
adenovirus from AAV preparations based on the different sizes of
the virion particles. It is well within the skill of those in the
art to select macroporous resins for their ability to separate AAV
from adenovirus based on discriminating pore size of the
resins.
[0078] In a particular embodiment of the invention, pressure lysis
of AAV-infected cells in the presence of detergent yields a cell
lysate which is clarified by filtration. The lysate is then applied
to a series of columns in order to separate AAV from cellular
proteins and contaminating adenovirus. A preferred series of column
separations includes the use of ceramic hydroxyapatite, DUE
ion-exchange, Cellufine.RTM. sulfate, and zinc chelate
chromatography. The AAV may be recovered from the columns as
follows: hydroxyapatite (at 100-135 mM phosphate, pH 6.4); DEAE-ion
exchange (at 200 mM salt in a phosphate buffer, pH 7.5);
Cellufine.RTM. sulfate (at 425 mM salt in phosphate-buffered
saline, pH 7.5) and in the flow-through from the zinc chelate
column (Hepes buffer, pH 7.5).
[0079] A particularly preferred embodiment for purification of AAV
includes the following steps: pressure lysis of AAV-infected 293
cells also infected with adenovirus in the presence of Tween-80 and
trypsin, and recovery of virus in the lysate; clarification of
lysate via filtration through an 0.45 .mu.m or 0.8 .mu.m cellulose
acetate filter; ceramic hydroxyapatite chromatography (CHA), where
bound AAV is eluted from the resin in 100-135 mM phosphate, pH 6.4;
DEAE ion-exchange chromatography of the CHA eluate using a
MemSep.RTM. cartridge, where bound AAV is eluted from the resin in
200 mM salt (phosphate buffer, pH 7.5); Cellufine.RTM. sulfate
chromatography of the DEAE-eluate, where bound AAV is eluted from
the resin in 425 mM salt (phosphate-buffered saline, pH 7.5); and,
optionally, zinc chelate chromatography where AAV is recovered in
the flow through fraction (Hepes buffer, pH 7.5).
[0080] AAV may also be separated from adenovirus using
Superdex.RTM. 200 resin (Pharmacia, Piscataway, N.J.), which
separates AAV from low molecular weight contaminants, and where AAV
is recovered in the void volume.
[0081] The methods described here permit retrieval of purified AAV
particles at high concentration in aqueous media without
centrifugal pelleting. Similarly the methods are suited to the
preparation of milligram quantities of virus without the use of
density centrifugation.
[0082] The use of other columns is also within the scope of the
invention which is directed to the use of column chromatography in
large scale purification of AAV.
[0083] In order to assess the integrity of a purification protocol,
one skilled in the art can use any number of assays to determine
whether AAV virus is recovered and whether cellular proteins and
contaminating helper virus (such as adenovirus) have been removed.
AAV recovery and purification can be monitored by determining the
levels of AAV DNA or AAV proteins in recovered fractions from the
various chromatography steps, or from the titer of infectious
virus.
[0084] The level of AAV DNA may be determined using a slot blot
apparatus which detects immobilized DNA using an AAV specific
probe. The number of viral particles can be determined with the use
of a standard curve generated from samples of known particle
number. Where recombinant AAV contains a marker gene, such as
.beta.-galactosidase, the amount of recovered virus can be
determined by an appropriate assay for the marker gene product
(e.g., X-gal) or by an assay that detects DNA copies of the gene
(e.g., PCR).
[0085] Alternatively, the presence of virus may be determined from
the level of AAV protein contained in recovered fractions. Viral
proteins may be assayed by Western blotting, immunoprecipitation,
Coomassie-stained SDS-PAGE gels, or any other methods for protein
characterization and quantitation known to those skilled in the
art. When an SDS-PAGE gel is stained with Coomassie Blue, the
presence of other non-AAV proteins may be determined as an index of
the concentration of the AAV fraction.
[0086] Purity of the isolated virus fraction is determined by
SDS-PAGE analysis of proteins in the fraction, followed by
Coomassie staining and densitometry. With respect to the AAV viral
proteins, VP3 usually accounts for about 80% of the viral protein,
while VP1 and VP2 together account for about 20% of total viral
protein. Purity is assessed by the absence of heterologous proteins
in assayed sample.
[0087] The purification methods of the invention may be applied to
naturally occurring or recombinant viruses.
[0088] The practice of the invention employs conventional
techniques of molecular biology, protein analysis and microbiology
which are within the skill of the art. Such techniques are
explained fully in, e.g., Current Protocols in Molecular Biology,
Ausubel et al., eds., John Wiley & Sons, New York, 1995, which
is incorporated herein by reference.
[0089] The invention is illustrated with reference to the following
examples.
Example 1
Extraction of Adenovirus from 293 Cells
[0090] A. Extraction of Adenovirus from Cells
[0091] The human embryonal cell line (293) was used to propagate
adenovirus. Virus-infected cells were incubated until the cell
monolayer exhibited extensive cytopathic effects (CPE). Usual
infection time was 48-60 hours. The cells were harvested into
phosphate-buffered saline (PBS) and collected by centrifugation at
1000.times.g. Cell pellets were frozen at -80.degree. C. for
further use or were resuspended in PBS containing 0.1% Tween-80,
10% glycerol, 2 mM MgCl.sub.2 and 50 .mu.M ZnCl.sub.2. Following
resuspension the cells were lysed using a Microfluidiser (Model
HC5000, Microfluidics, Newton, Mass.) at 1000 psi and the lysate
was incubated with benzonase (2500 units benzonase.RTM./10.sup.8
cells) for 1 hour at room temperature. To remove cellular debris,
the lysate was clarified by passing it through glass wool (without
vacuum) or by vacuum filtration through 3.0 .mu.m glass fiber
filters (MicroFiltration Systems #C300A090C, Sierra Court-Dublin,
Calif.). This was followed by filtration using an 0.8 .mu.m
cellulose acetate filter (MicroFiltration Systems 25 #CD80A090C,
Sierra Court-Dublin, Calif.). Following clarification the lysate
was either directly chromatographed or subjected to a further
filtration step using a Minitan ultrafiltration system (Millipore,
#XX42MT060, Bedford, Mass.) prior to chromatography.
