U.S. patent application number 11/509486 was filed with the patent office on 2008-02-28 for method for the production and purification of adenoviral vectors.
This patent application is currently assigned to Introgen Therapeutics, Inc.. Invention is credited to Toohyon Cho, Shawn Gallagher, Capucine Thwin, Zheng Wu, Shuyuan Zhang.
Application Number | 20080050770 11/509486 |
Document ID | / |
Family ID | 22752406 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080050770 |
Kind Code |
A1 |
Zhang; Shuyuan ; et
al. |
February 28, 2008 |
Method for the production and purification of adenoviral
vectors
Abstract
The present invention addresses the need to improve the yields
of viral vectors when grown in cell culture systems. In particular,
it has been demonstrated that for adenovirus, the use of low-medium
perfusion rates in an attached cell culture system provides for
improved yields. In other embodiments, the inventors have shown
that there is improved Ad-p53 production with cells grown in
serum-free conditions, and in particular in serum-free suspension
culture. Also important to the increase of yields is the use of
detergent lysis. Combination of these aspects of the invention
permits purification of virus by a single chromatography step that
results in purified virus of the same quality as preparations from
double CsCl banding using an ultracentrifuge.
Inventors: |
Zhang; Shuyuan; (Sugarland,
TX) ; Thwin; Capucine; (Spring, TX) ; Wu;
Zheng; (Sugarland, TX) ; Cho; Toohyon;
(US) ; Gallagher; Shawn; (Missouri City,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE., SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
Introgen Therapeutics, Inc.
Austin
TX
|
Family ID: |
22752406 |
Appl. No.: |
11/509486 |
Filed: |
August 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09880609 |
Jun 12, 2001 |
7125706 |
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11509486 |
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09203078 |
Dec 1, 1998 |
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09880609 |
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Current U.S.
Class: |
435/41 |
Current CPC
Class: |
C12N 7/00 20130101; A61K
38/00 20130101; C12N 2710/10351 20130101; C12N 15/86 20130101; C12N
2750/14143 20130101; A61K 35/761 20130101; C07K 14/4746 20130101;
C12N 2710/10352 20130101; C12N 2710/10343 20130101; A61K 48/00
20130101 |
Class at
Publication: |
435/41 |
International
Class: |
C12P 1/00 20060101
C12P001/00 |
Claims
1-43. (canceled)
44. A method for producing a purified adeno-associated virus
composition comprising: a) growing host cells, wherein the host
cells encode adeno-associated virus cap and rep genes; b) providing
nutrients to the host cells by perfusion or through a fed-batch
process; c) infecting the host cells with an adenovirus and an
adeno-associated virus; d) lysing the host cells to provide a cell
lysate comprising adeno-associated virus; and e) purifying
adeno-associated virus from the lysate to provide a purified
adeno-associated virus composition.
45. The method of claim 44, further comprising subjecting the
lysate to a clarification step.
46. The method of claim 44, further comprising subjecting the
lysate to a concentration step.
47. The method of claim 44, further comprising subjecting the
lysate to a diafiltration step.
48. The method of claim 44, further comprising treating the lysate
with a nuclease.
49. The method of claim 48, wherein the nuclease is
BENZONASE.TM..
50. The method of claim 48, wherein the nuclease is
PULMOZYME.TM..
51. The method of claim 44, wherein purifying the adeno-associated
virus from the lysate comprises using at least one chromatography
step.
52. The method of claim 51, wherein purifying the adeno-associated
virus from the lysate comprises using at least two chromatography
steps.
53. The method of claim 52, wherein purifying the adeno-associated
virus from the lysate comprises using at least three chromatography
steps.
54. A method for producing a purified adeno-associated virus
composition comprising: a) growing host cells, wherein the host
cells encode adeno-associated virus cap and rep genes; b) providing
nutrients to the host cells by perfusion or through a fed-batch
process; c) infecting the host cells with an adeno-associated virus
and a plasmid encoding at least a portion of an adenoviral gene
that assists with adeno-associated virus replication; d) lysing the
host cells to provide a cell lysate comprising adeno-associated
virus; and e) purifying adeno-associated virus from the lysate to
provide a purified adeno-associated virus composition.
55. The method of claim 54, wherein the adenovirus gene is E1, E2,
E4, or E5.
56. The method of claim 54, further comprising subjecting the
lysate to a clarification step.
57. The method of claim 54, further comprising subjecting the
lysate to a concentration step.
58. The method of claim 54, further comprising subjecting the
lysate to a diafiltration step.
59. The method of claim 54, further comprising treating the lysate
with a nuclease.
60. The method of claim 59, wherein the nuclease is
BENZONASE.TM..
61. The method of claim 59, wherein the nuclease is
PULMOZYME.TM..
62. The method of claim 54, wherein purifying the adeno-associated
virus from the lysate comprises using at least one chromatography
step.
63. The method of claim 62, wherein purifying the adeno-associated
virus from the lysate comprises using at least two chromatography
steps.
64. The method of claim 63, wherein purifying the adeno-associated
virus from the lysate comprises using at least three chromatography
steps.
Description
[0001] The present application is a continuation-in-part of
co-pending U.S. patent application Ser. No. 08/975,519 filed Nov.
29, 1997 which is based on U.S. Provisional Patent Application Ser.
No. 60/031,329 filed Nov. 20, 1996. The entire text of the
above-referenced disclosures are specifically incorporated by
reference herein without disclaimer.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the fields of
cell culture and virus production. More particularly, it concerns
improved methods for the culturing of mammalian cells, infection of
those cells with adenovirus and the production of infectious
adenovirus particles therefrom.
[0004] 2. Description of Related Art
[0005] Adenoviral vectors, which carry transgenes that can be
transcribed and translated to express therapeutic proteins, are
currently being evaluated in the clinic for the treatment of a
variety of cancer indications, including lung and head and neck
cancers. As the clinical trials progress, the demand for clinical
grade adenoviral vectors is increasing dramatically. The projected
annual demand for a 300 patient clinical trial could reach
approximately 1.08.times.10.sup.16 viral particles.
[0006] Traditionally, adenoviruses are produced in commercially
available tissue culture flasks, "cellfactories," or RB. Virus
infected cells are harvested and subjected to multiple freeze-thaws
to release the viruses from the cells in the form of crude cell
lysate. The produced crude cell lysate (CCL) is then purified by
multiple CsCl gradient ultracentrifugation steps. The typically
reported virus yield from 100 single tray cellfactories is about
1.times.10.sup.14 viral particles. Clearly, it becomes unfeasible
to produce the required amount of virus using this traditional
process. New scaleable and validatable production and purification
processes have to be developed to meet the increasing demand.
[0007] The purification throughput of CsCl gradient
ultracentrifugation is so limited that it cannot meet the demand
for adenoviral vectors for gene therapy applications. Therefore, in
order to achieve large scale adenoviral vector production,
purification methods other than CsCl gradient ultracentrifugation
have to be developed. Reports on the chromatographic purification
of viruses are very limited, despite the wide application of
chromatography for the purification of recombinant proteins. Size
exclusion, ion exchange and affinity chromatography have been
evaluated for the purification of retroviruses, tick-borne
encephalitis virus, and plant viruses with varying degrees of
success (Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al.,
1978, Smith and Lee, 1978; O'Neil and Balkovic, 1993). Even less
research has been done on the chromatographic purification of
adenovirus. This lack of research activity may be partially
attributable to the existence of the effective, albeit
non-scalable, CsCl gradient ultracentrifugation purification method
for adenoviruses.
[0008] Recently, Huyghe et al. (1996) reported adenoviral vector
purification using ion exchange chromatography in conjunction with
metal chelate affinity chromatography. Virus purity similar to that
from CsCl gradient ultracentrifugation was reported. Unfortunately,
only 23% of virus was recovered after the double column
purification process. Process factors that contribute to this low
virus recovery are the freeze/thaw step utilized by the authors to
lyse cells in order to release the virus from the cells and the two
column purification procedure.
[0009] Clearly, there is a demand for an effective and scaleable
method of adenoviral vector production that will result in a high
yield of product to meet the ever increasing demand for such
products. Recently Blanche et al in WO 98/00524, based on U.S. Ser.
No. 60/026,667, describe adenoviral production methods that are
useful as descriptive art. PCT publication No. WO 98/00524 and U.S.
Ser. No. 60/026,667 are specifically herein incorporated by
reference for their description of techniques for production and
purification of recombinant adenovirus.
SUMMARY OF THE INVENTION
[0010] The present invention describes a new large scale process
for the production and purification of adenovirus. This new
production process offers not only scalability and validatability
but also virus purity comparable to that achieved using CsCl
gradient ultracentrifugation.
[0011] The present invention relates to a process for preparing
large scale quantities of adenovirus. Indeed, it is believed that
very large quantities of adenovirus particles can be produced using
the processes of the present invention, quantities of up to about
1.times.10.sup.18 particles, and preferably at least about
5.times.10.sup.14 particles. This is highly desirable, as there are
currently no techniques available to produce the very large,
commercial quantities of adenovirus particles required for clinical
applications at the high level of purity needed.
[0012] In one embodiment, the process generally involves preparing
a culture of producer cells by seeding producer cells into a
culture medium, infecting cells in the culture after they have
reached a mid-log phase growth with a selected adenovirus (e.g., a
recombinant adenovirus), and harvesting the adenovirus particles
from the cell culture. This is because it has surprisingly been
discovered by the inventors that maximal virus production is
achieved in the producer cells when they are infected in the later
part of log phase growth and prior to stationary growth.
Preferably, the adenovirus particles so obtained are then subjected
to purification techniques either known in the art or set forth
herein.
[0013] In certain preferred embodiments of the present invention,
therefore, the producer cells are infected with adenovirus at
between about mid-log phase and stationary phase of growth. The log
phase of the growth curve is where the cells reach their maximum
rate of cell division (i.e. growth). The term mid-log phase of
growth refers to the transition mid-point of a logarithmic growth
curve. Stationary phase growth refers to the time on a growth curve
(i.e. a plateau) in which cell growth and cell death have come to
equilibrium.
[0014] In even more preferred embodiments, the producer cells are
infected with the adenovirus during or after late-log phase of
growth and before stationary phase. Late-log phase is defined as
cell growth approaching the end of logarithmic growth, and before
reaching the stationary phase of growth. Late-log phase can
typically be identified on a growth curve as a secondary or
tertiary point of inflection that occurs as the log-growth phase
slows, approaching stationary growth.
[0015] In a preferred embodiment of the present invention, the
producer cells are seeded into the cell culture medium using an
essentially homogeneous pool of cells. The inventors have
surprisingly discovered that the use of a homogeneous pool of cells
for seeding can provide much improved confluency and cell density
as well as better maturation of the virus, which in turn provides
for larger production quantities and ultimate purity of the virus
recovered. Indeed, seeding through the use of separate rather than
homogeneous cell populations, for example from individual cell
culture devices used in the cell expansion phase, can result in
uneven cell density, and therefore uneven confluency levels at the
time of infection. It is believed that the use of a homogeneous
cell pool for seeding overcomes these problems.
[0016] In another preferred embodiment of the present invention,
the culture medium is at least partially perfused during a portion
of time during cell growth of the producer cells or following
infection. Perfusion is used in order to maintain desired levels of
certain metabolites and to remove and thereby reduce impurities in
the culture medium. Perfusion rates can be measured in various
manners, such as in terms of replacement volumes/unit time or in
terms of levels of certain metabolites that are desired to be
maintained during times of perfusion. Of course, it is typically
the case that perfusion is not carried out at all times during
culturing, etc., and is generally carried out only from time to
time during culturing as desired. For example, perfusion is not
typically initiated until after certain media components such as
glucose begin to become exhausted and need to be replaced.
[0017] The inventors have discovered that low perfusion rates are
particularly preferred, in that low perfusion rates tend to improve
one's ability to obtain highly purified virus particles. The
inventors prefer to define perfusion rate in terms of the glucose
level that is achieved or maintained by means of the perfusion. For
example, in the present invention the glucose concentration in the
medium is preferably maintained at a concentration of between about
0.5 g/L and about 3.0 g/L. In a more preferred embodiment, the
glucose concentration is maintained at between about 0.70 g/L and
2.0 g/L. In a still more preferred embodiment, the glucose
concentration is maintained at between about 1.0 g/L and 1.5
g/L.
[0018] Also in certain preferred embodiments, the inventors prefer
to recirculate the cell culture media while carrying out processes
in accordance with the present invention, and even more preferably,
the recirculation is carried out continuously. Recirculation is
desirable in that it affords a more even distribution of nutrients
throughout the cell growth chamber.
[0019] In certain other embodiments, the cells are seeded into the
culture medium and allowed to attach to a culture surface for
between about 3 hours and about 24 hours prior to initiation of
medium recirculation. Attachment of cells to a cell surface
generally allows for a more consistent and uniform cell growth and
higher virus production rate, which in turn allows for the
production of higher quality virus. It has been found by the
inventors that recirculation can sometimes impede consistent and
uniform cell attachment, and that ceasing recirculation during cell
attachment phases can provide significant advantages.
[0020] With respect to seeding, in a preferred embodiment of the
present invention, the cell culture medium is seeded with between
about 0.5.times.10.sup.4 and about 3.times.10.sup.4 cells/cm.sup.2,
and more preferably with from about 1-2.times.10.sup.4
cells/cm.sup.2. The reason for this is that it has been found that
in order to achieve maximal cell expansion and growth, it is most
preferable to inoculate the selected growth chamber with a lower
number of cells that one might typically use in other cell growth
situations. The inventors have found that higher numbers of cells
used in the cell inoculation step results in a cell density that is
too high and can result in an over-confluence of cells at the time
of viral infection, thus lowering yields. It is well within one of
skill in the art to determine that in other types of cell culturing
systems, similar optimization of the seeding density for a
particular system could easily be determined. Nevertheless, in a
particularly preferred embodiment, the cell culture medium is
seeded with between about 7.5.times.10.sup.3 and about
2.0.times.10.sup.4 cells/cm.sup.2. In an even more preferred
embodiment, the cell culture medium is seeded with between about
9.times.10.sup.3 and 1.5.times.10.sup.4 cells/cm.sup.2.
[0021] In another preferred embodiment of the present invention,
the harvested adenovirus is purified and placed in a
pharmaceutically acceptable composition. A pharmaceutically
acceptable composition is defined as one that meets the minimal
safety required set forth by the FDA or other similar
pharmaceutical governing body, and can thus be administered safely
to a patient. The present invention provides processes for the
purification of the adenovirus. For example, the adenovirus is
purified by steps that include chromatographic separation. While
more than one chromatography step can be used in accordance with
the present invention to purify the adenovirus, this will often
result in significant losses in terms of yield. Thus, the inventors
have discovered that surprising levels of purity can be achieved
where only a single chromatography step is carried out,
particularly where that chromatography step is carried out using
ion-exchange chromatography. Ion-exchange chromatography is an
excellent choice for purification of adenovirus particles due to
the presence of a net negative charge on the surface of
adenoviruses at physiological pH, permitting high purity isolation
of adenovirus particles.
[0022] In particular embodiments of the present invention, the
recombinant adenovirus is a replication-deficient adenovirus
encoding a therapeutic gene operably linked to a promoter. A
replication deficient adenovirus carrying a therapeutic gene linked
to a promoter allows the controlled expression of the therapeutic
gene by activating the promoter. The precise choice of a promoter
further allows tissue specific regulation and expression of the
therapeutic gene. In particular embodiments, the promoter is an
SV40 IE, RSV LTR, .beta.-actin, CMV-IE, adenovirus major late,
polyoma F9-1, or tyrosinase promoter.
[0023] In other embodiments the replication deficient adenovirus is
lacking at least a portion of the E1 region of the adenoviral
genome. Replication deficient adenoviruses lacking a portion of the
E1 region are desired to reduce toxicity and immunologic reaction
to host cells. In another embodiment of the present invention, the
producer cells complement the growth of replication deficient
adenoviruses. This is an important feature of producer cells
required to maintain high viral particle number of the replication
deficient adenovirus. In certain such embodiments, the producer
cells are 293, PER.C6, 911 or IT293SF cells. In a preferred
embodiment, the producer cells are 293 cells. This allows
[0024] In a preferred embodiment of the present invention it is
contemplated that the recombinant adenovirus encodes a therapeutic
recombinant gene. For example, the therapeutic gene may encode
antisense ras, antisense myc, antisense raf antisense erb,
antisense src, antisense fms, antisense jun, antisense trk,
antisense ret, antisense gsp, antisense hst, antisense bcl
antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC,
CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1,
VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, mda-7,
thymidine kinase or p53. In an even more preferred embodiment, the
therapeutic gene is p53. One of the most frequent abnormalities
resulting in human cancer are mutations in p53, thus the ability to
replace a deficient p53 gene using the present invention is highly
desirable.
[0025] In another particular embodiment of the present invention,
the adenovirus is harvested by steps that include lysing the
producer cells by means other than freeze-thaw. The reason for this
is that the freeze-thaw method is somewhat cumbersome and not
particularly suited to production of commercial quantities. In
preferred embodiments the producer cells are lysed by means of
detergent lysis or autolysis. The harvesting of the adenovirus by
detergent lysis and autolysis results in a much higher virus
recovery than the freeze-thaw process and is therefore an
improvement in the large scale production of adenoviruses.
[0026] In a particular embodiment of the present invention the
purified recombinant adenovirus has one or more of the following
properties. For example, the property may be a virus titer of
between about 1.times.10.sup.9 and about 1.times.10.sup.13 pfu/ml,
a virus particle concentration between about 1.times.10.sup.10 and
about 2.times.10.sup.13 particles/ml, a particle:pfu ratio between
about 10 and about 60, less than 50 ng BSA per 1.times.10.sup.12
viral particles, between about 50 pg and 1 ng of contaminating
human DNA per 1.times.10.sup.12 viral particles or a single HPLC
elution peak consisting essentially of 97 to 99% of the area under
the peak. These criteria select for a highly purified
adenovirus.
[0027] To further impose limits on the purification process of the
adenovirus, between about 5.times.10.sup.14 and 1.times.10.sup.18
viral particles are desired. In addition, one or more of the
following properties further improve the selection for high purity
adenovirus particles. For example the property may be a virus titer
of between about 1.times.10.sup.9 and about 1.times.10.sup.13
pfu/ml, more preferably 1.times.10.sup.11 and about
1.times.10.sup.13 pfu/ml, and most preferably 1.times.10.sup.12 and
about 1.times.10.sup.13 pfu/ml. Further, a virus particle
concentration between about 1.times.10.sup.10 and about
2.times.10.sup.13 particles/ml, more preferably 1.times.10.sup.11
and about 2.times.10.sup.13 particles/ml, and most preferably
1.times.10.sup.12 and about 1.times.10.sup.13 particles/ml.
[0028] Additionally, a particle:pfu ratio between about 10 and
about 60, more preferably a particle:pfu ratio between about 10 and
about 50, even more preferable a particle:pfu ratio between about
10 and about 40, and most preferably a particle:pfu ratio between
about 20 and about 40.
[0029] To limit the BSA concentration, it is preferable to have
less than 50 ng BSA per 1.times.10.sup.12 viral particles, for
example, between about 1 ng to 50 ng BSA per 1.times.10.sup.12
viral particles, and more preferably between about 5 ng and 40 ng
of BSA per 1.times.10.sup.12 viral particles.
[0030] Low concentrations of DNA contamination are also desired.
Thus, between about 50 pg and 1 ng of contaminating human DNA per
1.times.10.sup.12 viral particles is acceptable, even more
preferable is between about 50 pg and 500 pg of contaminating human
DNA per 1.times.10.sup.12 viral particles, and most preferable is
between about 100 pg and 500 pg of contaminating human DNA per
1.times.10.sup.12 viral particles. Finally, an adenovirus that
elutes as a single HPLC peak is desired, more preferably is an
adenovirus that elutes as an HPLC peak that contains between about
97 and 99% of the total area under the peak.
[0031] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0033] FIG. 1A and FIG. 1B. HPLC profiles of the viral solutions
from production runs using medium perfusion rates characterized as
"high" (FIG. 1A) and "low" (FIG. 1B).
[0034] FIG. 2. The HPLC profile of crude cell lysate (CCL) from
CellCube.TM. (solid line A.sub.260; dotted line A.sub.280).
[0035] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E. The HPLC
profiles of lysis solutions from CellCube.TM. using different
detergents. FIG. 3A Thesit.RTM.. FIG. 3B Triton.RTM.X-100. FIG. 3C.
NP-40.RTM.. FIG. 3D. Brij.RTM.80. FIG. 3E. Tween.RTM.20. Detergent
concentration: 1% (w/v) lysis temperature: room temperature. (solid
line A.sub.260; dotted line A.sub.280).
[0036] FIG. 4A and FIG. 4B. The HPLC profiles of virus solution
before (FIG. 4A) and after (FIG. 4B) Benzonase treatment. (solid
line A.sub.260; dotted line A.sub.280).
[0037] FIG. 5. The HPLC profile of virus solution after Benzonase
treatment in the presence of 1 M NaCl. (solid line A.sub.260;
dotted line A.sub.280).
[0038] FIG. 6. Purification of AdCMVp53 virus under buffer A
condition of 20 mM Tris+1 mM MgCl.sub.2+0.2M NaCl, pH=7.5.
[0039] FIG. 7. Purification of AdCMVp53 virus under buffer A
condition of 20 mM Tris+1 mM MgCl.sub.2+0.2M NaCl, pH=9.0.
[0040] FIG. 8A, FIG. 8B, and FIG. 8C. HPLC analysis of fractions
obtained during purification FIG. 8A fraction 3. FIG. 8B fraction
4, FIG. 8C fraction 8. (solid line A.sub.260; dotted line
A.sub.280).
[0041] FIG. 9. Purification of AdCMVp53 virus under buffer A
condition of 20 mM Tris+1 mM MgCl.sub.2+0.3M NaCl, pH=9.
[0042] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E. HPLC
analysis of crude virus fractions obtained during purification and
CsCl gradient purified virus. FIG. 10A Crude virus solution. FIG.
10B Flow through. FIG. 10C. Peak number 1. FIG. 10D. Peak number 2.
FIG. 10E. CsCl purified virus. (solid line A.sub.260; dotted line
A.sub.280).
[0043] FIG. 11. HPLC purification profile from a 5 cm id
column.
[0044] FIG. 12. The major adenovirus structure proteins detected on
SDS-PAGE.
[0045] FIG. 13. The BSA concentration in the purified virus as
detected level of the western blot assay.
[0046] FIG. 14. The chromatogram for the crude cell lysate material
generated from the CellCube.TM..
[0047] FIG. 15. The elution profile of treated virus solution
purified using the method of the present invention using Toyopearl
SuperQ resin.
[0048] FIG. 16A and FIG. 16B. HPLC analysis of virus fraction from
purification protocol. FIG. 16A HPLC profiles of virus fraction
from first purification step. FIG. 16B HPLC profiles of virus
fraction from second purification. (solid line A.sub.260; dotted
line A.sub.280).
[0049] FIG. 17. Purification of 1% Tween.RTM. harvest virus
solution under low medium perfusion rate.
[0050] FIG. 18. HPLC analysis of the virus fraction produced under
low medium perfusion rate.
[0051] FIG. 19A, FIG. 19B and FIG. 19C. Analysis of column purified
virus. FIG. 19A SDS-PAGE analysis. FIG. 19B Western blot for BSA.
FIG. 19C nucleic acid slot blot to determine the contaminating
nucleic acid concentration.
[0052] FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E and FIG.
20F. Capacity study of the Toyopearl SuperQ 650M resin. FIG. 20A
Flow through from loading ratio of 1:1. FIG. 20B. Purified virus
from loading ratio of 1:1. FIG. 20C Flow through of loading ratio
of 2:1. FIG. 20D. Purified virus from the loading ratio of 2:1.
FIG. 20E Flow through from loading ratio of 3:1. FIG. 20F. Purified
virus from the loading ratio of 3:1. (solid line A.sub.260; dotted
line A280).
[0053] FIG. 21. Isopycnic CsCl ultracentrifugation column purified
virus.
[0054] FIG. 22. The HPLC profiles of intact viruses present in the
column purified virus. A. Intact virus B. Defective virus. (solid
line A.sub.260; dotted line A.sub.280).
[0055] FIG. 23. A production and purification flow chart for
AdCMVp53
[0056] FIG. 24. Kinetics of virus release in the supernatant in a
4.times.100 CellCube.TM..
[0057] FIG. 25. Chromatogram using Source 15Q resin for
purification.
[0058] FIG. 26. HPLC profile of purified Ad5CMV-p53 product from
Source 15Q resin.
[0059] FIG. 27. Comparison of bioactivity of original process vs.
optimized process to produce Ad5CMV-p53 product.
[0060] FIG. 28. Production and Purification flow chart for
Ad5CMV-p53 optimized process.
[0061] FIG. 29. Lyophilization cycle for adenovirus
formulations.
[0062] FIG. 30A and FIG. 30B. Storage stability data using
secondary drying at 0.degree. C. without N.sub.2 blanketing. FIG.
30A, secondary drying at 10.degree. C. without N.sub.2 blanketing
for formulation set 10. FIG. 30B, secondary drying at 10.degree. C.
without N.sub.2 blanketing for formulation set 11.
[0063] FIG. 31A and FIG. 31B. Storage stability data using
secondary drying at 30.degree. C. without N.sub.2 blanketing. FIG.
31A, secondary drying at 30.degree. C. without N.sub.2 blanketing
for formulation set 10. FIG. 31B, secondary drying at 30.degree. C.
without N.sub.2 blanketing for formulation set 11.
[0064] FIG. 32A and FIG. 32B. Storage stability data using
secondary drying at 30.degree. C. with N.sub.2 blanketing. FIG.
32A, secondary drying at 30.degree. C. with N.sub.2 blanketing for
formulation set 10. FIG. 32B, secondary drying at 30.degree. C.
with N.sub.2 blanketing for formulation set 11.
[0065] FIG. 33. Stability data for liquid formulation set #1.
[0066] FIG. 34. Stability data for liquid formulation set #2.
[0067] FIG. 35. Stability data for liquid formulation set #3.
[0068] FIG. 36. Stability data for liquid formulation set #4.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0069] It has been shown that adenoviral vectors can successfully
be used in eukaryotic gene expression and vaccine development.
Recently, animal studies have demonstrated that recombinant
adenovirus could be used for gene therapy. Successful studies in
administering recombinant adenovirus to different tissues have
proven the effectiveness of adenoviral vectors in therapy. This
success has led to the use of such vectors in human clinical
trials. There now is an increased demand for the production of
adenoviral vectors to be used in various therapies. The techniques
currently available are insufficient to meet such a demand. The
present invention provides methods for the production of large
amounts of adenovirus for use in such therapies.
[0070] The present invention involves a process that has been
developed for the production and purification of a replication
deficient recombinant adenovirus. The production process is based
on the use of a cell culture bioreactor for cell growth and virus
production. After viral infection of the producer cells, virus can
be harvested by any number of methods, including virus autolysis or
chemical lysis. The harvested crude virus solution can then be
purified using a single ion exchange chromatography run, after
concentration/diafiltration and nuclease treatment to reduce the
contaminating nucleic acid concentration in the crude virus
solution. The column purified virus has equivalent purity relative
to that of virus purified by cesium banding. The total process
recovery of the virus product is 70%.+-.10%. This is a significant
improvement over the results reported by Huyghe et al. (1996).
Compared to double CsCl gradient ultracentrifugation, column
purification has the advantage of being more consistent, scaleable,
validatable, faster and less expensive. This new process represents
a significant improvement in the technology for manufacturing of
adenoviral vectors for gene therapy.
[0071] Therefore, the present invention is designed to take
advantage of these improvements in large scale culturing systems
and purification for the purpose of producing and purifying
adenoviral vectors. The various components for such a system, and
methods of producing adenovirus therewith, are set forth in detail
below.
1. HOST CELLS
[0072] A) Cells
[0073] In a preferred embodiment, the generation and propagation of
the adenoviral vectors depend on a unique helper cell line,
designated 293, which was transformed from human embryonic kidney
cells by Adenovirus serotype 5 (Ad5) DNA fragments and
constitutively expresses E1 proteins (Graham et al., 1977). Since
the E3 region is dispensable from the Ad genome (Jones and Shenk,
1978), the current Ad vectors, with the help of 293 cells, carry
foreign DNA in either the E1, the E3 or both regions (Graham and
Prevec, 1991; Bett et al., 1994).
[0074] A first aspect of the present invention is the recombinant
cell lines which express part of the adenoviral genome. These cells
lines are capable of supporting replication of adenovirus
recombinant vectors and helper viruses having defects in certain
adenoviral genes, i.e., are "permissive" for growth of these
viruses and vectors. The recombinant cell also is referred to as a
helper cell because of the ability to complement defects in, and
support replication of, replication-incompetent adenoviral vectors.
The prototype for an adenoviral helper cell is the 293 cell line,
which contains the adenoviral E1 region. 293 cells support the
replication of adenoviral vectors lacking E1 functions by providing
in trans the E1-active elements necessary for replication. Other
cell lines which also support the growth of adenoviruses lacking E1
function include PER.C6 (IntroGene, NL), 911 (IntroGene, NL), and
IT293SF.
[0075] Helper cells according to the present invention are derived
from a mammalian cell and, preferably, from a primate cell such as
human embryonic kidney cell. Although various primate cells are
preferred and human or even human embryonic kidney cells are most
preferred, any type of cell that is capable of supporting
replication of the virus would be acceptable in the practice of the
invention. Other cell types might include, but are not limited to
Vero cells, HeLa cells or any eukaryotic cells for which tissue
culture techniques are established as long as the cells are
adenovirus permissive. The term "adenovirus permissive" means that
the adenovirus or adenoviral vector is able to complete the entire
intracellular virus life cycle within the cellular environment.
[0076] The helper cell may be derived from an existing cell line,
e.g.; from a 293 cell line, or developed de novo. Such helper cells
express the adenoviral genes necessary to complement in trans
deletions in an adenoviral genome or which support replication of
an otherwise defective adenoviral vector, such as the E1, E2, E4,
E5 and late functions. A particular portion of the adenovirus
genome, the E1 region, has already been used to generate
complementing cell lines. Whether integrated or episomal, portions
of the adenovirus genome lacking a viral origin of replication,
when introduced into a cell line, will not replicate even when the
cell is superinfected with wild-type adenovirus. In addition,
because the transcription of the major late unit is after viral DNA
replication, the late functions of adenovirus cannot be expressed
sufficiently from a cell line. Thus, the E2 regions, which overlap
with late functions (L1-5), will be provided by helper viruses and
not by the cell line. Typically, a cell line according to the
present invention will express E1 and/or E4.
[0077] As used herein, the term "recombinant" cell is intended to
refer to a cell into which a gene, such as a gene from the
adenoviral genome or from another cell, has been introduced.
Therefore, recombinant cells are distinguishable from
naturally-occurring cells which do not contain a
recombinantly-introduced gene. Recombinant cells are thus cells
having a gene or genes introduced through "the hand of man."
[0078] Replication is determined by contacting a layer of
uninfected cells, or cells infected with one or more helper
viruses, with virus particles, followed by incubation of the cells.
The formation of viral plaques, or cell free areas in the cell
layer, is the result of cell lysis caused by the expression of
certain viral products. Cell lysis is indicative of viral
replication.
[0079] Examples of other useful mammalian cell lines that may be
used with a replication competent virus or converted into
complementing host cells for use with replication deficient virus
are Vero and HeLa cells and cell lines of Chinese hamster ovary,
W138, BHK, COS-7, HepG2, 3T3, RUN, MDCK and A549 cells.
[0080] B) Growth in Selection Media
[0081] In certain embodiments, it may be useful to employ selection
systems that preclude growth of undesirable cells. This may be
accomplished by virtue of permanently transforming a cell line with
a selectable marker or by transducing or infecting a cell line with
a viral vector that encodes a selectable marker. In either
situation, culture of the transformed/transduced cell with an
appropriate drug or selective compound will result in the
enhancement, in the cell population, of those cells carrying the
marker.
[0082] Examples of markers include, but are not limited to, HSV
thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase
and adenine phosphoribosyltransferase genes, in tk-, hgprt- or
aprt-cells, respectively. Also, anti-metabolite resistance can be
used as the basis of selection for dhfr, that confers resistance to
methotrexate; gpt, that confers resistance to mycophenolic acid;
neo, that confers resistance to the aminoglycoside G418; and hygro,
that confers resistance to hygromycin.
[0083] C. Growth in Serum Weaning
[0084] Serum weaning adaptation of anchorage-dependent cells into
serum-free suspension cultures have been used for the production of
recombinant proteins (Berg, 1993) and viral vaccines (Perrin,
1995). There have been few reports on the adaptation of 293A cells
into serum-free suspension cultures until recently. Gilbert
reported the adaptation of 293A cells into serum-free suspension
cultures for adenovirus and recombinant protein production
(Gilbert, 1996). A similar adaptation method had been used for the
adaptation of A549 cells into serum-free suspension culture for
adenovirus production (Morris et al., 1996). Cell-specific virus
yields in the adapted suspension cells, however, are about
5-10-fold lower than those achieved in the parental attached
cells.
[0085] Using the similar serum weaning procedure, the inventors
have successfully adapted the 293A cells into serum-free suspension
culture (293SF cells). In this procedure, the 293 cells were
adapted to a commercially available 293 media by sequentially
lowering down the FBS concentration in T-flasks. Briefly, the
initial serum concentration in the media was approximately 10% FBS
DMEM media in T-75 flask and the cells were adapted to serum-free
IS 293 media in T-flasks by lowering down the FBS concentration in
the media sequentially. After 6 passages in T-75 flasks the FBS %
was estimated to be about 0.019% in the 293 cells. The cells were
subcultured two more times in the T flasks before they were
transferred to spinner flasks. The results described herein below
show that cells grow satisfactorily in the serum-free medium (IS293
medium, Irvine Scientific, Santa Ana, Calif.). Average doubling
time of the cells were 18-24 h achieving stationary cell
concentrations in the order of 4-10.times.10.sup.6 cells/ml without
medium exchange.
[0086] D. Adaptation of Cells for Suspension Culture
[0087] Two methodologies have been used to adapt 293 cells into
suspension cultures. Graham adapted 293A cells into suspension
culture (293N3S cells) by 3 serial passages in nude mice (Graham,
1987). The suspension 293N3S cells were found to be capable of
supporting El.sup.- adenoviral vectors. However, Garnier et al.
(1994) observed that the 293N3S cells had a relatively long initial
lag phase in suspension, a low growth rate, and a strong tendency
to clump.
[0088] The second method that has been used is a gradual adaptation
of 293A cells into suspension growth (Cold Spring Harbor
Laboratories, 293S cells). Garnier et al. (1994) reported the use
of 293S cells for production of recombinant proteins from
adenoviral vectors. The authors found that 293S cells were much
less clumpy in calcium-free media and a fresh medium exchange at
the time of virus infection could significantly increase the
protein production. It was found that glucose was the limiting
factor in culture without medium exchange.
[0089] In the present invention, the 293 cells adapted for growth
in serum-free conditions were adapted into a suspension culture.
The cells were transferred in a serum-free 250 mL spinner
suspension culture (100 mL working volume) for the suspension
culture at an initial cell density of between about 1.18 E+5 vc/mL
and about 5.22 E+5 viable cells/mL. The media may be supplemented
with heparin to prevent aggregation of cells. This cell culture
systems allows for some increase of cell density whilst cell
viability is maintained. Once these cells are growing in culture,
the cells are subcultured in the spinner flasks approximately 7
more passages. It may be noted that the doubling time of the cells
is progressively reduced until at the end of the successive
passages the doubling time is about 1.3 day, i.e. comparable to 1.2
day of the cells in 10% FBS media in the attached cell culture. In
the serum-free IS 293 media supplemented with heparin almost all
the cells existed as individual cells not forming aggregates of
cells in the suspension culture.
