Note: Descriptions are shown in the official language in which they were submitted.
WO 01/32838 CA 02389057 2002-04-26 pCT/US00/29549
METHODS FOR IMPROVING VIRAL VECTOR TITERS AND
REDUCING CELL DEATH IN CELL CULTURES
TECHNICAL FIELD
The present invention relates generally to methods and compositions for
improving the production of viral vectors in cell culture.
BACKGROUND OF INVENTION
The manufacture of products using animal cells is currently a major
component of the biotechnology industry. Such cells are currently being used
for the
production of viruses, vaccines, monoclonal antibodies, proteins, and
recombinant viral
vectors. Optimization of product expression and reduction of cost is important
for
economic and commercial feasibility of these products.
Unfortunately, the ability to express high amounts of praducts for long
periods of time is limited due to death of cells in culture. Prevention of
cell death
during culture is a high priority for the production of biopharmaceuticals,
however, very
little progress has been made in this direction especially for commercial
product
manufacturing.
One of the major limitations of present commercial production strategies
is their limited ability to delay apoptosis, particularly for cultures that do
not grow well
in suspension (i.e., are anchorage-dependent). This problem is particularly
apparent for
a large number of vaccine and viral vector applications where the cell lines
which are
utilized are anchorage dependent cultures.
One of the major components of most culture media, which significantly
increases the cost of manufacturing, is serum. However, removal or reduction
of serum
from a standard of 5-10% usually results in a decrease of product formation. A
number
of media have been developed for serum-free propagation, however, only a few
cell
types such CHO and 293 cells have been reliably grown under serum-free/reduced
serum conditions. Most commercial production of biological products still
require use
of serum, and production of viruses and viral vectors routinely require use of
higher
1
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
serum concentrations. Reduction in serum concentrations without sacrificing
product
yields or product titers is a challenge that has not been completely addressed
to date.
The present invention discloses compositions and methods for which
increase vector titer and decrease cell death, and further provides other
related
advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods
for the increasing the production of a recombinant viral vector in a cell
culture,
comprising culturing a recombinant viral vector producing cell line in a basal
media
which further comprises (a) greater than 1.5 g/L amino acids (or, a selected
amino acids
in a quantity and amount greater than present in a basal media such as DMEM),
and (b)
between 0.5% and 4% serum, such that the recombinant viral vectors are
produced.
Such methods can be utilized to increase the production of viral vector (or
viral titer) by
the cell line, and/or to decrease cell death during production. Moreover, the
effect of
such culturing conditions can be utilized to produce an anti-apoptotic effect
on cells
within the cell culture.
Representative examples of suitable basal media include RPMI or
DMEM. To such media is added amino acids (to a final concentration of greater
than
1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, or, 2.5 g/L) and serum
(between about
0.5% and 4%, preferably between about 1 or 2% and 3%. Within certain
embodiments,
the quantity and type of amino acids added to the basal media is based upon
the
utilization profile of the cell line to be cultured. For example, rather than
adding 1.5, 2,
2.5 or greater g/L of amino acids to a cell culture, selected amino acids can
be added in
order to supplement a given cell line's utilization profile.
Similarly, various types of serum can be used within the context of the
present invention, including for example, fetal calf serum and human serum.
Further
ingredients can also be added to the media, including for example metals such
as
selenium and zinc, and salts such as magnesium chloride.
2
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
The methods described herein may be utilized for a wide variety of
recombinant viral vector producing cell lines, including for example, cell
lines which
have been constructed to produce recombinant retroviral vectors, adenoviral
vectors,
adeno-associated viral vectors and alphavirus vectors. Such vectors may be
utilized for
a variety of purpose, including for example, for the in vivo or ex vivo
production of
proteins, or vaccine purposes. Within certain embodiments, the recombinant
viral
vector producing cell line is an anchorage-dependent cell line. Within further
embodiments, the cell line is cultured in a batch mode, or in a perfusion
culture system.