[0092] B. Ultrafiltration
[0093] Lysate from 293 cells infected with adenovirus (prepared as
discussed above) was passed through a Minitan Ultrafiltration
System (Millipore #XX42MT060, Bedford, Mass.) at flow rates varying
from 300-400 ml/min in the following buffer: phosphate buffered
saline (PBS), 0.05% Tween-80, 10% glycerol, 50 .mu.M ZnCl.sub.2. To
measure recovery of infectious units following ultrafiltration the
retentate was assayed for adenovirus infectivity using the virus
titer assay while recovery of protein in the retentate was measured
using a BCA assay (Pierce Chemical Co. #23220, Rockford, Ill.).
Results
[0094] Table 1 provides a comparison between recovery of active
adenovirus using both microfluidiser pressure lysis and
freeze-thawing as methods of lysis for adenovirus infected cells.
Using the microfluidiser and detergent-containing buffers, there
was a 96% recovery of active virus with pressure lysis compared to
lysis by freeze thawing. Thus, the microfluidiser provides an
alternative effective lysis procedure, which has the advantage of
allowing larger volumes of cells to be processed at one time. Also,
methods to scale up cell culture conditions, including growing
cells on microcarriers, are possible since the microfluidiser can
effectively lyse cells attached to microcarriers. Lysis of the
cells occurred in the presence of the nuclease Benzonase.RTM.,
which degrades host cell, nonencapsulated or incomplete adenoviral
nucleic acids.
[0095] Following lysis of the cells, the resulting lysate was
clarified to remove cellular debris by filtration through glass
wool or alternatively by using vacuum filtration through a 3.0
.mu.m glass fiber filter (MicroFiltration Systems #300A090C, Sierra
Court-Dublin, Calif.). A further filtration step using a 0.8 .mu.m
cellulose acetate filter (MicroFiltration Systems) was then carried
out. Typically, 84% of active adenovirus was recovered in the final
clarified lysate, while only 43% of total cell lysate protein was
recovered.
[0096] Ultrafiltration was then used to further purify the
clarified cell lysate prior to column chromatography. The molecular
size of adenovirus is 150.times.10.sup.6 daltons, while the
molecular size of the majority of host cell contaminating proteins
is expected to be lower. A Minitan.RTM. ultrafiltration system from
Millipore (molecular weight cut-off membrane of 300 kDa) was used.
Table 2 shows that the maximum recovery of infectious adenovirus
units was achieved when the flow rate through the membrane was 200
ml/min and the buffer contained glycerol and trypsin. Under these
conditions 100% of adenovirus activity was achieved while 55% of
host cell proteins was removed. Spinner cultures of 293 cells
(grown on microcarriers) and infected with adenovirus were used in
these studies. Therefore, effective cell lysis and release of
active adenovirus from 293 cells grown on microcarriers is possible
using the Microfluidiser.RTM. pressure cell.
TABLE-US-00001 TABLE 1 Comparison of different methods for lysis of
293 cells infected with adenovirus I.U. Total % Activity recovered
Freeze Thaw (3X) 4.3 .times. 10.sup.11 100 Microfluidiser 4.1
.times. 10.sup.11 96
TABLE-US-00002 TABLE 2 Ultrafiltration of Adenoviral Cell Lysates
Using a Minitan System (Millipore) Flow Rate (ml/min) 400 300 200
I.U. before ultrafiltration (.times.10.sup.10) 6.4 33.44 3.2 I.U.
after ultrafiltration (.times.10.sup.10) 2.42 11.73 3.2 %
Adenovirus activity recovered 38 70 100 % Total protein remaining
70 57.5 52
Example 2
Chromatographic Purification of Adenovirus
[0097] Column resins were tested for their separation
characteristics using a Pharmacia FPLC.
Methods
1. DEAE Chromatography
[0098] A. Adenovirus
[0099] A DEAE MemSep.RTM.1010 HP (Millipore) column (5 ml) was
equilibrated with phosphate buffered saline (PBS) (1.5 mM
KH.sub.2P0.sub.4, 150 mM NaCl, 5 mM Na.sub.2HP0.sub.4 pH 7.5)
containing 10% glycerol, 0.05% Tween-80, and 50 .mu.M ZnCl2.
Clarified lysate from 293 cells infected with adenovirus as
prepared in Example 1 was applied at a flow rate of 5 ml/min to the
pre-equilibrated column (in PBS, 10% glycerol, 0.05% Tween-80, 2 mM
MgCl.sub.2, 50 .mu.M ZnCl.sub.2). The column was washed with 10 mM
Na.sub.2HP0.sub.4, 100 mM NaCl, 100 mM KCl, and a linear gradient
(100 mM-1 M) of KCl and NaCl in 10 mM Na.sub.2HP0.sub.4 pH 7.5, 10%
glycerol, 0.05% Tween-80 and 50 .mu.M ZnCl.sub.2 was applied to the
resin at a flow rate of 5 ml/min. Bound proteins were eluted from
the resin and collected in 5 ml fractions. Each fraction was
monitored for a) adenoviral DNA and b) adenoviral proteins (as
described below). Fractions which were positive for both adenoviral
DNA and protein were assayed further for activity using the virus
titre assay.