2. CELL CULTURE SYSTEMS
[0090] In any cell culture system, there is a characteristic growth
pattern following inoculation that includes a lag phase, an
accelerated growth phase, an exponential or "log" phase, a negative
growth acceleration phase and a plateau or stationary phase. The
log and plateau phases give vital information about the cell line,
the population doubling time during log growth, the growth rate,
and the maximum cell density achieved in plateau. In the log phase,
as growth continues, the cells reach their maximum rate of cell
division. Numbers of cells increase in log relationship to time.
During this period of most active multiplication, the logarithms of
the numbers of cells counted at short intervals, plotted against
time, produce a straight line. By making one count at a specified
time and a second count after an interval during the log phase of
growth and knowing the number of elapsed time units, one can
calculate the total number of cell divisions or doublings, and both
the growth rate and generation time. Within a few hours or days
after the commencement of the log phase, the rate of cell division
begins to decline and some of the cells begin to die. This is
reflected on the growth curve by a gradual flattening out of the
line. Eventually the rate of cells dying is essentially equal to
the rate of cells dividing, and the total viable population remains
the same for a period of time. This is known as the stationary or
plateau phase and is represented on the growth curve as a
flattening out of the line where the slope approaches zero.
[0091] Measurement of the population doubling time can be used to
quantify the response of the cells to different inhibitory or
stimulatory culture conditions such as variations in nutrient
concentration, hormonal effects, or toxic drugs. It is also a good
monitor of the culture during serial passage and enables the
calculation of cell yields and the dilution factor required at
subculture.
[0092] The population doubling time is an average figure and
describes the net result of a wide range of cell division rates,
including zero, within the culture. The doubling time will also
differ with varying cell types, culture conditions, and culture
vessels. Single time points are unsatisfactory for monitoring
growth when the shape of the cell growth curve is not known. Thus
it is important to determine the growth curve for each cell type
being used in the conditions that are being used for the cell
culture. Typical growth curves are sigmoidal in shape, with the
first part of the curve representing the lag phase, the center part
of the curve representing the log phase, and the last part of the
curve representing the plateau phase. The log phase is when the
cells are growing at the highest rate, and as the cells reach their
saturation density, their growth will slow and the culture will
enter the plateau phase. A detailed description of cell culture
techniques and theory can be found in Freshney, 1992 and Freshney,
1987.
[0093] An important aspect of the present invention is infection of
the producer cells with recombinant adenovirus at an appropriate
time to achieve maximal virus production. The inventors have found
that maximal virus production is obtained when the producer cells
are infected between about when the cells reach the first
inflection point on the log phase of the cell growth curve, i.e.
mid-log phase, and before the 2.sup.nd inflection point on the
plateau phase of the cell growth curve, i.e. mid-plateau phase.
This range can be determined easily for any cell type and any
culture conditions with any cell culturing apparatus. The
inflection points on a cell growth curve are when the shape of the
line changes from a convex to a concave shape, or from a concave to
a convex shape.
[0094] For most growth curves plotted on semi-log scales, the log
phase of growth can be approximately represented by a linear
increase in the slope of the line over time. That is, at any short
interval between two points on the line of the logarithmic phase of
the curve, the log of cell number is increasing in a linear fashion
relative to time. Thus mid log phase can be approximately defined
as the point or interval within the log phase in which the cells
are dividing at their maximal rate, and the increase in logs of
cell number is linear with respect to time. Late log phase can be
defined as approximately the point or interval of time in which the
rate of cell division has slowed, and the log of number of cells is
no longer increasing in a linear fashion with respect to time. When
looking at a growth curve, this area would be represented by
gradual falling or flattening of the slope of the line. At early
stationary phase, the rate of cell growth is decreasing and getting
nearer the rate of cell death, and thus the slope of the line on
the growth curve is even less than that at late log phase. At
mid-stationary phase, the rate of cell growth is approximately
equal to the rate of cell division and thus the line on the growth
curve is relatively flat and has a slope approaching zero. It will
be understood that the skilled artisan can formulate growth curves
for any such cell line and identify the aforementioned regions on
the curve.
[0095] The ability to produce infectious viral vectors is
increasingly important to the pharmaceutical industry, especially
in the context of gene therapy. Over the last decade, advances in
biotechnology have led to the production of a number of important
viral vectors that have potential uses as therapies, vaccines and
protein production machines. The use of viral vectors in mammalian
cultures has advantages over proteins produced in bacterial or
other lower lifeform hosts in their ability to post-translationally
process complex protein structures such as disulfide-dependent
folding and glycosylation.
[0096] Development of cell culture for production of virus vectors
has been greatly aided by the development in molecular biology of
techniques for design and construction of vector systems highly
efficient in mammalian cell cultures, a battery of useful selection
markers, gene amplification schemes and a more comprehensive
understanding of the biochemical and cellular mechanisms involved
in procuring the final biologically-active molecule from the
introduced vector.
[0097] Frequently, factors which affect the downstream (in this
case, beyond the cell lysis) side of manufacturing scale-up were
not considered before selecting the cell line as the host for the
expression system. Also, development of bioreactor systems capable
of sustaining very high density cultures for prolonged periods of
time have not lived up to the increasing demand for increased
production at lower costs.
[0098] The present invention will take advantage of the recently
available bioreactor technology. Growing cells according to the
present invention in a bioreactor allows for large scale production
of fully biologically-active cells capable of being infected by the
adenoviral vectors of the present invention. By operating the
system at a low perfusion rate and applying a different scheme for
purification of the infecting particles, the invention provides a
purification strategy that is easily scaleable to produce large
quantities of highly purified product.
[0099] Bioreactors have been widely used for the production of
biological products from both suspension and anchorage dependent
animal cell cultures. The most widely used producer cells for
adenoviral vector production are anchorage dependent human
embryonic kidney cells (293 cells). Bioreactors to be developed for
adenoviral vector production should have the characteristic of high
volume-specific culture surface area in order to achieve high
producer cell density and high virus yield. Microcarrier cell
culture in stirred tank bioreactor provides very high
volume-specific culture surface area and has been used for the
production of viral vaccines (Griffiths, 1986). Furthermore,
stirred tank bioreactors have industrially been proven to be
scaleable. The multiplate Cellcube.TM. cell culture system
manufactured by Corning-Costar also offers a very high
volume-specific culture surface area. Cells grow on both sides of
the culture plates hermetically sealed together in the shape of a
compact cube. Unlike stirred tank bioreactors, the Cellcube.TM.
culture unit is disposable. This is very desirable at the early
stage production of clinical product because of the reduced capital
expenditure, quality control and quality assurance costs associated
with disposable systems. In consideration of the advantages offered
by the different systems, both the stirred tank bioreactor and the
Cellcube.TM. system were evaluated for the production of
adenovirus.
[0100] Table 1 list several exemplary techniques for cell culturing
and viral particle production. Currently, there are no methods
employed that result in both high purity and a high number of viral
particles. Thus, the following methods are considered in
combination with the large scale process for the production and
purification of adenovirus described in the present invention.
TABLE-US-00001 TABLE 1 Virus Particles 5 .times. 10.sup.14 1
.times. 10.sup.15 1 .times. 10.sup.16 1 .times. 10.sup.17 1 .times.
10.sup.18 Exemplary Cellcube .TM. Cellcube .TM. Packed Bed
1000-5000 L 10,000-20,000 L Techniques for per 10 L Stirred Tank
Stirred Tank Viral Particle Airlift Reactor -- Production Total
Cell 5 .times. 10.sup.10 1 .times. 10.sup.11 1 .times. 10.sup.12 1
.times. 10.sup.13 1 .times. 10.sup.14 Number
[0101] A) Anchorage-Dependent Versus Non-Anchorage-Dependent
Cultures.
[0102] Animal and human cells can be propagated in vitro in two
modes: as non-anchorage dependent cells growing freely in
suspension throughout the bulk of the culture; or as
anchorage-dependent cells requiring attachment to a solid substrate
for their propagation (i.e., a monolayer type of cell growth).
[0103] Non-anchorage dependent or suspension cultures from
continuous established cell lines are the most widely used means of
large scale production of cells and cell products. Large scale
suspension culture based on microbial (bacterial and yeast)
fermentation technology has clear advantages for the manufacturing
of mammalian cell products. The processes are relatively simple to
operate and straightforward to scale up. Homogeneous conditions can
be provided in the reactor which allows for precise monitoring and
control of temperature, dissolved oxygen, and pH, and ensures that
representative samples of the culture can be taken.
[0104] However, suspension cultured cells cannot always be used in
the production of biologicals. Suspension cultures are still
considered to have tumorigenic potential and thus their use as
substrates for production put limits on the use of the resulting
products in human and veterinary applications (Petricciani, 1985;
Larsson, 1987). Viruses propagated in suspension cultures as
opposed to anchorage-dependent cultures can sometimes cause rapid
changes in viral markers, leading to reduced immunogenicity
(Bahnemann, 1980). Finally, sometimes even recombinant cell lines
can secrete considerably higher amounts of products when propagated
as anchorage-dependent cultures as compared with the same cell line
in suspension (Nilsson and Mosbach, 1987). For these reasons,
different types of anchorage-dependent cells are used extensively
in the production of different biological products.
[0105] B) Reactors Aid Processes for Suspension.
[0106] Large scale suspension culture of mammalian cells in stirred
tanks was undertaken. The instrumentation and controls for
bioreactors adapted, along with the design of the fermentors, from
related microbial applications. However, acknowledging the
increased demand for contamination control in the slower growing
mammalian cultures, improved aseptic designs were quickly
implemented, improving dependability of these reactors.
Instrumentation and controls are basically the same as found in
other fermentors and include agitation, temperature, dissolved
oxygen, and pH controls. More advanced probes and autoanalyzers for
on-line and off-line measurements of turbidity (a function of
particles present), capacitance (a function of viable cells
present), glucose/lactate, carbonate/bicarbonate and carbon dioxide
are available. Maximum cell densities obtainable in suspension
cultures are relatively low at about 24.times.10.sup.6 cells/ml of
medium (which is less than 1 mg dry cell weight per ml), well below
the numbers achieved in microbial fermentation.
[0107] Two suspension culture reactor designs are most widely used
in the industry due to their simplicity and robustness of
operation--the stirred reactor and the airlift reactor. The stirred
reactor design has successfully been used on a scale of 8000 liter
capacity for the production of interferon (Phillips et al., 1985;
Mizrahi, 1983). Cells are grown in a stainless steel tank with a
height-to-diameter ratio of 1:1 to 3:1. The culture is usually
mixed with one or more agitators, based on bladed disks or marine
propeller patterns. Agitator systems offering less shear forces
than blades have been described. Agitation may be driven either
directly or indirectly by magnetically coupled drives. Indirect
drives reduce the risk of microbial contamination through seals on
stirrer shafts.
[0108] The airlift reactor, also initially described for microbial
fermentation and later adapted for mammalian culture, relies on a
gas stream to both mix and oxygenate the culture. The gas stream
enters a riser section of the reactor and drives circulation. Gas
disengages at the culture surface, causing denser liquid free of
gas bubbles to travel downward in the downcomer section of the
reactor. The main advantage of this design is the simplicity and
lack of need for mechanical mixing. Typically, the
height-to-diameter ratio is 10:1. The airlift reactor scales up
relatively easily, has good mass transfer of gasses and generates
relatively low shear forces.
[0109] Most large-scale suspension cultures are operated as batch
or fed-batch processes because they are the most straightforward to
operate and scale up. However, continuous processes based on
chemostat or perfusion principles are available.
[0110] A batch process is a closed system in which a typical growth
profile is seen. A lag phase is followed by exponential, stationary
and decline phases. In such a system, the environment is
continuously changing as nutrients are depleted and metabolites
accumulate. This makes analysis of factors influencing cell growth
and productivity, and hence optimization of the process, a complex
task. Productivity of a batch process may be increased by
controlled feeding of key nutrients to prolong the growth cycle.
Such a fed-batch process is still a closed system because cells,
products and waste products are not removed.
[0111] In what is still a closed system, perfusion of fresh medium
through the culture can be achieved by retaining the cells with a
variety of devices (e.g. fine mesh spin filter, hollow fiber or
flat plate membrane filters, settling tubes). Spin filter cultures
can produce cell densities of approximately 5.times.10.sup.7
cells/ml. A true open system and the simplest perfusion process is
the chemostat in which there is an inflow of medium and an outflow
of cells and products. Culture medium is fed to the reactor at a
predetermined and constant rate which maintains the dilution rate
of the culture at a value less than the maximum specific growth
rate of the cells (to prevent washout of the cell mass from the
reactor). Culture fluid containing cells and cell products and
byproducts is removed at the same rate.
[0112] C) Non-Perfused Attachment Systems.
[0113] Traditionally, anchorage-dependent cell cultures are
propagated on the bottom of small glass or plastic vessels. The
restricted surface-to-volume ratio offered by classical and
traditional techniques, suitable for the laboratory scale, has
created a bottleneck in the production of cells and cell products
on a large scale. In an attempt to provide systems that offer large
accessible surfaces for cell growth in small culture volume, a
number of techniques have been proposed: the roller bottle system,
the stack plates propagator, the spiral film bottles, the hollow
fiber system, the packed bed, the plate exchanger system, and the
membrane tubing reel. Since these systems are non-homogeneous in
their nature, and are sometimes based on multiple processes, they
suffer from the following shortcomings--limited potential for
scale-up, difficulties in taking cell samples, limited potential
for measuring and controlling key process parameters and difficulty
in maintaining homogeneous environmental conditions throughout the
culture.
[0114] Despite these drawbacks, a commonly used process for large
scale anchorage-dependent cell production is the roller bottle.
Being little more than a large, differently shaped T-flask,
simplicity of the system makes it very dependable and, hence,
attractive. Fully automated robots are available that can handle
thousands of roller bottles per day, thus eliminating the risk of
contamination and inconsistency associated with the otherwise
required intense human handling. With frequent media changes,
roller bottle cultures can achieve cell densities of close to
0.5.times.10.sup.6 cells/cm.sup.2 (corresponding to approximately
10.sup.9 cells/bottle or almost 10.sup.7 cells/ml of culture
media).
[0115] D) Cultures on Microcarriers
[0116] In an effort to overcome the shortcomings of the traditional
anchorage-dependent culture processes, van Wezel (1967) developed
the concept of the microcarrier culturing systems. In this system,
cells are propagated on the surface of small solid particles
suspended in the growth medium by slow agitation. Cells attach to
the microcarriers and grow gradually to confluency on the
microcarrier surface. In fact, this large scale culture system
upgrades the attachment dependent culture from a single disc
process to a unit process in which both monolayer and suspension
culture have been brought together. Thus, combining the necessary
surface for a cell to grow with the advantages of the homogeneous
suspension culture increases production.
[0117] The advantages of microcarrier cultures over most other
anchorage-dependent, large-scale cultivation methods are several
fold. First, microcarrier cultures offer a high surface-to-volume
ratio (variable by changing the carrier concentration) which leads
to high cell density yields and a potential for obtaining highly
concentrated cell products. Cell yields are up to
1-2.times.10.sup.7 cells/ml when cultures are propagated in a
perfused reactor mode. Second, cells can be propagated in one unit
process vessels instead of using many small low-productivity
vessels (i.e., flasks or dishes). This results in far better
nutrient utilization and a considerable saving of culture medium.
Moreover, propagation in a single reactor leads to reduction in
need for facility space and in the number of handling steps
required per cell, thus reducing labor cost and risk of
contamination. Third, the well-mixed and homogeneous microcarrier
suspension culture makes it possible to monitor and control
environmental conditions (e.g., pH, pO.sub.2, and concentration of
medium components), thus leading to more reproducible cell
propagation and product recovery. Fourth, it is possible to take a
representative sample for microscopic observation, chemical
testing, or enumeration. Fifth, since microcarriers settle out of
suspension quickly, use of a fed-batch process or harvesting of
cells can be done relatively easily. Sixth, the mode of the
anchorage-dependent culture propagation on the microcarriers makes
it possible to use this system for other cellular manipulations,
such as cell transfer without the use of proteolytic enzymes,
cocultivation of cells, transplantation into animals, and perfusion
of the culture using decanters, columns, fluidized beds, or hollow
fibers for microcarrier retainment. Seventh, microcarrier cultures
are relatively easily scaled up using conventional equipment used
for cultivation of microbial and animal cells in suspension.
[0118] E) Microencapsulation of Mammalian Cells
[0119] One method which has shown to be particularly useful for
culturing mammalian cells is microencapsulation. The mammalian
cells are retained inside a semipermeable hydrogel membrane. A
porous membrane is formed around the cells permitting the exchange
of nutrients, gases, and metabolic products with the bulk medium
surrounding the capsule. Several methods have been developed that
are gentle, rapid and non-toxic and where the resulting membrane is
sufficiently porous and strong to sustain the growing cell mass
throughout the term of the culture. These methods are all based on
soluble alginate gelled by droplet contact with a
calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,
incorporated herein by reference,) describes cells concentrated in
an approximately 1% solution of sodium alginate which are forced
through a small orifice, forming droplets, and breaking free into
an approximately 1% calcium chloride solution. The droplets are
then cast in a layer of polyamino acid that ionically bonds to the
surface alginate. Finally the alginate is reliquified by treating
the droplet in a chelating agent to remove the calcium ions. Other
methods use cells in a calcium solution to be dropped into a
alginate solution, thus creating a hollow alginate sphere. A
similar approach involves cells in a chitosan solution dropped into
alginate, also creating hollow spheres.
[0120] Microencapsulated cells are easily propagated in stirred
tank reactors and, with beads sizes in the range of 150-1500 .mu.m
in diameter, are easily retained in a perfused reactor using a
fine-meshed screen. The ratio of capsule volume to total media
volume can be maintained from as dense as 1:2 to 1:10. With
intracapsular cell densities of up to 10.sup.8, the effective cell
density in the culture is 1-5.times.10.sup.7.
[0121] The advantages of microencapsulation over other processes
include the protection from the deleterious effects of shear
stresses which occur from sparging and agitation, the ability to
easily retain beads for the purpose of using perfused systems,
scale up is relatively straightforward and the ability to use the
beads for implantation.
[0122] The current invention includes cells which are
anchorage-dependent in nature. 293 cells, for example, are
anchorage-dependent, and when grown in suspension, the cells will
attach to each other and grow in clumps, eventually suffocating
cells in the inner core of each clump as they reach a size that
leaves the core cells unsustainable by the culture conditions.
Therefore, an efficient means of large-scale culture of
anchorage-dependent cells is needed in order to effectively employ
these cells to generate large quantities of adenovirus.
[0123] F) Perfused Attachment Systems
[0124] Perfused attachment systems are a preferred form of the
present invention. Perfusion refers to continuous flow at a steady
rate, through or over a population of cells (of a physiological
nutrient solution). It implies the retention of the cells within
the culture unit as opposed to continuous-flow culture which washes
the cells out with the withdrawn media (e.g., chemostat). The idea
of perfusion has been known since the beginning of the century, and
has been applied to keep small pieces of tissue viable for extended
microscopic observation. The technique was initiated to mimic the
cells milieu in vivo where cells are continuously supplied with
blood, lymph, or other body fluids. Without perfusion, cells in
culture go through alternating phases of being fed and starved,
thus limiting full expression of their growth and metabolic
potential.
[0125] The current use of perfused culture is in response to the
challenge of growing cells at high densities (i.e.,
0.1-5.times.10.sup.8 cells/ml). In order to increase densities
beyond 2-4.times.10.sup.6 cells/ml, the medium has to be constantly
replaced with a fresh supply in order to make up for nutritional
deficiencies and to remove toxic products. Perfusion allows for a
far better control of the culture environment (pH, pO.sub.2,
nutrient levels, etc.) and is a means of significantly increasing
the utilization of the surface area within a culture for cell
attachment.
[0126] The development of a perfused packed-bed reactor using a bed
matrix of a non-woven fabric has provided a means for maintaining a
perfusion culture at densities exceeding 10.sup.8 cells/ml of the
bed volume (CelliGen.TM., New Brunswick Scientific, Edison, N.J.;
Wang et al., 1992; Wang et al., 1993; Wang et al., 1994). Briefly
described, this reactor comprises an improved reactor for culturing
of both anchorage- and non-anchorage-dependent cells. The reactor
is designed as a packed bed with a means to provide internal
recirculation. Preferably, a fiber matrix carrier is placed in a
basket within the reactor vessel. A top and bottom portion of the
basket has holes, allowing the medium to flow through the basket. A
specially designed impeller provides recirculation of the medium
through the space occupied by the fiber matrix for assuring a
uniform supply of nutrient and the removal of wastes. This
simultaneously assures that a negligible amount of the total cell
mass is suspended in the medium. The combination of the basket and
the recirculation also provides a bubble-free flow of oxygenated
medium through the fiber matrix. The fiber matrix is a non-woven
fabric having a "pore" diameter of from 10 .mu.m to 100 .mu.m,
providing for a high internal volume with pore volumes
corresponding to 1 to 20 times the volumes of individual cells.
[0127] In comparison to other culturing systems, this approach
offers several significant advantages. With a fiber matrix carrier,
the cells are protected against mechanical stress from agitation
and foaming. The free medium flow through the basket provides the
cells with optimum regulated levels of oxygen, pH, and nutrients.
Products can be continuously removed from the culture and the
harvested products are free of cells and can be produced in
low-protein medium which facilitates subsequent purification steps.
Also, the unique design of this reactor system offers an easier way
to scale up the reactor. Currently, sizes up to 30 liter are
available. One hundred liter and 300 liter versions are in
development and theoretical calculations support up to a 1000 liter
reactor. This technology is explained in detail in WO 94/17178
(Aug. 4, 1994, Freedman et al.), which is hereby incorporated by
reference in its entirety.
[0128] The Cellcube.TM. (Coming-Costar) module provides a large
styrenic surface area for the immobilization and growth of
substrate attached cells. It is an integrally encapsulated sterile
single-use device that has a series of parallel culture plate
joined to create thin sealed laminar flow spaces between adjacent
plates.
[0129] The Cellcube.TM. module has inlet and outlet ports that are
diagonally opposite each other and help regulate the flow of media.
During the first few days of growth the culture is generally
satisfied by the media contained within the system after initial
seeding. The amount of time between the initial seeding and the
start of the media perfusion is dependent on the density of cells
in the seeding inoculum and the cell growth rate. The measurement
of nutrient concentration in the circulating media is a good
indicator of the status of the culture. When establishing a
procedure it may be necessary to monitor the nutrients composition
at a variety of different perfusion rates to determine the most
economical and productive operating parameters.
[0130] Cells within the system reach a higher density of solution
(cells/ml) than in traditional culture systems. Many typically used
basal media are designed to support 1-2.times.10.sup.6
cells/ml/day. A typical Cellcube.TM., run with an 85,000 cm.sup.2
surface, contains approximately 6 L media within the module. The
cell density often exceeds 10.sup.7 cells/mL in the culture vessel.
At confluence, 24 reactor volumes of media are required per
day.
[0131] The timing and parameters of the production phase of
cultures depends on the type and use of a particular cell line.
Many cultures require a different media for production than is
required for the growth phase of the culture. The transition from
one phase to the other will likely require multiple washing steps
in traditional cultures. However, the Cellcube.TM. system employs a
perfusion system. On of the benefits of such a system is the
ability to provide a gentle transition between various operating
phases. The perfusion system negates the need for traditional wash
steps that seek to remove serum components in a growth medium.
[0132] In an exemplary embodiment of the present invention, the
CellCube.TM. system is used to grow cells transfected with
AdCMVp53. 293 cells were inoculated into the Cellcube.TM. according
to the manufacturer's recommendation. Inoculation cell densities
were in the range of 1-1.5.times.10.sup.4/cm.sup.2. Cells were
allowed to grow for 7 days at 37.degree. C. under culture
conditions of pH=7.20, DO=60% air saturation. The medium perfusion
rate was regulated according to the glucose concentration in the
Cellcube.TM.. One day before viral infection, medium for perfusion
was changed from a buffer comprising 10% FBS to a buffer comprising
0% FBS. On day 8, cells were infected with virus at a multiplicity
of infection (MOI) of 5. Medium perfusion was stopped for 1 hr
immediately after infection then resumed for the remaining period
of the virus production phase. Culture was harvested 45-48 hr
post-infection. Of course these culture conditions are exemplary
and may be varied according to the nutritional needs and growth
requirements of a particular cell line. Such variation may be
performed without undue experimentation and are well within the
skill of the ordinary person in the art.
[0133] G) Serum-Free Suspension Culture
[0134] In particular embodiments, adenoviral vectors for gene
therapy are produced from anchorage-dependent culture of 293 cells
(293A cells) as described above. Scale-up of adenoviral vector
production is constrained by the anchorage-dependency of 293A
cells. To facilitate scale-up and meet future demand for adenoviral
vectors, significant efforts have been devoted to the development
of alternative production processes that are amenable to scale-up.
Methods include growing 293A cells in microcarrier cultures and
adaptation of 293A producer cells into suspension cultures.
Microcarrier culture techniques have been described above. This
technique relies on the attachment of producer cells onto the
surfaces of microcarriers which are suspended in culture media by
mechanical agitation. The requirement of cell attachment may
present some limitations to the scaleability of microcarrier
cultures.
[0135] Until the present application there have been no reports on
the use of 293 suspension cells for adenoviral vector production
for gene therapy. Furthermore, the reported suspension 293 cells
require the presence of 5-10% FBS in the culture media for optimal
cell growth and virus production. Historically, presence of bovine
source proteins in cell culture media has been a regulatory
concerns, especially recently because of the outbreak of Bovine
Spongiform Encephalopathy (BSE) in some countries. Rigorous and
complex downstream purification process has to be developed to
remove contaminating proteins and any adventitious viruses from the
final product. Development of serum-free 293 suspension culture is
deemed to be a major process improvement for the production of
adenoviral vector for gene therapy.
[0136] Results of virus production in spinner flasks and a 3 L
stirred tank bioreactor indicate that cell specific virus
productivity of the 293SF cells was approximately
2.5.times.10.sup.4 vp/cell, which is approximately 60-90% of that
from the 293A cells. However, because of the higher stationary cell
concentration, volumetric virus productivity from the 293SF culture
is essentially equivalent to that of the 293A cell culture. The
inventors also observed that virus production increased
significantly by carrying out a fresh medium exchange at the time
of virus infection. The inventors are going to evaluate the
limiting factors in the medium. These findings allow for a
scaleable, efficient, and easily validatable process for the
production of adenoviral vector. This adaptation method is not
limited to 293A cells only and will be equally useful when applied
to other adenoviral vector producer cells.
3. METHODS OF CELL HARVEST AND LYSIS
[0137] Adenoviral infection results in the lysis of the cells being
infected. The lytic characteristics of adenovirus infection permit
two different modes of virus production. One is harvesting infected
cells prior to cell lysis. The other mode is harvesting virus
supernatant after complete cell lysis by the produced virus. For
the latter mode, longer incubation times are required in order to
achieve complete cell lysis. This prolonged incubation time after
virus infection creates a serious concern about increased
possibility of generation of replication competent adenovirus
(RCA), particularly for the current first generation adenoviral
vectors (E1-deleted vector). Therefore, harvesting infected cells
before cell lysis was chosen as the production mode of choice.
Table 2 lists the most common methods that have been used for
lysing cells after cell harvest.
TABLE-US-00002 TABLE 2 Methods used for cell lysis Methods
Procedures Comments Freeze-thaw Cycling between dry ice Easy to
carry out at lab and 37.degree. C. water bath scale. High cell
lysis efficiency Not scaleable Not recommended for large scale
manufacturing Solid Shear French Press Capital equipment Hughes
Press investment Virus containment concerns Lack of experience
Detergent lysis Non-ionic detergent Easy to carry out at both lab
solutions such as Tween, and manufacturing Triton, NP-40, etc.
scale Wide variety of detergent choices Concerns of residual
detergent in finished product Hypotonic water, citric buffer Low
lysis efficiency solution lysis Liquid Shear Homogenizer Capital
equipment Impinging Jet investment Microfluidizer Virus containment
concerns Scaleability concerns Sonication Ultrasound Capital
equipment investment Virus containment concerns Noise pollution
Scaleability concern
[0138] A) Detergents
[0139] Cells are bounded by membranes. In order to release
components of the cell, it is necessary to break open the cells.
The most advantageous way in which this can be accomplished,
according to the present invention, is to solubilize the membranes
with the use of detergents. Detergents are amphipathic molecules
with an apolar end of aliphatic or aromatic nature and a polar end
which may be charged or uncharged. Detergents are more hydrophilic
than lipids and thus have greater water solubility than lipids.
They allow for the dispersion of water insoluble compounds into
aqueous media and are used to isolate and purify proteins in a
native form.
[0140] Detergents can be denaturing or non-denaturing. The former
can be anionic such as sodium dodecyl sulfate or cationic such as
ethyl trimethyl ammonium bromide. These detergents totally disrupt
membranes and denature the protein by breaking protein-protein
interactions. Non denaturing detergents can be divided into
non-anionic detergents such as Triton.RTM.X-100, bile salts such as
cholates and zwitterionic detergents such as CHAPS. Zwitterionics
contain both cationic and anion groups in the same molecule, the
positive electric charge is neutralized by the negative charge on
the same or adjacent molecule.
[0141] Denaturing agents such as SDS bind to proteins as monomers
and the reaction is equilibrium driven until saturated. Thus, the
free concentration of monomers determines the necessary detergent
concentration. SDS binding is cooperative i.e. the binding of one
molecule of SDS increase the probability of another molecule
binding to that protein, and alters proteins into rods whose length
is proportional to their molecular weight.
[0142] Non-denaturing agents such as Triton.RTM.X-100 do not bind
to native conformations nor do they have a cooperative binding
mechanism. These detergents have rigid and bulky apolar moieties
that do not penetrate into water soluble proteins. They bind to the
hydrophobic parts of proteins. Triton.RTM.X100 and other
polyoxyethylene nonanionic detergents are inefficient in breaking
protein-protein interaction and can cause artifactual aggregations
of protein. These detergents will, however, disrupt protein-lipid
interactions but are much gentler and capable of maintaining the
native form and functional capabilities of the proteins.
[0143] Detergent removal can be attempted in a number of ways.
Dialysis works well with detergents that exist as monomers.
Dialysis is somewhat ineffective with detergents that readily
aggregate to form micelles because the micelles are too large to
pass through dialysis. Ion exchange chromatography can be utilized
to circumvent this problem. The disrupted protein solution is
applied to an ion exchange chromatography column and the column is
then washed with buffer minus detergent. The detergent will be
removed as a result of the equilibration of the buffer with the
detergent solution. Alternatively the protein solution may be
passed through a density gradient. As the protein sediments through
the gradients the detergent will come off due to the chemical
potential.
[0144] Often a single detergent is not versatile enough for the
solubilization and analysis of the milieu of proteins found in a
cell. The proteins can be solubilized in one detergent and then
placed in another suitable detergent for protein analysis. The
protein detergent micelles formed in the first step should separate
from pure detergent micelles. When these are added to an excess of
the detergent for analysis, the protein is found in micelles with
both detergents. Separation of the detergent-protein micelles can
be accomplished with ion exchange or gel filtration chromatography,
dialysis or buoyant density type separations.
[0145] Triton.RTM.X-Detergents: This family of detergents
(Triton.RTM.X-100, X114 and NP-40) have the same basic
characteristics but are different in their specific
hydrophobic-hydrophilic nature. All of these heterogeneous
detergents have a branched 8-carbon chain attached to an aromatic
ring. This portion of the molecule contributes most of the
hydrophobic nature of the detergent. Triton.RTM.X detergents are
used to solubilize membrane proteins under non-denaturing
conditions. The choice of detergent to solubilize proteins will
depend on the hydrophobic nature of the protein to be solubilized.
Hydrophobic proteins require hydrophobic detergents to effectively
solubilize them.
[0146] Triton.RTM.X-100 and NP-40 are very similar in structure and
hydrophobicity and are interchangeable in most applications
including cell lysis, delipidation protein dissociation and
membrane protein and lipid solubilization. Generally 2 mg detergent
is used to solubilize 1 mg membrane protein or 10 mg detergent/1 mg
of lipid membrane. Triton.RTM.X-114 is useful for separating
hydrophobic from hydrophilic proteins.
[0147] Brij.RTM. Detergents: These are similar in structure to
Triton.RTM.X detergents in that they have varying lengths of
polyoxyethylene chains attached to a hydrophobic chain. However,
unlike Triton.RTM.X detergents, the Brij.RTM. detergents do not
have an aromatic ring and the length of the carbon chains can vary.
The Brij.RTM. detergents are difficult to remove from solution
using dialysis but may be removed by detergent removing gels.
Brij.RTM.58 is most similar to Triton.RTM.X100 in its
hydrophobic/hydrophilic characteristics. Brij.RTM.-35 is a commonly
used detergent in HPLC applications.
[0148] Dializable Nonionic Detergents:
.eta.-Octyl-.beta.-D-glucoside (octylglucopyranoside) and
.eta.-Octyl-.beta.-D-thioglucoside (octylthioglucopyranoside, OTG)
are nondenaturing nonionic detergents which are easily dialyzed
from solution. These detergents are useful for solubilizing
membrane proteins and have low UV absorbances at 280 nm.
Octylglucoside has a high CMC of 23-25 mM and has been used at
concentrations of 1.1-1.2% to solubilize membrane proteins.
[0149] Octylthioglucoside was first synthesized to offer an
alternative to octylglucoside. Octylglucoside is expensive to
manufacture and there are some inherent problems in biological
systems because it can be hydrolyzed by .beta.-glucosidase.
[0150] Tween.RTM. Detergents: The Tween.RTM. detergents are
nondenaturing, nonionic detergents. They are polyoxyethylene
sorbitan esters of fatty acids. Tween.RTM. 20 and Tween.RTM. 80
detergents are used as blocking agents in biochemical applications
and are usually added to protein solutions to prevent nonspecific
binding to hydrophobic materials such as plastics or
nitrocellulose. They have been used as blocking agents in ELISA and
blotting applications. Generally, these detergents are used at
concentrations of 0.01-1.0% to prevent nonspecific binding to
hydrophobic materials.
[0151] Tween.RTM. 20 and other nonionic detergents have been shown
to remove some proteins from the surface of nitrocellulose.
Tween.RTM. 80 has been used to solubilize membrane proteins,
present nonspecific binding of protein to multiwell plastic tissue
culture plates and to reduce nonspecific binding by serum proteins
and biotinylated protein A to polystyrene plates in ELISA.
[0152] The difference between these detergents is the length of the
fatty acid chain. Tween.RTM. 80 is derived from oleic acid with a
C.sub.18 chain while Tween.RTM. 20 is derived from lauric acid with
a C.sub.12 chain. The longer fatty acid chain makes the Tween.RTM.
80 detergent less hydrophilic than Tween.RTM. 20 detergent. Both
detergents are very soluble in water.
[0153] The Tween.RTM. detergents are difficult to remove from
solution by dialysis, but Tween.RTM. 20 can be removed by detergent
removing gels. The polyoxyethylene chain found in these detergents
makes them subject to oxidation (peroxide formation) as is true
with the Triton.RTM. X and Brij.RTM. series detergents.
[0154] Zwitterionic Detergents: The zwitterionic detergent, CHAPS,
is a sulfobetaine derivative of cholic acid. This zwitterionic
detergent is useful for membrane protein solubilization when
protein activity is important. This detergent is useful over a wide
range of pH (pH 2-12) and is easily removed from solution by
dialysis due to high CMCs (8-10 mM). This detergent has low
absorbances at 280 nm making it useful when protein monitoring at
this wavelength is necessary. CHAPS is compatible with the BCA
Protein Assay and can be removed from solution by detergent
removing gel. Proteins can be iodinated in the presence of
CHAPS
[0155] CHAPS has been successfully used to solubilize intrinsic
membrane proteins and receptors and maintain the functional
capability of the protein. When cytochrome P450 is solubilized in
either Triton.RTM. X-100 or sodium cholate aggregates are
formed.