The method according to the present invention may further comprise a
first metal salt comprising a metal selected from the group consisting of
Cobalt (Co),
Copper (Cu), Manganese (Mn), Molybdenum (Mo), and Selenium (Se) and a second
metal salt comprising a metal selected from the group consisting of Iron (Fe),
Calcium
(Ca), Magnesium (Mg), and Zinc (Zn). Within certain embodiments, the first
metal salt
may be selected from the group consisting of CoC12.6H20, CuC12.2Hz0, CuS04,
MnC12.4H20, (NH4)6 Mo~02.4H20, and NazSe03. Additionally or alternatively, the
second metal salt may be selected from the group consisting of FeCl3, FeS04,
CaCl2,
CaN03, MgCl2, MgS04, ZnCl2, ZnS04, FeN03.9HZ0.
The present invention also provides compositions for increasing the
production of a recombinant viral vector and/or decreasing cell death in a
cell culture.
Exemplary compositions comprise a basal media; greater than 1.5 g/L amino
acids;
between 0.5% and 4% serum; a first metal salt wherein said first metal salt
comprises a
metal selected from the group consisting of Cobalt (Co), Copper (Cu),
Manganese
(Mn), Molybdenum (Mo), and Selenium (Se); and a second metal salt wherein said
second metal salt comprises a metal selected from the group consisting of Iron
(Fe),
Calcium (Ca), Magnesium (Mg), and Zinc (Zn).
Further provided are compositions wherein the basal media is selected
from the group consisting of BME, MEM, DMEM, DMEM-F-12, IMDM, McCoy's
SA, Media 199, Ham's F-10, Ham's F-12, MS-162, MS-174, and RPMI. Within
certain
embodiments, the basal media is selected from the group consisting of DMEM, MS
162, and MS-174.
3
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
Alternative embodiments of the present invention provide compositions
comprising greater than 1.7 g/L amino acids, greater than 2.0 g/L amino acids,
or
greater than 2.5 g/L amino acids. By certain embodiments, one of the amino
acids may
be L-Cystine which may be present at a concentration of between 75 and 300
mg/L or,
more preferably, at a concentration of between 100 and 200 mg/L. By still
further
embodiments, one of said amino acids is L-Serine which may be present at a
concentration of between 75 and 1000 mg/L or, more preferably, at a
concentration of
between 200 and 700 mg/L. Other embodiments provide that one of the amino
acids
may be L-Methionine at a concentration of between 75 and 300 mg/L or, more
preferably, at a concentration of between 100 and 200 mg/L.
Still further embodiments of the present invention ,provide compositions
comprising between 1% and 3% serum or, alternatively, between 2% and 3% serum.
By certain embodiments, the serum is fetal calf serum or human serum.
By certain embodiments, compositions according to the present
invention may comprise a first metal salt selected from the group consisting
of
CoC12.6H20, CuC12.2H20, CuS04, MnClz.4H20, (NH4)6 Mo~02.4H20, and NazSe03.
By preferred embodiments, the first metal salt may be present at a
concentration of
between 0.003 and 1.0 mg/L or, more preferably, at a concentration of between
0.01
and 0.1 mg/L.
Further embodiments provide compositions that may comprise a second
metal salt is selected from the group consisting of FeCl3, FeS04, CaCl2,
CaN03, MgClz,
MgSOa, ZnCl2, ZnS04, FeN03.9H20. By preferred embodiments, the second metal
salt
may be MgCl2 at a concentration of between 50 and S00 mg/L or, more
preferably, at a
concentration of between 125 and 175 mg/L. Alternatively, the second metal
salt may
be ZnClz at a concentration of between 1 and 5 mg/L or, more preferably, at a
concentration of between 2 and 4 mg/L.
These and other aspects of the present invention will become evident
upon reference to the following detailed description and attached drawings. In
addition,
various references are set forth herein which describe in more detail certain
procedures
4
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
or compositions (e.g., plasmids, etc.), and are therefore incorporated by
reference in
their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph which shows vector product produced from
DMEM 10% (FBS) vs. MS-162 10% (FBS) for 7 and 8 day cultures.
Figure 2 is a bar graph which shows cell death in cultures of DMEM
10% (FBS) vs. MS-162 10% (FBS) for 7 and 8 day cultures.