[0100] B. AAV
[0101] Cell lysates from 293 cells infected with adeno-associated
virus AAV were also chromatographed using DEAE 25 MemSep.RTM.
chromatography as above. However, the buffer used to lyse the cells
and equilibrate the column was 10 mM sodium phosphate, pH 7.5,
containing 50 mM NaCl and 1% NP-40. Bound proteins were eluted from
the resin using a salt gradient as described above for adenovirus
purification. Fractions collected from the resin were assayed for
AAV DNA using a slot blot assay and AAV proteins by immunoblotting
using an antibody (Catalog 03-65158, of American Research Products,
Belmont, Mass.) against the three capsid proteins of AAV, VP1, VP2
and VP3.
2. Gel Filtration Chromatography
[0102] A. Adenovirus
[0103] Gel filtration chromatography was then performed using a
Superdex.RTM. 200 HR 26/60 column (Pharmacia) equilibrated with
PBS, 10% glycerol, 2 mM MgCl.sub.2, 50 .mu.M ZnCl.sub.2, and 0.05%
Tween-80. Fractions eluted from the DEAE resin which showed the
presence of both adenoviral proteins and DNA were pooled and
concentrated using a stir cell (Amicon) to a volume of 15 ml. The
sample was applied to the Superdex.RTM. resin at a flow rate of 1
ml/min and 1.5 ml fractions were collected during elution.
Fractions were assayed for adenoviral DNA and proteins as described
below.
[0104] B. AAV
[0105] Adenovirus purified by the cesium chloride method (described
below) was applied directly to the Superdexm resin following the
final cesium chloride density centrifugation. This was to reduce
the concentration of cesium chloride in the sample which normally
was removed by dialysis.
[0106] In some experiments gel filtration chromatography of whole
cell lysates of AAV was performed using a Superdex.RTM. 200 HR
26/60 prior to chromatography on a DEAE column.
[0107] Additional polymeric materials useful according to this
aspect of the invention include cross-linked cellulose polymers
such as Sulfate Spherilose (ISCO).
Results
[0108] FIG. 2 shows a typical elution profile of adenovirus from
DEAE MemSep.RTM. resin following chromatography of a 293 cell
lysate containing adenovirus. All of the adenovirus bound to the
DEAE resin and was eluted with a salt gradient applied to the resin
(represented by the sloping line). Adenovirus eluted from the resin
between 500-700 mM NaCl as indicated on the elution profile. This
peak contained less than 10% of the total protein in the initial
whole cell lysate, while typically 60-100% of the adenovirus
activity was recovered. Further purification of this eluted
fraction was achieved by gel filtration chromatography using a
Superdex.RTM. 200 resin. Fractions from the DEAE column which had
the highest virus titers were pooled, concentrated using an Amicon
stir cell and applied to the Superdex.RTM. resin. Adenovirus eluted
in the void volume of the resin (FIG. 3). Approximately 50-70% of
the adenovirus activity was recovered in this fraction. Protein
estimation (BCA) (Pierce Chemical Co.) on all of the fractions
eluted from the column indicated that the gel filtration step
removed approximately 70% of contaminating cellular proteins. FIG.
4 shows an SDS-PAGE analysis of adenovirus purified by a
combination of DEAE and gel filtration column chromatography
compared to adenovirus purified by a prior art cesium chloride
method. There were some additional protein bands present in the
adenovirus purified by column chromatography. To achieve further
purification of the adenovirus other resins were evaluated for
their ability to remove these contaminating proteins.
Example 3
Hydrophobic Chromatography
Methods
[0109] Four different types of hydrophobic resins were tested for
their ability to remove contaminants from the adenovirus
preparations of Example 2: BioRad Macroprep.RTM. columns (butyl and
methyl) and Tosohaus.RTM. 650 M, 65 .mu.m (phenyl and ether).
Adenovirus was applied to each hydrophobic resin in 10 mM sodium
phosphate buffer, pH 7.5 containing 2 M NaCl. Bound proteins were
eluted from the resin using 0.15 mM KH.sub.2P0.sub.4, 15 mM NaCl,
0.5 mM Na.sub.2HP0.sub.4 pH 7.5.
Results
[0110] SDS-PAGE analysis of the flow-through and eluted fractions
showed that there was little separation of the adenovirus from
other cellular components using these resins.
Example 4
Cation Exchanse Resins
Methods
[0111] CM and SP MemSep.RTM. Cartridges (Millipore, Bedford,
Mass.), Fractogel.RTM. SO.sub.3 (a tentacle ion-exchange resin, EM
Sciences), and BioRad Macroprep S.RTM. were separately equilibrated
in 10 mM sodium phosphate buffer pH 7.5, containing 25 mM NaCl, 2
mM MgCl.sub.2, 10% glycerol and 0.05% Tween-80. Cell lysates from
293 cells infected with adenovirus were applied to each of the
resins in the same buffer containing 0.25% Tween-80. The columns
were washed with 10 mM Na.sub.2HP0.sub.4, 100 mM NaCl, 100 mM KCl,
and bound proteins were eluted from the resin using a linear
gradient (100 mM-1 M) of KCl and NaCl in 10 mM Na.sub.2HP0.sub.4,
pH 7.5, 10% glycerol, 0.05% Tween-80 and 50 .mu.M ZnCl.sub.2. The
results of the Macroprep S.RTM. chromatography are shown in Table
5.