[0156] B) Non-Detergent Methods
[0157] Various non-detergent methods, though not preferred, may be
employed in conjunction with other advantageous aspects of the
present invention:
[0158] Freeze-Thaw: This has been a widely used technique for lysis
cells in a gentle and effective manner. Cells are generally frozen
rapidly in, for example, a dry ice/ethanol bath until completely
frozen, then transferred to a 37.degree. C. bath until completely
thawed. This cycle is repeated a number of times to achieve
complete cell lysis.
[0159] Sonication: High frequency ultrasonic oscillations have been
found to be useful for cell disruption. The method by which
ultrasonic waves break cells is not fully understood but it is
known that high transient pressures are produced when suspensions
are subjected to ultrasonic vibration. The main disadvantage with
this technique is that considerable amounts of heat are generated.
In order to minimize heat effects specifically designed glass
vessels are used to hold the cell suspension. Such designs allow
the suspension to circulate away from the ultrasonic probe to the
outside of the vessel where it is cooled as the flask is suspended
in ice.
[0160] High Pressure Extrusion: This is a frequently used method to
disrupt microbial cell. The French pressure cell employs pressures
of 10.4.times.10.sup.7 Pa (16,000 p.s.i) to break cells open. These
apparatus consists of a stainless steel chamber which opens to the
outside by means of a needle valve. The cell suspension is placed
in the chamber with the needle valve in the closed position. After
inverting the chamber, the valve is opened and the piston pushed in
to force out any air in the chamber. With the valve in the closed
position, the chamber is restored to its original position, placed
on a solid based and the required pressure is exerted on the piston
by a hydraulic press. When the pressure has been attained the
needle valve is opened fractionally to slightly release the
pressure and as the cells expand they burst. The valve is kept open
while the pressure is maintained so that there is a trickle of
ruptured cell which may be collected.
[0161] Solid Shear Methods: Mechanical shearing with abrasives may
be achieved in Mickle shakers which oscillate suspension vigorously
(300-3000 time/min) in the presence of glass beads of 500 nm
diameter. This method may result in organelle damage. A more
controlled method is to use a Hughes press where a piston forces
most cells together with abrasives or deep frozen paste of cells
through a 0.25 mm diameter slot in the pressure chamber. Pressures
of up to 5.5.times.10.sup.7 Pa (8000 p.s.i.) may be used to lyse
bacterial preparations.
[0162] Liquid Shear Methods: These methods employ blenders, which
use high speed reciprocating or rotating blades, homogenizers which
use an upward/downward motion of a plunger and ball and
microfluidizers or impinging jets which use high velocity passage
through small diameter tubes or high velocity impingement of two
fluid streams. The blades of blenders are inclined at different
angles to permit efficient mixing. Homogenizers are usually
operated in short high speed bursts of a few seconds to minimize
local heat. These techniques are not generally suitable for
microbial cells but even very gentle liquid shear is usually
adequate to disrupt animal cells.
[0163] Hypotonic/Hypertonic Methods: Cells are exposed to a
solution with a much lower (hypotonic) or higher (hypertonic)
solute concentration. The difference in solute concentration
creates an osmotic pressure gradient. The resulting flow of water
into the cell in a hypotonic environment causes the cells to swell
and burst. The flow of water out of the cell in a hypertonic
environment causes the cells to shrink and subsequently burst.
[0164] Viral Lysis Methods: In some situations, the method of viral
lysis may be advantageous to use, and with modifications to the
experimental protocol, the formation of RCA may be minimized. Since
adenoviruses are lytic viruses, after infection of the host cells
the mature viruses lyse the cell and are released into the
supernatant and then can be harvested by conventional methods. One
of the advantages to using the viral lysis method is the generation
of more mature viral particles, since early lysis by mechanical or
chemical means may lead to increased numbers of defective
particles. In addition, the process permits an easier and more
precise follow-up of the production kinetics directly on the
homogeneous samples of supernatant, which produces better
reproducibility of the production runs. Chemical lysis also
presents an additional step in the process and requires the removal
of the lysis agent, both of which may lead to potential losses of
product and/or diminished activity.
[0165] In utilizing the viral lysis method, the kinetics of the
liberation of virions can be followed in different ways and will be
able to indicate the optimal time for supernatant harvest. For
example, HPLC, IEC, PCR, dye exclusion, spectrophotometry, ELISA,
RIA or nephelometric methods may be used. Harvesting is preferably
performed when approximately 50% of the virions have been released.
More preferably, the supernatant is harvested when at least 70% of
the virions are released, and most preferably, the supernatant is
harvested when at least 90% of the virions are released, or when
the viral release reaches a plateau as measured by one of the
methods indicated above. Variations in the time needed for the
virus release to reach a plateau may be observed when using
modification of gene transfer vector, however the harvest schedule
can easily be modified by the skilled artisan when using one or
more of the methods above to follow the kinetics of virus
release.
4. METHODS OF CONCENTRATION AND FILTRATION
[0166] One aspect of the present invention employs methods of crude
purification of adenovirus from a cell lysate. These methods
include clarification, concentration and diafiltration. The initial
step in this purification process is clarification of the cell
lysate to remove large particulate matter, particularly cellular
components, from the cell lysate. Clarification of the lysate can
be achieved using a depth filter or by tangential flow filtration.
In a preferred embodiment of the present invention, the cell lysate
is passed through a depth filter, which consists of a packed column
of relatively non-adsorbent material (e.g. polyester resins, sand,
diatomaceous earth, colloids, gels, and the like). In tangential
flow filtration (TFF), the lysate solution flows across a membrane
surface which facilitates back diffusion of solute from the
membrane surface into the bulk solution. Membranes are generally
arranged within various types of filter apparatus including open
channel plate and frame, hollow fibers, and tubules.
[0167] After clarification and prefiltration of the cell lysate,
the resultant virus supernatant is first concentrated and then the
buffer is exchanged by diafiltration. The virus supernatant is
concentrated by tangential flow filtration across an
ultrafiltration membrane of 100-300 K nominal molecular weight
cutoff. Ultrafiltration is a pressure-modified convective process
that uses semi-permeable membranes to separate species by molecular
size, shape and/or charge. It separates solvents from solutes of
various sizes, independent of solute molecular size.
Ultrafiltration is gentle, efficient and can be used to
simultaneously concentrate and desalt solutions. Ultrafiltration
membranes generally have two distinct layers: a thin (0.1-1.5
.mu.m), dense skin with a pore diameter of 10400 angstroms and an
open substructure of progressively larger voids which are largely
open to the permeate side of the ultrafilter. Any species capable
of passing through the pores of the skin can therefore freely pass
through the membrane. For maximum retention of solute, a membrane
is selected that has a nominal molecular weight cut-off well below
that of the species being retained. In macromolecular
concentration, the membrane enriches the content of the desired
biological species and provides filtrate cleared of retained
substances. Microsolutes are removed convectively with the solvent.
As concentration of the retained solute increases, the
ultrafiltration rate diminishes.
[0168] Diafiltration, or buffer exchange, using ultrafilters is an
ideal way for removal and exchange of salts, sugars, non-aqueous
solvents separation of free from bound species, removal of material
of low molecular weight, or rapid change of ionic and pH
environments. Microsolutes are removed most efficiently by adding
solvent to the solution being ultrafiltered at a rate equal to the
ultrafiltration rate. This washes microspecies from the solution at
constant volume, purifying the retained species. The present
invention utilizes a diafiltration step to exchange the buffer of
the virus supernatant prior to Benzonase.RTM. treatment.
5. VIRAL INFECTION
[0169] The present invention employs, in one example, adenoviral
infection of cells in order to generate therapeutically significant
vectors. Typically, the virus will simply be exposed to the
appropriate host cell under physiologic conditions, permitting
uptake of the virus. Though adenovirus is exemplified, the present
methods may be advantageously employed with other viral vectors, as
discussed below.
[0170] A) Adenovirus
[0171] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized DNA genome, ease of
manipulation, high titer, wide target-cell range, and high
infectivity. The roughly 36 kB viral genome is bounded by 100-200
base pair (bp) inverted terminal repeats (ITR), in which are
contained cis-acting elements necessary for viral DNA replication
and packaging. The early (E) and late (L) regions of the genome
that contain different transcription units are divided by the onset
of viral DNA replication.
[0172] The E1 region (E1A and E1B) encodes proteins responsible for
the regulation of transcription of the viral genome and a few
cellular genes. The expression of the E2 region (E2A and E2B)
results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene
expression, and host cell shut off (Renan, 1990). The products of
the late genes (L1, L2, L3, L4 and L5), including the majority of
the viral capsid proteins, are expressed only after significant
processing of a single primary transcript issued by the major late
promoter (MLP). The MLP (located at 16.8 map units) is particularly
efficient during the late phase of infection, and all the mRNAs
issued from this promoter possess a 5' tripartite leader (TL)
sequence which makes them preferred mRNAs for translation.
[0173] In order for adenovirus to be optimized for gene therapy, it
is necessary to maximize the carrying capacity so that large
segments of DNA can be included. It also is very desirable to
reduce the toxicity and immunologic reaction associated with
certain adenoviral products. Elimination of large potions of the
adenoviral genome, and providing the delete gene products in trans,
by helper virus and/or helper cells, allows for the insertion of
large portions of heterologous DNA into the vector. This strategy
also will result in reduced toxicity and immunogenicity of the
adenovirus gene products.
[0174] The large displacement of DNA is possible because the cis
elements required for viral DNA replication all are localized in
the inverted terminal repeats (ITR) (100-200 bp) at either end of
the linear viral genome. Plasmids containing ITR's can replicate in
the presence of a non-defective adenovirus (Hay et al., 1984).
Therefore, inclusion of these elements in an adenoviral vector
should permit replication.
[0175] In addition, the packaging signal for viral encapsidation is
localized between 194-385 bp (0.5-1.1 map units) at the left end of
the viral genome (Hearing et al., 1987). This signal mimics the
protein recognition site in bacteriophage .lamda. DNA where a
specific sequence close to the left end, but outside the cohesive
end sequence, mediates the binding to proteins that are required
for insertion of the DNA into the head structure. E1 substitution
vectors of Ad have demonstrated that a 450 bp (0-1.25 map units)
fragment at the left end of the viral genome could direct packaging
in 293 cells (Levrero et al., 1991).
[0176] Previously, it has been shown that certain regions of the
adenoviral genome can be incorporated into the genome of mammalian
cells and the genes encoded thereby expressed. These cell lines are
capable of supporting the replication of an adenoviral vector that
is deficient in the adenoviral function encoded by the cell line.
There also have been reports of complementation of replication
deficient adenoviral vectors by "helping" vectors, e.g., wild-type
virus or conditionally defective mutants.
[0177] Replication-deficient adenoviral vectors can be
complemented, in trans, by helper virus. This observation alone
does not permit isolation of the replication-deficient vectors,
however, since the presence of helper virus, needed to provide
replicative functions, would contaminate any preparation. Thus, an
additional element was needed that would add specificity to the
replication and/or packaging of the replication-deficient vector.
That element, as provided for in the present invention, derives
from the packaging function of adenovirus.
[0178] It has been shown that a packaging signal for adenovirus
exists in the left end of the conventional adenovirus map
(Tibbetts, 1977). Later studies showed that a mutant with a
deletion in the E1A (194-358 bp) region of the genome grew poorly
even in a cell line that complemented the early (E1A) function
(Hearing and Shenk, 1983). When a compensating adenoviral DNA
(0-353 bp) was recombined into the right end of the mutant, the
virus was packaged normally. Further mutational analysis identified
a short, repeated, position-dependent element in the left end of
the Ad5 genome. One copy of the repeat was found to be sufficient
for efficient packaging if present at either end of the genome, but
not when moved towards the interior of the Ad5 DNA molecule
(Hearing et al., 1987).
[0179] By using mutated versions of the packaging signal, it is
possible to create helper viruses that are packaged with varying
efficiencies. Typically, the mutations are point mutations or
deletions. When helper viruses with low efficiency packaging are
grown in helper cells, the virus is packaged, albeit at reduced
rates compared to wild-type virus, thereby permitting propagation
of the helper. When these helper viruses are grown in cells along
with virus that contains wild-type packaging signals, however, the
wild-type packaging signals are recognized preferentially over the
mutated versions. Given a limiting amount of packaging factor, the
virus containing the wild-type signals are packaged selectively
when compared to the helpers. If the preference is great enough,
stocks approaching homogeneity should be achieved.
[0180] B) Retrovirus
[0181] Although adenoviral infection of cells for the generation of
therapeutically significant vectors is a preferred embodiments of
the present invention, it is contemplated that the present
invention may employ retroviral infection of cells for the purposes
of generating such vectors. The retroviruses are a group of
single-stranded RNA viruses characterized by an ability to convert
their RNA to double-stranded DNA in infected cells by a process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes--gag, pol
and env--that code for capsid proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene, termed Y, functions as a signal for packaging of the
genome into virions. Two long terminal repeat (LTR) sequences are
present at the 5' and 3' ends of the viral genome. These contain
strong promoter and enhancer sequences and are also required for
integration in the host cell genome (Coffin, 1990).
[0182] In order to construct a retroviral vector, a nucleic acid
encoding a promoter is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol and env genes but without the LTR
and Y components is constructed (Mann et al., 1983). When a
recombinant plasmid containing a human cDNA, together with the
retroviral LTR and Y sequences is introduced into this cell line
(by calcium phosphate precipitation for example), the Y sequence
allows the RNA transcript of the recombinant plasmid to be packaged
into viral particles, which are then secreted into the culture
media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al.,
1983). The media containing the recombinant retroviruses is then
collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell
types. However, integration and stable expression require the
division of host cells (Paskind et al., 1975).
[0183] A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovirus by the chemical addition of galactose
residues to the viral envelope. This modifications could permit the
specific infection of cells such as hepatocytes via
asialoglycoprotein receptors, should this be desired.
[0184] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens, the
infection of a variety of human cells that bore those surface
antigens was demonstrated with an ecotropic virus in vitro (Roux et
al., 1989).
[0185] C) Adeno Associated Virus
[0186] AAV utilizes a linear, single-stranded DNA of about 4700
base pairs. Inverted terminal repeats flank the genome. Two genes
are present within the genome, giving rise to a number of distinct
gene products. The first, the cap gene, produces three different
virion proteins (VP), designated VP-1, VP-2 and VP-3. The second,
the rep gene, encodes four non-structural proteins (NS). One or
more of these rep gene products is responsible for transactivating
AAV transcription.
[0187] The three promoters in AAV are designated by their location,
in map units, in the genome. These are, from left to right, p5, p19
and p40. Transcription gives rise to six transcripts, two initiated
at each of three promoters, with one of each pair being spliced.
The splice site, derived from map units 42-46, is the same for each
transcript. The four non-structural proteins apparently are derived
from the longer of the transcripts, and three virion proteins all
arise from the smallest transcript.
[0188] AAV is not associated with any pathologic state in humans.
Interestingly, for efficient replication, AAV requires "helping"
functions from viruses such as herpes simplex virus I and II,
cytomegalovirus, pseudorabies virus and, of course, adenovirus. The
best characterized of the helpers is adenovirus, and many "early"
functions for this virus have been shown to assist with AAV
replication. Low level expression of AAV rep proteins is believed
to hold AAV structural expression in check, and helper virus
infection is thought to remove this block.
[0189] The terminal repeats of the AAV vector can be obtained by
restriction endonuclease digestion of AAV or a plasmid such as
p201, which contains a modified AAV genome (Samulski et al. 1987),
or by other methods known to the skilled artisan, including but not
limited to chemical or enzymatic synthesis of the terminal repeats
based upon the published sequence of AAV. The ordinarily skilled
artisan can determine, by well-known methods such as deletion
analysis, the minimum sequence or part of the AAV ITRs which is
required to allow function, i.e., stable and site-specific
integration. The ordinarily skilled artisan also can determine
which minor modifications of the sequence can be tolerated while
maintaining the ability of the terminal repeats to direct stable,
site-specific integration.
[0190] AAV-based vectors have proven to be safe and effective
vehicles for gene delivery in vitro, and these vectors are being
developed and tested in pre-clinical and clinical stages for a wide
range of applications in potential gene therapy, both ex vivo and
in vivo (Carter and Flotte, 1996;
[0191] Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et
al., 1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et
al., 1994; 1996, Kessler et al., 1996; Koeberl et al.,
[0192] AAV-mediated efficient gene transfer and expression in the
lung has led to clinical trials for the treatment of cystic
fibrosis (Carter and Flotte, 1996; Flotte et al., 1993). Similarly,
the prospects for treatment of muscular dystrophy by AAV-mediated
gene delivery of the dystrophin gene to skeletal muscle, of
Parkinson's disease by tyrosine hydroxylase gene delivery to the
brain, of hemophilia B by Factor IX gene delivery to the liver, and
potentially of myocardial infarction by vascular endothelial growth
factor gene to the heart, appear promising since AAV-mediated
transgene expression in these organs has recently been shown to be
highly efficient (Fisher et al., 1996; Flotte et al., 1993; Kaplitt
et al., 1994;
[0193] D) Herpesvirus
[0194] Because herpes simplex virus (HSV) is neurotropic, it has
generated considerable interest in treating nervous system
disorders. Moreover, the ability of HSV to establish latent
infections in non-dividing neuronal cells without integrating in to
the host cell chromosome or otherwise altering the host cell's
metabolism, along with the existence of a promoter that is active
during latency makes HSV an attractive vector. And though much
attention has focused on the neurotropic applications of HSV, this
vector also can be exploited for other tissues given its wide host
range.
[0195] Another factor that makes HSV an attractive vector is the
size and organization of the genome. Because HSV is large,
incorporation of multiple genes or expression cassettes is less
problematic than in other smaller viral systems. In addition, the
availability of different viral control sequences with varying
performance (temporal, strength, etc.) makes it possible to control
expression to a greater extent than in other systems. It also is an
advantage that the virus has relatively few spliced messages,
further easing genetic manipulations.
[0196] HSV also is relatively easy to manipulate and can be grown
to high titers. Thus, delivery is less of a problem, both in terms
of volumes needed to attain sufficient MOI and in a lessened need
for repeat dosings. For a review of HSV as a gene therapy vector,
see Glorioso et al. (1995).
[0197] HSV, designated with subtypes 1 and 2, are enveloped viruses
that are among the most common infectious agents encountered by
humans, infecting millions of human subjects worldwide. The large,
complex, double-stranded DNA genome encodes for dozens of different
gene products, some of which derive from spliced transcripts. In
addition to virion and envelope structural components, the virus
encodes numerous other proteins including a protease, a
ribonucleotides reductase, a DNA polymerase, a ssDNA binding
protein, a helicase/primase, a DNA dependent ATPase, a dUTPase and
others.
[0198] HSV genes form several groups whose expression is
coordinately regulated and sequentially ordered in a cascade
fashion (Honess and Roizman, 1974; Honess and Roizman 1975; Roizman
and Sears, 1995). The expression of .alpha. genes, the first set of
genes to be expressed after infection, is enhanced by the virion
protein number 16, or .alpha.-transinducing factor (Post et al.,
1981; Batterson and Roizman, 1983; Campbell, et al., 1983). The
expression of P genes requires functional .beta. gene products,
most notably ICP4, which is encoded by the .alpha.4 gene (DeLuca et
al., 1985). .gamma. genes, a heterogeneous group of genes encoding
largely virion structural proteins, require the onset of viral DNA
synthesis for optimal expression (Holland et al., 1980).
[0199] In line with the complexity of the genome, the life cycle of
HSV is quite involved. In addition to the lytic cycle, which
results in synthesis of virus particles and, eventually, cell
death, the virus has the capability to enter a latent state in
which the genome is maintained in neural ganglia until some as of
yet undefined signal triggers a recurrence of the lytic cycle.
Avirulent variants of HSV have been developed and are readily
available for use in gene therapy contexts (U.S. Pat. No.
5,672,344).
[0200] E) Vaccinia Virus
[0201] Vaccinia virus vectors have been used extensively because of
the ease of their construction, relatively high levels of
expression obtained, wide host range and large capacity for
carrying DNA. Vaccinia contains a linear, double-stranded DNA
genome of about 186 kb that exhibits a marked "A-T" preference.
Inverted terminal repeats of about 10.5 kb flank the genome. The
majority of essential genes appear to map within the central
region, which is most highly conserved among poxviruses. Estimated
open reading frames in vaccinia virus number from 150 to 200.
Although both strands are coding, extensive overlap of reading
frames is not common.
[0202] At least 25 kb can be inserted into the vaccinia virus
genome (Smith and Moss, 1983). Prototypical vaccinia vectors
contain transgenes inserted into the viral thymidine kinase gene
via homologous recombination. Vectors are selected on the basis of
a tk-phenotype. Inclusion of the untranslated leader sequence of
encephalomyocarditis virus, the level of expression is higher than
that of conventional vectors, with the transgenes accumulating at
10% or more of the infected cell's protein in 24 h (Elroy-Stein et
al., 1989).
[0203] F) SV40 Virus
[0204] Simian virus 40 (SV40) was discovered in 1960 as a
contaminant in polio vaccines prepared from rhesus monkey kidney
cell cultures. It was found to cause tumors when injected into
newborn hamsters. The genome is a double-stranded, circular DNA of
about 5000 bases encoding large (708 AA) and small T antigens (174
AA), agnoprotein and the structural proteins VP1, VP2 and VP3. The
respective size of these molecules is 362, 352 and 234 amino
acids.
[0205] Little is known of the nature of the receptors for any
polyoma virus. The virus is taken up by endocytosis and transported
to the nucleus where uncoating takes place. Early mRNA's initiate
viral replication and is necessary, along with DNA replication, for
late gene expression. Near the origin of replication, promoters are
located for early and late transcription. Twenty-one base pair
repeats, located 40-103 nucleotides upstream of the initiation
transcription site, are the main promoting element and are binding
sites for Sp1, while 72 base pair repeats act as enhancers.
[0206] Large T antigen, one of the early proteins, plays a critical
role in replication and late gene expression and is modified in a
number of ways, including N-terminal acetylation, phosphorylation,
poly-ADP ribosylation, glycosylation and acylation. The other T
antigen is produced by splicing of the large T transcript. The
corresponding small T protein is not strictly required for
infection, but it plays a role in the accumulation of viral
DNA.
[0207] DNA replication is controlled, to an extent, by a
genetically defined core region that includes the viral origin of
replication. The SV40 element is about 66 bp in length and has
subsequences of AT motifs, GC motifs and an inverted repeat of 14
bp on the early gene side. Large T antigen is required for
initiation of DNA replication, and this protein has been shown to
bind in the vicinity of the origin. It also has ATPase, adenylating
and helicase activities.
[0208] After viral replication begins, late region expression
initiates. The transcripts are overlapping and, in some respect,
reflect different reading frames (VP1 and VP2/3). Late expression
initiates is the same general region as early expression, but in
the opposite direction. The virion proteins are synthesized in the
cytoplasm and transported to the nucleus where they enter as a
complex. Virion assembly also takes place in the nucleus, followed
by lysis and release of the infectious virus particles.
[0209] It is contemplated that the present invention will encompass
SV40 vectors lacking all coding sequences. The region from about
5165-5243 and about 0-325 contains all of the control elements
necessary for replication and packaging of the vector and
expression of any included genes. Thus, minimal SV40 vectors are
derived from this region and contain at least a complete origin of
replication.
[0210] Because large T antigen is believed to be involved in the
expression of late genes, and no large T antigen is expressed in
the target cell, it will be desired that the promoter driving the
heterologous gene be a polyomavirus early promoter, or more
preferably, a heterologous promoter. Thus, where heterologous
control elements are utilized, the SV40 promoter and enhancer
elements are dispensable.
[0211] D) Other Viral Vectors
[0212] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from viruses such as
papillomaviruses, papovaviruses and lentivirus may be employed.
These viruses offer several features for use in gene transfer into
various mammalian cells, and it will be understood that various
modifications to such viruses can be made to enhance for example
infectivity and targeting. Chimeric viruses, employing advantageous
portions of different viruses, may also be constructed by one of
skill in the art.
6. ENGINEERING OF VIRAL VECTORS
[0213] In certain embodiments, the present invention further
involves the manipulation of viral vectors. Such methods involve
the use of a vector construct containing, for example, a
heterologous DNA encoding a gene of interest and a means for its
expression, replicating the vector in an appropriate helper cell,
obtaining viral particles produced therefrom, and infecting cells
with the recombinant virus particles. The gene could simply encode
a protein for which large quantities of the protein are desired,
i.e., large scale in vitro production methods. Alternatively, the
gene could be a therapeutic gene, for example to treat cancer
cells, to express immunomodulatory genes to fight viral infections,
or to replace a gene's function as a result of a genetic defect. In
the context of the gene therapy vector, the gene will be a
heterologous DNA, meant to include DNA derived from a source other
than the viral genome which provides the backbone of the vector.
Finally, the virus may act as a live viral vaccine and express an
antigen of interest for the production of antibodies thereagainst.
The gene may be derived from a prokaryotic or eukaryotic source
such as a bacterium, a virus, a yeast, a parasite, a plant, or even
an animal. The heterologous DNA also may be derived from more than
one source, i.e., a multigene construct or a fusion protein. The
heterologous DNA may also include a regulatory sequence which may
be derived from one source and the gene from a different
source.
[0214] A) Therapeutic Genes
[0215] p53 currently is recognized as a tumor suppressor gene
(Montenarh, 1992). High levels of mutant p53 have been found in
many cells transformed by chemical carcinogenesis, ultraviolet
radiation, and several viruses, including SV40. The p53 gene is a
frequent target of mutational inactivation in a wide variety of
human tumors and is already documented to be the most
frequently-mutated gene in common human cancers (Mercer, 1992). It
is mutated in over 50% of human NSCLC (Hollestein et al., 1991) and
in a wide spectrum of other tumors.
[0216] The p53 gene encodes a 393-amino-acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The protein is found in normal tissues and cells, but at
concentrations which are generally minute by comparison with
transformed cells or tumor tissue. Interestingly, wild-type p53
appears to be important in regulating cell growth and division.
Overexpression of wild-type p53 has been shown in some cases to be
anti-proliferative in human tumor cell lines. Thus, p53 can act as
a negative regulator of cell growth (Weinberg, 1991) and may
directly suppress uncontrolled cell growth or directly or
indirectly activate genes that suppress this growth. Thus, absence
or inactivation of wild-type p53 may contribute to transformation.
However, some studies indicate that the presence of mutant p53 may
be necessary for full expression of the transforming potential of
the gene.
[0217] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0218] Casey and colleagues have reported that transfection of DNA
encoding wild-type p53 into two human breast cancer cell lines
restores growth suppression control in such cells (Casey et al.,
1991). A similar effect has also been demonstrated on transfection
of wild-type, but not mutant, p53 into human lung cancer cell lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene
and will select against proliferation when transfected into cells
with the mutant gene. Normal expression of the transfected p53 is
not detrimental to normal cells with endogenous wild-type p53.
Thus, such constructs might be taken up by normal cells without
adverse effects. It is thus proposed that the treatment of
p53-associated cancers with wild-type p53 expression constructs
will reduce the number of malignant cells or their growth rate.
Furthermore, recent studies suggest that some p53 wild-type tumors
are also sensitive to the effects of exogenous p53 expression.
[0219] The major transitions of the eukaryotic cell cycle are
triggered by cyclin-dependent kinases, or CDK's. One CDK,
cyclin-dependent kinase 4 (CDK4), regulates progression through the
G.sub.1 phase. The activity of this enzyme may be to phosphorylate
Rb at late G.sub.1. The activity of CDK4 is controlled by an
activating subunit, D-type cyclin, and by an inhibitory subunit,
e.g. p16.sup.INK4, which has been biochemically characterized as a
protein that specifically binds to and inhibits CDK4, and thus may
regulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,
1995). Since the p16.sup.INK4 protein is a CDK4 inhibitor (Serrano,
1993), deletion of this gene may increase the activity of CDK4,
resulting in hyperphosphorylation of the Rb protein. p16 also is
known to regulate the function of CDK6.
[0220] p16.sup.INK4 belongs to a newly described class of
CDK-inhibitory proteins that also includes p16.sup.B, p21.sup.WAF1,
CIP1, SDI1, and p27.sup.KIP1. The p16.sup.INK4 gene maps to 9p21, a
chromosome region frequently deleted in many tumor types.
Homozygous deletions and mutations of the p16.sup.INK4 gene are
frequent in human tumor cell lines. This evidence suggests that the
p16.sup.INK4 gene is a tumor suppressor gene. This interpretation
has been challenged, however, by the observation that the frequency
of the p16.sup.INK4 gene alterations is much lower in primary
uncultured tumors than in cultured cell lines (Caldas et al., 1994;
Cheng et al., 1994; Hussussian et al., 1994; Kamb et al., 1994a;
Kamb et al., 1994b; Mori et al., 1994; Okamoto et al., 1994; Nobori
et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration
of wild-type p16.sup.INK4 function by transfection with a plasmid
expression vector reduced colony formation by some human cancer
cell lines (Okamoto, 1994; Arap, 1995).
[0221] C-CAM is expressed in virtually all epithelial cells (Odin
and Obrink, 1987). C-CAM, with an apparent molecular weight of 105
kD, was originally isolated from the plasma membrane of the rat
hepatocyte by its reaction with specific antibodies that neutralize
cell aggregation (Obrink, 1991). Recent studies indicate that,
structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily
and its sequence is highly homologous to carcinoembryonic antigen
(CEA) (Lin and Guidotti, 1989). Using a baculovirus expression
system, Cheung et al. (1993a; 1993b and 1993c) demonstrated that
the first Ig domain of C-CAM is critical for cell adhesion
activity.
[0222] Cell adhesion molecules, or CAMs are known to be involved in
a complex network of molecular interactions that regulate organ
development and cell differentiation (Edelman, 1985). Recent data
indicate that aberrant expression of CAMs may be involved in the
tumorigenesis of several neoplasms; for example, decreased
expression of E-cadherin, which is predominantly expressed in
epithelial cells, is associated with the progression of several
kinds of neoplasms (Edelman and Crossin, 1991; Frixen et al., 1991;
Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al.,
1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing expression of .alpha..sub.5.beta..sub.1 integrin by gene
transfer can reduce tumorigenicity of Chinese hamster ovary cells
in vivo. C-CAM now has been shown to suppress tumor growth in vitro
and in vivo.
[0223] Other tumor suppressors that may be employed according to
the present invention include RB, APC, DCC, NF-1, NF-2, WT-1,
MEN-I, MEN-II, zac1, p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21,
p57, pTEN, C-CAM, p27, mda-7 and BRCA2. Inducers of apoptosis, such
as Bax, Bak, Bcl-X.sub.5, Bik, Bid, Harakiri, Ad E1B, Bad and
ICE-CED3 proteases, similarly could find use according to the
present invention.
[0224] Various enzyme genes are of interest according to the
present invention. Such enzymes include cytosine deaminase,
hypoxanthine-guanine phosphoribosyltmmsferase,
galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,
glucocerbrosidase, sphingomyelinase, .alpha.-L-iduronidase,
glucose-6-phosphate dehydrogenase, HSV thymidine kinase and human
thymidine kinase.
[0225] Hormones are another group of gene that may be used in the
vectors described herein. Included are growth hormone, prolactin,
placental lactogen, luteinizing hormone, follicle-stimulating
hormone, chorionic gonadotropin, thyroid-stimulating hormone,
leptin, adrenocorticotropin (ACTH), angiotensin I and II,
.beta.-endorphin, .beta.-melanocyte stimulating hormone
(.beta.-MSH), cholecystokinin, endothelin I, galanin, gastric
inhibitory peptide (GIP), glucagon, insulin, lipotropins,
neurophysins, somatostatin, calcitonin, calcitonin gene related
peptide (CGRP), .beta.-calcitonin gene related peptide,
hypercalcemia of malignancy factor (140), parathyroid
hormone-related protein (107-139) (PTH-rP), parathyroid
hormone-related protein (107-111) (PTH-rP), glucagon-like peptide
(GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,
secretin, vasoactive intestinal peptide (VIP), oxytocin,
vasopressin (AVP), vasotocin, enkephalinamide, metorphinamide,
alpha melanocyte stimulating hormone (alpha-MSH), atrial
natriuretic factor (5-28) (ANF), amylin, amyloid P component
(SAP-1), corticotropin releasing hormone (CRH), growth hormone
releasing factor (GHRH), luteinizing hormone-releasing hormone
(LHRH), neuropeptide Y, substance K (neurokinin A), substance P and
thyrotropin releasing hormone (TRH).
[0226] Other classes of genes that are contemplated to be inserted
into the vectors of the present invention include interleukins and
cytokines. Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.
[0227] Examples of diseases for which the present viral vector
would be useful include, but are not limited to, adenosine
deaminase deficiency, human blood clotting factor IX deficiency in
hemophilia B, and cystic fibrosis, which would involve the
replacement of the cystic fibrosis transmembrane receptor gene. The
vectors embodied in the present invention could also be used for
treatment of hyperproliferative disorders such as rheumatoid
arthritis or restenosis by transfer of genes encoding angiogenesis
inhibitors or cell cycle inhibitors. Transfer of prodrug activators
such as the HSV-TK gene can be also be used in the treatment of
hyperploiferative disorders, including cancer.
[0228] B) Antisense Constructs
[0229] Oncogenes such as ras, myc, neu, raf erb, src, fms, jun,
trk, ret, gsp, hst, bcl and abl also are suitable targets. However,
for therapeutic benefit, these oncogenes would be expressed as an
antisense nucleic acid, so as to inhibit the expression of the
oncogene. The term "antisense nucleic acid" is intended to refer to
the oligonucleotides complementary to the base sequences of
oncogene-encoding DNA and RNA. Antisense oligonucleotides, when
introduced into a target cell, specifically bind to their target
nucleic acid and interfere with transcription, RNA processing,
transport and/or translation. Targeting double-stranded (ds) DNA
with oligonucleotide leads to triple-helix formation; targeting RNA
will lead to double-helix formation.
[0230] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. Antisense RNA constructs, or DNA encoding
such antisense RNAs, may be employed to inhibit gene transcription
or translation or both within a host cell, either in vitro or in
vivo, such as within a host animal, including a human subject.
Nucleic acid sequences comprising "complementary nucleotides" are
those which are capable of base-pairing according to the standard
Watson-Crick complementarity rules. That is, that the larger
purines will base pair with the smaller pyrimidines to form only
combinations of guanine paired with cytosine (G:C) and adenine
paired with either thymine (A:T), in the case of DNA, or adenine
paired with uracil (A:U) in the case of RNA.
[0231] As used herein, the terms "complementary" or "antisense
sequences" mean nucleic acid sequences that are substantially
complementary over their entire length and have very few base
mismatches. For example, nucleic acid sequences of fifteen bases in
length may be termed complementary when they have a complementary
nucleotide at thirteen or fourteen positions with only single or
double mismatches. Naturally, nucleic acid sequences which are
"completely complementary" will be nucleic acid sequences which are
entirely complementary throughout their entire length and have no
base mismatches.
[0232] While all or part of the gene sequence may be employed in
the context of antisense construction, statistically, any sequence
17 bases long should occur only once in the human genome and,
therefore, suffice to specify a unique target sequence. Although
shorter oligomers are easier to make and increase in vivo
accessibility, numerous other factors are involved in determining
the specificity of hybridization. Both binding affinity and
sequence specificity of an oligonucleotide to its complementary
target increases with increasing length. It is contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 or more base pairs will be used. One can readily determine
whether a given antisense nucleic acid is effective at targeting of
the corresponding host cell gene simply by testing the constructs
in vitro to determine whether the endogenous gene's function is
affected or whether the expression of related genes having
complementary sequences is affected.
