U.S. patent application number 10/365632 was filed with the patent office on 2003-08-21 for compositions comprising viruses and methods for concentrating virus preparations.
Invention is credited to Bondoc, Laureano L. JR., Frei, Andreas, Ihnat, Peter, Kwan, Henry K.H., Porter, Frederick William IV, Sandweiss, Varda E., Tang, John Chu-Tay, Vellekamp, Gary J., Yuen, Pui-Ho.
Application Number | 20030157066 10/365632 |
Document ID | / |
Family ID | 27739438 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157066 |
Kind Code |
A1 |
Frei, Andreas ; et
al. |
August 21, 2003 |
Compositions comprising viruses and methods for concentrating virus
preparations
Abstract
A composition is disclosed comprising virus in a formulation
comprising a polyhydroxy hydrocarbon buffered to maintain a pH in a
range from about 7 to about 8.5 at a temperature in the range from
about 2.degree. C. to 27.degree. C. Methods for concentrating and
purifying virus preparations are also disclosed.
Inventors: |
Frei, Andreas; (Freehold,
NJ) ; Kwan, Henry K.H.; (Summit, NJ) ;
Sandweiss, Varda E.; (Forest Hills, NY) ; Vellekamp,
Gary J.; (Glen Ridge, NJ) ; Yuen, Pui-Ho;
(Princeton Junction, NJ) ; Bondoc, Laureano L. JR.;
(Piscataway, NJ) ; Porter, Frederick William IV;
(Edison, NJ) ; Tang, John Chu-Tay; (Livingston,
NJ) ; Ihnat, Peter; (Brooklyn, NY) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Family ID: |
27739438 |
Appl. No.: |
10/365632 |
Filed: |
April 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10365632 |
Apr 4, 2003 |
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09249646 |
Feb 12, 1999 |
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6544769 |
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09249646 |
Feb 12, 1999 |
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08989227 |
Dec 11, 1997 |
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6261823 |
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60033176 |
Dec 13, 1996 |
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60074873 |
Feb 17, 1998 |
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60085559 |
May 15, 1998 |
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Current U.S.
Class: |
424/93.2 ;
435/456; 514/53 |
Current CPC
Class: |
C12N 2710/10351
20130101; A61K 48/0091 20130101; Y10S 514/937 20130101; C12N 7/00
20130101; C12N 2710/10343 20130101; C12N 2710/10334 20130101; A61K
39/12 20130101; A61K 39/235 20130101; C12N 15/86 20130101 |
Class at
Publication: |
424/93.2 ;
435/456; 514/53 |
International
Class: |
A61K 048/00; C12N
015/861 |
Claims
What is claimed is:
1. A composition comprising virus in a formulation comprising a
polyhydroxy hydrocarbon buffered to maintain a pH in a range from
about 7 to about 8.5 at a temperature in the range from about
2.degree. C. to 27.degree. C.
2. The composition of claim 1 wherein the virus is a recombinant
virus.
3. The composition of claim 1 wherein the polyhydroxy hydrocarbon
is glycerol.
4. The composition of claim 1 further comprising a
disaccharide.
5. The composition of claim 1 wherein the composition comprises a
buffer system comprising sodium phosphate monobasic dihydrate in a
concentration of about 0.5 to 10 mg/mL and tromethamine in a
concentration of about 0.5 to 10 mg/mL.
6. The composition of claim 1 further comprising a divalent metal
salt in a concentration of about 0.1 to 1 mg/mL.
7. The composition of claim 1 which further comprises a diluent
comprising water.
8. The composition of claim 1 wherein the virus is present in a
concentration of about 1.times.10.sup.9 to 1.times.10.sup.13
particles/mL.
9. The composition of claim 1 wherein the virus is adenovirus.
10. The composition of claim 2 wherein the recombinant virus
comprises a wild-type p53 gene.
11. The composition of claim 10 wherein the recombinant virus is
A/C/N153.
12. The composition of claim 1 wherein the virus is adenovirus; the
polyhydroxy hydrocarbon is glycerol; the buffer system maintains
the pH in a range from about 7.3 to about 7.9 at a temperature
ranging from 20.degree. C. to 27.degree. C.; and the composition
further comprises a disaccharide.
13. The composition of claim 12 wherein the adenovirus is A/C/N/53;
the disaccharide is sucrose; the buffer system comprises sodium
phosphate monobasic dihydrate and tromethamine; and the composition
further comprises a divalent metal salt and water.
14. A method for concentrating a virus preparation comprising: (a)
adding a polyhydroxy hydrocarbon to a virus preparation to a final
polyhydroxy hydrocarbon concentration of about 20% or more; and (b)
subjecting the virus preparation to a filtration process wherein
the concentration of virus is increased by applying pressure to the
preparation such that diluent is removed from the virus preparation
through a filter while the virus is retained.
15. The method of claim 14 wherein the filtration process comprises
ultrafiltration.
16. The method of claim 14 wherein the filtration process comprises
tangential flow filtration.
17. A method of purifying a virus preparation comprising: (a)
subjecting the virus preparation to anion-exchange chromatography,
wherein the virus is eluted as a virus preparation product from an
anion-exchange chromatographic medium; (b) adding a polyhydroxy
hydrocarbon to the virus preparation product of step (a) so that
the concentration of polyhydroxy hydrocarbon in the preparation
reaches a level of about 25% or more; and (c) increasing the
concentration of virus in the virus preparation product of step (b)
by applying pressure to the preparation such that diluent is
removed from the virus preparation through a filter while the virus
is retained; and (d) subjecting the concentrated virus preparation
product of step (c) to one or more additional processing steps.
18. The method of claim 14 wherein the virus is a recombinant
virus.
19. The method of claim 14 wherein the virus is a recombinant virus
carrying a therapeutic transgene for use in gene therapy.
20. The method of claim 17 wherein an additional processing step
comprises size exclusion chromatography.
21. The method of claim 17 wherein the process of step (c)
comprises tangential flow filtration.
22. The method of claim 21 wherein the process of step (c) is
carried out using apparatus comprising a Pellicon XL filtration
system.
23. A virus preparation concentrated by the method of claim 14.
24. A virus preparation purified by the method of claim 17.
25. A method for concentrating a virus preparation comprising: (a)
centrifuging a composition which comprises a first layer comprising
a polyhydroxy hydrocarbon in a concentration of 35% to 80% (v/v),
the first layer overlaid with a second layer comprising a
polyhydroxy hydrocarbon in a concentration of 5% to 30% (v/v), the
second layer overlaid with a third layer comprising virus; and (b)
recovering the virus from the first layer.
26. The composition of claim 1, wherein the composition is
subjected to an additional processing step of agitation, followed
by microfiltration, for the prevention of particulate formation
during storage of the composition.