Figure 3 is a graph which compares the titer of cells grown in DMEM
11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11% (FBS).
Figure 4 is a graph which compares the vector production rate of cells
grown in DMEM 11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11%
(FBS).
Figure 5 is a graph which compares LDH production per day of cells
grown in DMEM 11% (FBS), DMEM 2% (FBS), MS-174 2% (FBS), and MS-174 11%
(FBS).
Figure 6 is a graph which compares total vector production (on a
manufacturing scale) for cells grown in DMEM 2% (FBS) and MS-174 2% (FBS).
Figure 7 is a graph which compares cell death (on a manufacturing scale)
for cells grown in DMEM 2% (FBS) and MS-174 2% (FBS).
Figure 8 is a bar graph which shows vector titer from cells grown on
DMEM 10% (FBS) vs. MS-162 10% (FBS).
Figure 9 is a bar graph which shows vector production from cells grown
on DMEM 10% (FBS) vs. MS-162 10% (FBS).
5
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Prior to setting forth the invention, it may be helpful to an understanding
thereof to first set forth definitions of certain terms that will be used
hereinafter.
"Recombinant viral vector" refers to a construct which is capable of
delivering, and, within preferred embodiments expressing, one or more genes)
or
sequences) of interest in a host cell. Such recombinant viral vectors may be
constructed from or derived from a variety of viruses, such as, for example,
retroviruses, adenoviruses, adeno-associated viruses, alphaviruses and the
like.
"Recombinant adeno-associated virus vector"or "rAAV vector" refers to
a gene delivery vector based upon an adeno-associated virus. The rAAV vectors,
should contain 5' and 3' adeno-associated virus inverted terminal repeats
(ITRs), and a
transgene or gene of interest operatively linked to sequences which regulate
its
expression in a target cell. Within certain embodiments, the transgene may be
operably
linked to a heterologous promoter (such as CMV), or, an inducible promoter
such as
(tet). In addition, the rAAV vector may have a polyadenylation sequence.
"Recombinant Viral Vector Producing Cell Line" refers to a cell line
which is used to produce recombinant viral particles, representative examples
of such
cell lines include retroviral packaging or producer cell lines, and alphavirus
packaging
or producer cell lines.
"Basal media" refers to a minimal media that, when supplemented with
serum, is sufficient to support the growth and/or proliferation of a desired
cell line.
Representative examples of such media include BME, MEM, DMEM, DMEM-F-12,
IMDM, McCoy's SA, Media 199, Ham's F-10, Ham's F-12, and RPMI. Such media
typically provide a mixture of sugar (e.g., glucose), vitamins (e.g., B12,
etc.), and one
or more salts or buffers, and may be obtained from a variety of commercial
sources,
including for example, Hyclone, Inc. (Logan, UT), Irvine Scientific (Irvine,
CA),
6
WO 01/32838 CA 02389057 2002-04-26 pCT~S00/29549
Biowhittaker (Walkersville, MD), Gibco-LTI (Gaithersburg, MD), and Sigma
Chemical
Co. (St. Louis, MO).
As noted above, the present invention provides compositions and
methods for the production of a recombinant viral vector in a cell culture,
comprising
the step of culturing a recombinant viral vector producing cell line in a
basal media
which further comprises (a) greater than 1.5 g/L amino acids and (b) between
0.5% and
4% serum, such that the recombinant viral vectors are produced.
Such methods can be utilized to increase the production of viral vector
(or viral titer) by the cell line, and / or to decrease cell death during
production, and
result in a variety of advantages. For example, increasing cell viability and
product titer
can result in (a) a decrease in the resultant cost of recombinant viral
particles due to a
decreased need for serum; (b) an increase in purity (due to less contaminating
dead-cell
products); (c) ease in use of anchorage dependent cell lines; and (d) an
increase in cell
density, as compared to other standard media, thereby resulting in a higher
product
concentration and/or higher specific activity.