Example 5
Cellufine.RTM. Sulfate Resin (Amicon)
Methods
[0112] For all experiments with the Cellufine.RTM. sulfate resins
(Amicon, Beverly, Mass.), cell lysate from 293 cells infected with
adenovirus was applied to the resin in a solution of 25 mM NaCl and
10 mM sodium phosphate, pH 7.5, containing also 10% glycerol (w/v),
0.05% Tween-80, 2 mM MgCl.sub.2, 50 .mu.M ZnCl.sub.2. Bound
proteins were eluted from the resin using a linear salt gradient
(100 mM-1 M) of NaCl and KCl in 10 mM Na.sub.2HP0.sub.4 pH 7.5, 10%
glycerol, 0.05% Tween-80 and 50 .mu.M ZnCl.sub.2. Both the flow
through and eluted fractions were assayed for adenoviral DNA and
immunoblotted using an anti-adenoviral antibody as described
below.
Results
[0113] The resin which gave the most significant purification,
Cellufine.RTM. sulfate, did not however lead to a purified product
in which most of the adenovirus was present in an active form.
Cellufine.RTM. sulfate comprises a cellulose matrix with sulfonate
groups esterified at the number -6 carbon of the repeating glucose
subunits (P. F. O'Neil et al., Biotechnology, 11, 1993, pp.
173-178). Binding of proteins to this resin is thought to occur
through the polysaccharide moieties thereof Because adenovirus is a
non-enveloped virus with no surface glycoproteins, it was thought
that it should not bind to this resin while most cellular
glycoproteins, present as contaminants, would.
[0114] Table 3 shows the results for recovery of activity of
adenovirus following chromatography on the Cellufine.RTM. sulfate.
Adenoviral protein and DNA were recovered in the flow through
volume, as predicted, but less than about 10% of adenoviral
activity was recovered in this fraction. Inactivation of the virus
during chromatography on Cellufine.RTM. sulfate may have been a
result of adverse interaction/partitioning involving the pores of
the beads of the resin, which have a mean diameter of about 80
nm.
Example 6
Heparin Resins
Methods
[0115] Each of the following heparin resins were assessed for their
ability to purify adenovirus: Heparin Sepharose.RTM. CL6B
(Pharmacia), Heparin Agarose (4% cross linkage, Sigma), HiTrap
Heparin.RTM. (Pharmacia), Heparin Superflow Plus.RTM. (6% cross
linkage, Sterogene), Heparin, ACTI-MOD.RTM. cartridge.RTM.
(American International Chemical Inc.).
[0116] For all experiments with the heparin resins, cell lysate
from 293 cells infected with adenovirus was applied to the resin in
a solution of 25 mM NaCl and 10 mM sodium phosphate, pH 7.5,
containing also 10% glycerol (w/v), 0.05% Tween-80, 2 mM
MgCl.sub.2, 50 .mu.M ZnCl.sub.2. Bound proteins were eluted from
the resin using a linear salt gradient (100 mM-1 M) of NaCl and KCl
in 10 mM Na.sub.2HP0.sub.4 pH 7.5, 10% glycerol, 0.05% Tween-80 and
50 .mu.M ZnCl.sub.2. Both the flow through and eluted fractions
were assayed for adenoviral DNA and immunoblotted using an
anti-adenoviral antibody as described below.
Results
[0117] In order to further understand the causes for the
disappointing performance of Cellufine.RTM. sulfate, the
performance of heparinated polymers (resins) was also examined.
Heparin, like Cellufine.RTM. sulfate, is a sulfonated
polysaccharide and would be predicted to have certain binding
characteristics in common. Unlike Cellufine.RTM. sulfate, however,
it is commercially available in one or more forms cross-linked to a
variety of different beaded resins of various pore size. Table 3
shows the results of screening various heparin-linked resins. All
of the resins tested bound >40% of the cellular proteins (as
determined by BCA) that contaminated the virus samples, but the
only resin which gave 100% recovery of active virus was the
Sterogenee heparin agarose, a 6% cross-linked agarose. Generally
speaking, a 6% agarose matrix with an exclusion limit of about 6
million daltons would have smaller pores than, for example, an
agarose gel with 4% cross-linkage and an exclusion limit of about
20 million daltons. It is possible that, because of its average
pore size, the 6% cross-linked agarose excluded the adenovirus
completely during chromatography. As a result, active adenovirs was
recovered in the flow-through fraction.
TABLE-US-00003 TABLE 3 Recovery of Active Adenovirus following
Chromatography with Cellufine .RTM. Sulfate and Heparin Resins I.U.
before I.U. after % Activity Resin Chromatography Chromatography
Recovered Heparin Sepharose .RTM. 2.1 .times. 10.sup.10 5.7 .times.
10.sup.9 27 CL6B HiTrap .RTM. Heparin 5.8 .times. 10.sup.10 3.8
.times. 10.sup.9 7 Heparin Agarose, 5.8 .times. 10.sup.10 9.2
.times. 10.sup.9 16 4% X-link Heparin Superflow .RTM. 5.1 .times.