27. The composition of claim 1, wherein the composition is
subjected to one or more freeze/thaw cycles, followed by agitation,
and then microfiltration, for the prevention of particulate
formation during storage of the composition.
28. The composition of claim 12 further comprising a monovalent
metal salt in a concentration of about 0.6 to 10.0 mg/mL.
Description
I. FIELD OF THE INVENTION
[0001] The present invention relates to compositions comprising
viruses, especially viral vectors, having significantly improved
stability. The compositions of the present invention are useful in
maintaining the stability of viruses during storage, and
virus-containing compositions of the present invention are
particularly useful for therapeutic uses such as gene therapy. New
methods for concentrating and purifying virus preparations are also
provided.
II. BACKGROUND
[0002] Viruses have become-increasingly important for therapeutic
uses, such as vaccinations and gene therapy, and there is a need to
develop and prepare stable virus-containing compositions that can
easily be stored and transported, yet retain sufficient safety and
efficacy. In particular, given the extensive use of viral vectors
in gene therapy, it is important to develop and prepare
formulations that can stably preserve live recombinant viruses when
they carry therapeutic transgenes.
[0003] Moreover, there is a critical need for formulations that can
stabilize viral preparations at temperatures above -80.degree. C.
for extended periods of time. Virus-containing compositions
normally require storage at -80.degree. C. and cannot be stored at
standard refrigeration temperatures (e.g., 2.degree. C. to
8.degree. C., or higher) for substantial periods of time. This
limitation represents a serious impediment not only to storage, but
also to processing, distribution, and widespread clinical use.
[0004] There is also a need to develop virus-containing
compositions that can maintain pH in the range of about 7 to about
8.5 for extended periods despite being exposed to refrigeration
temperatures, and despite being subjected to harsh conditions such
as freeze/thaw, especially the slow rates of freeze/thaw that can
occur in connection with larger scale production, handling, or
distribution. Maintenance of pH is important for viral preparations
because at pH below 7.0 and above 8.5 the live virus particles are
vulnerable to losing viability due to physical and biological
instability.
[0005] Additional problems relate to increasing virus
concentrations. In particular, high virus concentration contributes
significantly to virus instability. However, increasingly higher
concentrations of virus and viral vectors are required for
therapeutic use. Therefore, there is a critical need to develop
formulations that stabilize relatively high concentrations of
virus, especially under the harsh conditions mentioned above. And
in addition, there is a particular need to develop new methods of
concentrating an existing virus preparation to achieve stable
preparations at higher concentration levels. The problems of
instability associated with higher virus concentrations are
exacerbated significantly if one tries to concentrate an existing
virus preparation. This is in part due to the additional mechanical
shear forces that come to bear during efforts to increase the
concentration of an existing virus preparation. If one could find a
method to concentrate a virus preparation without substantial
impairment to virus stability, then clinical dosages at any desired
concentration could be readily prepared (even when starting with
material having a lower concentration) and, importantly, the
ability to concentrate virus could eliminate problematic
bottlenecks and other scale-up problems during the purification
process by allowing significantly higher throughput during various
processing steps such as size exclusion chromatography.
[0006] There is thus a need for materials and methods to accomplish
the foregoing objectives.
III. SUMMARY OF INVENTION
[0007] The present invention fills the above-mentioned needs by
providing a stable composition comprising virus in a formulation
comprising a polyhydroxy hydrocarbon buffered to maintain a pH in a
range from about 7 to about 8.5 at a temperature in the range from
about 2.degree. C. to 27.degree. C.
[0008] Also provided are new methods of concentrating an existing
virus preparation that allow one to readily select and prepare
clinical dosages in a wide range of desired concentrations. A
preferred method of concentrating a virus preparation
comprises:
[0009] (a) adding a polyhydroxy hydrocarbon to a virus preparation
to a final polyhydroxy hydrocarbon concentration of about 20% or
more; and
[0010] (b) subjecting the virus preparation to a filtration process
wherein the concentration of virus is increased by applying
pressure to the preparation such that diluent is removed from the
virus preparation through a filter while the virus is retained.
[0011] Also provided herein is a method for concentrating a virus
preparation comprising:
[0012] (a) centrifuging a composition which comprises a first layer
comprising a polyhydroxy hydrocarbon in a concentration of 35% to
80% (v/v), the first layer overlaid with a second layer comprising
a polyhydroxy hydrocarbon in a concentration of 5% to 30% (v/v),
the second layer overlaid with a third layer comprising virus;
and
[0013] (b) recovering the virus from the first layer.
[0014] Furthermore, the present inventors found that their new
method of increasing virus concentration has the additional
advantage of enhancing further processing (e.g. by reducing or
eliminating problematic bottlenecks--during subsequent purification
by allowing significantly higher throughput during processing steps
such as size exclusion chromatography). Thus, in a preferred
embodiment, the method of concentrating virus preparations in
accordance with present invention further comprises a subsequent
purification step (e.g., size exclusion chromatography). In this
regard, the method of the present invention is particularly useful
when a step of size exclusion chromatography is performed
subsequent to ion exchange chromatography, and the virus
preparation is concentrated (in accordance with the present
invention) after the ion exchange chromatography but prior to the
size exclusion chromatography. Viral fractions obtained from anion
exchange chromatography, for example, typically contain high levels
of salts and possibly other impurities that further compromise
virus stability during concentration procedures. Thus, in a
particularly preferred embodiment, the present invention provides a
method of purifying a virus preparation comprising:
[0015] (a) subjecting the virus preparation to anion-exchange
chromatography, wherein the virus is eluted as a virus preparation
product from an anion-exchange chromatographic medium;
[0016] (b) adding a polyhydroxy hydrocarbon to the virus
preparation product of step (a) so that the concentration of
polyhydroxy hydrocarbon in the preparation reaches a final
concentration of about 25% or more; and
[0017] (c) increasing the concentration of virus in the virus
preparation product of step (b) by applying pressure to the
preparation such that diluent is removed from the virus preparation
through a filter while the virus is retained; and
[0018] (d) subjecting the concentrated virus preparation product of
step (c) to one or more additional processing steps.
[0019] The present invention also provides virus preparations
concentrated and/or purified by the foregoing methods.
IV. DETAILED DESCRIPTION
[0020] As noted above, the present application discloses novel
virus-containing compositions, as well as novel methods of
concentrating and purifying virus-containing compositions.
[0021] With regard to compositions, the present inventors have
developed a novel buffered formulation that can preserve viral
preparations with enhanced stability. In particular, the
formulation can stabilize viral preparations at temperatures well
above -80.degree. C. More important still, the compositions of the
present invention are stable at typical refrigeration temperatures
of, e.g., 2.degree. to 8.degree. C., or higher, for substantial
periods of time, preferably for several months or more. This is a
critical advantage because, as mentioned above, in order to meet
clinical needs it is impractical to keep viral preparations frozen
at -80.degree. C. during storage and transportation.