In order to further the understanding of the invention, described in more
detail below is: (A) Construction of Recombinant Viral Vector Producing Cell
Lines,
including methods for generating recombinant viral vector producing cell
lines; and
(B) Methods for Culturing Recombinant Viral Vector Producing Cell Lines,
including
methods for selecting appropriate media.
A. Recombinant Viral Vector Producing Cell Lines
1. Construction of retroviral vector producing cell lines
Within one aspect of the present invention, retroviral vectors are
provided which are constructed to carry or express a selected genes) or
sequences) of
interest. Briefly, retroviral gene delivery vehicles of the present invention
may be
readily constructed from a wide variety of retroviruses, including for
example, B, C,
and D type retroviruses as well as spumaviruses and lentiviruses (see RNA
Tumor
Viruses, 2d ed., Cold Spring Harbor Laboratory, 1985). Such retroviruses may
be
readily obtained from depositories or collections such as the American Type
Culture
7
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
Collection ("ATCC"; Rockville, Maryland), or isolated from known sources using
commonly available techniques.
Any of the above retroviruses may be readily utilized in order to
assemble or construct retroviral gene delivery vehicles given the disclosure
provided
herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular
Cloning:
A Laboratory Manual, Zd ed., Cold Spring Harbor Laboratory Press, 1989;
Kunkle,
PNAS 82:488, 1985). In addition, within certain embodiments of the invention,
portions of the retroviral gene delivery vehicles may be derived from
different
retroviruses. For example, within one embodiment of the invention, retrovector
LTRs
may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous
Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin
of
second strand synthesis from an Avian Leukosis Virus.
Within one aspect of the present invention, retroviral vectors are
provided comprising a 5' LTR, a tRNA binding site, a packaging signal, one or
more
heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR,
wherein the vector construct lacks gaglpol or env coding sequences.
Other retroviral vectors may likewise be utilized within the context of
the present invention, including for example EP 0,415,731; WO 90/07936; WO
91/0285, WO 9403622; WO 9325698; WO 9325234; U.S. Patent No. 5,219,740; WO
9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and
Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;
Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.
Neurosurg. 79:729-
735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and
W091/02805).
Packaging cell lines suitable for use with the above described retroviral
vectors may be readily prepared (see U.S. Serial No. 08/240,030, filed May 9,
1994; see
also U.S. Serial No. 07/800,921, filed November 27, 1991), and utilized to
create
producer cell lines (also termed vector cell lines or "VCLs") for the
production of
recombinant vector particles.
Two particularly preferred packaging cell lines, HA-II and HA-LB, were
developed based upon the expression of MLV gag/pol and amphotropic env
sequences,
8
WO 01/32838 CA 02389057 2002-04-26 pCT~S00/29549
in the HT-1080 (human fibrosarcoma) cell line. These packaging cell lines were
developed using the principle of splitting the retroviral genome, removing the
long term
repeat (LTR) sequences and replacing them with cytomegalovirus (CMV) immediate
early promoter to reduce the potential of generating replication competent
retrovirus
(RCR). HA-II and HA-LB differed in the number of overlapping sequences. Vector
producing cell lines were generated by transduction of VSV-G pseudotyped
provector
encoding appropriate gene into the appropriate packaging cell line.
2. Recombinant Adeno-Associated Virus Vectors
As noted above, a variety of rAAV vectors may be utilized to direct the
expression of one or more desired sequence of interests. Briefly,. the rAAV
should be
comprised of, in order, a 5' adeno-associated virus inverted terminal repeat,
a transgene
or gene of interest operatively linked to a sequence which regulates its
expression in a
target cell, and a 3' adeno-associated virus inverted terminal repeat. In
addition, the
rAAV vector may preferably have a polyadenylation sequence.
Generally, rAAV vectors should have one copy of the AAV ITR at each
end of the transgene or gene of interest, in order to allow replication,
packaging, and
efficient integration into cell chromosomes. The ITR consists of nucleotides 1
to 145 at
the 5' end of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same
sequence) at the 3' end of the AAV DNA genome. Preferably, the rAVV vector may
also include at least 10 nucleotides following the end of the ITR (i.e., a
portion of the
"D region").