[0233] In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain C-5
propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of
gene expression (Wagner et al., 1993).
[0234] As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" refers to an RNA-based
enzyme capable of targeting and cleaving particular base sequences
in oncogene DNA and RNA. Ribozymes can either be targeted directly
to cells, in the form of RNA oligo-nucleotides incorporating
ribozyme sequences, or introduced into the cell as an expression
construct encoding the desired ribozymal RNA. Ribozymes may be used
and applied in much the same way as described for antisense nucleic
acids.
[0235] C) Antigens for Vaccines
[0236] Other therapeutics genes might include genes encoding
antigens such as viral antigens, bacterial antigens, fungal
antigens or parasitic antigens. Viruses include picornavirus,
coronavirus, togavirus, flavirviru, rhabdovirus, paramyxovirus,
orthomyxovirus, bunyavirus, arenvirus, reovirus, retrovirus,
papovavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and
spongiform virus. Preferred viral targets include influenza, herpes
simplex virus 1 and 2, measles, small pox, polio or HIV. Pathogens
include trypanosomes, tapeworms, roundworms, helminths. Also, tumor
markers, such as fetal antigen or prostate specific antigen, may be
targeted in this manner. Preferred examples include HIV env
proteins and hepatitis B surface antigen. Administration of a
vector according to the present invention for vaccination purposes
would require that the vector-associated antigens be sufficiently
non-immunogenic to enable long term expression of the transgene,
for which a strong immune response would be desired. Preferably,
vaccination of an individual would only be required infrequently,
such as yearly or biennially, and provide long term immunologic
protection against the infectious agent.
[0237] D) Control Regions
[0238] In order for the viral vector to effect expression of a
transcript encoding a therapeutic gene, the polynucleotide encoding
the therapeutic gene will be under the transcriptional control of a
promoter and a polyadenylation signal. A "promoter" refers to a DNA
sequence recognized by the synthetic machinery of the host cell, or
introduced synthetic machinery, that is required to initiate the
specific transcription of a gene. A polyadenylation signal refers
to a DNA sequence recognized by the synthetic machinery of the host
cell, or introduced synthetic machinery, that is required to direct
the addition of a series of nucleotides on the end of the mRNA
transcript for proper processing and trafficking of the transcript
out of the nucleus into the cytoplasm for translation. The phrase
"under transcriptional control" means that the promoter is in the
correct location in relation to the polynucleotide to control RNA
polymerase initiation and expression of the polynucleotide.
[0239] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0240] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0241] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either cooperatively or independently to activate
transcription.
[0242] The particular promoter employed to control the expression
of a nucleic acid sequence of interest is not believed to be
important, so long as it is capable of directing the expression of
the nucleic acid in the targeted cell. Thus, where a human cell is
targeted, it is preferable to position the nucleic acid coding
region adjacent to and under the control of a promoter that is
capable of being expressed in a human cell. Generally speaking,
such a promoter might include either a human or viral promoter.
[0243] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, .beta.-actin, rat insulin
promoter and glyceraldehyde-3-phosphate dehydrogenase can be used
to obtain high-level expression of the coding sequence of interest.
The use of other viral or mammalian cellular or bacterial phage
promoters which are well-known in the art to achieve expression of
a coding sequence of interest is contemplated as well, provided
that the levels of expression are sufficient for a given purpose.
By employing a promoter with well-known properties, the level and
pattern of expression of the protein of interest following
transfection or transformation can be optimized.
[0244] Selection of a promoter that is regulated in response to
specific physiologic or synthetic signals can permit inducible
expression of the gene product. For example in the case where
expression of a transgene, or transgenes when a multicistronic
vector is utilized, is toxic to the cells in which the vector is
produced in, it may be desirable to prohibit or reduce expression
of one or more of the transgenes. Examples of transgenes that may
be toxic to the producer cell line are pro-apoptotic and cytokine
genes. Several inducible promoter systems are available for
production of viral vectors where the tansgene product may be
toxic.
[0245] The ecdysone system (Invitrogen, Carlsbad, Calif.) is one
such system. This system is designed to allow regulated expression
of a gene of interest in mammalian cells. It consists of a tightly
regulated expression mechanism that allows virtually no basal level
expression of the transgene, but over 200-fold inducibility. The
system is based on the heterodimeric ecdysone receptor of
Drosophila, and when ecdysone or an analog such as muristerone A
binds to the receptor, the receptor activates a promoter to turn on
expression of the downstream transgene high levels of mRNA
transcripts are attained. In this system, both monomers of the
heterodimeric receptor are constitutively expressed from one
vector, whereas the ecdysone-responsive promoter which drives
expression of the gene of interest is on another plasmid.
Engineering of this type of system into the gene transfer vector of
interest would therefore be useful. Cotransfection of plasmids
containing the gene of interest and the receptor monomers in the
producer cell line would then allow for the production of the gene
transfer vector without expression of a potentially toxic
transgene. At the appropriate time, expression of the transgene
could be activated with ecdysone or muristeron A.
[0246] Another inducible system that would be useful is the
Tet-Off.TM. or Tet-On.TM. system (Clontech, Palo Alto, Calif.)
originally developed by Gossen and Bujard (Gossen and Bujard, 1992;
Gossen et al., 1995). This system also allows high levels of gene
expression to be regulated in response to tetracycline or
tetracycline derivatives such as doxycycline. In the Tet-On.TM.
system, gene expression is turned on in the presence of
doxycycline, whereas in the Tet-Off.TM. system, gene expression is
turned on in the absence of doxycycline. These systems are based on
two regulatory elements derived from the tetracycline resistance
operon of E. coli. The tetracycline operator sequence to which the
tetracycline repressor binds, and the tetracycline repressor
protein. The gene of interest is cloned into a plasmid behind a
promoter that has tetracycline-responsive elements present in it. A
second plasmid contains a regulatory element called the
tetracycline-controlled transactivator, which is composed, in the
Tet-Off.TM. system, of the VP16 domain from the herpes simplex
virus and the wild-type tertracycline repressor. Thus in the
absence of doxycycline, transcription is constitutively on. In the
Tet-On.TM. system, the tetracycline repressor is not wild type and
in the presence of doxycycline activates transcription. For gene
therapy vector production, the Tet-Off.TM. system would be
preferable so that the producer cells could be grown in the
presence of tetracycline or doxycycline and prevent expression of a
potentially toxic transgene, but when the vector is introduced to
the patient, the gene expression would be constitutively on.
[0247] In some circumstances, it may be desirable to regulate
expression of a transgene in a gene therapy vector. For example,
different viral promoters with varying strengths of activity may be
utilized depending on the level of expression desired. In mammalian
cells, the CMV immediate early promoter if often used to provide
strong transcriptional activation. Modified versions of the CMV
promoter that are less potent have also been used when reduced
levels of expression of the transgene are desired. When expression
of a transgene in hematopoetic cells is desired, retroviral
promoters such as the LTRs from MLV or MMTV are often used. Other
viral promoters that may be used depending on the desired effect
include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters
such as from the E1A, E2A, or MLP region, AAV LTR, cauliflower
mosaic virus, HSV-TK, and avian sarcoma virus.
[0248] Similarly tissue specific promoters may be used to effect
transcription in specific tissues or cells so as to reduce
potential toxicity or undesirable effects to non-targeted tissues.
For example, promoters such as the PSA, probasin, prostatic acid
phosphatase or prostate-specific glandular kallikrein (hK2) may be
used to target gene expression in the prostate. Similarly, the
following promoters may be used to target gene expression in other
tissues (Table 3).
TABLE-US-00003 TABLE 3 Tissue specific promoters Tissue Promoter
Pancreas Insulin elastin amylase pdr-1 pdx-1 glucokinase Liver
albumin PEPCK HBV enhancer alpha fetoprotein apolipoprotein C
alpha-1 antitrypsin vitellogenin, NF-AB Transthyretin Skeletal
muscle myosin H chain muscle creatine kinase dystrophin calpain p94
skeletal alpha-actin fast troponin 1 Skin keratin K6 keratin K1
Lung CFTR human cytokeratin 18 (K18) pulmonary surfactant proteins
A, B and C CC-10 P1 Smooth muscle sm22 alpha SM-alpha-actin
Endothelium endothelin-1 E-selectin von Willebrand factor TIE
(Korhonen et al., 1995) KDR/flk-1 Melanocytes Tyrosinase Adipose
tissue lipoprotein lipase (Zechner et al., 1988) adipsin
(Spiegelman et al., 1989) acetyl-CoA carboxylase (Pape and Kim,
1989) glycerophosphate dehydrogenase (Dani et al., 1989) adipocyte
P2 (Hunt et al., 1986) Blood .beta.-globin
[0249] In certain indications, it may be desirable to activate
transcription at specific times after administration of the gene
therapy vector. This may be done with such promoters as those that
are hormone or cytokine regulatable. For example in gene therapy
applications where the indication is a gonadal tissue where
specific steroids are produced or routed to, use of androgen or
estrogen regulated promoters may be advantageous. Such promoters
that are hormone regulatable include MMTV, MT-1, ecdysone and
RuBisco. Other hormone regulated promoters such as those responsive
to thyroid, pituitary and adrenal hormones are expected to be
useful in the present invention. Cytokine and inflammatory protein
responsive promoters that could be used include K and T Kininogen
(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein
(Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum
amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989),
Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid
glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin,
lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et
al., 1991), fibrinogen, c-jun (inducible by phorbol esters,
TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),
collagenase (induced by phorbol esters and retinoic acid),
metallothionein (heavy metal and glucocorticoid inducible),
Stromelysin (inducible by phorbol ester, interleukin-1 and EGF),
alpha-2 macroglobulin and alpha-1 antichymotrypsin.
[0250] It is envisioned that cell cycle regulatable promoters may
be useful in the present invention. For example, in a bi-cistronic
gene therapy vector, use of a strong CMV promoter to drive
expression of a first gene such as p16 that arrests cells in the G1
phase could be followed by expression of a second gene such as p53
under the control of a promoter that is active in the G1 phase of
the cell cycle, thus providing a "second hit" that would push the
cell into apoptosis. Other promoters such as those of various
cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1 could be used.
[0251] Tumor specific promoters such as osteocalcin,
hypoxia-responsive element (HRE), MAGE4, CEA, alpha-fetoprotein,
GRP78/BiP and tyrosinase may also be used to regulate gene
expression in tumor cells. Other promoters that could be used
according to the present invention include Lac-regulatable,
chemotherapy inducible (e.g. MDR), and heat (hyperthermia)
inducible promoters, Radiation-inducible (e.g., EGR (Joki et al.,
1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acid
promoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, -actin and
alpha-globin. Many other promoters that may be useful are listed in
Walther and Stein (1996).
[0252] It is envisioned that any of the above promoters alone or in
combination with another may be useful according to the present
invention depending on the action desired. In addition, this list
of promoters is should not be construed to be exhaustive or
limiting, those of skill in the art will know of other promoters
that may be used in conjunction with the promoters and methods
disclosed herein. A further list of promoters is provided in the
Table 4.
TABLE-US-00004 TABLE 4 PROMOTER Immunoglobulin Heavy Chain
Immunoglobulin Light Chain T-Cell Receptor HLA DQ .alpha. and DQ
.beta. .beta.-Interferon Interleukin-2 Interleukin-2 Receptor MHC
Class II 5 MHC Class II HLA-DR.alpha. .beta.-Actin Muscle Creatine
Kinase Prealbumin (Transthyretin) Elastase I Metallothionein
Collagenase Albumin Gene .alpha.-Fetoprotein .tau.-Globin
.beta.-Globin c-fos c-HA-ras Insulin Neural Cell Adhesion Molecule
(NCAM) .alpha.1-Antitrypsin H2B (TH2B) Histone Mouse or Type I
Collagen Glucose-Regulated Proteins (GRP94 and GRP78) Rat Growth
Hormone Human Serum Amyloid A (SAA) Troponin I (TN I)
Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40
Polyoma Retroviruses Papilloma Virus Hepatitis B Virus Human
Immunodeficiency Virus Cytomegalovirus Gibbon Ape Leukemia
Virus
[0253] The promoter further may be characterized as an inducible
promoter. An inducible promoter is a promoter which is inactive or
exhibits low activity except in the presence of an inducer
substance. Some examples of promoters that may be included as a
part of the present invention include, but are not limited to, MT
II, MMTV, Colleganse, Stromelysin, SV40, Murine M gene,
.alpha.-2-Macroglobulin, MHC class I gene h-2 kb, HSP70,
Proliferin, Tumor Necrosis Factor, or Thyroid Stimulating Hormone a
gene. The associated inducers are shown in Table 5. It is
understood that any inducible promoter may be used in the practice
of the present invention and that all such promoters would fall
within the spirit and scope of the claimed invention.
TABLE-US-00005 TABLE 5 Element Inducer MT II Phorbol Ester (TPA)
Heavy metals MMTV (mouse mammary tumor Glucocorticoids virus)
.beta.-Interferon poly(rI).times.poly(rc) Adenovirus 5 E2 Ela c-jun
Phorbol Ester (TPA), H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene
A23187 .alpha.-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene
H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol
Ester-TPA Tumor Necrosis Factor FMA Thyroid Stimulating Hormone
.alpha. Thyroid Hormone Gene
[0254] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter and the Rous
sarcoma virus long terminal repeat can be used to obtain high-level
expression of the polynucleotide of interest. The use of other
viral or mammalian cellular or bacterial phage promoters which are
well-known in the art to achieve expression of polynucleotides is
contemplated as well, provided that the levels of expression are
sufficient to produce a growth inhibitory effect.
[0255] By employing a promoter with well-known properties, the
level and pattern of expression of a polynucleotide following
transfection can be optimized. For example, selection of a promoter
which is active in specific cells, such as tyrosinase (melanoma),
alpha-fetoprotein and albumin (liver tumors), CC10 (lung tumor) and
prostate-specific antigen (prostate tumor) will permit
tissue-specific expression of the therapeutic gene.
[0256] Enhancers were originally detected as genetic elements that
increased transcription from a promoter located at a distant
position on the same molecule of DNA. This ability to act over a
large distance had little precedent in classic studies of
prokaryotic transcriptional regulation. Subsequent work showed that
regions of DNA with enhancer activity are organized much like
promoters. That is, they are composed of many individual elements,
each of which binds to one or more transcriptional proteins.
[0257] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0258] Additionally any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base (EPDB)) could also be used to drive
expression of a particular construct. Use of a T3, T7 or SP6
cytoplasmic expression system is another possible embodiment.
Eukaryotic cells can support cytoplasmic transcription from certain
bacteriophage promoters if the appropriate bacteriophage polymerase
is provided, either as part of the delivery complex or as an
additional genetic expression vector.
[0259] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed. Such polyadenylation signals as that from SV40, bovine
growth hormone, and the herpes simplex virus thymidine kinase gene
have been found to function well in a number of target cells.
7. METHODS OF GENE TRANSFER
[0260] In order to create the helper cell lines of the present
invention, and to create recombinant adenovirus vectors for use
therewith, various genetic (i.e. DNA) constructs must be delivered
to a cell. One way to achieve this is via viral transductions using
infectious viral particles, for example, by transformation with an
adenovirus vector of the present invention. Alternatively,
retroviral or bovine papilloma virus may be employed, both of which
permit permanent transformation of a host cell with a gene(s) of
interest. In other situations, the nucleic acid to be transferred
is not infectious, i.e., contained in an infectious virus particle.
This genetic material must rely on non-viral methods for
transfer.
[0261] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells also are contemplated by
the present invention. These include calcium phosphate
precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;
Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation
(Tur-Kaspa et al., 1986; Potter et al., 1984), direct
microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes
(Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication
(Fechheimer et al., 1987), gene bombardment using high velocity
microprojectiles (Yang et al., 1990), and receptor-mediated
transfection (Wu and Wu, 1987; Wu and Wu, 1988).
[0262] Once the construct has been delivered into the cell the
nucleic acid encoding the therapeutic gene may be positioned and
expressed at different sites. In certain embodiments, the nucleic
acid encoding the therapeutic gene may be stably integrated into
the genome of the cell. This integration may be in the cognate
location and orientation via homologous recombination (gene
replacement) or it may be integrated in a random, non-specific
location (gene augmentation). In yet further embodiments, the
nucleic acid may be stably maintained in the cell as a separate,
episomal segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle. How
the expression construct is delivered to a cell and where in the
cell the nucleic acid remains is dependent on the type of
expression construct employed.
[0263] In one embodiment of the invention, the expression construct
may simply consist of naked recombinant DNA or plasmids. Transfer
of the construct may be performed by any of the methods mentioned
above which physically or chemically permeabilize the cell
membrane. This is particularity applicable for transfer in vitro,
however, it may be applied for in vivo use as well. Dubensky et al.
(1984) successfully injected polyomavirus DNA in the form of
CaPO.sub.4 precipitates into liver and spleen of adult and newborn
mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) also demonstrated that direct
intraperitoneal injection of CaPO.sub.4 precipitated plasmids
results in expression of the transfected genes. It is envisioned
that DNA encoding a CAM may also be transferred in a similar manner
in vivo and express CAM.
[0264] Another embodiment of the invention for transferring a naked
DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate DNA
coated microprojectiles to a high velocity allowing them to pierce
cell membranes and enter cells without killing them (Klein et al.,
1987). Several devices for accelerating small particles have been
developed. One such device relies on a high voltage discharge to
generate an electrical current, which in turn provides the motive
force (Yang et al., 1990). The microprojectiles used have consisted
of biologically inert substances such as tungsten or gold
beads.
[0265] In a further embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and an
inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991).
[0266] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Using the
.beta.-lactamase gene, Wong et al. (1980) demonstrated the
feasibility of liposome-mediated delivery and expression of foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et
al (1987) accomplished successful liposome-mediated gene transfer
in rats after intravenous injection. Also included are various
commercial approaches involving "lipofection" technology.
[0267] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al, 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear nonhistone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression constructs have been successfully employed in
transfer and expression of nucleic acid in vitro and in vivo, then
they are applicable for the present invention.
[0268] Other expression constructs which can be employed to deliver
a nucleic acid encoding a therapeutic gene into cells are
receptor-mediated delivery vehicles. These take advantage of the
selective uptake of macromolecules by receptor-mediated endocytosis
in almost all eukaryotic cells. Because of the cell type-specific
distribution of various receptors, the delivery can be highly
specific (Wu and Wu, 1993).
[0269] Receptor-mediated gene targeting vehicles generally consist
of two components: a cell receptor-specific ligand and a
DNA-binding agent. Several ligands have been used for
receptor-mediated gene transfer. The most extensively characterized
ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and
transferrin (Wagner et al., 1990). Recently, a synthetic
neoglycoprotein, which recognizes the same receptor as ASOR, has
been used as a gene delivery vehicle (Ferkol et al., 1993; Perales
et al., 1994) and epidermal growth factor (EGF) has also been used
to deliver genes to squamous carcinoma cells (Myers, EPO
0273085).
[0270] In other embodiments, the delivery vehicle may comprise a
ligand and a liposome. For example, Nicolau et al. (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that a
nucleic acid encoding a therapeutic gene also may be specifically
delivered into a cell type such as prostate, epithelial or tumor
cells, by any number of receptor-ligand systems with or without
liposomes. For example, the human prostate-specific antigen (Watt
et al., 1986) may be used as the receptor for mediated delivery of
a nucleic acid in prostate tissue.
8. REMOVING NUCLEIC ACID CONTAMINANTS
[0271] The present invention employs nucleases to remove
contaminating nucleic acids. Exemplary nucleases include
Benzonase.RTM., Pulmozyme.RTM.; or any other DNase or RNase
commonly used within the art.
[0272] Enzymes such as Benzonaze.RTM. degrade nucleic acid and have
no proteolytic activity. The ability of Benzonase.RTM. to rapidly
hydrolyze nucleic acids makes the enzyme ideal for reducing cell
lysate viscosity. It is well known that nucleic acids may adhere to
cell derived particles such as viruses. The adhesion may interfere
with separation due to agglomeration, change in size of the
particle or change in particle charge, resulting in little if any
product being recovered with a given purification scheme.
Benzonase.RTM. is well suited for reducing the nucleic acid load
during purification, thus eliminating the interference and
improving yield.
[0273] As with all endonucleases, Benzonase.RTM. hydrolyzes
internal phosphodiester bonds between specific nucleotides. Upon
complete digestion, all free nucleic acids present in solution are
reduced to oligonucleotides 2 to 4 bases in length.
9. PURIFICATION TECHNIQUES
[0274] The present invention employs a number of different
purification to purify adenoviral vectors of the present invention.
Such techniques include those based on sedimentation and
chromatography and are described in more detail herein below.
[0275] A) Density Gradient Centrifugation
[0276] There are two methods of density gradient centrifugation,
the rate zonal technique and the isopycnic (equal density)
technique, and both can be used when the quantitative separation of
all the components of a mixture of particles is required. They are
also used for the determination of buoyant densities and for the
estimation of sedimentation coefficients.
[0277] Particle separation by the rate zonal technique is based
upon differences in size or sedimentation rates. The technique
involves carefully layering a sample solution on top of a performed
liquid density gradient, the highest density of which exceeds that
of the densest particles to be separated. The sample is then
centrifuged until the desired degree of separation is effected,
i.e., for sufficient time for the particles to travel through the
gradient to form discrete zones or bands which are spaced according
to the relative velocities of the particles. Since the technique is
time dependent, centrifugation must be terminated before any of the
separated zones pellet at the bottom of the tube. The method has
been used for the separation of enzymes, hormones, RNA-DNA hybrids,
ribosomal subunits, subcellular organelles, for the analysis of
size distribution of samples of polysomes and for lipoprotein
fractionations.
[0278] The sample is layered on top of a continuous density
gradient which spans the whole range of the particle densities
which are to be separated. The maximum density of the gradient,
therefore, must always exceed the density of the most dense
particle. During centrifugation, sedimentation of the particles
occurs until the buoyant density of the particle and the density of
the gradient are equal (i.e., where p.sub.p=p.sub.m in equation
2.12). At this point no further sedimentation occurs, irrespective
of how long centrifugation continues, because the particles are
floating on a cushion of material that has a density greater than
their own.
[0279] Isopycnic centrifugation, in contrast to the rate zonal
technique, is an equilibrium method, the particles banding to form
zones each at their own characteristic buoyant density. In cases
where, perhaps, not all the components in a mixture of particles
are required, a gradient range can be selected in which unwanted
components of the mixture will sediment to the bottom of the
centrifuge tube whilst the particles of interest sediment to their
respective isopycnic positions. Such a technique involves a
combination of both the rate zonal and isopycnic approaches.
[0280] Isopycnic centrifugation depends solely upon the buoyant
density of the particle and not its shape or size and is
independent of time. Hence soluble proteins, which have a very
similar density (e.g., p=1.3 g cm.sup.-3 in sucrose solution),
cannot usually be separated by this method, whereas subcellular
organelles (e.g., Golgi apparatus, p=1.11 g cm.sup.-1,
mitochondria, p=1.19 g cm.sup.-1 and peroxisomes, p=1.23 g
cm.sup.-1 in sucrose solution) can be effectively separated.
[0281] As an alternative to layering the particle mixture to be
separated onto a preformed gradient, the sample is initially mixed
with the gradient medium to give a solution of uniform density, the
gradient `self-forming`, by sedimentation equilibrium, during
centrifugation. In this method (referred to as the equilibrium
isodensity method), use is generally made of the salts of heavy
metals (e.g., caesium or rubidium), sucrose, colloidal silica or
Metrizamide.
[0282] The sample (e.g., DNA) is mixed homogeneously with, for
example, a concentrated solution of caesium chloride.
Centrifugation of the concentrated caesium chloride solution
results in the sedimentation of the CsCl molecules to form a
concentration gradient and hence a density gradient. The sample
molecules (DNA), which were initially uniformly distributed
throughout the tube now either rise or sediment until they reach a
region where the solution density is equal to their own buoyant
density, i.e. their isopycnic position, where they will band to
form zones. This technique suffers from the disadvantage that often
very long centrifugation times (e.g., 36 to 48 hours) are required
to establish equilibrium. However, it is commonly used in
analytical centrifugation to determine the buoyant density of a
particle, the base composition of double stranded DNA and to
separate linear from circular forms of DNA.
[0283] Many of the separations can be improved by increasing the
density differences between the different forms of DNA by the
incorporation of heavy isotopes (e.g., 15N) during biosynthesis, a
technique used by Leselson and Stahl to elucidate the mechanism of
DNA replication in Esherichia coli, or by the binding of heavy
metal ions or dyes such as ethidium bromide. Isopycnic gradients
have also been used to separate and purify viruses and analyze
human plasma lipoproteins.
[0284] B) Chromatography
[0285] In certain embodiments of the invention, it will be
desirable to produce purified adenovirus. Purification techniques
are well known to those of skill in the art. These techniques tend
to involve the fractionation of the cellular milieu to separate the
adenovirus particles from other components of the mixture. Having
separated adenoviral particles from the other components, the
adenovirus may be purified using chromatographic and
electrophoretic techniques to achieve complete purification.
Analytical methods particularly suited to the preparation of a pure
adenovrial particle of the present invention are ion-exchange
chromatography, size exclusion chromatography; polyacrylamide gel
electrophoresis. A particularly efficient purification method to be
employed in conjunction with the present invention is HPLC.
[0286] Certain aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an adenoviral particle. The term "purified" as
used herein, is intended to refer to a composition, isolatable from
other components, wherein the adenoviral particle is purified to
any degree relative to its naturally-obtainable form. A purified
adenoviral particle therefore also refers to an adenoviral
component, free from the environment in which it may naturally
occur.
[0287] Generally, "purified" will refer to an adenoviral particle
that has been subjected to fractionation to remove various other
components, and which composition substantially retains its
expressed biological activity. Where the term "substantially
purified" is used, this designation will refer to a composition in
which the particle, protein or peptide forms the major component of
the composition, such as constituting about 50% or more of the
constituents in the composition.
[0288] Various methods for quantifying the degree of purification
of a protein or peptide will be known to those of skill in the art
in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number". The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0289] There is no general requirement that the adenovirus, always
be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater-fold purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0290] Of course, it is understood that the chromatographic
techniques and other purification techniques known to those of
skill in the art may also be employed to purify proteins expressed
by the adenoviral vectors of the present invention. Ion exchange
chromatography and high performance liquid chromatography are
exemplary purification techniques employed in the purification of
adenoviral particles and are described in further detail herein
below.
[0291] Ion-Exchange Chromatography. The basic principle of
ion-exchange chromatography is that the affinity of a substance for
the exchanger depends on both the electrical properties of the
material and the relative affinity of other charged substances in
the solvent. Hence, bound material can be eluted by changing the
pH, thus altering the charge of the material, or by adding
competing materials, of which salts are but one example. Because
different substances have different electrical properties, the
conditions for release vary with each bound molecular species. In
general, to get good separation, the methods of choice are either
continuous ionic strength gradient elution or stepwise elution. (A
gradient of pH alone is not often used because it is difficult to
set up a pH gradient without simultaneously increasing ionic
strength.) For an anion exchanger, either pH and ionic strength are
gradually increased or ionic strength alone is increased. For a
cation exchanger, both pH and ionic strength are increased. The
actual choice of the elution procedure is usually a result of trial
and error and of considerations of stability. For example, for
unstable materials, it is best to maintain fairly constant pH.
[0292] An ion exchanger is a solid that has chemically bound
charged groups to which ions are electrostatically bound; it can
exchange these ions for ions in aqueous solution. Ion exchangers
can be used in column chromatography to separate molecules
according to charge; actually other features of the molecule are
usually important so that the chromatographic behavior is sensitive
to the charge density, charge distribution, and the size of the
molecule.
[0293] The principle of ion-exchange chromatography is that charged
molecules adsorb to ion exchangers reversibly so that molecules can
be bound or eluted by changing the ionic environment. Separation on
ion exchangers is usually accomplished in two stages: first, the
substances to be separated are bound to the exchanger, using
conditions that give stable and tight binding; then the column is
eluted with buffers of different pH, ionic strength, or composition
and the components of the buffer compete with the bound material
for the binding sites.
[0294] An ion exchanger is usually a three-dimensional network or
matrix that contains covalently linked charged groups. If a group
is negatively charged, it will exchange positive ions and is a
cation exchanger. A typical group used in cation exchangers is the
sulfonic group, SO.sub.3.sup.-. If an H.sup.+ is bound to the
group, the exchanger is said to be in the acid form; it can, for
example, exchange on H.sup.+ for one Na.sup.+ or two H.sup.+ for
one Ca.sup.2+. The sulfonic acid group is called a strongly acidic
cation exchanger. Other commonly used groups are phenolic hydroxyl
and carboxyl, both weakly acidic cation exchangers. If the charged
group is positive--for example, a quaternary amino group--it is a
strongly basic anion exchanger. The most common weakly basic anion
exchangers are aromatic or aliphatic amino groups.
[0295] The matrix can be made of various material. Commonly used
materials are dextran, cellulose, agarose and copolymers of styrene
and vinylbenzene in which the divinylbenzene both cross-links the
polystyrene strands and contains the charged groups. Table 6 gives
the composition of many ion exchangers.
[0296] The total capacity of an ion exchanger measures its ability
to take up exchangeable groups per milligram of dry weight. This
number is supplied by the manufacturer and is important because, if
the capacity is exceeded, ions will pass through the column without
binding.
TABLE-US-00006 TABLE 6 Matrix Exchanger Functional Group Tradename
Dextran Strong Cationic Sulfopropyl SP-Sephadex Weak Cationic
Carboxymethyl CM-Sephadex Strong Anionic Diethyl-(2- QAE-Sephadex
hydroxypropyl)- aminoethyl Weak Anionic Diethylaminoethyl
DEAE-Sephadex Cellulose Cationic Carboxymethyl CM-Cellulose
Cationic Phospho P-cel Anionic Diethylaminoethyl DEAE-cellulose
Anionic Polyethylenimine PEI-Cellulose Anionic Benzoylated-
DEAE(BND)-cellulose naphthoylated, deiethylaminoethyl Anionic
p-Aminobenzyl PAB-cellulose Styrene- Strong Cationic Sulfonic acid
AG 50 divinyl- Strong Anionic AG 1-Source15Q benzene Strong
Cationic + Sulfonic acid + AG 501 Strong Anionic
Tetramethylammonium Acrylic Weak Cationic Carboxylic Bio-Rex 70
Strong Anionic Trimethylamino- E. Merk ethyl Strong Anionic
Trimethylamino Toso Haas TSK-Gel-Q- group 5PW Phenolic Strong
Cationic Sulfonic acid Bio-Rex 40 Expoxyamine Weak Anionic Tertiary
amino AG-3
[0297] The available capacity is the capacity under particular
experimental conditions (i.e., pH, ionic strength). For example,
the extent to which an ion exchanger is charged depends on the pH
(the effect of pH is smaller with strong ion exchangers). Another
factor is ionic strength because small ions near the charged groups
compete with the sample molecule for these groups. This competition
is quite effective if the sample is a macromolecule because the
higher diffusion coefficient of the small ion means a greater
number of encounters. Clearly, as buffer concentration increases,
competition becomes keener.
[0298] The porosity of the matrix is an important feature because
the charged groups are both inside and outside the matrix and
because the matrix also acts as a molecular sieve. Large molecules
may be unable to penetrate the pores; so the capacity will decease
with increasing molecular dimensions. The porosity of the
polystyrene-based resins is determined by the amount of
cross-linking by the divinylbenzene (porosity decreases with
increasing amounts of divinylbenzene). With the Dowex and AG
series, the percentage of divinylbenzene is indicated by a number
after an X--hence, Dowex 50-X8 is 8% divinylbenzene
[0299] Ion exchangers come in a variety of particle sizes, called
mesh size. Finer mesh means an increased surface-to-volume ration
and therefore increased capacity and decreased time for exchange to
occur for a given volume of the exchanger. On the other hand, fine
mesh means a slow flow rate, which can increase diffusional
spreading. The use of very fine particles, approximately 10 .mu.m
in diameter and high pressure to maintain an adequate flow is
called high-performance or high-pressure liquid chromatography or
simply HPLC.
[0300] Such a collection of exchangers having such different
properties--charge, capacity, porosity, mesh--makes the selection
of the appropriate one for accomplishing a particular separation
difficult. How to decide on the type of column material and the
conditions for binding and elution is described in the following
Examples.
[0301] There are a number of choices to be made when employing ion
exchange chromatography as a technique. The first choice to be made
is whether the exchanger is to be anionic or cationic. If the
materials to be bound to the column have a single charge (i.e.,
either plus or minus), the choice is clear. However, many
substances (e.g., proteins, viruses), carry both negative and
positive charges and the net charge depends on the pH. In such
cases, the primary factor is the stability of the substance at
various pH values. Most proteins have a pH range of stability
(i.e., in which they do not denature) in which they are either
positively or negatively charged. Hence, if a protein is stable at
pH values above the isoelectric point, an anion exchanger should be
used; if stable at values below the isoelectric point, a cation
exchanger is required.
[0302] The choice between strong and weak exchangers is also based
on the effect of pH on charge and stability. For example, if a
weakly ionized substance that requires very low or high pH for
ionization is chromatographed, a strong ion exchanger is called for
because it functions over the entire pH range. However, if the
substance is labile, weak ion exchangers are preferable because
strong exchangers are often capable of distorting a molecule so
much that the molecule denatures. The pH at which the substance is
stable must, of course, be matched to the narrow range of pH in
which a particular weak exchanger is charged. Weak ion exchangers
are also excellent for the separation of molecules with a high
charge from those with a small charge, because the weakly charged
ions usually fail to bind. Weak exchangers also show greater
resolution of substances if charge differences are very small. If a
macromolecule has a very strong charge, it may be impossible to
elute from a strong exchanger and a weak exchanger again may be
preferable. In general, weak exchangers are more useful than strong
exchangers.
[0303] The Sephadex and Bio-gel exchangers offer a particular
advantage for macromolecules that are unstable in low ionic
strength. Because the cross-links in these materials maintain the
insolubility of the matrix even if the matrix is highly polar, the
density of ionizable groups can be made several times greater than
is possible with cellulose ion exchangers. The increased charge
density means increased affinity so that adsorption can be carried
out at higher ionic strengths. On the other hand, these exchangers
retain some of their molecular sieving properties so that sometimes
molecular weight differences annul the distribution caused by the
charge differences; the molecular sieving effect may also enhance
the separation.
[0304] Small molecules are best separated on matrices with small
pore size (high degree of cross-linking) because the available
capacity is large, whereas macromolecules need large pore size.
However, except for the Sephadex type, most ion exchangers do not
afford the opportunity for matching the porosity with the molecular
weight.
[0305] The cellulose ion exchangers have proved to be the best for
purifying large molecules such as proteins and polynucleotides.
This is because the matrix is fibrous, and hence all functional
groups are on the surface and available to even the largest
molecules. In may cases however, beaded forms such as DEAE-Sephacel
and DEAE-Biogel P are more useful because there is a better flow
rate and the molecular sieving effect aids in separation.
[0306] Selecting a mesh size is always difficult. Small mesh size
improves resolution but decreases flow rate, which increases zone
spreading and decreases resolution. Hence, the appropriate mesh
size is usually determined empirically.
[0307] Because buffers themselves consist of ions, they can also
exchange, and the pH equilibrium can be affected. To avoid these
problems, the rule of buffers is adopted: use cationic buffers with
anion exchangers and anionic buffers with cation exchangers.
Because ionic strength is a factor in binding, a buffer should be
chosen that has a high buffering capacity so that its ionic
strength need not be too high. Furthermore, for best resolution, it
has been generally found that the ionic conditions used to apply
the sample to the column (the so-called starting conditions) should
be near those used for eluting the column.
[0308] High Performance Liquid Chromatography (HPLC) is
characterized by a very rapid separation with extraordinary
resolution of peaks. This is achieved by the use of very fine
particles and high pressure to maintain an adequate flow rate.