[0022] An important feature of the compositions of the present
invention is the addition of a polyhydroxy hydrocarbon. As used
herein, a polyhydroxy hydrocarbon means a branched, linear, or
cyclic compound substituted with 2 or more (preferably 2 to 6, more
preferably 2 to 4) hydroxy groups. Polyhydroxy hydrocarbons for use
in the present invention preferably are polyhydroxy-substituted
alkyl compounds (branched or unbranched), preferably having 2 to 7
carbon atoms, and can include, e.g., glycerol, sorbitol and
polypropanol. Glycerol is particularly preferred. As shown by data
provided below, the present inventors found that glycerol allows
for surprisingly high levels of stability for extended periods of
time even under standard refrigeration conditions.
[0023] An effective amount of polyhydroxy hydrocarbon for
compositions of the present invention is an amount sufficient to
stabilize the virus in the formulation of the present invention
without adversely affecting the effectiveness of the virus for
further use, especially in cases where the virus contains a
transgene for use in gene therapy. The polyhydroxy hydrocarbon is
preferably present at a final concentration of about 20 to 200
mg/mL. A narrower range can be 80 to 120 mg/mL. More than one
polyhydroxy hydrocarbon can be used to achieve the desired total
amount of polyhydroxy hydrocarbon in the composition of the present
invention.
[0024] The polyhydroxy hydrocarbon in compositions of the present
invention can optionally contain an aldehyde group. In particular,
the polyhydroxy hydrocarbon can be a disaccharide such as sucrose.
Furthermore, even if the polyhydroxy hydrocarbon selected for the
composition does not contain an aldehyde group, the composition can
additionally include a disaccharide, such as sucrose, as a
stabilizer and tonicity-adjusting agent. When the composition of
the present invention already contains a suitable polyhydroxy
hydrocarbon (such as glycerol) and a disaccharide is employed in
preferred embodiments as an additional stabilizer or
tonicity-adjusting agent, the disaccharide is preferably present in
a range of 5 to 25 mg/mL, more preferably 20 mg/mL, and preferably
the disaccharide is sucrose.
[0025] Pharmaceutically acceptable divalent metal salt stabilizers,
such as magnesium salts, zinc salts and calcium salts, are used in
preferred embodiments of the composition of the present invention.
Preferably, the salt is a chloride salt or a magnesium salt,
magnesium salt being particularly preferred. Preferably, the salt
(e.g., the magnesium salt) is present in an amount of from about
0.1 to 1 mg/mL, more preferably in an amount of about 0.4
mg/mL.
[0026] Pharmaceutically acceptable monovalent metal salt
stabilizers such as potassium, sodium, lithium and cesium salts may
be included in preferred embodiments of the present invention as
optional stabilizers. Preferably, the salt is sodium chloride
present in the amount of 0.6 to 10.0 mg/ml, more preferably in an
amount of about 5.8 mg/ml.
[0027] In addition to stabilizing the composition, sodium chloride
may suppress the rate and extent of the appearance of by-products
of fermentation, resulting in a more pharmaceutically elegant
presentation that may have reduced antigenicity potential due to
protein aggregates. The addition of sodium chloride does not affect
the pH of the formulation.
[0028] The composition of the present invention is capable of
maintaining a pH in the range of about 7 to about 8.5 for extended
periods of time, even when subjected to harsh conditions such as
refrigeration and freeze/thaw. Moreover, the compositions can
remain stable and maintain the required pH range even when
subjected to the relatively slow rate of freeze/thaw that can occur
in connection with larger scale production, distribution, and
handling. As noted above, maintenance of pH is important for viral
preparations, because at pH below 7.0 and above 8.5 the live virus
particles can become unstable and degrade. The particular
composition of viruses makes viruses difficult to stabilize and
preserve.
[0029] To accomplish pH maintenance under harsh conditions, the
present invention preferably comprises a buffer system that can
maintain an optimal pH in a range from about 7.0 to about 8.5
despite storage between -80.degree. C. and 27.degree. C. and
despite being subjected to freeze/thaw conditions. Since pH can
vary depending on temperature, pH ranges of the present invention
are more specifically illustrated below with reference to specific
temperature ranges. For instance, at refrigeration temperatures
(e.g., about 2.degree. C. to 8.degree. C.) a preferred pH range is
about 7.7 to about 8.3, more preferably about 7.9 to about 8.2. At
room temperature (e.g., about 20.degree. C. to 27.degree. C.,
preferably 22.degree. C.-25.degree. C.), a preferred pH range is
about 7.3 to about 8.2, more preferably about 7.4 to about 7.9.
[0030] A preferred buffer system of the present invention comprises
sodium phosphate monobasic dihydrate in a concentration of about
0.5 to 10 mg/mL and tromethamine in a concentration of about 0.5 to
10 mg/mL. (Tromethamine is also known as TRIS or "Trizma" available
from Sigma Chemical Co.). However, other buffer systems can be
used. For example, sodium phosphate dibasic dihydrate can be used
if coupled with an acidic form of tris buffer. In a particularly
preferred embodiment, the buffer system comprises sodium phosphate
monobasic dihydrate in a concentration of about 1.7 mg/mL and
tromethamine in a concentration of about 1.7 mg/mL, and has the
ability to maintain the formulation in an optimal pH range of about
7.3 to about 7.9 at 25.degree. C.
[0031] The formulation of the present invention has the additional
advantage of having the ability to stabilize high concentrations of
virus at the above-mentioned harsh conditions (such as
refrigeration temperatures and freeze/thaw processing). In
particular, the formulation of the present invention can maintain
stability of the virus at concentrations ranging up to
1.times.10.sup.13 particles/mL. A preferable range of virus
concentrations for use in the present invention is in an amount of
1.times.10.sup.9 to 1.times.10.sup.13 particles/mL., more
preferably, up to 1.times.10.sup.12 particles/mL, e.g.
1.times.10.sup.9 (or 1.times.10.sup.10) to 1.times.10.sup.12.
[0032] The term "diluent" as used herein can comprise a solvent
(e.g., water, preferably sterile water) or a mixture of a solvent
and other ingredients such as additional solvents, additional
stabilizers, additional buffers, and/or other substances that do
not adversely affect safety, efficacy and stability of the
formulation. With regard to diluents, stabilizers, buffers and the
like, reference may be made, e.g., to Remington's Pharmaceutical
Science, 15th Ed., Mack Publishing Company, Easton, Pa.