Within preferred embodiments of the invention, the transgene sequence
will be of about 2 to 5 kb in length (or alternatively, the transgene may
additionally
contain a "stuffer" or "filler" sequence to bring the total size of the
nucleic acid
sequence between the two ITRs to between 2 and 5 kb). Alternatively, the
transgene
may be composed of same heterologous sequence several times (e.g., two nucleic
acid
molecules which encode FGF-2 separated by a ribosome readthrough, or
alternatively,
by an Internal Ribosome Entry Site or "IRES"), or several different
heterologous
sequences (e.g., FGF-2 and FGF-5, separated by a ribosome readthrough or an
IRES).
9
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
Recombinant AVV vectors of the present invention may be generated
from a variety of adeno-associated viruses, including for example, serotypes 1
through
6. For example, ITRs from any AAV serotype are expected to have similar
structures
and functions with regard to replication, integration, excision and
transcriptional
mechanisms.
Within certain embodiments of the invention, expression of the
transgene may be accomplished by a separate promoter (e.g., a viral promoter).
Representative examples of suitable promoters in this regard include a CMV
promoter,
RSV promoter, SV40 promoter, or MoMLV promoter. Other promoters that may
similarly be utilized within the context of the present invention include cell
or tissue
specific promoters (e.g., a rod, cone, or ganglia derived promoter), or
inducible
promoters. Representative examples of suitable inducible promoters include
tetracycline-response promoters ("Tet", see, e.g., Gossen and Bujard, Proc.
Natl. Acad.
Sci. USA. 89:5547-5551, 1992; Gossen et al., Science 268:1766-1769, 1995;
Baron et
al., Nucl. Acids Res. 25:2723-2729, 1997; Blau and Rossi, Proc. Natl. Acad.
Sci. USA.
96:797-799, 1999; Bohl et al., Blood 92:1512-1517, 1998; and Haberman et al.,
Gene
Therapy 5:1604-1611, 1998), the ecdysone system (see, e.g., No et al., Proc.
Natl.
Acad. Sci. USA. 93:3346-3351, 1996), and other regulated promoters or promoter
systems (see, e.g., Rivera et al., Nat. Med. 2:1028-1032, 1996;).
The rAVV vector may also contain additional sequences, for example
from an adenovirus, which assist in effecting a desired function for the
vector. Such
sequences include, for example, those which assist in packaging the rAVV
vector in
adenovirus particles.
Packaging cell lines suitable for producing adeno-associated viral
vectors may be readily prepared given readily available techniques (see, e.g.,
U.S.
Patent No. 5,872,005).
3. Recombinant Alphavirus vectors
The present invention also provides a variety of Alphavirus vectors
which may function as gene delivery vehicles. For example, the Sindbis virus
is the
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
prototype member of the alphavirus genus of the togavirus family. The
unsegmented
genomic RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides in
length, contains a 5' cap and a 3' poly-adenylated tail, and displays positive
polarity.
Infectious enveloped Sindbis virus is produced by assembly of the viral
nucleocapsid
proteins onto the viral genomic RNA in the cytoplasm and budding through the
cell
membrane embedded with viral encoded glycoproteins. Entry of virus into cells
is by
endocytosis through clatharin coated pits, fusion of the viral membrane with
the
endosome, release of the nucleocapsid, and uncoating of the viral genome.
During viral
replication the genomic 49S RNA serves as template for synthesis of the
complementary negative strand. This negative strand in turn serves as template
for
genomic RNA and an internally initiated 26S subgenomic RNA. The Sindbis viral
nonstructural proteins are translated from the genomic RNA while structural
proteins
are translated from the subgenomic 26S RNA. All viral genes are expressed as a
polyprotein and processed into individual proteins by post translational
proteolytic
cleavage. The packaging sequence resides within the nonstructural coding
region,
therefore only the genomic 49S RNA is packaged into virions.
Several different alphavirus vector systems and packaging cell lines can
be readily generated given the disclosure provided herein. Representative
examples of
such systems include those described within U.S. Patent Nos. 5,843,723, and
5,789,245,
and PCT Publication No. WO 95/07994.