10.sup.9 1.3 .times. 10.sup.10 100 Plus 6% x-link agarose Cellufine
.RTM. Sulfate -- -- 10
[0118] Lysate from 293 cells infected with adenovirus was
chromatographed using a heparin ACTI-MOD.RTM. disc as described
above. Adenovirus was recovered in the flow through fraction and
purified further using a combination of DEAE ion exchange and gel
filtration chromatography. SDS-PAGE analysis of the adenovirus
fraction from the GF column showed that the purity of the
adenovirus (purified by column chromatography) was as pure as the
control adenovirus purified by CsCl centrifugation (FIG. 5). FIG. 6
shows a densitometric analysis of the fractions analyzed in FIG.
5.
Example 7
Biorad Ceramic Hydroxyapatite (80 .mu.m Pore Size)
[0119] The BioRad hydroxyapatite column was equilibrated with 10 mM
sodium phosphate pH 7.5 containing 25 mM NaCl, 1 mM MgCl.sub.2, and
10% glycerol. Adenovirus or AAV was applied to the resin in the
same buffer. Bound proteins were eluted from the resin using an
increasing linear salt gradient from 10 to 300 mM of sodium
phosphate, all at pH 7.5.
Example 8
Partitioning Polymers
[0120] A series of partitioning polymers (resins) were screened for
their ability to purify active adenovirus (Table 4). It was found
that the majority of the polymers tested gave significant
purification, but use of only a few led to recovery of purified
adenovirus in an active form. In general, the membrane-based
cartridge polymers (resins), such as the MemSep.RTM. cartridge from
Millipore or the ACTI-MOD.RTM. cartridge (American Chemical
International) gave a better recovery of active virus. The superior
performance of these products is believed attributable to the open
macroporous structure of the membrane matrix in these cartridges
[in these preferred examples, DEAE groups are covalently linked to
the polymer matrix cellulose in the case of MemSep.RTM., or to
silicate in the case of ACTI-MOD.RTM.]. This type of macroporous
structure [having openings (pores) of about 12,000 A.degree., or
1.2 .mu.m, in width] allows rapid passage of the adenovirus virions
which have diameters of about 140 nm (including fibre).
[0121] Table 5 is a summary chart of various chromatographic
methods for adenovirus purification.
TABLE-US-00004 TABLE 4 Recovery of Active Adenovirus from Different
Types of Resins Type of Resin Pore Size Activity Recovered Membrane
Based 1 .mu.m 100% (MemSep .RTM. or ACTI-MOD .RTM.) Macroporous
(BioRad) 0.08-0.1 .mu.m 10% Tentacle (EM Science) ND <10% 4%
Agarose ND <20% 6% Agarose ND 100%
TABLE-US-00005 TABLE 5 Results of Resin Screen CLEAN- RESIN TYPE UP
ACTIVITY Sperilose Sulfate (ISCO) Cross-linked good 62% cellulose
Heparin Superflow Plus 6% cross-linked good 80% POROS PI 6000-8000
A fair 5% thru-pores Fractogel DEAE (EM "tentacles" rather good 35%
Sciences) than pores DEAE MemSep .RTM. Membrane based good 80%
SartoBind DEAE Membrane based good 30% (Sartorius) PolyFlo .RTM.
(Puresyn) non-porous excellent 4%* Heparin Agarose (Sigma) 4%
cross-linked good 16% agarose HiTrap Heparin cross-linked agarose
good 7% (Pharmacia) Heparin CL6B cross-linked agarose good 16%
Cellufine .RTM. Sulfate MacroPorous good <10% (Amicon)
Hydroxyapatite (BioRad) calcium phosphate good <10% MacroPrep S
(BioRad) Macroporous 40%** *Purification of adenovirus on the
PolyFlo resin involves low salt and organic elution. **Batch
Process
Example 9
Purification of Adeno-Associated Virus (AAV)
[0122] One of the main problems associated with using AAV as a
vector in gene therapy is production of sufficient quantities of
the virus. Currently AAV is purified by density gradient
ultracentrifugation techniques, which generally results in very low
yields (0.3-5%) of active virus. However density gradient
ultracentrifugation is very effective in separating AAV from
adenovirus, which is used as a helper virus in propagating AAV in
293 cells. The present invention provides combining an improved
method for extraction of AAV from infected cells with column
chromatography steps to increase the yield of AAV.
[0123] Improved extraction of AAV from infected 293 cells was
achieved by pressure lysis of the cell in the presence of detergent
(Tween-80). Following clarification of the lysate as provided above
in Example 1, AAV was separated from other cellular proteins by gel
filtration chromatography. FIG. 7a shows the elution profile from a
Superdex.RTM. 200 resin Pharmacia) following chromatography of AAV
infected 293 cell lysate. The void volume peak contains the
majority of the AAV as detected by slot blot analysis and
immunoblotting of this fraction.
[0124] Further purification of this peak was achieved by
ion-exchange chromatography using a DEAE-MemSep.RTM. cartridge. The
DEAE column was very effective in separating AAV from adenovirus.