[0033] A surfactant, preferably a nonionic detergent such as a
polyoxyethylene fatty acid ester (e.g., polyoxyethylenesorbitans
such as Polysorbate 20, Polysorbate 40, Polysorbate 60, or
Polysorbate 80 from ICI Americas, Inc., Wilmington Del., or. Tween
20, Tween, 40, Tween 60 and Tween 80 from Sigma, St. Louis, Mo.),
can optionally be included in the composition of the present
invention. Preferably, the nonionic detergent is a polyoxyethylene
fatty acid ester, and the polyoxyethylene fatty acid ester is
preferably Polysorbate 80, which can act as a stabilizer in the
composition of the present invention. The concentration of
non-ionic detergent is preferably in a range of 0.03 to 0.3 mg/mL;
more preferably, 0.15 mg/mL.
[0034] Compositions of the present invention can further contain
one or more "delivery-enhancing agents". A "delivery-enhancing
agent" refers to any agent which enhances delivery of a therapeutic
gene, such as a tumor suppressor gene to a cancerous tissue or
organ. Examples of such delivery-enhancing agents include but are
not limited to detergents, alcohols, glycols, surfactants, bile
salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic
salt solutions, and acetates.
[0035] Detergents (as the term is used herein) can include anionic,
cationic, zwitterionic, and nonionic detergents. Exemplary
detergents include but are not limited to taurocholate,
deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium
chloride, ZWITTERGENT.RTM. 3-14 detergent, CHAPS
(3-[3-Cholamidopropyl) dimethylammoniol]-1-propanes- ulfonate
hydrate, Aldrich), Big CHAP, Deoxy Big CHAP, TRITON.RTM.-X-100
detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC.RTM.-F68
detergent, TWEEN.RTM. 20 detergent, and TWEEN.RTM. 80 detergent
(CALBIOCHEM.RTM. Biochemicals).
[0036] The use of delivery-enhancing agents is described in detail
in copending U.S. patent application U.S. Ser. No. 08/889,335 filed
on Jul. 8, 1997, and International Application Publication No. WO
97/25072, Jul. 17, 1997, and in U.S. patent application U.S. Ser.
No. 09/112,074 filed on Jul. 8, 1998, International Application
PCT/US 98/14241. In addition, use of calpain inhibitors in
conjunction with viral vectors to increase transduction efficiency
is described in U.S. patent applications U.S. Ser. No. 09/172,685
and 60/104,321 filed on Oct. 15, 1998.
[0037] A wide range of viruses can be used in the compositions of
the present invention, including but not limited to adenoviruses,
pox viruses, iridoviruses, herpes viruses, papovaviruses,
paramyxoviruses, orthomyxoviruses, retroviruses, adeno-associated
virus, vaccinia virus, rotaviruses, etc. (see, e.g., Anderson,
Science (1992) 256: 808-813); adenoviruses being particularly
preferred. The viruses are preferably recombinant viruses, but can
include clinical isolates, attenuated vaccine strains, and so on.
Thus, for example, an exemplary recombinant adenovirus that can be
used in compositions of the invention is A/C/N153, which is
disclosed in PCT patent application no. WO 95/11984.
[0038] The formulation of the present invention is particularly
well suited for stabilizing a recombinant virus, such as a live
recombinant adenovirus (or "viral vector"), for therapeutic use in
gene therapy. For instance, the virus used in the present invention
can comprise a tumor suppressor gene, such as a wild-type p53 gene
or an Rb gene (e.g., p110.sup.RB or p56 .sup.RB), and with
transgenes such as wild-type p53 inserted in a viral vector, the
composition of the present invention can be used as a
pharmaceutical composition for treatment of cancer.
[0039] In this regard, the formulations of the present invention
have a remarkable ability to maintain the viability of live virus,
in particular a viral vector into which a nucleotide sequence
encoding a transgene such as p53 has been inserted. This feature
allows the virus to maintain its ability to infect target cells so
that the therapeutic protein encoded by the inserted transgene is
adequately produced.
[0040] With specific regard to p53 and its uses, it is noted that
mutation of the p53 gene is the most common genetic alteration in
human cancers (Bartek (1991) Oncogene, 6: 1699-1703, Hollstein
(1991) Science, 253: 49-53). Introduction of wild-type p53 in
mammalian cancer cells lacking endogenous wild-type p53 protein
suppresses the neoplastic phenotype of those cells (see, e.g., U.S.
Pat. No. 5,532,220).
[0041] In the examples below, the virus is a live recombinant
adenovirus containing wild-type p53 gene. The particular viral
vector construct used in these examples is referred to herein as
"A/C/N153". A/C/N/53 (also referred to as "ACN53") is a
particularly preferred viral vector construct described in
copending application U.S. Ser. No. 08/328,673 filed on Oct. 25,
1994, and in WO 95/11984 (May 4, 1995), expressly incorporated
herein by reference.
[0042] A representative formula for preferred embodiments of the
present invention that contain Polysorbate 80 is set forth
below:
1 Representative Formula Active Substance A/C/N/53 1 .times.
10.sup.9 to 1 .times. 10.sup.13 particles/mL Buffer Sodium
Phosphate 0.5 to 10 mg/mL Monobasic Tromethamine 0.5 to 10 mg/mL
Stabilizer/tonicity Sucrose 5 to 25 mg/mL agent Stabilizers
Glycerol 20 to 200 mg/mL Magnesium Chloride 0.1 to 1 mg/mL
Polysorbate 80 0.03 to 0.3 mg/mL Solvent Water for Injection q.s.
ad 1 mL
[0043] (The compositions are typically stored in 1.0 mL dosages.
"q.s. ad" in the formula above means adding sufficient solvent to
reach the 1 mL total volume).
[0044] Four particularly preferred embodiments are set forth below.
(Polysorbate 80 is present in Examples 1 and 2, but absent in
Examples 3 and 4).
2 Example 1 Example 2 A/C/N/53 7.5 .times. 10.sup.11 7.5 .times.
10.sup.10 particles/mL particles/mL Sodium Phosphate Monobasic
Dihydrate 1.7 mg/mL 1.7 mg/mL Tromethamine 1.7 mg/mL 1.7 mg/mL
Magnesium Chloride Hexahydrate 0.4 mg/mL 0.4 mg/mL Sucrose 20 mg/mL
20 mg/mL Polysorbate 80 0.15 mg/mL 0.15 mg/mL Glycerol 100 mg/mL
100 mg/mL Water for Injection q.s. ad 1 mL 1 mL pH 7.4 to 7.8 7.4
to 7.8
[0045]
3 Example 3 Example 4 A/C/N/53 7.5 .times. 10.sup.11 7.5 .times.
10.sup.10 particles/mL particles/mL Sodium Phosphate Monobasic
Dihydrate 1.7 mg/mL 1.7 mg/mL Tromethamine 1.7 mg/mL 1.7 mg/mL
Magnesium Chloride Hexahydrate 0.4 mg/mL 0.4 mg/mL Sucrose 20 mg/mL
20 mg/mL Glycerol 100 mg/mL 100 mg/mL Water for Injection q.s. ad 1
mL 1 mL pH 7.4 to 7.9 7.3 to 7.8
[0046] The following ingredients: sodium phosphate monobasic
dihydrate, tromethamine, magnesium chloride hexahydrate, sucrose,
and glycerol can all be obtained from, e.g., EM Industries, INC., 7
Skyline Drive, Hawthorne, N.Y. 10532. Polysorbate 80 is available
from, e.g., ICI Americas, Inc., Wilmington Del., 19897.