Separation can be accomplished in a matter of minutes, or at most
an hour. Moreover, only a very small volume of the sample is needed
because the particles are so small and close-packed that the void
volume is a very small fraction of the bed volume. Also, the
concentration of the sample need not be very great because the
bands are so narrow that there is very little dilution of the
sample.
10. QUALITY CONTROL ASSAYS
[0309] Recombinant adenovirus vectors made according to the present
invention are tested to ensure that they meet desired product
release specifications. These specifications are defined by assays
for biological activity, virus titer, final product purity,
identity and physico-chemical characteristics. These assays are
performed at various stages of production including analysis of the
crude cell lysate, in-process bulk (pre-filter), in-process bulk
(post-filter), and the final product. Crude cell lysate is defined
as the material that is removed from the cell culture apparatus
before any processing has been done. In-process bulk (prefilter) is
defined as the material that has been processed through the HPLC
purification step, but has not been sterile filtered prior to
vialing. In-process bulk (post filter) is defined as the material
that has been sterile filtered and is ready to be vialed. Final
product is defined as the material that has been placed into
individual vials and is ready for storage or use. It will be
understood that similar protocols may be used as tests for
Ad5CMV-p53 as well as other adenoviral vectors containing the same
or different transgenes. The following section describes
representative assays used for testing the recombinant adenovirus
product.
A. Safety Assays
[0310] General Safety Assay
[0311] The general safety assay test (C.F.R. 610.11) is performed
to detect the presence of extraneous toxic contaminants. Guinea
pigs (Hartley albino, either sex) and mice (Swiss outbred, either
sex) are inoculated intraperitoneally with the test article diluted
in sterile water for injection and observed for overt signs of ill
health, weight loss, or death for the test period. Their weights
are measured just prior to and upon completion of the test period
of 7 days. A passing test is one in which the controls perform as
expected and the animals inoculated with the test article have
satisfactory responses.
[0312] PCR Assay for the Detection of Adeno-Associated Virus (AAV)
in Biological Samples
[0313] This assay detects the presence of AAV nucleic acid
sequences by PCR amplification with a set of primers targeted to a
conserved region in the capsid gene. The amplified DNA from the
test article is run on an agarose gel containing ethidium bromide
and visualized by photography. Briefly, the DNA is extracted from
the test sample, and 0.5 micrograms is analyzed by PCR. PCR
amplification is performed using AAV oligonucleotides primers
specific for the capsid region of AAV. Negative and positive
control DNA is also analyzed. Assay acceptance is determined by the
absence of any bands in the negative control sample, and the
expected size band in the positive control sample. For the present
assay, a specific 459 bp band is the expected size. A passing test
for the test article is the absence of the 459 base pair band.
[0314] In Vitro Assay for the Presence of Viral Contaminants
[0315] This assay determines whether adventitious viral
contaminants are present in the test article through the
inoculation and observation of three types of indicator cells. The
presence of viral contamination is determined by observations for
cytopathic effect (CPE) or other visually discernible effects,
hemagglutination, and hemadsorption. The indicator cells include
MRC-5, a diploid human lung line; Vero, an African green monkey
kidney line; and HeLa, a human epithelioid carcinoma cell line.
Briefly, the three indicator cell lines are seeded into 6-well
plates and maintained for approximately 24 hours. The cultures are
then inoculated with 0.5 ml of the adenoviral sample or virus
controls and allowed to absorb for 1 hours at 36 degrees Celsius.
The virus is then removed and replaced with culture medium, and the
wells observed for 14 days for evidence of CPE. Each well is also
tested for hemadsorption and hemagglutination using three types of
erythrocytes. All culture fluids are blind passaged onto additional
culture plates of indicator cells and observed for CPE for another
14 days.
[0316] To accept this assay, certain criteria should preferably be
met. These include: 1) each of the positive control viruses should
preferably cause CPE in the indicator cell lines into which it is
inoculated; 2) each of the positive control viruses should
preferably produce hemadsorption and/or hemagglutination with at
least one type of erythrocyte at 4 degrees Celsius and/or 36
degrees Celsius at one or more time points with each of the
indicator cells lines into which it is inoculated; 3) The indicator
cells lines inoculated with the negative control should preferably
not exhibit any CPE, hemadsorption, or hemagglutination. A passing
test for the test article is preferably the absence of CPE,
hemadsorption and hemagglutination.
[0317] In Vivo Adventitious Virus Assay
[0318] This assay is designed to detect the presence of viruses
which do not cause a discernible effect in in vitro cell culture
systems, but may cause unwanted effects in vivo. The experimental
design utilizes inoculations of adult and suckling mice, guinea
pigs, and embryonated hens' eggs, and is similar to that used by
the British Institute for Biological Standards and Control. This
test includes blind passages of homogenates to successive animals
and/or hens eggs to increase the likelihood of detection of low
level viral contaminants.
[0319] The test method is as follows. Suckling mice will be
inoculated intraperitoneally, per os, and intracranially and
observed for 14 days. A single pool of emulsified tissue (minus
skin and gastrointestinal tract) of all surviving mice will be used
to inoculate additional mice using the same routes. Sham control
mice will also be inoculated. Adult mice of both sexes will be
inoculated intraperitoneally, per os, intradermally, and
intracranially and observed for 28 days. Sham controls will also be
inoculated. Adult guinea pigs of both sexes will be inoculated
intraperitoneally and intracranially and observed for 28 days. Sham
control guinea pigs will also be inoculated. The yolk sac of 6-7
days old embryonated hens' eggs will be inoculated and incubated at
least nine days. The yolk sacs will be harvested, pooled, and a 10%
suspension will be sub-passaged into new embryonated hens' eggs.
Nine days later, the eggs are evaluated for viability.
[0320] Acceptance criteria for the assay include healthy animals at
the start of the testing, and the tests will be considered valid if
about 90% of control adult mice, about 80% of control suckling
mice, about 80% of the embryonated hens' eggs, and about 75% of the
control guinea pigs survive the incubation period and show no
lesions at the site of inoculation or show no signs of viral
infection. The test article will be considered not contaminated if
about 80% of the animals remain healthy and survive the observation
period and if about 95% the animals used in the test fail to show
any lesions of any kind at the site of injection and fail to show
any signs of viral infection.
B. Purity Assays
[0321] BCA Assay for Total Protein
[0322] This assay allows for a quantitative determination of total
protein in the final product. The assay uses the Pierce BCA kit
procedure. Briefly, replicate samples are prepared and placed in a
microtiter plate. A Bovine Serum Albumin (BSA) standard is prepared
and placed in a microtiter plate as a control. For a negative
control, diluent is placed in a microtiter plate. The BCA reagent
is dispensed into the microtiter plates and the plates are
incubated to allow color development. The plates are then read
spectrophotometrically at 550 nm, and the test sample
concentrations are calculated based on the BSA standard. Preferred
protein content by BCA is 250 to 500 micrograms per 1.times.10e12
viral particles. Most preferable protein content is 260 to 320
micrograms per 1.times.10e12 viral particles. The protein
concentration determined by this assay is used to calculate the
amount of protein to load on the SDS-PAGE gel for restriction
analysis.
[0323] Sterility Assay
[0324] Sterility assays (documented in U.S.P. XXIII <71>) are
used at both the bulk and final product stage. Sterility testing is
via membrane filtration and is performed in a soft-wall isolator
system to minimize laboratory contamination of samples tested. All
test articles should preferably pass the sterility test.
[0325] Bioburden Test
[0326] The bioburden test is used to detect microbial load in a
test sample by filtering the test sample onto a membrane filter,
placing the membrane filter onto Tryptic Soy agar and Sabourad agar
plates and observing for growth after 2-5 days incubation.
Suspensions with known levels of Bacillus subtilis and Candid
albicans are also assayed to confirm assay suitability.
[0327] Briefly the test method is as follows. Test samples may be
stored up to 24 hours at 2-8 degree Celsius before testing. Reserve
samples that are not to be tested within 24 hours may be frozen at
less than 60 degrees Celsius. Negative controls (sterile diluent)
are prepared by filtering 100 mL of sterile diluent through an
analytical filter unit using a vacuum. The membrane filter is
removed from the unit and placed on a pre-warmed Tryptic Soy agar
plate. The process is repeated using a second filter unit and the
filter is placed on a pre-warmed Sabouraud agar plate. In-process
test samples are tested by filtering 5.times.10 mL of crude cell
lysate onto 5 separate filters or 10 mL of prefiltered bulk product
onto a single filter. Each membrane filter is removed from the unit
and placed on a pre-warmed Tryptic Soy agar plate. The process is
repeated using a second set of filter units and the filter is
placed on a pre-warmed Sabouraud agar plate. Bacillus subtilis
positive controls are prepared by filtering 50 mL of sterile
diluent through an analytical filter unit using a vacuum. The
membrane filter is removed from the unit and placed on a pre-warmed
Tryptic Soy agar plate. The process is repeated using a Candida
albicans positive control using a second filter unit and the filter
is placed on a pre-warmed Sabouraud agar plate. Tryptic Soy agar
plates are incubated at 30-35 degrees Celsius for 2-5 days.
Sabouraud agar plates are incubated at 22-27 degrees Celsius for
5-7 days. Colonies on the membrane filters are counted after the
incubation period. The assays are acceptable when the negative
controls exhibit no growth and positive controls exhibit 1-100
colonies per membrane filter. The test article should preferably
contain less than or equal to 1000 colony forming units per 100 mL
of the crude cell lysate. It is more preferable that the crude cell
lysate contain less than or equal to 500 colony forming units per
100 mL, and most preferable that the crude cell lysate contain less
than or equal to 10 colony forming units per 100 mL. It is most
preferable that the prefiltered bulk product contain less than or
equal to one colony forming unit per 10 mL. Using purification
techniques in accordance with the present disclosure, bioburden
values less than 1 have been obtained at the crude cell lysate
step, and less than 1 at the prefiltered bulk product step.
[0328] Bacterial Endotoxin Test
[0329] The purpose of this test is to measure the amount of gram
negative bacterial endotoxin in a given sample. The Limulus
Amebocyte Lysate (LAL) assay is performed in accordance with
USPXXIII using a commercial chromogenic test kit. It is used to
quantify the gram-negative bacterial endotoxin level in test
samples. Dilutions of samples are run with and without a spike of
endotoxin for evaluation of inhibition or enhancement effects.
[0330] The test method is performed according to the directions
outlined in the test kit insert, and is as follows. The assay is
performed in 96 well plates and LAL-free water is used as an assay
blank. A standard curve ranging from 0.01 to 5.0 endotoxin units/mL
is made using commercially available exdotoxin standard. Test
samples are tested either neat or diluted appropriately in
endotoxin free water. Positive controls are prepared by spiking
test samples at each dilution with 0.05 EU/mL. All manipulations
are performed in pyrogen free glass or polystyrene tubes using
pyrogen free pipette tips. The 96 well plate is incubated with
blank, standard curve, test samples, and positive control for 10
minutes, after which the LAL reagent is added to each well. The
plate is read in a kinetic reader at 405 nm for 150 seconds and the
results are expressed in EU/mL.
[0331] For the assay to be acceptable, the standard curve should
preferably be linear with an r value of -0.980 to 1.000, the slope
of the curve should preferably be -0.0. to -0.100, the Y-intercept
should preferably be 2.5000 to 3.5000 and endotoxin recovery in the
positive control should preferably be 5-150% of the spike. It is
preferable that the sample have less than five (5) EU/mL, more
preferably the sample have less than 3 EU/mL, and most preferable
that the sample have less than 0.05 EU/mL. Using purification
techniques in accordance with the present disclosure, endotoxin
values as low as 0.15 have been obtained at the prefiltered bulk
product step and as low as 0.3 at the final product step.
[0332] Test for the Presence of Agar-Cultivable and Non-Cultivable
Mycoplasmas
[0333] This assay detects the presence of Mycoplasma in a test
article based on the ability of Mycoplasma to grow in any one of
the test systems: Agar isolation and Vero cell culture system.
Growth is signified by colony formation, shift in pH indicators, or
presence of Mycoplasma by staining, depending on the system used.
The assay is performed using a large sample volume. The test
methods are as follows. The test article and positive controls are
inoculated directly onto Mycoplasma agar plates and into Mycoplasma
semi-solid broth which is subcultured three times onto agar plates.
The samples are incubated both aerobically and anaerobically. At 14
days post-infection the agar plates are examined for evidence of
growth. The test article is also inoculated directly onto Vero cell
cultures and incubated for 3-5 days. The cultures are stained with
a DNA-binding fluorochrome and evaluated microscopically by
epifluorescence for the presence of Mycoplasma.
[0334] For the Agar isolation assay, the positive controls should
preferably show Mycoplasma growth in at least two out of five
direct plates for each media type and for each incubation
condition, and in the semi-broth. The negative control plates and
bottles should preferably show absence of Mycoplasma growth. For
the Vero cell culture assay, positive controls should preferably
show the presence of Mycoplasma, negative controls should
preferably show no presence of Mycoplasma, and all of the controls
should preferably show the absence of bacterial or fungal
contaminants. The test article will preferably be negative for the
presence of Mycoplasma.
[0335] Contaminating Host Cell DNA Assay
[0336] This method allows evaluation of contaminating host cell DNA
in a final product. Test samples are extracted and examined for
contaminating DNA. The test method is as follows. Samples are
extracted and transferred to nitrocellulose. Diluted reference
samples are spiked with human DNA and transferred to
nitrocellulose. Positive controls are prepared by spiking human DNA
into aliquots of BSA and transferred to nitrocellulose. The
nitrocellulose with all samples and controls is probed with a
.sup.32P-labeled human DNA probe. The filter is rinsed and the
hybridized radioactivity is measured using an AMBIS Radioanalytic
Imaging System. Acceptable performance of the assay is determined
by the controls performing as expected, and a test article should
preferably have less than or equal to 10 ng contaminating host cell
DNA per 1.times.10.sup.12 viral particles. It is more preferable
that the level of contaminating human DNA be less than 7
ng/1.times.10.sup.12 viral particles, even more preferable that the
level of contaminating human DNA be less than 5
ng/1.times.10.sup.12 viral particles, even more preferable that the
level of contaminating human DNA be less than 3
ng/1.times.10.sup.12 viral particles and most preferable that the
level of contaminating human DNA be less than 5
pg/1.times.10.sup.12 viral particles. Using purification techniques
in accordance with the present disclosure, contaminating DNA values
as low as 200 pg/mL have been obtained at the final product step
and 80 pg/mL in a developmental batch.
[0337] Quantitative Replication Competent Adenovirus (RCA)
Assay.
[0338] The RCA present in a recombinant-defective adenovirus
population such as Ad5CMV-p53 are detected by infection of
non-competent A549 human carcinoma cells. A549 cells are grown in
cell culture dishes to give a monolayer of cells and are then
infected with the adenovirus sample to be tested. After 4 hours of
infection time, the supernatant is discarded and the A549 monolayer
is covered by a mixture containing both culture media and agarose.
After solidification, the agarose limits any infected cell to
formation of a single plaque. After 14 days at 37 degrees Celsius,
agarose is stained with neutral red and the visualized plaques are
counted. Positive controls are run concomitantly and contain either
wild type adenovirus alone or the test article spiked with wild
type adenovirus such that any inhibitory effect coming from the
sample could be detected. In order to characterize any observed
RCA, all plaques are subcultured and PCR characterized. PCR
analysis is performed using probes targeted against the E1 region
in order to demonstrate the presence of E1 region in the vector,
and against the E3 region to exclude the presence of wild type
viruses. It has been demonstrated that the presence of E1 excludes
the presence of the p53 gene and that the RCA consist of only
double homologous constructions.
[0339] The test methodology is as follows. A human lung carcinoma
line, A549, is grown to sub-confluence in cell culture dishes and
then infected with the Ad5CMV-p53 sample to be tested at an MOI of
less than 200 viral particles per cell. The cells are then exposed
to the virus for a 4 hour infection time, the supernatant is
discarded, and the cell monolayer is covered with a media/agarose
overlay. One positive control containing wild type adenovirus and
one containing the test sample spiked with wild type adenovirus are
run concomitantly to assure assay sensitivity. After a 14 day
incubation at 37 degrees Celsius the overlay is stained with
neutral red to allow visualization of any plaques. Plaques are
counted, picked and transferred to 0.8 mL of culture media and
subjected to three freeze-thaw cycles to release virus. The plaque
supernatant is then used to infect additional multi well dishes of
A549 cells. The cells are observed for CPE and the supernatant from
those dishes is harvested. The harvested supernatant is subjected
to amplification by PCR using probes directed against the E1 region
of the wild type adenovirus genome and against the E3 region of the
wild type adenovirus virus. If the E3 region is present the RCA is
scored as wild type. If only the E1 region is present the RCA is
scored as a double homologous recombination product. For the assay
to be considered valid, all controls must perform as expected. It
is preferable that the test article contain less than 40 plaque
forming units in 1.times.10.sup.11 viral particles. It is more
preferable that the test article contain less than 4 plaque forming
units in 1.times.10.sup.11 viral particles, and most preferable
that the test article contain less than 0.4 plaque forming units in
1.times.10.sup.11 viral particles. Using purification techniques in
accordance with the present disclosure, RCA values .ltoreq.1 in
2.5.times.10.sup.11 virus particles have been obtained at the final
product step.
[0340] Determination of BSA Levels
[0341] This assay is used to determine levels of contaminating
bovine serum albumin (BSA) in adenoviral preparations. In certain
recombinant adenovirus production runs, the vector is produced in a
cell culture system containing bovine serum. This assay is an
enzyme linked immunosorbent assay (ELISA) that detects the presence
and quantity of low levels of BSA that remain in the final
product.
[0342] The test method is as follows. A standard curve ranging from
1.9 ng to 1125 ng/mL of purified BSA is prepared. A positive
control is prepared by spiking 0.2% gelatin with 3.9, 15, and 62.5
ng/mL BSA. A negative control sample is 0.2% gelatin in Tris
buffered saline. The test sample is tested neat and at dilutions of
1:10 through 1:320. All samples and controls are transferred to an
ELISA assay plate, and the BSA content is detected with a probe
antibody specific for BSA. The plates are read at 492 nm. For the
assay to be considered valid, the blank OD.sub.492 should
preferably be less than 0.350. The test article should preferably
contain less than 100 ng BSA per 1.times.10.sup.12 viral particles.
It is more preferable that the test article contain less than 85 ng
BSA per 1.times.10.sup.12 viral particles, even more preferable
that the test article contain less than 75 ng BSA per
1.times.10.sup.12 viral particles, even more preferable that the
test article contain less than 65 ng BSA per 1.times.10.sup.12
viral particles and most preferable that the test article contain
less than 1 ng BSA per 1.times.10.sup.12 viral particles. Using
purification techniques in accordance with the present disclosure,
BSA values <1.9 ng/1.times.10.sup.12 virus particles have been
obtained at the final product step.
[0343] P53 Mutation Assay
[0344] This assay is to demonstrate the ability of p53 expressed
from AdSCMV-p53 final product to activate transcription. The
critical biochemical function of p53, which underlies its tumor
suppressor activity, is the ability to activate transcription.
Mutant proteins fail to activate transcription in mammalian cells.
The transcriptional activity of human p53 is conserved in yeast,
and mutant which are inactive in human cells are also inactive in
yeast. The detection of p53 mutations is possible in yeast by
testing the transcriptional competence of human p53 expressed in a
Saccharomyces cerevisiae defective in adenine synthesis due to a
mutation in ADE2 but which contains a second copy of ADE2 in an
open reading frame controlled by a p53 responsive promoter. The
Saccharomyces cerevisiae strain is cotransformed with a linearized
plasmid and the isolated p53 fragment from Ad5CMV-p53. Recombinants
will constitutively express p53. When grown on adenine poor media,
the yeast strain will appear red. If the yeast carries a wild-type
p53 gene the colonies will appear white.
[0345] The test method is as follows. DNA from the test article is
extracted using a phenol/chloroform/isoamyl alcohol procedure and
the p53 DNA insert from the adenoviral genome is isolated following
restriction digestion. An expression vector containing the ADH1
promoter is linearized. Yeast (strain yIG397) is co-transformed
with the DNA fragment bearing the p53 gene from the test article
and the linearized expression vector. A p53 expression vector is
formed in vivo by homologous recombination. The yeast cultures are
grown for two to three days at 30 degrees Celsius. The ADH1
promoter causes recombinants to constitutively express p53. The
yIG397 strain of yeast is defective in adenine synthesis because of
a mutation in the endogenous ADE2 gene, but it contains a second
copy of the ADE2 open reading frame controlled by the
p53-responsive ADH1 promoter. The colonies of yIG397 that are ADE2
mutant turn red when grown on low adenine plates. Colonies of
yIG397 with mutant p53 are also red, and colonies containing wild
type p53 are white. Red and white colonies are counted at the end
of the assay. The assay is considered valid if all the controls
perform as expected, and the test article should preferably contain
p53 mutations at a frequency of less than 3% to pass product
release specifications. It is more preferable that the test article
contain less than 2% p53 mutations, and most preferable that the
test article contain 0% p53 mutations. Using purification
techniques in accordance with the present disclosure, p53 mutations
values .ltoreq.1% have been obtained at the final product step.
[0346] Plaque Assay for Adenoviral Vectors
[0347] This assay is used to determine the titer of adenoviral
material in the final product by measuring the development of
plaques on human 293 cells, which are derived from human embryonic
kidney. Ad5CMV-p53 is replication deficient on normal cells due to
deletion of the E1 region. The E1 function is provided in trans in
293 cells which contain the E1 region of adenovirus type 5. Five
fold dilutions of the test article are utilized to quantify the
titer.
[0348] The test method is as follows. Hunan 293 cells are seeded in
66 well tissue culture plates and the cells are allowed to grow to
greater than 90% confluence before infection. Vector dilutions are
made to target 5-80 plaques per well. A reference virus is used as
a control. Two concentrations are tested for the positive control
using six replicates. Four concentrations are tested for each
sample using six replicates. The vector is allowed to infect for
one hour during which the plates are rocked every 15 minutes to
ensure even coverage of the virus. After the incubation period, the
cells are overlaid with a 0.5% agarose solution, and the
virus-infected cells are incubated for six days at which time they
are stained with Neutral Red. The plaques are counted between four
and 25 hours after staining, depending on the size of the plaques.
Wells which contain greater than 80 plaques are scored TNTC (Too
Numerous To Count), and wells that cannot be counted are marked as
NC (Not Counted) and the reason is noted on the record. Plaque
counts and their respective dilutions are used to calculate the
sample titer. For the assay to be considered valid, the negative
control wells should preferably contain no plaques, the titer of
the positive control should preferably be within one quarter (0.25)
log of the official titer of the virus being used as the positive
control, the % CV for the positive control should preferably be
less than or equal to 25%, and for any one dilution in the positive
control, there should preferably be no more than three wells
designated "NC".
[0349] At the final product testing step, the test article should
preferably have a titer of 1.times.10.sup.7 to 1.times.10.sup.12
pfu/mL. It is more preferable to have a titer of 1.times.10.sup.9
to 1.times.10.sup.12 pfu/mL, even more preferable to have a titer
of 1.times.10.sup.10 to 1.times.10.sup.12 pfu/mL, even more
preferable to have a titer of 5.times.10.sup.10 to
1.times.10.sup.12 pfu/mL, and most preferable to have a titer of
8.times.10.sup.10 to 1.times.10.sup.12 pfu/ml. Using purification
techniques in accordance with the present disclosure, titer values
as high as 5.times.10.sup.12 virus particles/mL have been obtained
at the final product step.
[0350] Determination of Viral Particle Concentration and
Particle/PFU Ratio
[0351] This assay measures the concentration, in viral
particles/mL, for a sample of adenoviral material. This assay is a
spectrophotometric assay that determines the total number of
particles in a sample based on absorbance at 260 nm. The extinction
coefficient used to convert to viral particles is 1
OD.sub.260=10.sup.12 viral particles.
[0352] The test method is as follows. Three replicates are prepared
for each sample using an appropriate dilution to fall within the
linear range of the spectrophotometer. The virus sample is combined
with 1% SDS (or 10% SDS for dilute test samples) and water to
achieve a total volume of 150 microliters. The sample is incubated
at room temperature for 15-30 minutes to disrupt the virion. Each
sample is read at A.sub.260 and A.sub.280 and the mean optical
density for replicate samples is multiplied by the dilution factor
to determine viral particles/mL. The Particle/PFU ratio is
determined using the titer determined by the plaque assay described
previously. For the assay to be considered valid, the % CV for the
three sample replicates should preferably be less than or equal to
10%. The test sample should preferably contain 1.times.10.sup.7 to
2.times.10.sup.13 viral particles/mL at the final product step. It
is more preferable that the test sample contain between about
0.8.times.10.sup.12 and 2.times.10.sup.13 viral particles/mL, and
most preferable that the sample contain between about
1.2.times.10.sup.12 and 2.times.10.sup.13 particles/mL It is most
preferable that the A.sub.260/A.sub.280 is 1.2 to 1.4. It is
preferable that the Particle/PFU ratio is less than 100, even more
preferable that it is less than 75, and most preferable that it is
10 to 60. Using purification techniques in accordance with the
present disclosure, viral particle concentration values as high as
5.times.10.sup.12 virus particles/mL have been obtained at the
final product step.
[0353] Adenoviral p53 Bioactivity Assay
[0354] The SAOS LM assay is a bioactivity assay which is conducted
for the purpose of determining the activity of the p53 component of
Ad5CMV-p53. The assay measures the inhibition of growth of SAOS-LM
cells (human osteocareinoma cell line with a homozygous p53
deletion). Any significant loss of inhibitory activity compared
with a standard would indicate the presence of an unacceptable
amount of inactive vector. The inhibition of growth of SAOS cells
is followed using the Alamar Blue indicator dye, which is used to
quantitatively measure cell proliferation. This dye contains a
colorimetric oxidation/reduction (REDOX) indicator. As cellular
activity results in chemical reduction of the cellular environment,
inhibition of growth results in an oxidized environment that allows
the measurement of p53 activity.
[0355] The test method is as follows. SAOS cells are plated in 96
well plates and grown overnight at 37 degrees Celsius to greater
than 75% confluence. Media is removed from the wells and the cells
are challenged with either a media control, positive control virus
(MOI=1000) or varying dilutions of the test sample. Following
challenge, the cells are incubated at 37 degrees Celsius for four
days. Alamar Blue is added to the wells and the plates are
incubated approximately eight hours at 37 degrees Celsius. Cell
density is determined by reading the plates at 570 nm. To accept
the assay the OD.sub.570 of the positive control must be less than
0.1 and the media control cell density must be at least 75%
confluent. It is preferable that the MOI of the test article that
causes 50% cell death is less than 1000 viral particles. It is more
preferable that the test article have an MOI that causes 50% cell
death of less than 700 viral particles, and most preferable that
the MOI that causes 50% cell death is less than 400 viral
particles. Using purification techniques in accordance with the
present disclosure, bioactivity values as high as 250-260 have been
obtained at the final product step.
[0356] HPLC Assay for p53.
[0357] This assay is a quantitative evaluation of Ad5CMV-p53
particle number and purity of in-process samples and of final
product stability samples. The method allows quantitation of
Ad5CMV-p53 particles by an ion exchange HPLC method.
[0358] The test method is as follows. A Toso Haas TSK-Gel-Q-5PW
column is used with a buffered salt gradient mobile phase for
separation of virus particles and impurities. A reference control
calibration curve is run on a newly installed column and scanned at
A.sub.260. A blank is prepared and run using the same column and
method. The sample to be tested is prepared by dilution with the
same low salt buffer used in gradient formation. The sample
absorbance is detected at 260 and 280 nm wavelengths, and the total
are for all peaks detected is determined. The ratio of the area for
the A.sub.260/A.sub.280 peak is determined, and the concentration
for the 260 nm peak is determined by comparison to the reference
calibration curve. Assay acceptance criteria include similar
profile to historical samples, and a A.sub.260/A.sub.280 ratio of
1.3+/-0.1 The test sample should preferably have a purity of
greater than or equal to 98%. It is more preferable that the purity
be greater than 99%, and most preferable that the purity is greater
than 99.9%. Using purification techniques in accordance with the
present disclosure, virus purity values as high as 99.8% have been
obtained at the final product step.
C. Identity Assays
[0359] Restriction Enzyme Mapping Assay for Ad5CMV-p53
[0360] This method allows evaluation of Ad5CMV-p53 DNA by
restriction enzyme analysis. Restriction enzymes recognize specific
base pair sequences on DNA, cutting the DNA at these restriction
sites. There are a limited number of recognition sites within a
vector for any particular restriction enzyme. Test sample DNA is
digested with two restriction enzymes and the fragments separated
electrophoretically in an agarose gel matrix. The DNA fragments are
checked for number and size.
[0361] The test method is as follows. DNA is extracted from vector
particles using a commercially available ion exchange spin column.
The extracted DNA is quantified and checked for purity by analyzing
the A.sub.260/A.sub.280 ratio. Approximately 0.4-5 micrograms of
the extracted DNA is digested with a cocktail of two restriction
enzymes, Eco RI and Cla I. The digested DNA is loaded onto a 1%
agarose gel containing ethidium bromide alongside an equal amount
of unrestricted DNA from the same sample. The samples are separated
by electrophoresis and visualized using an ultraviolet light
source. Data is captured by photography. The assay acceptance
criteria that should preferably be met for the assay to be
considered valid is a A.sub.260/A.sub.280 ratio of extracted DNA of
greater than 1.6. The test article should preferably have
restriction fragment sizes that match the theoretical fragment
sizes expected from the sequence of Ad5CMV-p53. The expected band
sizes are 486, 2320, 8494 and 24008 base pairs.
[0362] SDS Page Assay
[0363] This method allows evaluation of total proteins in final
product ranging in size from 5 to 100 kDa by separation according
to molecular weight.
[0364] The test method is as follows. Total proteins are determined
using a Pierce BCA method according to the protocol described
previously in this section. The test sample, internal standard and
molecular weight standards are prepared in sample buffer and
denatured by heating. All samples and standards are loaded into
wells of a pre-cast Tris-glycine gel and set in an electrophoresis
tank containing running buffer. The gel is run on a constant
current setting for approximately 90 minutes. The gel is then
removed from the cassette, stained using Coomassie Brilliant Blue
stain and destained. The gel is then analyzed using a densitometric
scanning instrument, and the data captured by photography.
Alternatively, the gel is dried for archiving. In all controls, the
presence of expected proteins is preferable and there should
preferably be no contaminating proteins. In the test sample, the
expected bands should preferably be observed, with no significant
extra bands.
[0365] Western Blot Assay:
[0366] This method tests for the presence of p53 protein in
Ad5CMV-p53 transduced cells. The test method is as follows.
Individual 60 mm tissue culture dishes for product samples and
control samples are seeded at a density of 7.times.10.sup.5 cells
and grown to greater than 80% confluence. The test article is
diluted in media to provide 3.5.times.10.sup.8 viral particles/mL.
A reference control is diluted to 3.5.times.10.sup.8 vp/mL and a
negative control with no vector is also prepared. The cells are
exposed to media containing product for one hour during which the
plates are rocked to ensure even distribution of vector. At the end
of the hour, additional media is added to the dishes and they are
incubated for approximately five hours to allow time for expression
of p53. At the end of the incubation period, the cells are treated
with trypsin to allow harvest, washed with DPBS and solubilized
with a detergent buffer. The total amount of protein in each sample
and control is determined by a colorimetric quantitation method
(Pierce BCA). For each sample and method, 3-5 micrograms of protein
are loaded onto a gel alongside a commercially purchased p53
protein reference and separated by polyacrylamide gel
electrophoresis (PAGE). The proteins in the gel are transferred to
a PVDF membrane and the membrane is exposed to a milk buffer to
block non-specific binding sites and then sequentially exposed to
antibodies. The primary antibody, a mouse anti-human p53 antibody
specifically binds to p53. The secondary antibody is a goat
anti-mouse IgG with horseradish peroxidase (HRP) covalently bound.
A colorimetric substrate is exposed to the bound HRP enzyme which
enables visualization of p53 protein on the blot. For the assay to
be considered valid, the control p53 band should preferably be
visible, and the negative control should preferably show no
expression of p53. The test article should preferably show
expression of p53.
[0367] Recoverable Fill Volume Assay
[0368] This method is a gravimetric determination of volume
recoverable from the container closure for Ad5CMV-p53 final
product. Product is recovered from seven vials using tared 3 cc
syringes and 21G 1.5 inch needles. The product is weighed, and the
weights are converted to volume using the specific gravity of the
product of 1.03 g/mL. The balance calibration must be met before
weighing of the samples and it is preferable that all seven vials
tested must meet specification of 1.0 to 1.4 mL of recoverable fill
volume. It is more preferable that the recoverable fill volume be
1.1 to 1.3 .mu.L. It will be understood by those of skill in the
art that this assay is an example for the Ad5CMVp53 product, and
that those of skill in the art will be able to modify this assay
for other products in other types of container closures.
[0369] Physical Description Assay
[0370] This method allows evaluation of the physical description of
final product. Approximately seven milliliters of product are
pooled in a clear plastic tube. The product is inspected by an
analyst to document the color, transparency, and the presence of
any gross particulate matter. The test article should preferably be
clear to opalescent and contain no gross particulate matter by
visual inspection.
[0371] pH Assay
[0372] This method is a pH determination of the Ad5CMV-p53 final
product. Approximately 0.5 mL of the product is placed in a tube.
The pH is determined using a calibrated pH meter at a temperature
of 25+/-5 degrees Celsius. The pH standard solutions should
preferably demonstrate a slope range of 80-120%. The pH of the
final product should preferably be between about 6 and about 9. It
is more preferable that the pH is between about 6.5 and about 8.8,
even more preferable that the pH is between about 7.0 and about
8.6, and most preferable that the pH is between about 7.5 and about
8.5.
[0373] Restriction Enzyme Mapping for Identity Testing of Master
Viral Bank or Working Viral Bank
[0374] The goal of this test is to assess the identity of the
Ad5CMV-p53 genome through measurement of the DNA fragments
generated after cleavage of the whole viral genome (approximately
35308 base pairs). When the unpurified viruses are contained in a
cell mixture such as a virus bank, the viral DNA first has to be
extracted from the crude cell lysate. An aliquot of the sample is
digested by proteinase K in the presence of SDS. The DNA is then
extracted using a mixture of phenol/chloroform/isoamyl alcohol and
precipitated with ethanol. The DNA concentration is measured by UV
spectrometry. Approximately one microgram of the viral DNA is then
submitted to restriction enzyme digestion. Four individual digests
are performed utilizing a battery of three restriction enzymes in
different combinations. The digests and DNA size markers are then
separated on an agarose gel using electrophoresis and stained with
Syb-Green. The gels are integrated using a camera and a calibration
curve calculated from the standards. The size of the fragments
greater than 500 bp and less than 8000 bp is then determined. The
size of the fragments obtained should preferably correspond to the
theoretical size of the fragments obtained from the expected
theoretical sequence. The fragment sizes of the test sample should
preferably correspond to those expected from the DNA sequence.
[0375] PCR to Detect E1 DNA Sequences in 293 MCB and WCB
[0376] This assay is used to determine the identity of the 293
Master and Working Cell Banks by demonstrating the presence of the
E1 region. Using two specific pairs of PCR primers, one targeted
against the E1 region present in both 293 cells and wild-type
adenovirus and another one targeted against the E1 region only
present in the wild type adenovirus. The method should demonstrate
the identity of the 293 cell line contained in the test
article.