Under the conditions used (10 mM sodium phosphate pH 7.5 containing
25 mM NaCl, 10% glycerol and 0.05% Tween-80), both AAV and
adenovirus bound to the DEAE-resin. When a linear salt (KCl and
NaCl) gradient was applied to the resin, AAV eluted at 200 mM salt
(FIG. 7, panel B), while the adenovirus remained more tightly bound
to the resin and was eluted later in the gradient at 500-700 mM
NaCl (FIG. 7, panel B). Therefore, AAV and adenovirus can be
effectively separated from one another using DEAE ion-exchange
chromatography. SDS-PAGE analysis of two DEAE fractions containing
AAV is shown in FIG. 8. The activity of AAV in the DEAE fraction #7
was 6.5.times.10.sup.7 i.u./ml or a total of 3.8.times.10.sup.8
i.u. The activity of AAV in the DEAE pool fraction was
1.38.times.10.sup.7 i.u./ml or a total of 2.76.times.10.sup.8 i.u.
Collectively, these fractions provide recovery of approximately
100%, of the AAV infectious units applied to the DEAE resin.
Example 10
Extraction of AAV from 293 Cells
[0125] The human embryonal cell line (293) was also used to
propagate the AAV. Virally infected cells were incubated until the
cell monolayer exhibited extensive cytopathic effects (CPE). The
cells were harvested and collected by centrifugation at
1000.times.g. Cell pellets were frozen at 80.degree. C. for further
use or were resuspended in 10 mM NaPi, 10 mM NaCl, 10% glycerol, 2
mM MgCl.sub.2, pH 6.4.
[0126] Following resuspension, the cells were treated with
benzonase.RTM. for 1 hour at room temperature followed by trypsin
treatment in the presence of 1% Tween-80. The cells were then lysed
using a Microfluidiser (Microfluidics, Newton, Mass.) at 1000 psi.
The resulting lysate was clarified to remove cellular debris by
vacuum filtration through a 3.0 .mu.m glass fiber filter
(Microfiltration Systems), followed by a further filtration step
using a 0.8 .mu.m cellulose acetate filter (Microfiltration
Systems) or filtered through a 0.45 .mu.m 15 Millex.RTM. HV
(Millipore) filter unit before chromatography.
Example 11
Chromatographic Purification of AAV
[0127] Various chromatography resins were tested for effective AAV
purification characteristics using a Pharmacia FPLC. The following
series of chromatography steps were found particularly useful.
[0128] a) BioRad Ceramic Hydroxyapatite (80 .mu.m Pore Size)
[0129] Cell lysates from 293 cells infected with AAV (in the
presence of adenovirus) (Example 10) were chromatographed on a
BioRad a hydroxyapatite column, which was pre-equilibrated with 10
mM Na.sub.2HP0.sub.4, pH 6.4, containing 10 mM NaCl and 10%
glycerol. AAV was applied to the resin in the same buffer. Bound
proteins were eluted from the resin using an increasing gradient
from 10 to 400 mM sodium phosphate, at pH 6.4. Fractions collected
from the resin were assayed for AAV DNA using a slot blot assay and
AAV proteins by immunoblotting using an antibody (Catalog 03-65158,
from American Research Products, Belmont, Mass.) against the three
capsid proteins of AAV--VP1, VP2 and VP3. Fractions eluted from the
resin were also analyzed for adenoviral contaminating proteins by
immunoblotting using an anti-adenoviral antibody.
[0130] FIG. 9A shows a typical elution profile of a ceramic
hydroxyapatite (CHA) resin following chromatography of a 293 cell
lysate containing AAV and adenovirus. All of the AAV and adenovirus
bound to the CHA resin and was eluted when a phosphate gradient was
applied to the resin. AAV eluted from the resin at 125 mM phosphate
as indicated on the elution profile. This peak contained less than
20% of the total protein in the initial whole cell lysate while
typically 80% of the AAV activity was recovered. The eluted AAV
peak also contained some contaminating adenoviral proteins as
measured by immunoblotting and by titre analysis (Table 5).
[0131] b) DEAE Chromatography (Anion Exchange)
[0132] In order to separate AAV from the adenovirus, ion exchange
chromatography using a DEAE-MemSep.RTM. column was used. A DEAE
MemSep.RTM. 1010 HP (Millipore) column (5 ml) was equilibrated with
10 mM phosphate buffer containing 50 mM NaCl, 10% glycerol, pH 7.5.
AAV-containing fractions eluted from the hydroxyapatite column
(above) were pooled and dialyzed into the same buffer used for
equilibration of the DEAE resin. A linear salt gradient (50 mM-2 M)
of KCl and NaCl in 10 mM Na.sub.2HP0.sub.4 pH 7.5, and 10% glycerol
was applied to the resin at a flow rate of 5 ml/min. Bound proteins
were eluted from the resin and collected in 2.5 ml fractions. AAV
eluted at 200 mM salt while adenovirus eluted at 500-700 mM salt.
Each fraction was monitored for a) AAV proteins (Coomassie blue
staining and immunoblotting); b) AAV DNA; c) contaminating
adenoviral proteins; and (d) infectivity.
[0133] Under the conditions used (10 mM sodium phosphate, pH 7.5,
containing 50 mM NaCl, 10% glycerol and 0.05% Tween-80) both AAV
and adenovirus bound to the DEAE-resin. When a linear salt gradient
was applied to the resin, AAV eluted at 200 mM salt (FIG. 9B) while
the adenovirus remained more tightly bound to the resin and was
eluted later in the salt gradient at 500-700 mM NaCl (FIG. 9B).