[0047] Compositions of the present invention can be prepared during
purification of the virus in a gel filtration chromatography column
by combining the ingredients (excluding Polysorbate-80) at the
desired concentrations in the gel filtration column. (With regard
to gel filtration methods, reference can be made, e.g., to Section
V below). Then, if it is desired to dilute the concentration of the
virus, or to incorporate Polysorbate-80, then diluents can be
prepared by standard techniques. An illustrative example is set
forth below:
[0048] Charge and dissolve sodium phosphate monobasic dihydrate,
tromethamine, sucrose, magnesium chloride hexahydrate and glycerol
in approximately 75% of batch volume of water for injection at room
temperature in a stainless steel vessel equipped with agitator.
Bring the batch of the resulting diluent to final volume with water
for injection. Check the pH. Calculate the required volume of
A/C/N/53 (adenovirus with wild-type p53 as a transgene) Drug
Substance in Suspension and the required volume of diluent to make
A/C/N/53 Injection. If the final A/C/N/53 Injection will contain
Polysorbate 80, prepare a stock solution that contains 10% excess
Polysorbate 80 in diluent. Charge the calculated amounts of
A/C/N/53 Drug Substance in Suspension and diluent into a stainless
steel container and mix. Charge the Polysorbate 80 solution, prior
to adding all of the Diluent, based upon 10% of the total A/C/N/53
Injection batch volume if required. Aseptically filter the
suspension through a sterilized filter (0.22 .mu.m or equivalent).
Test the filter integrity after filtration. Collect and fill the
sterilized suspension into vials having the appropriate volume.
Stopper and seal the vials.
[0049] Stability data for Examples 1, 2, 3, and 4 are set forth,
respectively, in Tables 1, 2, 3, and 4 below.
[0050] In the Tables below, the antiproliferation assay is a
bioassay used to measure the product's ability to suppress cancer
cells and is based generally on procedures used by Wills, et al.,
1994, Human Gene Therapy, 5:1079-1088. The numbers listed indicate
activity whereas the control has no activity.
[0051] The "Plaque Assay" measures virus particles in culture by
scoring the number of viral plaques as a function of dilution and
is based generally on procedures described in Graham, F. L., and
Prevec, L., Methods in Molecular Biology, vol. 7: Gene Transfer and
Expression Protocols, E. J. Murray, ed. (Humana Press Inc., Clifton
N.J.) pp. 109-128 (1991); see also Graham, F. L., Smiley, J.,
Russel, W. C., and Nairn, R., J. Gen. Virol. vol. 36, pp 59-74
(1977).
[0052] The "FACS" assay shows the ability of the virus to infect
cells, and these measurements are based generally on methods
described in, e.g., International Patent Application PCT/US97/11865
(WO 98/01582, published Jan. 15, 1998). In the next column to the
right, the numbers presented under the heading "Concentration"
represent the concentration of the total number of virus particles.
Finally, the numbers under the heading "Particle/FACS ratio"
represent the ratio of the total number of virus particles as
compared to the number of infectious virus particles, thus
indicating the relative potency of the virus preparation.
[0053] The data under the heading "UV" indicate the aggregation of
the virus particles as shown by the UV absorbance ratio for the
wavelengths A.sub.320/A.sub.260 as an indication of light scatter.
Basically, the absorbance at 320 nanometer wavelength measures the
amount of light scatter, whereas the absorbance at 260 nanometer
wavelength correlates with amount of DNA.
[0054] The temperatures listed in the second column of the tables
under "condition" represent the storage temperature. The
physiological assays are performed at 37.degree. C. and the pH in
the last column is measured at room temperature, approximately
25.degree. C.
4TABLE 1 Stability Data on Example 1 Antiproliferation Plaque Assay
Assay FACS Concentration Particles Stability Condition
.times.10.sup.5 .times.10.sup.8 .times.10.sup.10 .times.10.sup.11
FACS UV Time .degree. C. SPU/mL PFU/mL U/mL part./mL Ratio
A.sub.320/A.sub.260 pH initial 3.3 5.8 3.86 7.95 21 0.23 7.53 1
week 25 4.9 10 2.27 8.06 36 0.24 7.53 2 weeks 4 3.1 6.6 3.41 7.84
23 0.23 7.49 4 weeks 4 3.4 17 3.91 7.79 20 0.23 7.63 8 weeks 4 5.8
8.8 3.38 7.80 23 0.24 7.61 12 weeks 4 3.9 36 2.24 7.80 35 0.24 7.72
5 months 4 not tested not tested 1.57 8.21 52 0.26 7.60 6 months 4
3.0 7.6 2.74 7.68 28 0.28 7.58 9 months 4 3.1 19 3.47 7.19* 21 0.28
7.58 11 months 4 not tested not tested nt 6.71 -- 0.29 nt 12 months
4 2.6 10 0.81 6.13 76 0.30 7.62 *Retest of 9 month UV samples: 6.89
.times. 10.sup.11 particles/mL; A.sub.320/A.sub.260 = 0.28.
[0055]
5TABLE 2 Stability Data on Example 2 Antiproliferation Plaque Assay
Assay FACS Concentration Particles Stability Condition
.times.10.sup.4 .times.10.sup.7 .times.10.sup.9 .times.10.sup.10
FACS UV Time .degree. C. SPU/mL PFU/mL U/mL part./mL Ratio
A.sub.320/A.sub.260 pH initial 3.3 4.8 1.99 10.0 50 0.24 7.36 1
week 25 2.4 7.1 1.64 nd* -- 0.46 7.42 2 weeks 4 2.4 6.9 2.13 9.79
46 0.24 7.51 4 weeks 4 3.2 8.4 2.50 9.24 37 0.22 7.55 8 weeks 4 6.9
8.6 2.60 8.10 31 0.25 7.54 12 weeks 4 5.5 8.3 1.09 8.60 79 0.24
7.64 5 months 4 not tested not tested 1.74 8.03 46 0.27 7.55 6
months 4 3.3 7.3 2.10 8.25 39 0.23 7.52 9 months 4 2.3 13 1.97 7.59
39 0.24** 7.53 11 months 4 not tested (nt) not tested nt 7.48 --
0.23 nt 12 months 4 1.8 9.4 0.60 4.94 82 0.26 7.59 *Not determined
due to assay interference. **Retest of 9 month UV samples: 7.37
.times. 10.sup.10 particles/mL, A.sub.320A.sub.260 = 0.24.