[0377] The test method is as follows. After thawing, cells from the
test article are grown using standard conditions in a cell culture
dish until a monolayer of cells is obtained. The cells are then
digested with proteinase K to remove the proteins, and DNA isolated
using phenol/chloroform/isoamyl alcohol extractions followed by
ethanol precipitation. The extracted DNA is quantified and checked
for purity by an absorbance scan from OD260-OD280. The PCR reaction
is performed using the two E1 targeted pairs of primers on the test
article and on both positive and negative DNA controls. The
negative control is a mammalian cell line which does not contain
the E1 region. The positive control is a wild type adenovirus. The
PCR products from each reaction are loaded onto an agarose gel and
the size of the fragments obtained after electrophoresis and
staining are recorded using photography. The non-bearing E1
mammalian cell line must exhibit no amplification product with both
pairs of PCR primers, while the wild type adenovirus must show the
correct amplification product with both pairs of PCR primers. The
test article must demonstrate the correct amplification product
with the pair of primers located in the E1 region described to be
present in the 293 cell, and must be negative with the second pair
of primers known only to be present in the wild type adenoviral
genome.
11. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS
[0378] When purified according to the methods set forth above, it
is contemplated that the viral particles of the present invention
may be administered in vitro, ex vivo or in vivo. Thus, it will be
desirable to prepare the complex as a pharmaceutical composition
appropriate for the intended application. Generally this will
entail preparing a pharmaceutical composition that is essentially
free of pyrogens, as well as any other impurities that could be
harmful to humans or animals. One also will generally desire to
employ appropriate salts and buffers to render the complex stable
and allow for complex uptake by target cells.
[0379] Aqueous compositions of the present invention comprise an
effective amount of the expression construct and nucleic acid,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. Such compositions can also be referred to as
inocula. The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or a human, as appropriate. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
[0380] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0381] The viral particles of the present invention may include
classic pharmaceutical preparations for use in therapeutic
regimens, including their administration to humans. Administration
of therapeutic compositions according to the present invention will
be via any common route so long as the target tissue is available
via that route. This includes oral, nasal, buccal, rectal, vaginal
or topical. Alternatively, administration will be by orthotopic,
intradermal subcutaneous, intramuscular, intraperitoneal, or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions that
include physiologically acceptable carriers, buffers or other
excipients. For application against tumors, direct intratumoral
injection, inject of a resected tumor bed, regional (i.e.,
lymphatic) or general administration is contemplated. It also may
be desired to perform continuous perfusion over hours or days via a
catheter to a disease site, e.g., a tumor or tumor site.
[0382] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain about
100 mg of human serum albumin per milliliter of phosphate buffered
saline. Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like may be used. Examples of non-aqueous solvents
are propylene glycol, polyethylene glycol, vegetable oil and
injectable organic esters such as ethyloleate. Aqueous carriers
include water, alcoholic/aqueous solutions, saline solutions,
parenteral vehicles such as sodium chloride, Ringer's dextrose,
etc. Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial agents, anti-oxidants,
chelating agents and inert gases. The pH and exact concentration of
the various components the pharmaceutical composition are adjusted
according to well known parameters.
[0383] Additional formulations which are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. When the route is topical, the form may be
a cream, ointment, salve or spray.
[0384] An effective amount of the therapeutic agent is determined
based on the intended goal, for example (i) inhibition of tumor
cell proliferation, (ii) elimination or killing of tumor cells,
(iii) vaccination, or (iv) gene transfer for long term expression
of a therapeutic gene. The term "unit dose" refers to physically
discrete units suitable for use in a subject, each unit containing
a predetermined-quantity of the therapeutic composition calculated
to produce the desired responses, discussed above, in association
with its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, both according to number
of treatments and unit dose, depends on the subject to be treated,
the state of the subject and the result desired. Multiple gene
therapeutic regimens are expected, especially for adenovirus.
[0385] In certain embodiments of the present invention, an
adenoviral vector encoding a tumor suppressor gene will be used to
treat cancer patients. Typical amounts of an adenovirus vector used
in gene therapy of cancer is 10.sup.3-10.sup.15 viral
particles/dose, (10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13,
10.sup.14, 10.sup.15) wherein the dose may be divided into several
injections at different sites within a solid tumor. The treatment
regimen also may involve several cycles of administration of the
gene transfer vector over a period of 3-10 weeks. Administration of
the vector for longer periods of time from months to years may be
necessary for continual therapeutic benefit.
[0386] In another embodiment of the present invention, an
adenoviral vector encoding a therapeutic gene may be used to
vaccinate humans or other mammals. Typically, an amount of virus
effective to produce the desired effect, in this case vaccination,
would be administered to a human or mammal so that long term
expression of the transgene is achieved and a strong host immune
response develops. It is contemplated that a series of injections,
for example, a primary injection followed by two booster
injections, would be sufficient to induce an long term immune
response. A typical dose would be from 10.sup.6 to 10.sup.15
PFU/injection depending on the desired result. Low doses of antigen
generally induce a strong cell-mediated response, whereas high
doses of antigen generally induce an antibody-mediated immune
response. Precise amounts of the therapeutic composition also
depend on the judgment of the practitioner and are peculiar to each
individual.
12. EXAMPLES
[0387] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Materials and Methods
[0388] A) Cells
[0389] 293 cells human epithelial embryonic kidney cells) from the
Master Cell Bank were used for the studies.
[0390] B) Media
[0391] Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L
glucose)+10% fetal bovine serum (FBS) was used for the cell growth
phase. For the virus production phase, the FBS concentration in
DMEM was lowered to 2%.
[0392] C) Virus
[0393] AdCMVp53 is a genetically engineered,
replication-incompetent human type 5 adenovirus expressing the
human wild type p53 protein under control of the cytomegalovirus
(CMV) immediate early promoter.
[0394] D) Celligen Bioreactor
[0395] A Celligen bioreactor (New Brunswick Scientific, Co. Inc.)
with 5 L total volume (3.5 L working volume) was used to produce
virus supernatant using microcarrier culture. 13 g/L glass coated
microcarrier (SoloHill) was used for culturing cells in the
bioreactor.
[0396] E) Production of Virus Supernatant in the Celligen
Bioreactor
[0397] 293 cells from master cell bank (MCB) were thawed and
expanded into Cellfactories (Nunc). Cells were generally split at a
confluence of about 85-90%. Cells were inoculated into the
bioreactor at an inoculation concentration of 1.times.10.sup.5
cells/ml. Cells were allowed to attach to the microcarriers by
intermittent agitation. Continuous agitation at a speed of 30 rpm
was started 6-8 hr post cell inoculation. Cells were cultured for 7
days with process parameters set at pH=7.20, dissolved oxygen
(DO)=60% of air saturation, temperature=37.degree. C. On day 8,
cells were infected with AdCMVp53 at an MOI of 5. Fifty hr post
virus infection, agitation speed was increased from 30 rpm to 150
rpm to facilitate cell lysis and release of the virus into the
supernatant. The virus supernatant was harvested 74 hr
post-infection. The virus supernatant was then filtered for further
concentration/diafiltration.
[0398] F) Cellcube.TM. Bioreactor System
[0399] A Cellcube.TM. bioreactor system (Corning-Costar) was also
used for the production of AdCMVp53 virus. It is composed of a
disposable cell culture module, an oxygenator, a medium
recirculation pump and a medium pump for perfusion. The cell
culture module used has a culture surface area of 21,550 cm.sup.2
(1 mer).
[0400] G) Production of Virus in the Cellcube.TM.
[0401] 293 cells from master cell bank (MCB) were thawed and
expanded into Cellfactories (Nunc). Cells were generally split at a
confluence of about 85-90%. Cells were inoculated into the
Cellcube.TM. according to the manufacturer's recommendation.
Inoculation cell densities were in the range of
1-1.5.times.10.sup.4/cm.sup.2. Cells were allowed to grow for 7
days at 37.degree. C. under culture conditions of pH=7.20, DO=60%
air saturation. Medium perfusion rate was regulated according to
the glucose concentration in the Cellcube.TM.. One day before viral
infection, medium for perfusion was changed from DMEM+10% FBS to
DMEM+2% FBS. On day 8, cells were infected with AdCMVp53 virus at a
multiplicity of infection (MOI) of 5. Medium perfusion was stopped
for 1 hr immediately after infection then resumed for the remaining
period of the virus production phase. Culture was harvested 45-48
hr post-infection.
[0402] H) Lysis Solution
[0403] Tween-20 (Fisher Chemicals) at a concentration of 1% (v/v)
in 20 mM Tris+0.25 M NaCl+1 mM MgCl.sub.2, pH=7.50 buffer was used
to lyse cells at the end of the virus production phase in the
Cellcube.TM..
[0404] I) Clarification and Filtration
[0405] Virus supernatant from the Celligen bioreactor and virus
solution from the Cellcube.TM. were first clarified using a depth
filter (Preflow, GelmanSciences), then was filtered through a
0.8/0.22 .mu.m filter (Supor Cap 100, GelmanSciences).
[0406] J) Concentration/Diafiltration
[0407] Tangential flow filtration (TFF) was used to concentrate and
buffer exchange the virus supernatant from the Celligen bioreactor
and the virus solution from the Cellcube.TM.. A Pellicon II mini
cassette (Millipore) of 300 K nominal molecular weight cut off
(NMWC) was used for the concentration and diafiltration. Virus
solution was first concentrated 10-fold. This was followed by 4
sample volume of buffer exchange against 20 mM Tris+1.0 M NaCl+1 mM
MgCl.sub.2, pH=9.00 buffer using the constant volume diafiltration
method.
[0408] Similar concentration/diafiltration was carried out for the
column purified virus. A Pellicon II mini cassette of 100 K NMWC
was used instead of the 300 K NMWC cassette. Diafiltration was done
against 20 mM Tris+0.25 M NaCl+1 mM MgCl.sub.2, pH=9.00 buffer or
Dulbecco's phosphate buffered saline (DPBS).
[0409] K) Benzonase Treatment
[0410] The concentrated/diafiltrated virus solution was treated
with Benzonase.TM. (American International Chemicals) at a
concentration of 100 u/ml, room temperature overnight to reduce the
contaminating nucleic acid concentration in the virus solution.
[0411] L) CsCl gradient ultracentrifugation
[0412] Crude virus solution was purified using double CsCl gradient
ultracentrifugation using a SW40 rotor in a Beckman ultracentrifuge
(XL-90). First, 7 ml of crude virus solution was overlaid on top of
a step CsCl gradient made of equal volume of 2.5 ml of 1.25 g/ml
and 1.40 g/ml CsCl solution, respectively. The CsCl gradient was
centrifuged at 35,000 rpm for 1 hr at room temperature. The virus
band at the gradient interface was recovered. The recovered virus
was then further purified through a isopicnic CsCl gradient. This
was done by mixing the virus solution with at least 1.5-fold volume
of 1.33 g/ml CsCl solution. The CsCl solution was centrifuged at
35,000 rpm for at least 18 hr at room temperature. The lower band
was recovered as the intact virus. The virus was immediately
dialyzed against 20 mM Tris+1 mM MgCl.sub.2, pH=7.50 buffer to
remove CsCl. The dialyzed virus was stored at -70.degree. C. for
future use.
[0413] M) Ion Exchange Chromatography (IEC) Purification
[0414] The Benzonase treated virus solution was purified using IEC.
Strong anionic resin Toyopearl SuperQ 650M (Tosohaas) was used for
the purification. A FPLC system (Pharmacia) with a XK16 column
(Pharmacia) were used for the initial method development. Further
scale-up studies were carried out using a BioPilot system
(Pharmacia) with a XK 50 column (Pharmacia). Briefly, the resin was
packed into the columns and sanitized with 1 N NaOH, then charged
with buffer B which was followed by conditioning with buffer A.
Buffers A and B were composed of 20 mM Tris+0.25 M NaCl+1 mM
MgCl.sub.2, pH=9.00 and 20 mM Tris+2M NaCl+1 mM MgCl.sub.2,
pH=9.00, respectively. Viral solution sample was loaded onto the
conditioned column, followed by washing the column with buffer A
until the UV absorption reached base line. The purified virus was
eluted from the column by using a 10 column volume of linear NaCl
gradient.
[0415] N) HPLC Analysis
[0416] A HPLC analysis procedure was developed for evaluating the
efficiency of virus production and purification.
Tris(hydroxymethyl)aminomethane (tris) was obtained from
FisherBiotech (Cat# BP154-1; Fair Lawn, N.J., U.S.A.); sodium
chloride (NaCl) was obtained from Sigma (Cat# S-7653, St. Louis,
Mo., U.S.A.). Both were used directly without further purification.
HPLC analyses were performed on an Analytical Gradient System from
Beckman, with Gold Workstation Software (126 binary pump and 168
diode array detector) equipped with an anion-exchange column from
TosoHaas (7.5 cm.times.7.5 mm ID, 10 .mu.m particle size, Cat#
18257). A 1-ml Resource Q (Pharmacia) anion-exchange column was
used to evaluate the method developed by Huyghe et al. using their
HEPES buffer system. This method was only tried for the Bioreactor
system.
[0417] The buffers used in the present HPLC system were Buffer A:
10 nM tris buffer, pH 9.0. Buffer B: 1.5 M NaCl in buffer A, pH
9.0. The buffers were filtered through a 0.22 .mu.m bottle top
filter by Corning (Cat# 25970-33). All of the samples were filtered
through a 0.8/0.22 .mu.m Acrodisc PF from Gelman Sciences (Cat#
4187) before injection.
[0418] The sample is injected onto the HPLC column in a 60-100
.mu.l volume. After injection, the column (TosoHaas) is washed with
20% B for 3 min at a flow rate of 0.75 ml/min. A gradient is then
started, in which B is increased from 20% to 50% over 6 min. Then
the gradient is changed from 50% to 100% B over 3 min, followed by
100% B for 6 min. The salt concentration is then changed back
stepwise to 20% again over 4 min, and maintained at 20% B for
another 6 min. The retention time of the Adp53 is 9.5.+-.0.3 min
with A.sub.260/A.sub.280.apprxeq.1.26.+-.0.03. Cleaning of the
column after each chromatographic run is accomplished by injecting
100 .mu.l of 0.15 M NaOH and then running the gradient.
Example 2
Effect of Medium Perfusion Rate in Cellcube.TM. on Virus Production
and Purification
[0419] For a perfusion cell culture system, such as the
Cellcube.TM., medium perfusion rate plays an important role on the
yield and quality of product. Two different medium perfusion
strategies were examined. One strategy was to keep the glucose
concentration in the Cellcube.TM. .gtoreq.2 g/L (high perfusion
rate). The other one was to keep the glucose concentration
.gtoreq.1 g/L (low medium perfusion rate).
[0420] No significant changes in the culture parameters, such as
pH, DO, was observed between the two different perfusion rates.
Approximately equivalent amount of crude viruses (before
purification) were produced after harvesting using 1% Tween-20
lysis solution as shown in Table 7. However, dramatic difference
was seen on the HPLC profiles of the viral solutions from the high
and low medium perfusion rate production runs.
TABLE-US-00007 TABLE 7 Effect of medium glucose concentration on
virus yield Glucose concentration (g/L) .gtoreq.2.0 .gtoreq.1.0
Crude virus yield (PFU) 4 .times. 10.sup.12 4.9 .times.
10.sup.12
[0421] As shown in FIG. 1, a very well separated virus peak
(retention time 9.39 min) was produced from viral solution using
low medium perfusion rate. It was found that virus with adequate
purity and biological activity was attained after a single step ion
exchange chromatographic purification of the virus solution
produced under low medium perfusion rate. On the other hand, no
separated virus peak in the retention time of 9.39 min was observed
from viral solution produced using high medium perfusion rate. This
suggests that contaminants which have the same elution profile as
the virus were produced under high medium perfusion rate. Although
the nature of the contaminants is not yet clear, it is expected
that the contaminants are related to the increased extracellular
matrix protein production under high medium perfusion rate (high
serum feeding) from the producer cells. This poor separation
characteristic seen on the HPLC created difficulties for process
IEC purification as shown in the following Examples. As a result,
medium perfusion rate used during the cell growth and the virus
production phases in the Cellcube.TM. has a significant effect on
the downstream IEC purification of the virus. Low medium perfusion
rate is recommended. This not only produces easy to purify crude
product but also offers more cost-effective production due to the
reduced medium consumption.
Example 3
Methods of Cell Harvest and Lysis
[0422] Based on previous experience, the inventors first evaluated
the freeze-thaw method. Cells were harvested from the Cellcube.TM.
45-48 hr post-infection. First, the Cellcube.TM. was isolated from
the culture system and the spent medium was drained. Then, 50 mM
EDTA solution was pumped into the Cube to detach the cells from the
culture surface. The cell suspension thus obtained was centrifuged
at 1,500 rpm (Beckman GS-6KR) for 10 min. The resultant cell pellet
was resuspended in Dulbecco's phosphate buffered saline (DPBS). The
cell suspension was subjected to 5 cycles of freeze/thaw between
37.degree. C. water bath and dry-ice ethanol bath to release virus
from the cells. The crude cell lysate (CCL) thus generated was
analyzed on HPLC.
[0423] FIG. 2 shows the HPLC profile. No virus peak is observed at
retention time of 9.32 min. Instead, two peaks at retention times
of 9.11 and 9.78 min are produced. This profile suggests that the
other contaminants having similar elution time as the virus exist
in the CCL and interfere with the purification of the virus. As a
result, very low purification efficiency was observed when the CCL
was purified by IEC using FPLC.
[0424] In addition to the low purification efficiency, there was a
significant product loss during the cell harvest step into the EDTA
solution as indicated in Table 8. Approximately 20% of the product
was lost into the EDTA solution which was discarded. In addition,
about 24% of the crude virus product is present in the spent medium
which was also discarded. Thus, only 56% of the crude virus product
is in the CCL. Furthermore, freeze-thaw is a process of great
variation and very limited scaleability. A more efficient cell
lysis process with less product loss needed to be developed.
TABLE-US-00008 TABLE 8 Loss of virus during EDTA harvest of cells
from Cellcube .TM. Waste EDTA Crude product Spent harvest Crude
cell Total crude Medium Solution lysate product (PFU) Volume (ml)
2800 2000 82 -- Titer (PFU/ml) 2.6 .times. 10.sup.8 3 .times.
10.sup.8 2 .times. 10.sup.10 -- Total virus 7.2 .times. 10.sup.11 6
.times. 10.sup.11 1.64 .times. 10.sup.12 3 .times. 10.sup.12 (PFU)
Percentage 24% 20% 56% Data was generated from 1 mer Cellcube
.TM..
TABLE-US-00009 TABLE 9 Evaluation of non-ionic detergents for cell
lysis Concentrations Detergents (w/v) Chemistry Comments Thesit 1%
Dodecylpoly(ethylene glycol ether).sub.n, Large 0.5% n = 9-10
Precipitate 0.1% NP-40 1% Ethylphenolpoly
(ethylene-glycolether).sub.n Large 0.5% n = 9-11 precipitate 0.1%
Tween-20 1% Poly(oxyethylene).sub.n-sorbitan-monolaurate Small 0.5%
n = 20 precipitate 0.1% Brij-58 1% Cetylpoly
(ethyleneglycolether).sub.n n = 20 Cloudy 0.5% Solution 0.1% Triton
X-100 1% Octylphenolpoly(ethyleneglycolether).sub.n n = 10 Large
0.5% precipitate 0.1%
[0425] Detergents have been used to lyse cells to release
intracellular organelles. Consequently, the inventors evaluated the
detergent lysis method for the release of adenovirus. Table 9 lists
the 5 different non-ionic detergents that were evaluated for cell
lysis. Cells were harvested from the Cellcube.TM. 48 hr
post-infection using 50 mM EDTA. The cell pellet was resuspended in
the different detergents at various concentrations listed in Table
9.
[0426] Cell lysis was carried out at either room temperature or on
ice for 30 min. Clear lysis solution was obtained after
centrifugation to remove the precipitate and cellular debris. The
lysis solutions were treated with Benzonase and then analyzed by
HPLC. FIG. 3 shows the HPLC profiles of lysis solutions from the
different detergents. Thesit and NP-40 performed similarly as
Triton X-100. Lysis solution generated from 1% Tween-20 gave the
best virus resolution with the least virus resolution being
observed with Brij-58. More efficient cell lysis was found at
detergent concentration of 1% (w/v). Lysis temperature did not
contribute significantly to the virus resolution under the
detergent concentrations examined. For the purpose of process
simplicity, lysis at room temperature is recommended. Lysis
solution composed of 1% Tween-20 in 20 mM Tris+0.25M NaCl+1 mM
MgCl.sub.2, pH=7.50 was employed for cell lysis and virus harvest
in the Cellcube.TM..
Example 4
Effects of Concentration/Diafiltration on Virus Recovery
[0427] Virus solution from the lysis step was clarified and
filtered before concentration/diafiltration. TFF membranes of
different NMWCs, including 100 K, 300 K, 500 K, and 1000 K, were
evaluated for efficient concentration/diafiltration. The highest
medium flux with minimal virus loss to the filtrate was obtained
with a membrane of 300 K NMWC. Bigger NMWC membranes offered higher
medium flux, but resulted in greater virus loss to the filtrate,
while smaller NMWC membranes achieved an insufficient medium flux.
Virus solution was first concentrated 10-fold, which was followed
by 4 sample volumes of diafiltration against 20 mM Tris+0.25 M
NaCl+1 mM MgCl.sub.2, pH=9.00 buffer using the constant volume
method. During the concentration/diafiltration process, pressure
drop across the membrane was kept .ltoreq.5 psi. Consistent, high
level virus recovery was demonstrated during the
concentration/diafiltration step as indicated in Table 10.
TABLE-US-00010 TABLE 10 Concentration/diafiltration of crude virus
solution Titer (PFU/ml) Volume (ml) Total virus (PFU) Recovery Run
#1 Run #2 Run #1 Run #2 Run #1 Run #2 Run #1 Run #2 Before 2.6
.times. 10.sup.9 2 .times. 10.sup.9 1900 2000 4.9 .times. 10.sup.12
.sup. 4 .times. 10.sup.12 conc./diafl. Post .sup. 2.5 .times.
10.sup.10 1.7 .times. 10.sup.10 200 200 5 .times. 10.sup.12 3.4
.times. 10.sup.12 102% 85% conc./diafl. Conc. 9.5 10 Factor
Filtrate 5 .times. 10.sup.5 1 .times. 10.sup.6 3000 3000 1.5
.times. 10.sup.9 3 .times. 10.sup.9
Example 5
Effect of Salt Addition on Benzonase Treatment
[0428] Virus solution after concentration/diafiltration was treated
with Benzonase (nuclease) to reduce the concentration of
contaminating nucleic acid in virus solution. Different working
concentrations of Benzonase, which included 50, 100, 200, 300
units/ml, were evaluated for the reduction of nucleic acid
concentrations. For the purpose of process simplicity, treatment
was carried out at room temperature overnight. Significant
reduction in contaminating nucleic acid that is hybridizable to
human genomic DNA probe was seen after Benzonase treatment.
[0429] Table 11 shows the reduction of nucleic acid concentration
before and after Benzonase treatment. Virus solution was analyzed
on HPLC before and after Benzonase treatment. As shown in FIG. 4A
and FIG. 4B, dramatic reduction in the contaminating nucleic acid
peak was observed after Benzonase treatment. This is in agreement
with the result of the nucleic acid hybridization assay. Because of
the effectiveness, a Benzonase concentration of 100 u/ml was
employed for the treatment of the crude virus solution.
TABLE-US-00011 TABLE 11 Reduction of contaminating nucleic acid
concentration in virus solution Before Treatment After Treatment
Reduction Contaminating 200 .mu.g/ml 10 ng/ml 2 .times.
10.sup.4-fold nucleic acid concentration Treatment condition:
Benzonase concentration: 100 u/ml, temperature: room temperature,
time: overnight.
[0430] Considerable change in the HPLC profile was observed pre-
and post-Benzonase treatment. No separated virus peak was detected
at retention time of 9.33 min after Benzonase treatment. At the
same time, a major peak with high 260 nm adsorption at retention
time of 9.54 min was developed. Titer assay results indicated that
Benzonase treatment did not negatively affect the virus titer and
virus remained intact and infectious after Benzonase treatment. It
was reasoned that cellular nucleic acid released during the cell
lysis step interacted with virus and either formed aggregates with
the virus or adsorbed onto the virus surface during Benzonase
treatment.
[0431] To minimize the possible nucleic acid virus interaction
during Benzonase treatment, different concentrations of NaCl was
added into the virus solution before Benzonase treatment. No
dramatic change in the HPLC profile occurred after Benzonase
treatment in the presence of 1 M NaCl in the virus solution. FIG. 5
shows the HPLC profile of virus solution after Benzonase treatment
in the presence of 1M NaCl. Unlike that shown in FIG. 4B, virus
peak at retention time of 9.35 min still exists post Benzonase
treatment. This result indicates that the presence of 1M NaCl
prevents the interaction of nucleic acid with virus during
Benzonase treatment and facilitates the further purification of
virus from contaminating nucleic acid.
Example 6
Ion Exchange Chromatographic Purification
[0432] The presence of negative charge on the surface of adenovirus
at physiological pH conditions prompted evaluation of anionic ion
exchangers for adenovirus purification. The strong anionic ion
exchanger Toyopearl Super Q 650M was used for the development of a
purification method. The effects of NaCl concentration and pH of
the loading buffer (buffer A) on virus purification was evaluated
using the FPLC system.
[0433] A) Method Development
[0434] For ion exchange chromatography, buffer pH is one of the
most important parameters and can have dramatic influence on the
purification efficiency. In reference to the medium pH and
conductivity used during virus production, the inventors formulated
20 mM Tris+1 mM MgCl.sub.2+0.2M NaCl, pH=7.50 as buffer A. A XK16
column packed with Toyopearl SuperQ 650M with a height of 5 cm was
conditioned with buffer A.
[0435] A sample of 5 ml of Benzonase treated
concentrated/diafiltrated virus supernatant from the Celligen
bioreactor was loaded onto the column. After washing the column,
elution was carried out with a linear gradient of over 10 column
volumes to reach buffer B (20 mM Tris+1 mM MgCl.sub.2+2M NaCl,
pH=7.50).
[0436] FIG. 6 shows the elution profile. Three peaks were observed
during elution without satisfactory separation among them. Control
study performed with 293 cell conditioned medium (with no virus)
showed that the first two peaks are virus related. To further
improve the separation efficiency, the effect of buffer pH was
evaluated. Buffer pH was increased to 9.00 while keeping other
conditions constant. Much improved separation, as shown in FIG. 7,
was observed as compared to that of buffer pH of 7.50. Fractions
#3, #4, and #8 were analyzed on HPLC.
[0437] As shown in FIG. 8, the majority of virus was found in
fraction #4, with no virus being detected in fractions #3 and #8.
Fraction #8 was found to be mainly composed of contaminating
nucleic acid. However, the purification was still not optimal.
There is overlap between fractions #3 and #4 with contaminants
still detected in fraction #4.
[0438] Based on the chromatogram in FIG. 7, it was inferred that
further improvement of virus purification could be achieved by
increasing the salt concentration in buffer A. As a result, the
contaminants present in the fraction #3, which is prior to the
virus peak, can be shifted to the flow through faction. The NaCl
concentration in buffer A was increased to 0.3 M while keeping
other conditions constant. FIG. 9 shows the elution profile under
the condition of 0.3 M NaCl in buffer A.
[0439] Dramatic improvement in purification efficiency was
achieved. As expected the contaminant peak observed in FIG. 7 was
eliminated under the increased salt condition. Samples from crude
virus sup, flow through, peak #1, and peak #2 were analyzed on HPLC
and the results are shown in FIG. 10. No virus was detected in the
flow through fraction. The majority of the contaminants present in
the crude material were found in the flow through. HPLC analysis of
peak #1 showed a single well defined virus peak. This HPLC profile
is equivalent to that obtained from double CsCl gradient purified
virus. Peaks observed at retention times of 3.14 and 3.61 min in
CsCl gradient purified virus are glycerol related peaks. The
purified virus has a A260/A280 ratio of 1.27.+-.0.03. This similar
to the value of double CsCl gradient purified virus as well as the
results reported by Huyghe et al. (1996). Peak #2 is composed
mainly of contaminating nucleic acid. Based on the purification
result, the inventors proposed the following method for IEC
purification of adenovirus sup from the bioreactor.
TABLE-US-00012 Buffer A: 20 mM Tris + 1 mM MgCl.sub.2 + 0.3M NaCl,
pH = 9.00 Buffer B: 20 mM Tris + 1 mM MgCl.sub.2 + 2M NaCl, pH =
9.00 Elution: 10 column volume linear gradient
[0440] B) Method Scale-Up
[0441] Following the development of the method, purification was
scaled-up from the XK16 column (1.6 cm I.D.) to a XK50 column (5 cm
I.D., 10-fold scale-up) using the same purification method. A
similar elution profile was achieved on the XK50 column as shown in
FIG. 11. The virus fraction was analyzed on HPLC, which indicated
equivalent virus purity to that obtained from the XK16 column.
[0442] During the scale-up studies, it was found that it was more
convenient and consistent to use conductivity to quantify the salt
concentration in buffer A. The optimal conductivity of buffer A is
in the range of 25.+-.2 mS/cm at approximately room temperature
(21.degree. C.). Samples produced during the purification process
together with double CsCl purified virus were analyzed on
SDS-PAGE.
[0443] As shown in FIG. 12, all the major adenovirus structure
proteins are detected on the SDS-PAGE. The IEC purified virus shows
equivalent staining as that of the double CsCl purified virus.
Significant reduction in bovine serum albumin (BSA) concentration
was achieved during purification. The BSA concentration in the
purified virus was below the detection level of the western blot
assay as shown in FIG. 13.
[0444] The reduction of contaminating nucleic acid concentration in
virus solution during the purification process was determined using
nucleic acid slot blot. .sup.32P labeled human genomic DNA was used
as the hybridization probe (because 293 cells are human embryonic
kidney cells). Table 12 shows the nucleic acid concentration at
different stages of the purification process. Nucleic acid
concentration in the final purified virus solution was reduced to
60 pg/ml, an approximate 3.6.times.10.sup.6-fold reduction compared
to the initial virus supernatant. Virus titer and infectious to
total particle ratio were determined for the purified virus and the
results were compared to that from double CsCl purification in
Table 11. Both virus recovery and particle/PFU ratio are very
similar between the two purification methods. The titer of the
column purified virus solution can be further increased by
performing a concentration step.
TABLE-US-00013 TABLE 12 Removal of contaminating nucleic acids
during purification Contaminating nucleic acid Steps during
purification concentration Virus supernatant from bioreactor 220
.mu.g/ml Concentrated/diafiltrated sup 190 .mu.g/ml Sup post
Benzonase treatment (O/N, RT, 10 ng/ml 100 u/ml) Purified virus
from column 210 pg/ml Purified virus post 60 pg/ml
concentration/diafiltration CsCl purified virus 800 pg/ml
Example 7
Other Purification Methods
[0445] In addition to the strong anionic ion exchange
chromatography, other modes of chromatographic methods, were also
evaluated for the purification of AdCMVp53 virus (e.g. size
exclusion chromatography, hydrophobic interaction chromatography,
cation exchange chromatography, or metal ion affinity
chromatography). Compared to the Toyopearl Super Q, all those modes
of purification offered much less efficient purification with low
product recovery. Therefore, Toyopearl Super Q resin is recommended
for the purification of AdCMVp53. However, other quaternary
ammonium chemistry based strong anionic exchangers are likely to be
suitable for the purification of AdCMVp53 with some process
modifications.
Example 8
Purification of crude AdCMVp53 virus generated from
Cellcube.TM.
[0446] Two different production methods were developed to produce
AdCMVp53 virus. One was based on microcarrier culture in a stirred
tank bioreactor. The other was based on a Cellcube.TM. bioreactor.
As described above, the purification method was developed using
crude virus supernatant generated from the stirred tank bioreactor.
It was realized that although the same medium, cells and viruses
were used for virus production in both the bioreactor and the
Cellcube.TM., the culture surface onto which cells attached was
different.
[0447] In the bioreactor, cells were grown on a glass coated
microcarrier, while in the Cellcube.TM. cells were grown on
proprietary treated polystyrene culture surface. Constant medium
perfusion was used in the Cellcube.TM., on the other hand, no
medium perfusion was used in the bioreactor. In the Cellcube.TM.,
the crude virus product was harvested in the form of virally
infected cells, which is different from the virus supernatant
harvested from the bioreactor.
[0448] Crude cell lysate (CCL), produced after 5 cycles freeze-thaw
of the harvested virally infected cells, was purified by IEC using
the above described method. Unlike the virus supernatant from the
bioreactor, no satisfactory purification was achieved for the CCL
material generated from the Cellcube.TM.. FIG. 14 shows the
chromatogram. The result suggests that crude virus solution
generated from the Cellcube.TM. by freeze-thawing harvested cells
is not readily purified by the IEC method.
[0449] Other purification methods, including hydrophobic
interaction and metal chelate chromatography, were examined for the
purification of virus in CCL. Unfortunately, no improvement in
purification was observed by either method. Considering the
difficulties of purification of virus in CCL and the disadvantages
associated with a freeze-thaw step in the production process, the
inventors decided to explore other cell lysis methods.
[0450] A) Purification of Crude Virus Solution in Lysis Buffer
[0451] As described in Examples 1 and 3, HPLC analysis was used to
screen different detergent lysis methods. Based on the HPLC
results, 1% Tween-20 in 20 mM Tris+0.25 M NaCl+1 mM MgCl.sub.2,
pH=7.50 buffer was employed as the lysis buffer. At the end of the
virus production phase, instead of harvesting the infected cells,
the lysis buffer was pumped into the Cellcube.TM. after draining
the spent medium. Cells were lysed and virus released into the
lysis buffer by incubating for 30 min.
[0452] After clarification and filtration, the virus solution was
concentrated/diafiltrated and treated with Benzonase to reduce the
contaminating nucleic acid concentration. The treated virus
solution was purified by the method developed above using Toyopearl
SuperQ resin. Satisfactory separation, similar to that obtained
using virus supernatant from the bioreactor, was achieved during
elution. FIG. 15 shows the elution profile. However, when the virus
fraction was analyzed on HPLC, another peak in addition to the
virus peak was detected. The result is shown in FIG. 16A.
[0453] To further purify the virus, the collected virus fraction
was re-purified using the same method. As shown in FIG. 16B, purity
of the virus fraction improved considerably after the second
purification. Metal chelate chromatography was also evaluated as a
candidate for the second purification. Similar improvement in virus
purity as seen with the second IEC was achieved. However, because
of its simplicity, IEC is preferred as the method of choice for the
second purification.
[0454] As described above in Example 2, medium perfusion rate
employed during the cell growth and virus production phases has a
considerable impact on the HPLC separation profile of the Tween-20
crude virus harvest. For crude virus solution produced under high
medium perfusion rate, two ion exchange columns are required to
achieve the required virus purity.
[0455] Based on the much improved separation observed on HPLC for
virus solution produced under low medium perfusion rate, it is
likely that purification through one ion exchange column may
achieve the required virus purity. FIG. 17 shows the elution
profile using crude virus solution produced under low medium
perfusion rate. A sharp virus peak was attained during elution.
HPLC analysis of the virus fraction indicates virus purity
equivalent to that of CsCl gradient purified virus after one ion
exchange chromatography step. FIG. 18 shows the HPLC analysis
result.
[0456] The purified virus was further analyzed by SDS-PAGE, western
blot for BSA, and nucleic acid slot blot to determine the
contaminating nucleic acid concentration. The analysis results are
given in FIG. 19A, FIG. 19B and FIG. 19C, respectively. All those
analyses indicate that the column purified virus has equivalent
purity compared to the double CsCl gradient purified virus. Table
13 shows the virus titer and recovery before and after the column
purification. For comparison purposes, the typical virus recovery
achieved by double CsCl gradient purification was also included.
Similar virus recoveries were achieved by both methods.
TABLE-US-00014 TABLE 13 Comparison of IEC and double CsCl gradient
ultracentrifugation purification of AdCMVp53 from Cellcube .TM.