4. Other viral gene delivery vectors
In addition to retroviral vectors and alphavirus vectors, numerous other
viral vectors systems may also be utilized as a gene delivery vehicle.
Representative
examples of such gene delivery vehicles include viruses such as pox viruses,
such as
canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 86:317-321, 1989;
Flexner
et al., Ann. N. Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-
21, 1990;
U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40
(Mulligan
et al., Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell
59:1107-1113,
1989; McMicheal et al., N. Eng. J. Med. 309:13-17, 1983; and Yap et al.,
Nature
11
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
273:238-239, 1978); herpes (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S.
Patent
No. 5,288,641); HIV (Poznansky, J. Virol. 65:532-536, 1991); measles (EP 0
440,219);
Semliki Forest Virus, and coronavirus, as well as other viral systems (e.g.,
EP
0,440,219; WO 92/06693; U.S. Patent No. 5,166,057). In addition, viral Garners
may
be homologous, non-pathogenic (defective), replication competent virus (e.g.,
Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular
immune
responses, including CTL.
B. Methods for Culturing Recombinant Viral Vector Producing Cell Lines
In order to increase the production of viral vector (or viral titer) by the
cell line, and / or to decrease cell death during production, the viral vector
producing
cell line is cultured or incubated in a basal media to which is added (a)
greater than 1.5
g/L amino acids (or a specific amount of selected amino acids) and (b) between
0.5%
and 4% serum.
Briefly, as noted above basal media refers to a minimal media which,
when supplemented with serum, is sufficient to support the growth and/or
proliferation
of a desired cell line. Basal media typically contains a mixture of sugar
(e.g., glucose),
vitamins (e.g., B 12, etc.), and one or more salts or buffers. Representative
examples of
such media include BME, MEM, DMEl~'I, DMEM-F-12, IMDM, McCoy's SA, Media
199, Ham's F-10, Ham's F-12, and RPMI, and may be obtained from a variety of
commercial sources, including for example, Hyclone, Inc. (Logan, UT), Irvine
Scientific (Irvine, CA), Biowhittaker (Walkersville, MD), Gibco-LTI
(Gaithersburg,
MD), and Sigma Chemical Co. (St. Louis, MO).
To such basal media is added a selected quantity of preferably
monomeric amino acids. Within certain aspects of the invention, particular
amino acids
may be added to the basal media a higher proportion than other amino acids,
and
indeed, only certain amino acids may be specifically added to the basal media.
In order
to assess which amino acids are preferred, the desired viral-vector producing
cell line
may be analyzed based upon its amino acid utilization, and a quantity of amino
acids
added to the basal media to meet this need.
12
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
As a representative example, producer cell lines (HA-LB/CF8 or HA-
II/CF8) are grown in DMEM +10 % FBS at 37°C in 10% C02 incubators
until
confluence in a T-flask and culture media is replaced by fresh media (DMEM
+10%
FBS) on day 6. Media is replaced on a daily basis by fresh media, and spent
media is
stored at -80°C for further amino acid analysis. Cells are also
harvested from these
flasks on day 6, 7 and 8 using trypsin-EDTA for a cell counting using trypan
blue
exclusion. As a baseline, DMEM+10% FBS (unused) sample is also stored for
amino
acid analysis.
Amino acid analysis may be carried out by a variety of commercial
organizations, including for example, the Scientific Research Consortium, Inc.
(St.
Paul, MN). Briefly, amino acid analysis can be performed on Beckman
Instruments,
Inc., Models 6300 and 7300 dedicated amino acid analyzers, which incorporate a
10 cm
cation-ion exchange column, four sequential lithium-based eluents, and lithium
hydroxide for column regeneration. Absorbence is measured at 440 and 570 nm
following post-column color development by Ninhydrin reagent at 131 °C.
Data
acquisition and management is accomplished with a computer running Beckman
System Gold 8.10 chromatography software. Beckman reference solutions fulfills
standardization requirements. (S)-2-Aminoethyl-1-cysteine (S2AEC) or
Glucosamine
acid is added to the sample as the preferred internal standard.