[0056]
6TABLE 3 Stability Data on Example 3 Antiproliferation Plaque Assay
Assay FACS Concentration Particles Stability Condition
.times.10.sup.5 .times.10.sup.8 .times.10.sup.10 .times.10.sup.11
FACS UV Time .degree. C. SPU/mL PFU/mL U/mL part./mL Ratio
A.sub.320/A.sub.260 pH initial 3.2 6.3 2.59 7.45 29 0.24 7.67 1
week 25 4.4 4.3 1.65 7.49 45 0.24 7.68 2 weeks 4 2.3 8.0 3.62 7.27
20 0.24 7.49 4 weeks 4 2.7 7.6 4.08 6.97 17 0.24 7.75 8 weeks 4 5.9
8.7 2.69 7.00 26 0.25 7.82 12 weeks 4 3.0 15 0.70 7.10 101 0.25
7.80 6 months 4 2.4 6.4 2.45 7.12 29 0.25 7.76 9 months 4 2.8 9.4
2.51 7.06 28 0.26 7.81 11 months 4 not tested (nt) nt nt 6.71 --
0.25 nt 12 months 4 2.2 9.8 0.74 6.75 91 0.26 7.81
[0057]
7TABLE 4 Stability Data on Example 4 Antiproliferation Plaque Assay
Assay FACS Concentration Particles Stability Condition
.times.10.sup.4 .times.10.sup.7 .times.10.sup.9 .times.10.sup.10
FACS UV Time .degree. C. SPU/mL PFU/mL U/mL part./mL Ratio
A.sub.320/A.sub.260 pH initial 3.3 4.7 2.36 8.91 38 0.23 7.37 1
week 25 2.3 9.3 1.40 8.25 59 0.24 7.37 2 weeks 4 2.8 8.0 2.16 8.80
41 nd* 7.37 4 weeks 4 2.9 6.6 2.54 9.35 37 0.20 7.63 8 weeks 4 6.9
7.2 2.56 7.60 30 0.24 7.60 12 weeks 4 4.4 8.6 1.45 7.20 50 0.23
7.73 6 months 4 3.6 7.1 2.85 7.92 28 0.21 7.58 9 months 4 2.9 11
1.87 7.26 39 0.20 7.60 11 months 4 not tested (nt) nt nt 6.93 --
0.22 nt 12 months 4 2.4 22 0.70 7.15 102 0.23 7.61 *Not determined
due to assay interference.
EXAMPLE 5
[0058] Formulation for Example 5: A/C/N/53 (7.5.times.10.sup.11
Particles/mL), Tromethamine (TRIS) (1.7 mg/mL), Sodium Phosphate
Monobasic Dihydrate (1.7 mg/mL), Sucrose (20 mg/mL), Magnesium
Chloride Hexahydrate (0.4 mg/mL), Glycerol (100 mg/mL), Sodium
Chloride (5.8 mg/mL), Fill Volume=10 mL.
8TABLE 5 Stability Data on Example 5 Antiproliferation Assay FACS
Concentration Particles Stability Condition .times.10.sup.5
.times.10.sup.10 .times.10.sup.11 FACS UV Time .degree. C. SPU/mL
U/mL part./mL Ratio A.sub.320/A.sub.260 pH initial 4.5 1.87 7.81 42
0.23 7.80 1 month 4 8.0 1.67 7.83 47 0.23 7.80 4 month 4 13.0 1.58
7.84 50 0.23 7.70
[0059] In some cases, particulates have been observed to form in
the formulation during storage at 4.degree. C. Analysis of the
particulates by SDS-PAGE suggests that the particulates are
composed of minor impurities (i.e., additional proteins and some
immature viral particles), and thus these particulates do not
affect the viability of the formulation. Nonetheless, in a
preferred embodiment to further clarify the formulation (to prevent
possible particulate formation), an optional step of
microfiltration can be carried out to remove any potential
particulates with little loss of viral particles. (When carrying
out microfiltration, it should be noted that sufficient
microfiltration membrane surface area per filtration volume is
critical to avoid loss of virus as the particulate is removed.)
[0060] In addition, in a preferred embodiment, the present
inventors have found that agitation, such as stirring, can
accelerate particulate formation and is therefore an additional
optional step in the clarification process. Thus, gentle stirring
(e.g., overnight, 10.degree. C., using a magnetic stirbar) followed
by microfiltration was shown to remove the particulates such that
no more particulate would reform upon restirring.
[0061] It was also found that cycles of freeze/thaw could promote
particulate formation during restirring. Thus, in another preferred
procedure, one or more freeze/thaw cycles can optionally be carried
out, followed by stirring, and then microfiltration, for the
prevention of particulate formation during storage of the virus
final product at refrigeration temperatures (e.g. 4.degree.
C.).
Methods of Concentrating and Purifying
Virus-Containing Compositions
[0062] The present application also discloses a new method of
stably concentrating an existing virus preparation by employing
tangential flow filtration (hereafter sometimes referred to as
"TFF"), allowing one to readily select and prepare clinical dosages
in a wide range of desired concentrations. The new method of
concentrating a virus preparation comprises:
[0063] (a) adding a polyhydroxy hydrocarbon to a virus preparation
to a final polyhydroxy hydrocarbon concentration of about 20% or
more; and
[0064] (b) subjecting the virus preparation to a filtration process
wherein the concentration of virus is increased by applying
pressure to the preparation such that diluent is removed from the
virus preparation through a filter while the virus is retained.
[0065] 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, adeno-associated
virus, vaccinia virus, rotaviruses, etc.; adenoviruses being
particularly preferred. The viruses are preferably recombinant
viruses, but can include clinical isolates, attenuated vaccine
strains, and so on. The present invention is particularly useful
for concentrating recombinant viruses carrying a heterologous
transgene for use in gene therapy. Such viruses are especially
vulnerable to potentially destabilizing forces, such as the
additional shear mechanical forces accompanying methods of
concentrating virus preparations. An exemplary recombinant
adenovirus that can be concentrated by the method of the invention
is A/C/N/53, which is disclosed in PCT Patent Application No. WO
95/11984.