Titer (PFU/ml) A260/A280 Particle/PFU Recovery IEC 1 .times.
10.sup.10 1.27 36 63% Ultracentrifugation 2 .times. 10.sup.10 1.26
38 60%
[0457] A) Resin Capacity Study
[0458] The dynamic capacity of the Toyopearl Super Q resin was
evaluated for the purification of the Tween-20 harvested virus
solution produced under low medium perfusion rate. One hundred ml
of resin was packed in a XK50 column. Different amount of crude
virus solution was purified through the column using the methods
described herein.
[0459] Virus breakthrough and purification efficiency were analyzed
on HPLC. FIG. 20 shows the HPLC analysis results. At a column
loading factor greater than sample/column volume ratio of 2:1,
purity of the virus fraction was reduced. Contaminants co-eluted
with the virus. At a loading factor of greater than 3:1,
breakthrough of the virus into the flow through was observed.
Therefore, it was proposed that the working loading capacity of the
resin be in the range of sample/column volume ratio of 1:1.
[0460] B) Concentration/Diafiltration Post Purification
[0461] A concentration/diafiltration step after column purification
serves not only to increase the virus titer, if necessary, but also
to exchange to the buffer system specified for the virus product. A
300 K NMWC TFF membrane was employed for the concentration step.
Because of the absence of proteinacious and nucleic acid
contaminants in the purified virus, very high buffer flux was
achieved without noticeable pressure drop across the membrane.
[0462] Approximately 100% virus recovery was achieved during this
step by changing the buffer into 20 mM Tris+1 mM MgCl.sub.2+0.15 M
NaCl, pH=7.50. The purified virus was also successfully buffer
exchanged into DPBS during the concentration/diafiltration step.
The concentration factor can be determined by the virus titer that
is desired in the final product and the titer of virus solution
eluted from the column. This flexibility will help to maintain the
consistency of the final purified virus product.
[0463] C) Evaluation of Defective Adenovirus in the IEC Purified
AdCMVp53
[0464] Due to the less than 100% packaging efficiency of adenovirus
in producer cells, some defective adenoviruses generally exist in
crude virus solution. Defective viruses do not have DNA packaged
inside the viral capsid and therefore can be separated from intact
virus on CsCl gradient ultracentrifugation based the density
difference. It is likely that it would be difficult to separate the
defective from the intact viruses based bn ion exchange
chromatography assuming both viruses have similar surface
chemistry. The presence of excessive amount of defective viruses
will impact the quality of the purified product.
[0465] To evaluate the percentage of defective virus particles
present, the purified and concentrated viruses were subjected to
isopicnic CsCl ultracentrifugation. As shown in FIG. 21, a faint
band on top of the intact virus band was observed after
centrifugation. Both bands were recovered and dialyzed against 20
mM Tris+1 mM MgCl.sub.2, pH=7.50 buffer to remove CsCl. The
dialyzed viruses were analyzed on HPLC and the results are shown in
FIG. 22. Both viruses show similar retention time. However, the
defective virus has a smaller A260/A280 ratio than that of the
intact virus. This is indicative of less viral DNA in the defective
virus.
[0466] The peaks seen at retention times between 3.02 to 3.48 min
are produced by glycerol which is added to the viruses (10% v/v)
before freezing at -70.degree. C. The percentage of the defective
virus was less than 1% of the total virus. This low percentage of
defective virus is unlikely to impact the total particle to
infectious virus (PFU) ratio in the purified virus product. Both
viruses were analyzed by SDS-PAGE (shown in FIG. 19A). Compared to
the intact viruses, defective viruses lack the DNA associated core
proteins banded at 24 and 48.5 KD. This result is in agreement with
the absence of DNA in defective virus.
[0467] D) Process Overview of the Production and Purification of
AdCMVp53 Virus
[0468] Based on the above process development results, the
inventors propose a production and purification flow chart for
AdCMVp53 as shown in FIG. 23. The step and accumulative virus
recovery is included with the corresponding virus yield based on a
1 mer Cellcube.TM.. The final virus recovery is about 70.+-.10%.
This is about 3-fold higher than the virus recovery reported by
Huyghe et al. (1996) using a DEAE ion exchanger and a metal chelate
chromatographic purification procedure for the purification of p53
protein encoding adenovirus. Approximately 3.times.10.sup.12 PFU of
final purified virus product was produced from a 1 mer
Cellcube.TM.. This represents a similar final product yield
compared to the current production method using double CsCl
gradient ultracentrifugation for purification.
[0469] E) Scale-Up
[0470] Successful scale-up studies have been performed with the 4
mer Cellcube.TM. system, and are currently underway to evaluate
virus production in the 16 mer Cellcube.TM. system. The crude virus
solution produced will be filtered, concentrated and diafiltrated
using a bigger Pellicon cassette. The quality and recovery of the
virus will be determined. After Benzonase treatment, the crude
virus solution will be purified using a 20 cm and a 30 cm
BioProcess column for the 4 mer and 16 mer, respectively.
Example 9
Improved Ad-p53 Production in Serum-Free Suspension Culture
Adaptation of 293 Cells
[0471] 293 cells were adapted to a commercially available IS293
serum-free media (Irvine Scientific; Santa Ana, Calif.) by
sequentially lowering down the FBS concentration in T-flasks. The
frozen cells in one vial of PDWB were thawed and placed in 10% FBS
DMEM media in T-75 flask and the cells were adapted to serum-free
IS 293 media in T-flasks by lowering down the FBS concentration in
the media sequentially. After 6 passages in T-75 flasks the FBS %
was estimated to be about 0.019%. The cells were subcultured two
more times in the T flasks before they were transferred to spinner
flasks.
Serum-Free Adapted 293 Cells in T Flasks were Adapted to Suspension
Culture
[0472] The above serum-free adapted cells in T-flasks were
transferred to a serum-free 250 mL spinner suspension culture (100
mL working volume) for the suspension culture. The initial cell
density was 1.18 E+5 vc/mL. During the cell culture the viability
decreased and the big clumps of cells were observed. After 2 more
passages in T-flasks the adaptation to suspension culture was tried
again. In a second attempt the media was supplemented with heparin,
at a concentration of 100 mg/L, to prevent aggregation of cells and
the initial cell density was increased to 5.22 E+5 vc/mL. During
the cell culture there was some increase of cell density and cell
viability was maintained. Afterwards the cells were subcultured in
the spinner flasks for 7 more passages and during the passages the
doubling time of the cells was progressively reduced and at the end
of seven passages it was about 1.3 day which is comparable to 1.2
day of the cells in 10% FBS media in the attached cell culture. In
the serum-free IS 293 media supplemented with heparin almost all
the cells existed as individual cells not forming aggregates of
cells in the suspension culture (Table 14).
TABLE-US-00015 TABLE 14 Serum-Free Suspension Culture: Adaptation
to Suspension Passage No. Flask No. Average Doubling Time (days) 11
Viability decreased 13 3.4 14 3.2 15 1 Viability decreased Heparin
added 2 4.7 3 5.0 4 3.1 16 1 5.5 2 4.8 3 4.3 4 4.3 17 1 2.9 2 3.5 3
2.4 4 1.7 18 1 3.5 2 13.1 3 6.1 4 3.8 19 1 2.5 2 2.6 3 2.3 4 2.5 20
1 1.3 (97% viability) 2 1.5 (99% viability) 3 1.8 (92% viability) 4
1.3 (96% viability)
Viral Production and Growth of Cells in Serum-Free Suspension
Culture in Spinner Flask
[0473] To test the production of Ad5-CMVp53 vectors in the
serum-free suspension culture the above cells adapted to the
serum-free suspension culture were grown in 100 mL serum-free IS293
media supplemented with 0.1% Pluronic F-68 and Heparin (100 mg/L)
in 250 mL spinner flasks. the cells were infected at 5 MOI when the
cells reached 1.36 E+06 viable cells/mL on day 3. The supernatant
was analyzed everyday for HPLC viral particles/mL after the
infection. No viruses were detected other than day 3 sample. On day
3 it was 2.2 E+09 vps/mL. The pfu/mL on day 6 was 2.6+/-0.6 E+07
pfu/mL. The per cell pfu production was estimated to be 19 which is
approximately 46 times below the attached culture in the
serum-supplemented media. As a control the growth of cells was
checked in the absence of an infection.
TABLE-US-00016 TABLE 15 Serum-Free Suspension Culture: Viral
Production and Cell Growth Control Viral infection Viral infection
w/o viral w/o media w/media infection exchange exchange Initial
Density 2.1 .times. 10.sup.5 2.1 .times. 10.sup.5 2.1 .times.
10.sup.5 (vc/mL) Cell Density at infection 9.1 .times. 10.sup.5 1.4
.times. 10.sup.6 1.5 .times. 10.sup.6 (vc/mL) Volumetric viral
production NA 2.6 .times. 10.sup.7 2.8 .times. 10.sup.8 (pfu/mL) 6
days P.I. Volumetric viral production NA NA 1.3 .times. 10.sup.10
(HPLC vps/mL) 6 days P.I. Per cell viral production NA NA 1.3
.times. 10.sup.4 (HPLC vps/cell)
Preparation of Serum-Free Suspension Adapted 293 Cell Banks
[0474] As described above, after it was demonstrated the cells
produce the Ad-p53 vectors, the cells were propagated in the
serum-free IS293 media with 0.1% F-68 and 100 mg/L heparin in the
spinner flasks to make serum-free suspension adapted cell banks
which contain 1.0 E+07 viable cells/mL/vial. To collect the cells
they were centrifuged down when they were at mid-log phase growth
and the viability was' over 90% and resuspended in the serum-free,
supplemented IS293 media and centrifuged down again to wash out the
cells. Then the cells were resuspended again in the
cryopreservation media which is cold IS293 with 0.1% F-68, 100 mg/L
heparin, 10% DMSO and 0.1% methylcellulose resulting in 1 E+07
viable cells/mL. The cell suspension was transferred to sterile
cryopreservation vials and they were sealed and frozen in
cryocontainer at -70 C overnight. The vials were transferred to
liquid nitrogen storage. The mycoplasma test was negative.
[0475] To revive the frozen cells one vial was thawed into the 50
mL serum-free IS293 media with 0.1% F-68 and 100 mg/L heparin in a
T-150. Since then the cultures were subcultured three times in 250
mL spinner flasks. In the other study one vial was thawed into 100
mL serum-free, supplemented IS293 media in a 250 mL spinner flask.
Since then these were subcultured in serum-free spinner flasks 2
times. In both of the studies the cells grew very well.
Media Replacement and Viral Production in Serum-Free Suspension
Culture in Spinner Flask
[0476] In the previous serum-free viral production in the
suspension culture in the spinner flask the per cell viral
production was too low for the serum-free suspension production to
be practical. It was supposed that this might be due to the
depletion of nutrients and/or the production of inhibitory
byproducts. To replace the spent media with fresh serum-free,
supplemented IS293 media the cells were centrifuged down on day 3
and resuspended in a fresh serum-free IS-293 medium supplemented
with F-68 and heparin (100 mg/L) and the resulting cell density was
1.20 E+06 vc/mL and the cells were infected with Ad5-CMVp53 vectors
at 5 MOI. The extracellular HPLC vps/mL was 7.7 E+09 vps/mL on day
3, 1.18 E+10 vps/mL on day 4, 1.2 E+10 vps/mL on day 5 and 1.3 E+10
vps/mL on day 6 and the pfu/mL on day 6 was 2.75+/-0.86 E+08
tvps/mL. The ratio of HPLC viral particles to pfus was about 47.
Also the cells have been centrifuged down and lysed with the same
type of the detergent lysis buffer as used in the harvest of
CellCube. The cellular HPLC vps/mL was 1.6 E+10 vps/mL on day 2,
6.8 E+09 vps/mL on day 3, 2.2 E+09 vps/mL on day 4, 2.24 E+09
vps/mL on day 5 and 2.24 E+09 vps/mL on day 6.
[0477] The replacement of the spent media with a fresh serum-free,
supplemented IS 293 media resulted in the significant increase in
the production of Ad-p53 vectors. The media replacement increased
the production of extracellular HPLC viral particles 3.6 times
higher above the previous level on day 3 and the production of
extracellular pfu titer ten times higher above the previous level
on day 6. Per cell production of Ad-p53 vectors was estimated to be
approximately 1.33 E+04 HPLC vps.
[0478] The intracellular HPLC viral particles peaked on day 2
following the infection and then the particle numbers decreased. In
return the extracellular viral particles increased progressively to
the day 6 of harvest. Almost all the Ad-p53 vectors were produced
for the 2 days following the infection and intracellularly
localized and then the viruses were released outside of the cells.
Almost half of the viruses were released outside of the cells into
the supernatant between day 2 and day 3 following the infection and
the rate of release decreased as time goes on.
[0479] All the cells infected with Ad-p53 vectors lost their
viability at the end of 6 days after the infection while the cells
in the absence of infection was 97% viable. In the presence of
infection the pH of the spent media without the media exchange and
with the media exchange was 6.04 and 5.97, respectively, while the
one in the absence of the infection was 7.00 (Table 14).
Viral Production and Cell Culture in Stirred Bioreactor with Media
Replacement and Gas Overlay
[0480] To increase the production of Ad-p53 vectors, a 5 L CelliGen
bioreactor was used to provide a more controlled environment. In
the 5 L CelliGen bioreactor the pH and the dissolved oxygen as well
as the temperature were controlled. Oxygen and carbon dioxide gas
was connected to the solenoid valve for oxygen supply and the pH
adjustment, respectively. For a better mixing while generating low
shear environment a marine-blade impeller was implemented. Air was
supplied all the time during the operation to keep a positive
pressure inside the bioreactor.
[0481] To inoculate the bioreactor a vial of cells was thawed into
100 mL serum-free media in a 250 mL spinner flask and the cells
were expanded in 250 or 500 mL spinner flasks. 800 mL cell
inoculum, grown in 500 mL flasks, was mixed with 2700 mL fresh
media in a 10 L carboy and transferred to the CelliGen bioreactor
by gas pressure. The initial working volume of the CelliGen
bioreactor was about 3.5 L culture. The agitation speed of the
marine-blade impeller was set at 80 rpm, the temperature at
37.degree. C., pH at 7.1 at the beginning and 7.0 after the
infection and the DO at 40% all the time during the run.
[0482] The initial cell density was 4.3 E+5 vc/mL (97% viability)
and 4 days later when the cell density reached to 2.7 E+6 vc/mL
(93% viability) the cells were centrifuged down and the cells were
resuspended in a fresh media and transferred to the CelliGen
bioreactor. After the media exchange the cell density was 2.1 E+6
vc/mL and the cells were infected at MOI of 10. Since then the DO
dropped to below 40%. To keep the DO above 40%, about 500 mL of
culture was withdrawn from the CelliGen bioreactor to lower down
the oxygen demand by the cell culture and the upper marine-blade
was positioned close to the interface between the gas and the
liquid phase to improve the oxygen transfer by increasing the
surface renewal. Since then the DO could be maintained above 40%
until the end of the run.
[0483] For pH control, CO.sub.2 gas was used to acidify the cell
culture and 1 N NaHCO.sub.3 solution to make the cell culture
alkaline. The pH control was initially set at 7.10. The initial pH
of the cell culture was about pH 7.41. Approximately 280 mL 1N
NaHCO.sub.3 solution was consumed until the pH of cell culture
stabilized around pH 7.1. After the viral infection of the cell
culture, the pH control was lowered down to pH 7.0 and the CO.sub.2
gas supply line was closed off to reduce the consumption of
NaHCO.sub.3 solution. The consumption of too much NaHCO.sub.3
solution for pH adjustment would increase the cell culture volume
undesirably. Since then 70 mL 1N NaHCO.sub.3 solution was consumed
and the pH was in the range between 7.0 and 7.1 most of the time
during the run. The temperature was controlled between 35.degree.
C. and 37.degree. C.
[0484] After the infection the viability of the cells decreased
steadily until day 6 of harvest after the infection. On the harvest
day none of the cells was viable. The volumetric viral production
of the CelliGen bioreactor was 5.1 E+10 HPLC vps/mL compared to the
1.3 E+10 vps/mL in the spinner flask. The controlled environment in
the CelliGen bioreactor increased the production of Ad-p53 vectors
4-fold compared to the spinner flasks with media replacement. This
is both due to the increase of the cell density at the time of
infection from 1.2 E+6 to 2.1 E+6 vc/mL and the increase of per
cell viral production from 1.3 E+4 to 2.5 E+4 vps/mL. The 2.5 E+4
vps/mL is comparable to the 3.5 E+4 vps/cell in the
serum-supplemented, attached cell culture.
Viral Production and Cell Culture in Stirred and Sparged
Bioreactor
[0485] In the first study the cells were successfully grown in an
stirred bioreactor for viral production, and the oxygen and
CO.sub.2 were supplied by gas overlay in the headspace of a
bioreactor. However, this method will limit the scale-up of the
cell culture system because of its inefficient gas transfer.
Therefore in the second study, to test the feasibility of the scale
up of the serum-free suspension culture was investigated by growing
of cells and producing Ad-p53 in a sparged bioreactor. Pure oxygen
and CO.sub.2 gases were supplied by bubbling through the serum-free
IS293 media supplemented with F-68 (0.1%) and heparin (100
mg/L).
[0486] Pure oxygen was bubbled through the liquid media to supply
the dissolved oxygen to the cells and the supply of pure oxygen was
controlled by a solenoid valve to keep the dissolved oxygen above
40%. For efficient oxygen supply while minimizing the damage to the
cells, a stainless steel sintered air diffuser, with a nominal pore
size of approximately 0.22 micrometer, was used for the pure oxygen
delivery. The CO.sub.2 gas was also supplied to the liquid media by
bubbling from the same diffuser as the pure oxygen to maintain the
pH around 7.0. For pH control, Na.sub.2CO.sub.3 solution (106 g/L)
was also hooked up to the bioreactor. Air was supplied to the head
space of the bioreactor to keep a positive pressure inside the
bioreactor. Other bioreactor configuration was the same as the
first study.
[0487] Inoculum cells were developed from a frozen vial. One vial
of frozen cells (1.0 E+7 vc) was thawed into 50 mL media in a T-150
flask and subcultured 3 times in 200 mL media in 500 mL spinner
flasks. 400 mL of inoculum cells grown in 2 of 500 mL spinner
flasks were mixed with IS293 media with F-68 and heparin in a 10 L
carboy to make 3.5 L cell suspension and it was transferred to the
5 L CelliGen bioreactor.
[0488] The initial cell density in the bioreactor was 3.0 E+4
vc/mL. The initial cell density is lower than the first study. In
the first study four of 500 mL spinner flasks were used as the
inoculum. Even with the lower initial cell density the cells grew
up to 1.8 E+6 vc/mL on day 7 in the sparged environment and the
viability was 98%. During the 7 days' growth, glucose concentration
decreased from 5.4 g/L to 3.0 g/L and lactate increased from 0.3
g/L to 1.8 g/L.
[0489] On day 7, when the cell density reached 1.8 E+6 vc/mL, the
cells in the bioreactor were centrifuged down and resuspended in
3.5 L fresh serum-free IS293 media with F-68 and heparin in a 10 L
carboy. The 293 cells were infected with 1.25 E+11 pfu Ad-p53 and
transferred to the CelliGen bioreactor. In the bioreactor, cell
viability was 100% but the cell density was only 7.2 E+5 vc/mL.
There was a loss of cells during the media exchange operation. The
viral titer in the media was measured as 2.5 E+10 HPLC vps/mL on
day 2, 2.0 E+10 on day 3, 2.8 E+10 on day 4, 3.5 E+10 on day 5 and
3.9 E+10 HPLC vps/mL on day 6 of harvest. The first CelliGen
bioreactor study with gas overlay produced 5.1 E+10 HPLC vps/mL.
The lower virus concentration in the second run was likely due to
the lower cell density at the time of infection. Compared to the
7.2 E+5 vc/mL in the second run, 2.1 E+6 vc/mL was used in the
first run. Actually the per cell production of Ad-p53 in the second
sparged CelliGen bioreactor is estimated to be 5.4 E+4 vps/cell
which is the highest per cell production ever achieved so far. The
per cell production in the first serum-free CelliGen bioreactor
without sparging and the serum-supplemented T-flask was 2.5 E+4
vps/cell and 3.5 E+4 vps/cell, respectively.
[0490] After the viral infection, the viability of the cells
decreased from 100% to 13% on day 6 of harvest. During those 6 days
after the infection the glucose concentration decreased from 5.0
g/L to 2.1 g/L and the lactate increased from 0.3 g/L to 2.9 g/L.
During the entire period of operation about 20 mL of
Na.sub.2CO.sub.3 (106 g/L) solution was consumed.
[0491] The experimental result shows that it is technically and
economically feasible to produce Ad-p53 in the sparged and stirred
bioreactor. Scale-up and large-scale unit operation of sparged and
stirred bioreactor are well established.
Example 10
Blanche et al Production Process
[0492] The following example is text excerpted from pages 4-14 of
Blanche et al in U.S. Ser. No. 60/076,662. This text is descriptive
of the methods used by Blanche et al in production of recombinant
adenovirus.
[0493] Recombinant adenoviruses are usually produced by the
introduction of viral DNA into the encapsulation line, followed by
lysis of the cells after approximately 2 or 3 days (with the
kinetics of the adenoviral cycle being 24 to 36 hours). After lysis
of the cells, the recombinant viral particles are isolated by
centrifugation on a cesium chloride gradient.
[0494] For implementation of the process, the viral DNA introduced
may be the complete recombinant viral genome, possibly constructed
in a bacterium (ST 95010) or in a yeast (WO95/03400), transfected
in the cells. It may also be a recombinant virus used to infect the
encapsulation line. It is further possible to introduce the viral
DNA in the form of fragments, each carrying a portion of the
recombinant viral genome and a homology zone permitting the
recombinant viral genome to be reconstituted by homologous
recombination between the different fragments after introduction
into the encapsulation cell. Thus a classical adenovirus production
process includes the following steps: The cells (for example, cells
293) are infected in a culture plate with a viral prestock at the
rate of 3 to 5 viral particles per cell (Multiplicity of Infection
(MOI)=3 to 5), or transfected with viral DNA. The incubation then
lasts 40 to 72 hours. The virus is subsequently released from the
nucleus by lysis of the cells, generally by several successive thaw
cycles. The cellular lysate obtained is then centrifuged at low
speed (2000 to 4000 rpm), after which the supernatant (clarified
cellular lysate) is purified by centrifugation in the presence of
cesium chloride in two steps: [0495] A first rapid 1.5 hour
centrifugation on two layers of cesium chloride of densities 1.25
and 1.40 surrounding the density of the virus (1.34) in such a way
as to separate the virus from the proteins of the medium; [0496] A
second, longer centrifugation in a gradient (from 10 to 40 hours
according to the rotor used), which constitutes the true and only
purification step of the virus.
[0497] Generally, after the second centrifugation step, the band of
the virus is intensified. Nevertheless, two finer, less dense bands
are observed. Observation under the electron microscope has shown
that these bands are made up of empty or broken viral particles for
the denser band and of viral subunits (pentons, hexons) for the
less dense band. After this step, the virus is harvested by needle
puncture in the centrifugation tube and the cesium is eliminated by
dialysis or deionization.
[0498] Although the purity levels obtained are satisfactory, this
type of process presents certain drawbacks. In particular, it is
based on the use of cesium chloride, which is a reagent
incompatible with therapeutic use in man. Thus, it is imperative to
eliminate the cesium chloride at the end of purification. This
process also has certain other disadvantages mentioned below,
limiting its use to an industrial scale.
[0499] To remedy these problems, it has been proposed to purify the
virus obtained after lysis, not by gradient of cesium chloride, but
by chromatography. Thus the article of Huyghe et at. (Hum. Gen.
Ther. 6 (1996) 1403) describes a study of different types of
chromatographs applied to the purification of recombinant
adenoviruses. This article describes in particular a study of
recombinant adenovirus purification using weak anion exchange
chromatography (DEAE). Earlier studies already described the use of
this type of chromatography toward that goal (Klemperer et al.,
Virology 9 (1959) 536; Philipson, L., Virology 10 (1960) 459;
Haruna et al., Virology 13 (1961) 264). The results presented in
the article by Huyghe et al. show a rather poor efficacy of the ion
exchange chromatography protocol recommended. Thus, the resolution
obtained is average, with the authors indicating that virus
particles are present in several chromatographic peaks; the yield
is low (viral particle yield: 67%; infectious particle yield: 49%);
and the viral preparation obtained following this chromatographic
step is impure. In addition, pretreatment of the virus with
different enzymes/proteins is necessary. This same article also
describes a study of the use of gel permeation chromatography,
showing very poor resolution and very low yields (15-20%).
[0500] The present invention describes a new process for the
production of recombinant adenoviruses. The process according to
the invention results from changes in previous processes in the
production phase and/or in the purification phase. The process
according to the invention now makes it possible in a very rapid
and industrializable manner to obtain stocks of virus of very high
quantity and quality.
[0501] One of the first features of the invention concerns more
particularly a process for the preparation of recombinant
adenoviruses in which the viruses are harvested from the culture
supernatant. Another aspect of the invention concerns a process for
the preparation of adenoviruses including an ultrafiltration step.
According to yet another aspect, the invention concerns a process
for the purification of recombinant adenoviruses including an anion
exchange chromatography step. The present invention also describes
an improved purification process, using gel permeation
chromatography, possibly coupled with anion exchange
chromatography. The process according to the invention makes it
possible to obtain viruses of high quality in terms of purity,
stability, morphology, and infectivity, with very high yields and
under production conditions completely compatible with the
industrial requirements and with the regulations concerning the
production of therapeutic molecules.
[0502] In particular, in terms of industrialization, the process
according to the invention uses methods of the treatment of
supernatants of cultures tested on a large scale for recombinant
proteins, such as microfiltration or deep filtration, and
tangential ultrafiltration. Furthermore, because of the stability
of the virus at 37.degree. C., this process permits better
organization at the industrial stage inasmuch as, contrary to the
intracellular method, the harvesting time does not need to be
precise to within a half day. Moreover, it guarantees maximum
harvesting of the virus, which is particularly important in the
case of viruses defective in several regions. In addition, the
process according to the invention permits an easier and more
precise follow-up of the production kinetics directly on homogenous
samples of supernatant, without pretreatment, which permits better
reproducibility of the productions. The process according to the
invention also makes it possible to eliminate the cell lysis step.
The lysis of the cells presents a number of drawbacks. Thus, it may
be difficult to consider breaking the cells by freeze/thaw cycles
at the industrial level. Besides, the alternative lysis methods
(Dounce, X-press, sonification, mechanical shearing, etc.) present
drawbacks as well: they are potential generators of sprays that are
difficult to confine for L2 or L3 viruses (level of confinement of
the viruses, depending on their pathogenicity or their mode of
dissemination), with these viruses having a tendency to be
infectious through airborne means; they generate shear forces
and/or a liberation of heat that are difficult to control,
diminishing the activity of the preparations. The solution of using
detergents to lyse the cells would demand validation and would also
require that elimination of the detergent be validated. Finally,
cellular lysis leads to the presence in the medium of a large
quantity of cellular debris, which makes purification more
difficult. In terms of virus quality, the process according to the
invention potentially permits better maturation of the virus,
leading to a more homogenous population. In particular, provided
that the packing of the viral DNA is the last step in the viral
cycle, the premature lysis of the cells potentially liberates empty
particles which, although not replicative, are a priori infectious
and capable of participating in the distinctive toxic effect of the
virus and of increasing the ratio of specific activity of the
preparations obtained. The ratio of specific infectivity of a
preparation is defined as the ratio of the total number of viral
particles, measured by biochemical methods (OD 260 nm, HPLC, CRP,
immuno-enzymatic methods, etc.), to the number of viral particles
generating a biologic effect (formation of lysis plaques on cells
in culture and solid medium, translation of cells). In practice,
for a purified preparation, this ratio is determined by dividing
the concentration of particles measured by OD at 260 nm by the
concentration of plaque-forming units in the preparation. This
ratio should be less than 100.
[0503] The results obtained show that the process according to the
invention makes it possible to obtain a virus of a purity
comparable to the homologous one purified by centrifugation in
cesium chloride gradient, in a single step and without preliminary
treatment, starting from a concentrated viral supernatant.
[0504] A first goal of the invention thus concerns a process for
the production of recombinant adenoviruses characterized by the
fact that the viral DNA is introduced into a culture of
encapsulation cells and the viruses produced are harvested after
release into the culture supernatant. Contrary to the previous
processes in which the viruses are harvested following premature
cellular lysis performed mechanically or chemically, in the process
according to the invention the cells are not lysed by means of an
external factor. Culturing is pursued during a longer period of
time, and the viruses are harvested directly in the supernatant,
after spontaneous release by the encapsulation cells. In this way
the virus according to the invention is recovered in the cellular
supernatant, while in the previous processes it is an intracellular
and more particularly an intranuclear virus that is involved.
[0505] The applicant has now shown that despite that elongation in
duration of the culture and despite the use of larger volumes, the
process according to the invention makes it possible to generate
viral particles in large quantity and of better quality.
[0506] In addition, as indicated above, this process makes it
possible to avoid the lysis steps, which are cumbersome from the
industrial standpoint and generate numerous impurities.
[0507] The principle of the process thus lies in the harvesting of
the viruses released into the supernatant. This process may involve
a culture time longer than that used in the previous techniques
based on lysis of the cells. As indicated above, the harvesting
time does not have to be precise to within a half day. It is
essentially determined by the kinetics of release of the viruses
into the culture supernatant.
[0508] The kinetics of liberation of the viruses can be followed in
different ways. In particular, it is possible to use analysis
methods such as reverse-phase HPLC, ion exchange analytic
chromatography, semiquantitative PCR (example 4.3), staining of
dead cells with trypan blue, measurement of liberation of LDH type
intracellular enzymes, measurement of particles in the supernatant
by Coulter type equipment or by light diffraction, immunologic
(ELISA, RIA, etc.) or nephelometric methods, titration by
aggregation in the presence of antibodies, etc.
[0509] Harvesting is preferably performed when at least 50% of the
viruses have been released into the supernatant. The point in time
at which 50% of the viruses have been released can easily be
determined by doing a kinetic study according to the methods
described above. Even more preferably, harvesting is performed when
at least 70% of the viruses have been released into the
supernatant. It is particularly preferred to do the harvesting when
at least 90% of the viruses have been released into the
supernatant, i.e., when the kinetics reach a plateau. The kinetics
of liberation of the virus are essentially based on the replication
cycle of the adenovirus and can be influenced by certain factors.
In particular, they may vary according to the type of virus used,
and especially according to the type of deletion done in the
recombinant viral genome. In particular, deletion of region E3
seems to slow liberation of the virus. Thus, in the presence of
region E3, the virus can be harvested between 24 and 48 hours
post-infection. In contrast, in the absence of region E3, a longer
culturing time seems necessary. In this regard, the applicant has
had experience with the kinetics of liberation of an adenovirus
deficient in regions E1 and E3 into the supernatant of the cells,
and has shown that liberation begins approximately 4 to 5 days
post-infection and lasts up to about day 14. Liberation generally
reaches a plateau between day 8 and day 14, and the titer remains
stable for at least 20 days post-infection.
[0510] Preferably, in the process according to the invention, the
cells are cultured during a period ranging between 2 and 14 days.
Furthermore, liberation of the virus may be induced by expression
in the encapsulation cell of a protein, for example a viral one,
involved in the liberation of the virus. Thus, in the case of the
adenovirus, liberation may be modulated by expression of the Death
protein coded by region E3 of the adenovirus (protein E3-11.6K),
possibly expressed under the control of an inducible promoter.
Consequently, it is possible to reduce the virus liberation time
and to harvest in the culture supernatant more than 50% of the
viruses 24-48 hours post-infection.
[0511] To recover the viral particles, the culture supernatant is
advantageously first filtered. Since the adenovirus is
approximately 0.1 .mu.m (120 nm) in size, filtration is performed
with membranes whose pores are sufficiently large to let the virus
pass through, but sufficiently fine to retain the contaminants.
Preferably, filtration is performed with membranes having a
porosity greater than 0.2 .mu.m. According to a particularly
advantageous exemplified embodiment, filtration is performed by
successive filtrations on membranes of decreasing porosity.
Particularly good results have been obtained by doing filtration on
filters with a range of decreasing porosity -10 .mu.m, 1.0 .mu.m,
then 0.8-0.2 .mu.m. According to another preferred variant,
filtration is performed by tangential microfiltration on flat
membranes or hollow fibers. More particularly, it is possible to
use flat Millipore membranes or hollow fibers ranging in porosity
between 0.2 and 0.6 .mu.m. The results presented in the examples
show that this filtration step has a yield of 100% (no loss of
virus was observed by retention on the filter having the lowest
porosity).
[0512] According to another aspect of the invention, the applicant
has now developed a process making it possible to harvest and
purify the virus from the supernatant. Toward this goal, a
supernatant thus filtered (or clarified) is subjected to
ultrafiltration. This ultrafiltration makes is possible (i) to
concentrate the supernatant, with the volumes used being important;
(ii) to do a first purification of the virus and (iii) to adjust
the buffer of the preparation in the subsequent preparation steps.
According to a preferred exemplified embodiment, the supernatant is
subjected to tangential ultrafiltration. Tangential ultrafiltration
consists of concentrating and fractionating a solution between two
compartments, retentate and filtrate, separated by membranes of
specified cutoff thresholds, by producing a flow in the retentate
compartment of the apparatus and by applying a transmembrane
pressure between this compartment and the filtrate compartment. The
flow is generally produced with a pump in the retentate compartment
of the apparatus, and the transmembrane pressure is controlled by a
valve on the liquid channel of the retentate circuit or by a
variable-speed pump on the liquid channel of the filtrate circuit.
The speed of the flow and the transmembrane pressure are chosen so
as to generate low shear forces (Reynolds number less than 5000
sect.sup.-1, preferably below 3000 sect, pressure below 1.0 bar),
while preventing plugging of the membranes. Different systems can
be used to accomplish ultrafiltration, e.g., spiral membranes
(Millipore, Amicon), as well as flat membranes or hollow fibers
(Amicon, Millipore, Sartorius, Pall, GF, and Sepracor). Since the
adenovirus has a mass of ca. 1000 kDa, it is advantageous within
the scope of the invention to use membranes having a cutoff thresh
below 1000 kDa, and preferably ranging between 100 kDa and 1000
kDa. The use of membranes having a cutoff threshold of 1000 kDa or
higher in effect causes a large loss of virus at this stage. It is
preferable to use membranes having a cutoff threshold ranging
between 200 and 600 kDa, and even more preferable, between 300 and
500 kDa. The experiences presented in the examples show that the
use of a membrane having a cutoff threshold at 300 kDa permits more
than 90% of the viral particles to be retained, while eliminating
the contaminants from the medium (DNA, proteins in the medium,
cellular proteins, etc.). The use of a cutoff threshold of 500 kDa
offers the same advantages.
[0513] The results presented in the examples show that this step
makes it possible to concentrate large volumes of supernatant
without loss of virus (90% yield), with generation of a better
quality virus. In particular, concentration factors of 20- to
100-fold can easily be obtained.
[0514] This ultrafiltration step thus includes an additional
purification compared to the classical model inasmuch as the
contaminants of mass below the cutoff threshold (300 or 500 kDa)
are eliminated at least in part. A distinct improvement in the
quality of the viral preparation may be seen upon comparing the
appearance of the separation after the first ultracentrifugation
step according to the two processes. In the classical process
involving lysis, the viral preparation tube presents a cloudy
appearance with a coagulum (lipids, proteins) sometimes touching
the virus band, while in the process according to the invention,
following liberation and ultrafiltration, the preparation presents
a band that is already well resolved of the contaminants of the
medium that persist in the upper phase. An improvement in quality
is also demonstrated upon comparing the profiles on ion exchange
chromatography of a virus obtained by cellular lysis with a virus
obtained by ultrafiltration as described in the present invention.