Therefore AAV and adenovirus can be effectively separated using
anion exchange (DEAE) chromatography. The recovery of activity of
AAV from the DEAE-MemSep.RTM. was 75% (Table5). SDS-PAGE analysis
of the pooled-MV containing fraction from the DEAE resin (FIG. 10,
lane 3) showed that there were still some contaminating proteins
present so this fraction was purified further using a
Cellufine.RTM. sulfate resin. Lanes 1-4 represent equal percentages
of each fraction (0.5%) and show the recovery of MV proteins
throughout the purification. Lane 5 represents a larger percentage
of the final MV containing fraction and gives a more intense
staining of the MV proteins VP1, VP2 and VP3. Fractions which were
positive for both AAV DNA and protein were pooled and
chromatographed further using a Cellufine.RTM. sulfate resin
(below).
[0134] c) Cellufine.RTM. Sulfate Resin (Amicon)
[0135] Cellufine.RTM. sulfate resin was equilibrated with PBS
containing 10% glycerol. Fractions eluted from the DEAE resin which
contained AAV proteins and DNA were pooled and applied to the resin
at a flow rate of 1 ml/min. The resin was washed with 250 mM NaCl
and a linear salt gradient of 0.25-1 M NaCl in PBS/glycerol was
applied. The material eluted from the resin using this salt
gradient and the flow through fraction were both analyzed for a)
AAV proteins (immunoblotting); (b) AAV infectivity (titre
analysis); and c) adenovirus proteins (immunoblotting) and
adenovirus infectivity (titre analysis). Under the buffer
conditions used, AAV bound to the resin and was eluted at 475 mM
salt (FIG. 9C). Adenoviral protein and DNA were recovered in the
flow through fraction.
[0136] Table 5 shows the recovery of AAV activity from several
purification runs. FIG. 10 shows Coomassie Blue-stained SDS-PAGE
results of the purification from each column. The three column
purification procedure described above provided an AAV yield of 48%
and a purity of >90% pure (FIG. 11). FIG. 12 is a Coomassie
stained gel comparing AAV purified using the CsCl gradient method
and AAV purified using the CsCl gradient method and AAV purified
using the column purification procedure of the present invention.
This gel shows that the AAV purified using both methods is of
comparable purity. In addition, the AAV purified by the above
procedure was shown to be free of Rep proteins (FIG. 13). FIG. 13
shows an immunoblot (using an anti-Rep antibody) of the
column-purified AAV along with known Rep standards. Approximately
71% of the AAV activity is recovered in the Cellufine sulfate
eluate while less than 15 1% of the adenovirus activity is
recovered in the same fraction.
[0137] Cellufine.RTM. sulfate thus has two main uses: a) it reduces
the level of contaminating adenovirus in AAV preparations and b) it
concentrates the AAV containing fraction. However, despite the fact
that the level of contaminating adenovirus is reduced following
Cellufine.RTM. sulfate chromatography there still remains some low
level adenovirus activity (6.73.times.10.sup.3 IU/ml).
[0138] d) Zinc Chelate Chromatography
[0139] In order to completely remove all of the contaminating
adenovirus a further chromatography step using zinc chelate
chromatography was employed. The interaction of virions with metals
has been inferred from studies of viruses and bacteriophages.
Previous studies from the inventors' laboratory showed that
adenovirus can adsorb to a zinc metal affinity column.
[0140] The final fraction of purified AAV recovered from the
Cellufine.RTM. sulfate resin was analyzed by SDS-PAGE, followed by
immunoblotting using an anti-adenovirus antibody. This was to
determine the level of contamination of adenovirus in the final
AAV-containing fraction. Immunoblotting showed that there was no
detectable adenoviral proteins, while titre analysis showed that
there was some adenoviral activity in this fraction even though it
only accounted for 1% of the total activity (Table 6).
[0141] The immobilized zinc column was prepared for metal charging
by washing the column sequentially with one volume of 100 mM EDTA
and one volume of 0.2 M NaOH. The matrix was charged with zinc by
washing with 100 mM ZnCl.sub.2 in water acidified with glacial
acetic acid. The column was then washed with water and equilibrated
with 50 mM Hepes pH 7.5, containing 450 mM NaCl, 2 mM MgCl.sub.2
and 0.05% Tween-80. The AAV-containing fraction eluted from the
Cellufine.RTM. sulfate resin was applied to the zinc chelate resin.
After loading, the column was washed with a ten column volume
linear gradient from 50 mM Hepes, pH 7.5, containing 450 mM NaCl, 2
mM MgCl.sub.2, 10% glycerol and 0.05% Tween-80 to 50.times.nM Hepes
containing 150 mM NaCl, 2 mM MgCl, 10% glycerol and 0.05% Tween-80.
Elution was performed with a linear (0-500 mM) glycine gradient in
150 mM NaCl, 50 mM Hepes, pH 7.5, 2 mM MgCl.sub.2, over ten column
volumes.
[0142] The AAV containing fraction eluted from the Cellufine.RTM.
sulfate resin was applied to the zinc chelate resin in 450 mM NaCl.
The flow through fraction was collected and bound proteins were
eluted using a glycine gradient. SDS-PAGE analysis of the flow
through fraction showed that all of the AAV was recovered in the
flow through while immunoblots using an anti-adenovirus antibody
showed that the adenovirus had bound to the zinc chelate resin and
was eluted in the presence of an increasing gradient of glycine.
Initial experiments using zinc chelate chromatography indicates
that it can be a useful resin for the separation of AAV and
adenovirus. The purification procedure yielded AAV which was
greater than 70% pure with an overall yield of 30%-40%.