[0066] The filtration process used to concentrate the virus
according to the method of the present invention can include any
filtration process (e.g., ultrafiltration) where the concentration
of virus is increased by forcing diluent to be passed through a
filter in such a manner that the diluent is removed from the virus
preparation whereas the virus is unable to pass through the filter
and thereby remains, in concentrated form, in the virus
preparation. Ultrafiltration is described in detail in, e.g.,
Microfiltration and Ultrafiltration: Principles and Applications,
L. Zeman and A. Zydney (Marcel Dekkar, Inc., New York, N.Y., 1996).
A particularly preferred filtration process is Tangential Flow
Filtration ("TFF") as described in, e.g., MILLEPORE catalogue
entitled "Pharmaceutical Process Filtration Catalogue" pp. 177-202
(Bedford, Mass., 1995/96). Preferred TFF apparatus comprises either
a Pellicon II or Pellicon XL filter system from Millipore
Corporation, 80 Ashby Rd., Bedford, Mass. (internet address:
www.millipore.com), a Pellicon XL system being particularly
preferred. In a preferred embodiment, the methods of the present
invention are carried out at temperatures in a range from about
2.degree. C. to 27.degree. C.
[0067] Other concentration processes can be employed to concentrate
virus preparations in accordance with the present invention. For
instance, employment of polyhydroxy hydrocarbon can advantageously
be used to concentrate a virus preparation by centrifugation. Thus,
the present invention also provides a method for concentrating a
virus preparation comprising:
[0068] (a) centrifuging a composition which comprises a first layer
comprising a polyhydroxy hydrocarbon in a concentration of 35% to
80% (v/v), the first layer overlaid with a second layer comprising
a polyhydroxy hydrocarbon in a concentration of 5% to 30% (v/v),
the second layer overlaid with a third layer comprising virus;
and
[0069] (b) recovering the virus from the first layer.
[0070] By way of example, an adenovirus preparation can be
concentrated by low speed centrifugation at 3,200 g using swing
bucket rotors of a Beckman centrifuge. To accomplish this, the
virus preparation can be placed into multiple 5 ml tubes, each tube
containing 6.25% volume of 70% glycerol in a first layer at the
tube bottom, overlaid with 2.5% volume of 20% glycerol, with the
virus preparation laid on top. The preparation is then centrifuged
at 3,200 g at 4.degree. C. for approximately 16 hours to pellet the
concentrated virus into the glycerol layers and then the
newly-concentrated virus preparation is subsequently recovered from
the first layer. Virus concentrated by the procedures described
above had good light scattering characteristics and had suitable
infectivity properties.
[0071] With regard to the polyhydroxy hydrocarbon used in the
methods of the present invention, a "polyhydroxy hydrocarbon" means
a branched, linear, or cyclic compound substituted with 2 or more
(preferably 2 to 6, more preferably 2 to 4) hydroxy groups, and an
effective amount of polyhydroxy hydrocarbon is an amount sufficient
to stabilize the virus against potentially destabilizing forces,
such as the mechanical shear forces that occur during the
concentration process. Preferably, the polyhydroxy hydrocarbon in
the virus-concentrating methods of the present invention is present
in a minimum concentration of 20%, more preferably 25%. Polyhydroxy
hydrocarbons for use in the present invention preferably are
polyhydroxy-substituted alkyl compounds (branched or unbranched),
preferably having 2 to 7 carbon atoms, and can include glycerol,
sorbitol and polypropanol. Glycerol is particularly preferred.
[0072] The inventors' new method of increasing virus concentration
has the additional advantage of enhancing processing, e.g., by
eliminating problematic bottlenecks by allowing significantly
higher throughput during various processing steps such as size
exclusion chromatography. Thus, in a preferred embodiment, the
method of concentrating virus preparations in accordance with
present invention can be applied to methods of purifying viruses
where a size exclusion chromatography step (e.g., gel filtration)
is performed subsequent to anion exchange chromatography. In this
embodiment, there are additional threats to virus stability
stemming not only from the mechanical shear forces needed to
concentrate the virus prior to the rate-limiting size exclusion
chromatography step, but also due to the fact that the virus
preparation eluted from the anion exchange chromatography step
typically contains high levels of salts and other impurities that
further compromise virus stability. Thus, in a particularly
preferred embodiment, the present invention provides a method of
purifying a virus preparation comprising:
[0073] (a) subjecting the virus preparation to anion-exchange
chromatography, wherein the virus is eluted as a virus preparation
product from an anion-exchange chromatographic medium;
[0074] (b) adding a polyhydroxy hydrocarbon to the virus
preparation product of step (a) so that the concentration of
polyhydroxy hydrocarbon in the preparation reaches a final
concentration of about 25% or more; and
[0075] (c) increasing the concentration of virus in the virus
preparation product of step (b) by applying pressure to the
preparation such that diluent is removed from the virus preparation
through a filter while the virus is retained; and
[0076] (d) subjecting the concentrated virus preparation product of
step (c) to one or more additional processing steps.
[0077] In the preferred embodiment set forth above in connection
with anion exchange chromatography, the minimum level of glycerol
is 25% (rather than the 20% minimum level in general applications
of the concentration methods of the present invention) because this
particular application must take into account the additional threat
to stability posed by the high salt concentrations in the product
eluted from the anion exchange column The addition of 25% glycerol
(preferably 30%) results in stability of the salt-containing DEAE
pool for >10 days at, e.g., 4.degree. C.; therefore subsequent
steps of virus concentration and/or gel filtration can be performed
on separate days with substantial flexibility across a 10 day
period. As will be appreciated, the employment of polyhydroxy
hydrocarbon in the higher concentration of 25% or more can also be
used in methods of the present invention when the virus preparation
contains high salt content due to other processing conditions.
V. EXAMPLES OF METHODS OF THE PRESENT INVENTION
[0078] The following examples illustrate preferred embodiments of
the present invention; the scope of the invention is not to be
construed as limited thereby.
[0079] Brief Overview--A concentrated batch starts with frozen
crude viral materials originating from fermentation recovery. In
one embodiment, the adenovirus product is first purified by anion
exchange chromatography. Then, prior to loading the preparation
onto a size exclusion column, the anion exchange pool can be
concentrated by tangential flow filtration (TFF) in the presence of
30% (v/v) glycerol. Alternatively, in another embodiment, the TFF
concentration step can be carried out in the presence of 20% (v/v)
or more (preferably 25%) glycerol after size exclusion
chromatography.
[0080] Preparation of Starting Materials by Anion-Exchange
Chromatography Prior to TFF
[0081] In a preferred embodiment, an adenovirus anion exchange pool
is prepared for concentration as follows. Frozen viral material
from fermentation and recovery steps is thawed and filtered through
a 0.45 .mu.m hydrophilic membrane. The salt concentration of the
filtrate is adjusted by adding 4M sodium chloride. This feed
solution is then applied to a Fractogel EMD DEAE-650M column
pre-equilibrated with 50 mM sodium phosphate pH 7.5, 260 mM sodium
chloride, 2 mM magnesium chloride, 2% (w/v) sucrose (Buffer A). The
adenovirus binds to the anion exchange resin, whereas the majority
of media and host cell impurities pass through the column ending up
in the spent charge. The column is initially washed with 4 volumes
of buffer A followed by a second isocratic wash of 8 bed volumes of
94% buffer A and 6% buffer B (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 is eluted from the column
with a 30 bed volume linear gradient from 6% to 100% buffer B. The
Adenovirus peak of the elution profile as determined by A.sub.280
is collected. Then glycerol is added to the DEAE pool at a final
concentration of 30% (v/v) for further processing.