In addition, it is possible to further enhance the quality by
pursuing ultrafiltration with diafiltration of the concentrate.
This diafiltration is performed based on the same principle as
tangential ultrafiltration, and makes it possible to more
completely eliminate the large-sized contaminants at the cutoff
threshold of the membrane, while achieving equilibration of the
concentrate in the purification buffer.
[0515] In addition, the applicant has also shown that this
ultrafiltration makes it possible to purify the virus directly by
ion exchange chromatography or by gel permeation chromatography,
permitting excellent resolution of the viral particle peak without
requiring treatment of the preparation beforehand with
chromatography. This is particularly unexpected and advantageous.
In fact, as indicated in the article by Huyghe et al. mentioned
above, purification by chromatography of viral preparations gives
poor results and also requires pretreatment of the viral suspension
with benzonase and cyclodextrins.
Example 11
Optimization of Production Process
[0516] To arrive at an optimized process that may be used for
adenovirus production for clinical therapeutic production, a few
steps in the above process as well as that of Blanche et al in PCT
Publication No. WO 98/00524 (incorporated by reference) have been
modified to enhance large scale production. Those steps involve
modification to the virus harvest step, the nuclease treatment
step, and the resin used for purification. The optimized process is
depicted by the flow chart in FIG. 28.
Virus Harvest Step
[0517] In the process described above, virus was harvested by
lysing the 293 cells using a 1% Tween-20 lysis solution 2 days
post-viral infection. This harvest method required the introduction
of a lysis step into the process and the addition of one substance
(Tween-20) into the crude viral harvest. In consideration of the
lytic nature of the adenovirus life cycle, an alternative strategy
was used to harvest the virus-containing supernatant after complete
viral-mediated 293 cell lysis. Viral release kinetics were
determined by analyzing daily samples of supernatant from the
CellCube.TM. system after infection. Viral release into the
supernatant reached a plateau on day 5 post-infection. The kinetics
of viral release were found to be consistent. FIG. 24 shows the
typical viral release kinetics for Ad5CMV-p53. Equivalent viral
yield was obtained by using either the Tween-20 lysis or the
autolysis supernatant harvest methods. The supernatant harvest
method, however, simplified the production process by removing the
lysis step in the process and the added lysis agent (Tween-20) in
the crude viral harvest. As a result, the supernatant harvest
method will preferably be used for the optimized process.
Nuclease Treatment Step
[0518] In the above process and that of Blanch et al in PCT
Publication No. WO 98/00524, 1 M NaCl was included in the
Benzonase.TM. treatment buffer to prevent viral precipitation
during enzyme treatment. Unfortunately, the presence of 1 M NaCl in
the buffer was found to significantly inhibit the Benzonase.TM.
enzymatic activity. As a result, other buffers which could prevent
viral precipitation without retarding the Benzonase.TM. enzymatic
activity were examined. A 0.5M Tris/HCl+1 mM MgCl.sub.2, pH=8.0,
buffer was found to meet both criteria. In addition, this buffer
has a conductivity of 19 mS/cm, which makes it possible to load the
Benzonase.TM.-treated viral solution directly onto the
chromatographic column for purification As a result, changing to
the 0.5M Tris/HCl+1 mM MgCl.sub.2, pH=8.0, buffer will not only
improve the Benzonase.TM. treatment efficiency but also simplify
the downstream process.
Resin for Purification
[0519] The Fractogel TMAE(s) resin and Toyopearl SuperQ 650M resin
employed in the above process and that of Blanche et al performed
consistently well. However because of supply and technical support
problems, alternative resins were chosen for use in virus
purification. Source 15Q resin manufactured by Pharmacia Biotech
was found to perform as well as the Fractogel and Toyopearl resins.
FIG. 25 shows a typical chromatogram from the Source 15Q resin.
Surprisingly, viral material was found to interact slightly
stronger with the Source 15Q resin than with the Fractogel and
Toyopearl resin. As a result, a larger viral protein peak was seen
at the beginning of the gradient elution. The purified virus
fraction was also eluted relatively later in the gradient. However,
the overall purification profile was not significantly different
from that of the Fractogel or Toyopearl resin. HPLC analysis of the
purified viral fraction from the Source 15Q resin showed an
equivalent profile to that from the Fractogel resin. FIG. 26 shows
the HPLC profile.
[0520] Ad5CMV-p53 made by the optimized process was also assessed
for biological activity compared to material made by the above
process and that of Blanche et al. Two cell lines, H1299 and
SAOS-LM, which express no endogenous p53, were transduced with
materials made by the two processes at equal multiplicities of
infection (viral particles/cell). p53 expression was monitored at 6
hours post-transduction in H1299 and 24 hours post-transduction in
SAOS-2. The level of p53 expression mediated by the two materials
was equivalent and dose-dependent in both recipient cell lines
(FIG. 27).
Process Hold Points
[0521] The freeze and thaw stability of the purified viral fraction
eluted from the chromatography column (purified bulk) was
evaluated. The purified viral fraction eluted from the column was
frozen bulk at .ltoreq.-60.degree. C. after supplementing with
glycerol to a final concentration of 10% (v/v). The frozen bulk was
thawed successfully without detrimental effects on titer. The
freeze and thaw data are given in Table 16.
TABLE-US-00017 TABLE 16 Freeze and thaw of purified bulk Post
freeze-thaw Small volume No freeze Bulk freeze (45 ml) freeze (1
ml) Viral particles/ml 4.0 .times. 10.sup.11 3.8 .times. 10.sup.11
4.1 .times. 10.sup.11
[0522] Furthermore, no change in HPLC profiles was observed pre-
and post-freeze and thaw. Therefore, viral material at the
post-chromatography step can be held at <-60.degree. C. for
further processing, and a process hold can be introduced at the
post-chromatography step (purified bulk).
[0523] Similar freeze and thaw stability was observed for
formulated sterile bulk product. Table 17 shows the freeze and thaw
data.
TABLE-US-00018 TABLE 17 Freeze and thaw of formulated sterile bulk
product Post freeze-thaw Small volume freeze No freeze Bulk freeze
(45 ml) (1 ml) Viral particles/ml 1.3 .times. 10.sup.13 1.4 .times.
10.sup.13 1.3 .times. 10.sup.13
[0524] As a result, the formulated sterile bulk product can be held
at <-60.degree. C. before aseptic filling without damaging
effects on the viral titer and a process hold point can be
introduced at the post-formulation step (sterile bulk).
Example 12
Parameters for Large Scale Production
[0525] During the scale up and optimization of the large scale
process (16-mer), several parameters were found by the inventors to
be desirable for successful production runs and high virus yield.
These desirable parameters are centered around the cell culturing
system, the most upstream portion of the adenovirus production
process, and are believed to be applicable to other types of cell
culturing systems and at larger scales. In particular, it can be
easily envisioned that the changes described below which result in
functional changes to the system will be useful to enable
modification and optimization of other cell culture systems.
[0526] For the present example, the culture control parameters are
as follows. Cells are cultured at 37.degree. C. with 10% CO.sub.2.
Cell culture medium is DMEM+10% FBS, and the inoculation cell
density for cell expansion is <4.times.10.sup.4 cell/cm.sup.2.
The parameters that involve the set up and execution of the
CellCube.TM. system and are listed below.
[0527] CellCube.TM. setup: In the full scale set up (4.times.100 or
"16-mer"), it is desirable to use one separate cell culture medium
recirculation loop for each cube module (4-mer) to achieve even
medium perfusion. For example, in the present 16-mer set-up, the
16-mer is composed of four 4-mers linked together in a series, each
4-mer having it's own medium recirculation loop. The 16-mer is
considered one unit and is controlled by a single control module
that modulates the rate of medium perfusion and measures the
culture control parameters. Other setups such as using one medium
recirculation loop for every two 4-mer modules results in uneven
medium perfusion due to pressure drops in the system, and is
detrimental to the health of the cells in the second cube with
lower levels of nutrients and freshly oxygenated medium. Thus, in a
cell culture system used for adenovirus production, it is
preferable that the cell culture medium perfusion be maintained at
a constant pressure and rate, ensuring consistent and optimal
health of the producer cells. The perfusion rate is determined by
monitoring one or more of the cell culture control parameters, such
as glucose concentration.
[0528] Seeding Density: In order to achieve maximal cell expansion
and growth, it is most preferable to inoculate the CellCube.TM.
with 1-2.times.10.sup.4 cells/cm.sup.2. Higher numbers of cells
used in the cell inoculation step results in a cell density that is
too high and the result is an over-confluence of cells at the time
of viral infection, thus lowering yields. It is well within one of
skill in the art to determine that in other types of cell culturing
systems, similar optimization of the seeding density for a
particular system could easily be determined.
[0529] Seeding Method: It has been found that for full scale
production, it is advantageous to use one homogeneous cell pool for
seeding of all CellCube.TM. modules. Prior to seeding the cell
culture apparatus, producer cells from the working cell bank are
expanded from stock cultures. This cell expansion is accomplished
by growing the cells in tissue culture flasks or other similar cell
culture devices, and continual splitting of the cells into larger
tissue culture devices. Upon reaching the total number of needed
cells for inoculation of the large scale cell culture apparatus,
all of the cells from each of the cell culture devices used for
cell expansion are pooled together. This homogeneous cell pool is
used to inoculate each of the CellCube.TM. modules of the 16-mer.
Seeding of each of the modules using separate cell populations, for
example from individual cell culture devices used in the cell
expansion phase, can result in uneven cell density, and therefore
uneven confluency levels at the time of infection. It is believed
that the use of a homogeneous cell pool for seeding overcomes these
problems.
[0530] Length of Cell Inoculation: During inoculation of each of
the CellCube.TM. modules, cells are added to the module and allowed
a period of a few hours to attach to the surface of the module.
During this time there is no medium perfusion or recirculation. It
has been found by the inventors that it is advantageous to complete
this cell seeding in one day (24 hrs). Thus for example, one side
of the module is inoculated and left for a period of 6-8 hours to
allow cell attachment, and then the other side of the module is
inoculated and left overnight to allow the cells to attach to the
surface of the module. During this seeding process, the cell
culture medium from each side of the module is kept separate, and
not allowed to flow to the other side of the module. It has been
observed that if the cell inoculation procedure is done over a
period of time longer than one day, and/or with medium exchange
between sides of the module, that there is a greater likelihood of
cell detachment from the cell culture surface due to weak
attachment. Possible reasons for this weak attachment may include:
1) the medium exchange between sides of the module which may
produce shear forces with the potential to dislodge cells
undergoing the attachment process, and 2) the longer time period
before medium perfusion is started may result in low levels of
nutrients in the media, and therefore the health of the cells
deteriorates, leading to less efficient attachment.
[0531] Culture Control Parameters: The inventors have found that
glucose concentration of the cell culture medium should preferably
be maintained at 1-2 g/L. Previous studies using glucose
concentrations at higher levels has been shown to reduce product
yield.
[0532] Infection Method: Eight days post-cell seeding, the cells
are infected with adenovirus. During the infection process, medium
perfusion is stopped for one hour, however medium recirculation is
maintained, thus keeping high levels of fresh oxygen in the medium.
It has been found by the inventors that if medium recirculation is
also stopped during the infection step, there is an increased
possibility of cell death due to oxygen starvation.
Example 13
Optimized Large Scale Production and Purification of Adenovirus
[0533] The example described below is descriptive of the methods
and materials used in a large scale production and purification
process for recombinant Ad5CMV-p53 adenovirus. This process uses a
CellCube.TM. bioreactor apparatus as the cell culturing system, and
large scale in this example refers to a CellCube 4.times.100 set up
or multiples thereof. Total maximum virus yields that may be
obtained from one CellCube 4.times.100 system are about
1-5.times.101.sup.5 viral particles at harvest.
[0534] Cell Expansion and Culture
[0535] The CellCube.TM. 4.times.100 was set up as described above,
with 4 CellCube.TM. 100 modules in parallel, all in a medium
recirculation loop, and the whole system being controlled by a
single control unit. The producer cells, 293 cells from a working
cell bank (WCB), were thawed and expanded in T flasks and
Cellfactories (Nunc) seeding at densities from 1-8.times.10e4
cells/cm.sup.2. Cells were generally split at a confluence of about
85-90% and continually expanded until enough cells were obtained
for inoculation of the Cellcube.TM.. At the end of the cell
expansion phase, all the cells from each of the Cellfactories were
pooled to make one homogeneous mixture of 293 cells. This cell pool
was used to inoculate the Cellcube.TM. at a total cell number in
the range of 1-3.times.10e9 viable cells per side. During cell
inoculation, medium perfusion and recirculation is suspended for a
period of time to allow the cells to attach to the substrate. Cells
are allowed to attach to side one for 4-6 hours; then side two is
inoculated and the cells allowed to attach for no more than 18
hours before recirculation is restarted. After cell attachment,
medium perfusion and recirculation was restarted and the cells were
allowed to grow for 7 days at 37.degree. C. under culture
conditions of pH=6.90-7.45, DO=40-50% air saturation. Medium
perfusion rate is regulated according to the glucose concentration
in the Cellcube.TM., and was maintained at between 1-2 g/L. One day
before viral infection, medium for perfusion was changed from
DMEM+10% FBS to Basal DMEM (no FBS). On day 8, cells were infected
with AdCMVp53 virus at a multiplicity of infection (MOI) of 5-50
viral particles per cell based on 8.times.10.sup.10 cells total.
Medium perfusion was stopped for 1 hr at the time of infection and
then resumed for approximately two days. Medium recirculation was
maintained throughout the virus infection period.
[0536] Virus Harvest and Purification
[0537] Previous studies looking at virus release kinetics after
Ad5CMV-p53 infection of 293 cells determined that maximal virus
release from the producer cells due to the lytic nature of
adenovirus was obtained four to six days after infection. Thus,
four to six days after virus infection, the supernatant from the
Cellcube.TM. modules was removed as a pool. The virus supernatant
was then clarified by filtration through two Polyguard 5.0 micron
filters, followed by a 5.0 micron Polysep filter (Millipore). The
supernatant was then concentrated approximately 10-fold using
tangential flow filtration through a Pellicon cassette (Millipore)
of 300 K nominal molecular weight cut-off (NMWC). The buffer was
then exchanged by diafiltration against 0.5 M Tris+1 mM MgCl.sub.2,
pH=8. The supernatant was then treated at room temperature with 100
U/ml Benzonase.TM. in a buffer of 0.5M Tris/HCl+1 mM MgCl.sub.2,
pH=8.0; 0.2 micron filtered, and incubated overnight at room
temperature to remove contaminating cellular nucleic acids. The
crude virus preparation is then 0.2 micron filtered and loaded
directly onto an ion exchange column (BPG 200/500, Pharmacia)
containing Source 15Q resin equilibrated with 20 mM Tris+1 mM
MgCl.sub.2+250 mM NaCl, pH=8.0. The virus was eluted with a 40
column linear gradient using an elution buffer composed of 20 mM
Tris+1 mM MgCl.sub.2+2 M NaCl, pH=8.0. The purified virus was then
subjected to another concentration and diafiltration step to place
the virus in the final formulation for the virus product. The
concentration step used a 300 NMWC Pellicon TFF membrane, and for
diafiltration the buffer was exchanged using 8-10 column volumes of
Dulbecco's Phosphate Buffered Saline+10% Glycerol. The purified
virus was then sterile filtered through a 0.2 micron Millipak
(Millipore) filter. The formulated product was then filled into
sterile glass vials with stoppers. Flip off crimp caps were applied
prior to final product inspection and labeling.
[0538] Two process hold points may be introduced into the process
as described in Example 10. The first process hold may be
introduced after the IEC step, at which time 10% glycerol may be
added to the eluate and frozen for later processing. The second
process hold step may be introduced after the final product is
obtained but prior to sterile filtering and vialing. The final bulk
product can at this point can be frozen and held for final
filtering and vialing.
[0539] The following list of parameters was measured throughout the
production and purification process. The Specification is the
desired measurement that the test article should meet. The result
of each test is shown to the right on the table.
TABLE-US-00019 Test Specification Result Mycoplasma PTC Negative
PASS 1993 Bioburden .ltoreq.10 CFU/100 ML 0 CFU/100 ML In Vitro
Adventitious NEGATIVE PASS Virus In Vivo Adventitious NEGATIVE PASS
Virus Adeno-Associated NEGATIVE PASS Virus (PCR) Bioburden
.ltoreq.1 cfu/10 mL 0 cfu/10 mL Bacterial Endotoxins <5 EU/mL
<0.15 EU/mL Test Sterility STERILE Pass Sterility Sterile Pass
Bacterial Endotoxins <5 EU/mL <0.15 EU/mL Test Titration of 2
.times. 10.sup.10-8 .times. 10.sup.10 pfu/mL 5 .times. 10.sup.10
pfu/mL Adenovirus Vector Virus Particle 8.0 .times. 10.sup.11-1.2
.times. 10.sup.12 9.4 .times. 10.sup.11 Enumeration Viral
Particles/mL Viral Particles/mL Ratio 260/280 Ratio 260/280
Particle/pfu Ratio 10-60 20 Western Blot Express p53 Protein Pass
(anti-p53) Bioactivity (SAOS) MOI Causing 50% Cell <1000 vp/cell
Death is <1000 vp/cell Restriction Mapping Molecular Size as
Pass Expected Protein Content by .ltoreq.320 .mu.g/1 .times.
10.sup.12 245 .mu.g/1 .times. 10.sup.12 BCA Viral Particles Viral
Particles SDS-PAGE Bands as Expected Pass No Significant Extra
Bands HPLC .gtoreq.98% Purity .gtoreq.99.57% Ion Exchange Bovine
Serum .ltoreq.50 ng BSA/10.sup.12 <1.9 ng BSA/10.sup.12 Albumin
(ELISA) Viral Particles Viral Particles Recoverable Fill 1.0 to 1.4
mL 7 of 7 vials in the Volume range of 1.1 to 1.2 mL Physical
Description Clear to opalescent with Pass no gross particles by
visual inspection huDNA <10 ng/1 .times. 10.sup.12 0.4 ng/1
.times. 10.sup.12 Viral Particles Viral Particles General Safety
Pass Pass Replication <1 pfu in 2.5 .times. 10.sup.9 Viral
Report Value at 2.5 .times. 10.sup.9 Competent Particles and
Adenovirus 2.5 .times. 10.sup.10 p53 Mutation <3% <1%
Frequency pH 6.0-8.0 7.5
Example 14
Summary of Formulation Development for Adenovirus
[0540] Currently, clinical Adp53 product is stored frozen at
.ltoreq.60.degree. C. This deep frozen storage condition is not
only expensive, but also creates problems for shipment and
inconvenience for clinic use. The goal of the formulation
development effort is to develop either a liquid or a lyophilized
formulation for Adp53 that can be stored at refrigerated condition
and be stable for extended period of time. Formulation development
for Adp53 is focused on both lyophilization and liquid
formulations. From manufacturing and marketing economics point of
view, liquid formulation is preferred to a lyophilized formulation.
Preliminary results from both fronts of formulation development are
summarized here.
[0541] Materials and Equipment
[0542] Lyophilizer
[0543] A Dura-stop .mu.p lyophilizer (FTSsystems) with in process
sample retrieving device was used. The lyophilizer is equipped with
both thermocouple vacuum gauge and capacitance manometer for vacuum
measurement. Condenser temperature is programmed to reach to
-80.degree. C. Vials were stoppered at the end of each run with a
build-in mechanical stoppering device.
[0544] Residual Moisture Measurement
[0545] Residual moisture in freeze dried product was analyzed by a
Karl-Fisher type coulometer (Mettler DL37, KF coulometer).
[0546] HPLC Analysis
[0547] HPLC analysis of samples was done on a Beckman Gold HPLC
system.
[0548] Vials and Stoppers
[0549] Borosilicate 3 ml with 13 mm opening lyo vials and their
corresponding butyl rubber stoppers (both from Wheaton) were used
for both lyophilization and liquid formulation development. The
stoppered vials were capped with Flip-off aluminum caps using a
capping device (LW312 Westcapper, The West Company).
[0550] Results
[0551] Lyophilization
[0552] Initial Cycle and Formulation Development
[0553] There are three main process variables that can be
programmed to achieve optimal freeze-drying. Those are shelf
temperature, chamber pressure, and lyophilization step duration
time. To avoid cake collapse, shelf temperature need to be set at
temperatures 2-3.degree. C. below the glass transition or eutectic
temperature of the frozen formulation. Both the glass transition
and eutectic temperatures of a formulation can be determined by
differential scanning coloremetry (DSC) analysis. Chamber pressure
is generally set at below the ice vapor pressure of the frozen
formulation. The ice vapor pressure is dependent on the shelf
temperature and chamber pressure. Too high a chamber pressure will
reduce the drying rate by reducing the pressure differential
between the ice and the surrounding, while too low a pressure will
also slow down drying rate by reducing the heat transfer rate from
the shelf to the vials. The development of a lyophilization cycle
is closely related with the formulation and the vials chosen for
lyophilization. Formulation excipient selection was based on the
classical excipients found in most lyophilized pharmaceuticals. The
excipients in a lyophilization formulation should provide the
functions of bulking, cryoprotection, and lyoprotection. The
excipients chosen were mannitol (M, bulking agent), sucrose (S,
cryo- and lyoprotectant), and human serum albumin (HSA,
lyoprotectant). These excipients were formulated in 10 mM Tris+1 mM
MgCl.sub.2, pH=7.50 at various percentages and filled into the 3 ml
vials at a fill volume of 1 ml. To start with, a preliminary cycle
was programmed to screen a variety of formulations based on the
criteria of residual moisture and physical appearance after drying.
The cycle used is plotted in FIG. 29. Extensive screening was
carried out by variation of the percentages of the individual
excipients. Table 18 shows briefly some of the results.
TABLE-US-00020 TABLE 18 Evaluation of different formulations under
the same cycle Formulation M %/S %/HSA % Appearance Moisture (%
weight) 10/5/0.5 good cake 0.89 5/5/0.5 good cake 1.5 3/5/0.5 loose
cake 3.4 (partial collapse) 1/5/0.5 no cake (collapse) 6.4
[0554] The results suggest that a minimum amount of 3% mannitol is
required in the formulation in order to achieve pharmaceutically
elegant cake. The percentages of sucrose in the formulation were
also examined. No significant effect on freeze-drying was observed
at sucrose concentrations of .ltoreq.10%. HSA concentration was
kept constant to 0.5% during the initial screening stage.
[0555] After the evaluation of the formulations, freeze-drying
cycle was optimized by changing the shelf temperature, chamber
vacuum and the duration of each cycle step. Based on the extensive
cycle optimization, the following cycle (cycle #14) was used for
further virus lyophilization development. [0556] Load sample at
room temperature onto shelf [0557] Set shelf temperature to
45.degree. C. and freeze sample. Step time 2 h. [0558] Set shelf
temperature at -45.degree. C., turn vacuum pump and set vacuum at
400 mT. Step time 5 h [0559] Set shelf temperature at -35.degree.
C., set vacuum at 200 mT. Step time 13 h [0560] Set shelf
temperature at -22.degree. C., set vacuum at 100 mT. Step time 15 h
[0561] Set shelf temperature at -10.degree. C., set vacuum at 100
mT. Step time 5 h [0562] Set shelf temperature at 10.degree. C.,
set vacuum at 100 mT, Step time 4 h [0563] Vial stoppering under
vacuum
[0564] Cycle and Formulation Development with Virus in
Formulation
[0565] Effect of Sucrose Concentration in Formulation
[0566] Cycle and formulation were further optimized according to
virus recovery after lyophilization analyzed by both HPLC and
plaque forming unit (PFU) assays. Table 19 shows the virus
recoveries immediate after drying in different formulations using
the above drying cycle. Variation of the percentage of sucrose in
the formulation had significant effect on virus recoveries.
TABLE-US-00021 TABLE 19 Recoveries of virus after lyophilization
Formulation Residual Recovery M %/S %/HSA % Appearance moisture (%)
6/0/0.5 Good cake 0.44% 0 6/3.5/0.5 Good cake 2.2% 56 6/5/0.5 Good
cake 2.5% 81 6/6/0.5 Good cake 2.7% 120 6/7/0.5 Good cake 2.8% 120
6/8/0.5 Good cake 3.3% 93 6/9/0.5 Good cake 3.7% 120
[0567] Residual moisture in the freeze-dried product increased as
the sucrose percentage increased. A minimum sucrose concentration
of 5% is required in the formulation to maintain a good virus
recovery after lyophilization. Similar sucrose effects in
formulation that had 5% instead of 6% mannitol were observed.
However, good virus recovery immediately after drying does not
necessary support a good long-term storage stability. As a result,
formulations having 4 different sucrose concentrations of 6, 7, 8,
and 9%, were incorporated for further evaluation.
[0568] Effect of HSA in Formulation
[0569] The contribution of HSA concentrations in the formulation on
virus recovery after drying was examined using the same freeze
drying cycle. Table 20 shows the results
TABLE-US-00022 TABLE 20 Effects of HSA concentration on
lyophilization Formulation Residual Recovery M %/S %/HSA %
Appearance moisture (%) 6/7/0 Good cake 0.98 83 6/7/0.5 Good cake
1.24 120 6/7/2 Good cake 1.5 110 6/7/5 Good cake 1.7 102
[0570] The results indicate that inclusion of HSA in the
formulation had positive effect on virus recovery after drying.
Concentrations higher than 0.5% did not further improve the virus
recovery post drying. As a result, 0.5% HSA is formulated in all
the lyophilization formulations.
[0571] Cycle Optimization
[0572] As indicated in Table 19, relatively high residual moistures
were present in the dried product. Although there has not been a
known optimal residual moisture for freeze dried viruses, it could
be beneficial for long term storage stability to further reduce the
residual moisture in the dried product. After reviewing of the
drying cycle, it was decided to increase the secondary drying
temperature from 10.degree. C. to 30.degree. C. without increasing
the total cycle time. As indicated in Table 21, significant
reduction in residual moisture had been achieved in all the
formulations without negative effects on virus recoveries. With the
improved drying cycle, residual moisture was less than 2% in all
the formulations immediately after drying. It is expected that the
reduced residual moisture will improve the long-term storage
stability of the dried product.
TABLE-US-00023 TABLE 21 Effects of secondary drying temperature on
lyophilization Secondary Secondary drying at 10.degree. C. drying
at 30.degree. C. Formulation Residual Recovery Residual M %/S %/HAS
% moisture (w %) (%) moisture Recovery 6/6/0.5 2.2 100 0.8 93
6/7/0.5 2.5 86 1.1 100 6/8/0.5 2.7 83 1.3 87 6/9/0.5 3.3 93 1.5 86
5/6/0.5 2.3 110 1.0 94 5/7/0.5 2.7 88 1.2 85 5/8/0.5 3.5 97 1.6 88
5/9/0.5 4 90 1.9 86
[0573] N.sub.2 Backfilling (Blanketing)
[0574] Lyophilization was done similarly as above except that dry
N.sub.2 was used for gas bleeding for pressure control during the
drying and backfilling at the end of the cycle. At the end of a
drying run, the chamber was filled with dry N.sub.2 to about 80%
atmospheric pressure. Subsequently, the vials were stoppered. No
difference was noticed between the air and N.sub.2 blanketing runs
immediate after drying. However, if oxygen present in the vial
during air backfilling causes damaging effect (oxidation) on the
virus or excipients used during long-term storage, backfilling with
dry N.sub.2 is likely to ameliorate the damaging effects and
improve long term storage stability of the virus.
[0575] Removal of Glycerol from Formulation
[0576] During the preparation of virus containing formulations,
stock virus solution was added to the pre-formulated formulations
at a dilution factor of 10. Because of the presence of 10% glycerol
in the stock virus solution, 1% glycerol was introduced into the
formulations. To examine any possible effect of the presence of 1%
glycerol on lyophilization, a freeze drying run was conducted using
virus diafiltered into the formulation of 5% (M)/7% (S)/0.5% (HSA).
Diafiltration was done with 5 vol of buffer exchange using a
constant volume buffer exchange mode to ensure adequate removal of
residual glycerol (99% removal). After diafiltration, virus
solution was filled into vials and then lyophilized similarly.
Table 22 shows the lyophilization results
TABLE-US-00024 TABLE 22 Lyophilization without glycerol Formulation
M %/S %/HSA % Residual moisture Recovery (%) 5/7/0.5 1.0 80
[0577] No significant difference after freeze drying was observed
between formulations with and without 1% glycerol. Possible
implications of this change on long term storage will be
evaluated.
[0578] Long Term Storage Stability
[0579] Adp53 virus lyophilized under different formulations and
different cycles was placed at -20.degree. C., 4.degree. C., and
room temperature (RT) under dark for long term storage stability
evaluation. Parameters measured during the stability study were
PFU, HPLC viral particles, residual moisture, and vacuum inside
vial (integrity). FIG. 30A and FIG. 30B show the data after
12-month storage with secondary drying at 10.degree. C. without
N.sub.2 blanketing. Lyophilized virus is stable at both -20.degree.
C. and 4.degree. C. storage for up to 12 months. However, virus was
not stable at room temperature storage. More than 50% loss in
infectivity was observed at RT after 1-month storage. The reason
for the quick loss of infectivity at RT is not clear. However, it
is likely that RT is above the glass transition temperature of the
dried formulation and results in the accelerated virus degradation.
A differential scanning colorimitry (DSC) analysis of the
formulation could provide very useful information. Pressure change
inside the vials during storage was not detected, which indicates
that the vials maintained their integrity. The slight increase in
residual moisture during storage can be attributed to the release
of moisture from the rubber stopper into the dried product.
[0580] FIG. 31 and FIG. 32 show the storage stability data with
secondary drying at 30.degree. C. without and with N.sub.2
backfilling, respectively. Because of the nearly identical
stability observed at -20.degree. C. and 4.degree. C. storage
conditions, and to reduce the consumption of virus, -20.degree. C.
was not included in the long-term storage stability study. Similar
to the samples dried with secondary drying at 10.degree. C., virus
is stable at 4.degree. C. but not stable at RT. However, relative
better stability was observed at RT storage than those dried at
10.degree. C. secondary drying. This is likely to be the result of
the lower residual moisture attained at 30.degree. C. secondary
drying. This result suggests that residual moisture is an important
parameter that affects storage stability during long term storage.
Longer time storage is needed to reveal any beneficial effects of
doing N.sub.2 blanketing during lyophilization since no significant
effect was observed for up to 3 months storage. During storage,
HPLC analysis indicates that virus is stable at both -20.degree. C.
and 4.degree. C. storage and not stable at RT, which is consistent
with the results from PFU assay.
[0581] HSA Alternatives
[0582] The presence of HSA in the formulations could be a potential
regulatory concern. As a result, a variety of excipients have been
evaluated to substitute HSA in the formulation.
[0583] The substitutes examined included PEG, amino acids (glycine,
arginine), polymers (polyvinylpyrrolidone), and surfactants
(Tween-20 and Tween-80).
[0584] Liquid Formulation
[0585] Concurrent with the development of lyophilization of Adp53
product, experimentation was carried out to examine the possibility
of developing a liquid formulation for Adp53 product. The goal was
to develop a formulation that can provide enough stability to the
virus when stored at above freezing temperatures. Four sets of
liquid formulations have been evaluated. In the first set of
formulation, the current 10% glycerol formulation was compared to
HSA and PEG containing formulations. In the second set of
formulation, various amino acids were examined for formulating
Adp53. In the third set of formulation, the optimal formulation
developed for lyophilization was used to formulate Adp53 in a
liquid form. In the fourth set of formulation, detergents were
evaluated for formulating Adp53. Viruses formulated with all those
different formulations are being tested for long term storage
stability at -20.degree. C., 4.degree. C., and RT.
[0586] Liquid Formulation Set #1
[0587] HSA containing formulation (5% sucrose+5% HSA in 10 mM Tris
buffer, 150 mM NaCl, and 1 mM MgCl.sub.2, pH=8.20 buffer) was
compared with 10% glycerol in DPBS buffer and sucrose/PEG and
Trehalose/PEG formulations. PEG has been recommended as a good
preferential exclusion agent in formulations (Wong and
Parasrampurita, Pharmaceutical excipients for the stabilization of
proteins, BioPharm, 10(11) 52-61, 1997). It is included in this set
of formulation to examine whether it can provide stabilization
effect on Adp53. Formulations were filled into the 3 ml lyo vials
at a fill volume of 0.5 ml. Vials were capped under either
atmospheric or N.sub.2 blanketing conditions to examine any
positive effects N.sub.2 blanketing may have on long term storage
stability of Adp53. To ensure adequate degassing from the
formulation and subsequent N.sub.2 blanketing, the filled vials was
partially stoppered with lyo stoppers and loaded onto the shelf of
the lyophilizer under RT. The lyophilizer chamber was closed and
vacuum was established by turning on the vacuum pump. The chamber
was evacuated to 25 in. Hg. Then the chamber was purged completely
with dry N.sub.2. The evacuation and gassing were repeated twice to
ensure complete N.sub.2 blanketing. N.sub.2 blanketed vials were
placed with the non-N.sub.2 blanketed vials at various storage
conditions for storage stability evaluation. FIG. 33 shows the
analysis data for up to 9 months storage at 4.degree. C. and
RT.
[0588] Statistically significant drops in virus PFU and HPLC viral
particles were observed for 10% glycerol formulation after 3 months
storage at both 4.degree. C. and RT. No statistically significant
virus degradation was observed for all other formulations at
4.degree. C. storage. However, decrease in virus infectivity was
observed when stored at RT. Longer time storage is needed to
evaluate the effectiveness of the different formulations.
[0589] Liquid Formulation #2
[0590] Various combinations of amino acids, sugars, PEG and urea
were evaluated for Adp53 stabilization during long storage. FIG. 34
shows the 6-month stability data. The results indicate that
combination of 5% mannitol and 5% sucrose with other excipients
gave better storage stability at RT. In this set of formulation, no
human or animal derived excipients were included.
[0591] Liquid Formulation Set #3
[0592] The optimal formulations developed for lyophilization was
evaluated for formulating Adp53 in a liquid form. This approach
would be a good bridging between liquid formulation and
lyophilization if satisfactory Adp53 stability can be achieved
using lyophilization formulation for liquid fill. Filled samples
were stored at -20.degree. C. and 4.degree. C. for stability study.
FIG. 35 shows the 3-month stability data. Virus is stable at both
-20.degree. C. and 4.degree. C. for the four different
formulations. This is in agreement with the results from
formulation set #2, which suggests that better virus stability is
expected with the presence of both mannitol and sucrose in the
formulation. Longer time storage stability data is being
accrued.
[0593] Liquid Formulation Set #4
[0594] Detergents have been used in the formulations for a variety
of recombinant proteins. In this set of formulation, various
concentrations of detergents were examined for formulating Adp53.
The detergents used were non-ionic (Tween-80) and zwitterionic
(Chaps). FIG. 36 shows the 6-month stability data. Virus is stable
at 4.degree. C. storage. Better virus stability is observed in
Tween-80 containing formulations. Further accumulation of stability
data will help to optimize the detergent concentration. Similar to
formulation set#2, no exogenous protein is included in this set of
formulations.
[0595] Both lyophilization and liquid formulation have produced
very interesting and promising data and information. A
lyophilization cycle and corresponding formulations have been
developed to produce lyophilized Adp53 that is stable at 4.degree.
C. for at least 12 months. Longer time storage stability is being
collected. Because of the conservative approach taken in the
initial development of the lyophilization cycle, we are
investigating further to significantly reduce the lyophilization
cycle time and to improve the lyophilization process efficiency.
Somewhat to our surprise, very promising stability data was
generated for liquid formulation at 4.degree. C. storage. However,
longer time storage data is needed to evaluate the feasibility of
developing a liquid formulation for Adp53.
[0596] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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