TABLE-US-00006 TABLE 6 Summary of AAV Chromatographic Purification
Column Performance: Total Protein % Protein % Protein Sample Total
Protein Remaining.sup.1 (cumulative).sup.2 HA Load 180 mg 110 100
HA Eluate 20 mg 11 11 DEAE Eluate 6 mg 30 3 CS Eluate 0.5 mg 8 0.4
.sup.1Individual column performance .sup.2Overall performance
Column Performance: Recovery of AAV Activity Sample HA Load #1 #2
#3 #4 #5* Average HA Eluate 84% 86% 60% 100% 100% 88% EAE Eluate
75% 100% 100% 56% 18% 70% CS Eluate 71% 24% 30% 22% 8% 48% Zinc FT
9% 9% 20% *not included in the average Column Performance:
Adenovirus Activity Removed Sample HA Load #1 #2 average HA Eluate
90% 0* 90% DEAE Eluate 95% 70% 80% CS Eluate >99% >99% 50%
Total 99.99% of contaminating adenovirus activity removed *not
included in the average Average Activity Recovered during
Purification Runs (n = 2): DEAE HA Load HA Eluate Eluate CS Eluate
Total mgs 236 13 5 0.6 AAV IU/ml 3.58E+07 1.31E+08 5.4E+08 2.42E+08
Total AAV Ius 3.92E+09 7.24E+09 2.47E+09 1.11E+09 AV Ius/ug
1.66E+04 5.57E+05 4.94E+05 1.85E+06 The average adenovirus activity
remaining (calculated from 2 runs) after 3 columns = 4.8E+04 (+/-
4E+04). Adenovirus activity represents 0.03% +/- 0.015% of the
total AAV activity.
Example 12
Density Gradient Purification of Virus
[0143] Standard recombinant adenovirus or AAV virus was prepared by
a three step centrifugation procedure. Infected cells were lysed by
three cycles of freeze-thaw in the presence of benzonase.RTM..
Lysate was centrifuged in a table top centrifuge for 15 min at 3500
rpm at 4.degree. C. The pellet was discarded and the supernatant
was layered onto a 1.27 g/cm.sup.3 CsCl and 1.4 g/cm.sup.3 CsCl
discontinuous step gradient and centrifuged at 26,000 rpm for 1.5
hours. The virus band was collected and mixed with 1.34 g/ml CsCl
and centrifuged for at least two hours at 60,000 rpm. The viral
band from this first equilibrium gradient was collected mixed with
1.34 g/ml CsCl and recentrifuged at 30,000 rpm overnight. The final
virus pool from this step was dialyzed extensively against
phosphate buffered saline (PBS) supplemented with 1% sucrose.
Alternatively the CsCl was removed by gel filtration on a
Superdex.RTM. resin (Pharmacia) as described above.
Example 13
Detection of Adenoviral DNA Using Agarose Gels
[0144] Column fractions were first treated with 0.1% SDS for 15
minutes then digested with Pronase (Sigma) for 1 hour at room
temperature. After digestion was complete, the samples were
extracted twice with one volume of phenol: CHCL.sub.3: isoamyl
alcohol, then precipitated with two volumes of ice-cold 95% ethanol
for 20 minutes at -20.degree. C. The precipitate was pelleted at
13,000.times.g for 20 min at 4.degree. C. Samples were resuspended
in TE buffer (Tris, EDTA). Restriction enzyme digestion of the
adenoviral DNA was performed and the digest was analyzed for
diagnostic adenoviral fragments on a 0.8% agarose gel at 120 V for
4 hours or overnight at 35 V.
Example 14
Slot Blot Analysis of Column Fractions for Detection of AAV DNA
[0145] Column fractions were assayed for AAV DNA by slot blot
analysis (AAV DNA was present in a vector construct provided by Dr.
N. Muzyczka, University of Florida, Miami, also containing lacZ as
reporter gene, see FIG. 14). Samples were incubated at 56.degree.
C. for 20 minutes to inactivate the adenovirus followed by
treatment with DNaseI at 37.degree. C. for 15 min to degrade
nonvirion DNA. DNaseI was then inactivated by heat treatment at
68.degree. C. for 10 minutes. Following Proteinase K treatment of
the samples, the DNA was extracted using phenol/chloroform and
precipitated with 3 M Na0Ac. DNA was applied to a Gene Screen Plus
membrane and, following a prehybridization and hybridization step,
the membrane was probed with a P.sup.32 random label CMV .beta.-gal
Pvu II fragment. The number of particles of AAV in the sample was
calculated using a pTRlacZ DNA standard curve.
Example 15
Virus Protein Detection
[0146] One dimensional SDS-PAGE was performed using either a 4-20%
or 10-20% gradient (Daiichi) gels. Proteins in the gel were
detected using Coomassie blue. For immunoblotting, PVDF membranes
(Novex) were prewetted with methanol and soaked in 10 mM CAPS, pH
11, containing 10% methanol. Gels were equilibrated in this
transfer buffer for 10 minutes and then blotted at 30 V for 1 hour
in a Novex Blot Module. After transfer membranes were blocked with
1% dried milk in TBS (20 mM Tris-HCl, pH 7.5, containing 150 mM
NaCl) for one hour. After blocking, the membranes were probed with
anti-adenovirus antibody (Lee Biomolecular) or anti-VP1, VP2, VP3
(AAV) antibody in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and 0.05%
Tween 20 (TBST) containing 0.1% BSA for 2 hours. The membranes were
incubated with horseradish peroxidase labeled anti-mouse IgG for 20
minutes and the immunoreactive bands visualized by
chemiluminescence using the BM Chemiluminescent Western Blotting
Detection System (Boehringer Mannheim).
* * * * *