[0082] Concentration of DEAE Pool Using Tangential Flow
Filtration
[0083] The DEAE pool (prepared in accordance with the above
description) is concentrated to 10- to 20-fold by using a Millipore
TFF unit (Pellicon XL System) with 1 million molecular weight
cut-off Biomax membranes. The process is carried out either at
2-10.degree. C or room temperature (25.degree. C.). The following
filtration parameters are used in this procedure: average inlet
pressure=14 psi; average permeate pressure=o psi; average flux
rate=13 liters/hour-square meter. The final concentration of
adenovirus achieves approximately 1.0-2.0.times.10.sup.13 particles
per ml. Based on the Resource Q-HPLC and UV absorbance (A.sub.260)
analysis, the recovery of concentration step is >80% with no
significant aggregation (light scattering assay by
A.sub.320/A.sub.260).
[0084] Buffer Exchange by Size Exclusion Chromatography (Gel
Filtration)
[0085] The concentrated adenovirus preparation is applied to a
Superdex-200 size exclusion column pre-equilibrated with 20 mM
sodium phosphate pH 8.0, 100 mM sodium chloride, 2 mM magnesium
chloride, 2% (w/v) sucrose, 10% glycerol (Buffer C) or 11 mM sodium
phosphate, 14 mM Tris, 2 mM magnesium chloride, 2% (w/v) sucrose,
10% glycerol, pH 7.8 (Buffer D). The column is eluted with
equilibration buffer. The Adenovirus peak of the elution profile as
determined by A.sub.280 is collected and pooled. The concentrated
adenovirus preparation is filtered through a 0.2 .mu.m hydrophilic
Durapore membrane (Stericup, Millipore) at 2 to 10.degree. C., and
can be stored at -80.degree. C., or at higher temperatures (such as
2 to 10.degree. C.).
[0086] Concentration of Superdex-200 Pool Using Tangential Flow
Filtration
[0087] As discussed above, a preferred embodiment of the present
invention involves concentrating the virus after anion exchange
chromatography, but before gel filtration. However, in another
embodiment, the disclosed methods of concentrating virus
preparations can also be used after the gel filtration step (even
if no virus concentration step was employed in between the anion
exchange step and the gel filtration step). In this case, the
filtration parameters are the same as those for concentration of a
DEAE pool, except that the polyhydroxy hydrocarbon (e.g., glycerol)
can be added to the Superdex-200 pool at a final concentration as
low as 20% (v/v) since it is no longer necessary to deal with the
high salt concentrations in the DEAE pool. In this regard, it
should be noted that in cases where the addition of polyhydroxy
hydrocarbon is postponed until after gel filtration, the DEAE pool
should be applied immediately to the gel filtration column (due to
the vulnerability of the DEAE pool--with its high salt
concentration). Thus, it can be seen that an additional advantage
of adding polyhydroxy hydrocarbon to the DEAE pool (in accordance
with the present invention) is increased flexibility in terms of
the time interval and storage options during the period of time
between anion-exchange chromatography and subsequent
processing.
[0088] The methods of concentrating virus preparations can be
applied in connection with a variety of purification methods. For
additional information on purification methods, reference can be
made, e.g., to Huyghe et al., Human Gene Therapy, Vol. 6, pp.
1403-1416 (1995) and U.S. patent application Ser. No. 08/989,227,
expressly incorporated herein by reference.
VI. STABILITY DATA FOR METHODS OF CONCENTRATING VIRUS PREPARATIONS
USING TANGENTIAL FLOW FILTRATION
[0089] As shown by the experimental data below, the methods of the
present invention allow for greatly enhanced virus stability,
despite the mechanical shear
[0090] B. Concentrating Virus Subsequent to Gel Filtration
Example S-1
[0091] In the following example, the virus preparation was
concentrated in 20% glycerol subsequent to gel filtration.
[0092] Final Formulation: 16 mM NaPi, 80 mM NaCl, 1.6 .mu.M
MgCl.sub.2, 1.6% sucrose, 20% glycerol, pH 8 at 2-10.degree. C.
[0093] Results: Particles/FACS=72;
[0094] Light Scattering (A320/A260)=0.26
[0095] Conc.=1.66.times.10.sup.13 particles/ml
[0096] All publications, patents and patent applications cited
herein are incorporated in their entirety by reference to the same
extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
[0097] 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. forces of
concentrating the virus, and despite harsh conditions such as high
salt levels in a DEAE pool. Thus, methods of the present invention
allow for, inter alia, (1) ready preparation of clinical dosages at
any desired concentration (even when starting with material having
a lower concentration), (2) enhancement of processing (e.g., by
allowing significantly higher throughput during size exclusion
chromatography), and (3) stability of the salt-containing DEAE pool
for >10 days at 2-10.degree. C. (thus allowing for subsequent
steps of virus concentration and/or gel filtration to be performed
on separate days with substantial flexibility across a 10 day
period.
[0098] A. Concentrating Virus Subsequent to DEAE Chromatography
[0099] In the following three examples, stable concentrations of
adenovirus were prepared by concentrating DEAE Pools in 30%
glycerol (in accordance with the methods of the present invention).
The preparations were then subjected to further purification by
Superdex-200 gel filtration chromatography to obtain the final
formulation for testing.
9 Example D-1 Final 20 mM NaPi, 100 mM NaCl, 2 mM MgCl.sub.2, 2%
sucrose, Formulation: 10% glycerol, pH 8 at 2-10.degree. C.
Results: Particles/FACS = 24 Light Scattering (A320/A260) = 0.22
Conc. = 1.6 .times. 10.sup.13 particles/ml Example D-2 Final 14 mM
Tris base, 11 mM NaPi, 2 mM MgCl.sub.2, 2% sucrose, Formulation:
10% glycerol, pH 7.8 at 2-10.degree. C. Results: Particles/FACS =
17 Light Scattering (A320/A260) = 0.25 Conc. = 1.5 .times.
10.sup.13 particles/ml Example D-3 Final 20 mM NaPi, 100 mM NaCl, 2
mM MgCl.sub.2, 2% sucrose, Formulation: 10% glycerol, pH 8 at
2-10.degree. C.. Results: Particles/FACS = 24 Light Scattering
(A320/A260) = 0.25 Conc. = 1.3 .times. 10.sup.13 particles/ml.
* * * * *
References