Based on the amino acid analysis, consumption rates for each amino acid
is a calculated by subtracting the amount present in used media from the
amount present
in unused media and divided by the time spent between refeeds. The specific
consumption rate is calculated by dividing amino acid consumption rate by the
viable
cell number. A positive value indicates a net consumption and a negative value
indicates a net accumulation of the specific amino acid. Based on intended
media
usage, amino acid concentration in media is calculated by multiplying desired
cell
concentration and specific amino acid consumption rates. All the amino acids
that are
produced by the culture, can be deleted from the media composition.
Utilizing this method, amino acid concentrations based on nutrient
selection method combined with a typical basal media compositions of vitamins,
buffer,
13
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
metal salts and other salts is used to design a high density culture media. As
an
example, a comparison of MS-162 and MS-174 is provided below, versus the amino
acid profile of the basal media DMEM ( mg/L). The resultant media MS-162 and
MS-
174 had approximately 2.9 or 3 times increase in total amino acid content as
compared
to DMEM, and did not contain certain amino acids such as L-Alanine or L-
Glutamate.
MS-162 MS-174 DMEM
Amino Acid
L-Alanine 0 0 0
L-Arginine, free 302 400 84
base
L-Asparagine 39 100 0
L-Aspartic acid 29 88 0
L-Cystine 260 141 62.6
L-Glycine 40 0 30
L-Glutamate 0 0 0
L-Histidine, free85 49 42
base
L-Isoleucine 253 283 105
L-Leucine 353 429 105
L-Lysine.HCl 218 203 146
L-Methionine 143 137 30
L-Phenylalanine 116 112 66
L-Proline 39 0 0
L-Serine 461 458 42
L-Threonine 131 150 95
L-Tryptophan 80 59 16
L-Tyrosine 134 95 103.79
L-Valine 185 217 94
Sum of AA 2867 2921 1021
In addition, certain metal salts may also be added to the culture media in
order to enhance vector titer production and decrease cell death.
Representative
examples of such metal salts include: CoC12.6H20, CuC12.2H20, CuS04,
MnC12.4Hz0,
(NH4)6 Mo~02.4H20, and Na2Se03.
14
CA 02389057 2002-04-26
WO 01/32838 PCT/L1S00/29549
Metal Salts (mg_/L) MS-162 MS-174 DMEM
CoC12.6H20 237.95 0.003 0.03 0
CuC12.2H20 135.03 0.003 0.03 0
CuS04 0 0 0
MnC12.4H20 197.9 0.003 0.03 0
(NH4)6 Mo7 024.4H201235.95 0.003 0.03 0
Na2Se03 172.95 0.01 0.01 0
Other metals may also be included such as, e.g., Iron (Fe), Calcium (Ca),
Magnesium (Mg), and Zinc (Zn). Exemplary metal salts are presented in the
following
table:
Metal Salts (m~/L) MS-162 MS-174 DMEM
FeCl3 162.22 0 0
FeS04 0 0
CaCl2 110.99 265 200 265
CaN03 0 0
MgCl2 95.23 155 155
MgS04 0 0 97.6
ZnCl2 136.29 3 3
ZnS04 0 0
FeN03.9H20 0.1 0.1 0.1
MS-162 media was designed to support 1.7 e7 cells/ml of HA-II/CF8
vector producing cell lines and MS-174 was designed to support 1.7 e7 cells/ml
of -
LB/CF8 VPCL.
MS-174 media was also designed based on rational media development
principles. Amino acid consumption rates for HA-LB/CF8 cell lines were
calculated
and a media was designed to support HA-LB/CF8 cell lines at 1.7 x 10'
cells/ml. This
cell concentration is 10 times higher than the cell concentration that DMEM
can
support without exhausting any of its nutrients. In addition to higher amino
acids, this
media contains metal salts to support growth at low serum, and MgCl2 to
enhance
retroviral vector stability.
Utilizing such media, viral vector producing cell lines can be cultured
under a variety of conditions. For example, HA-LB/CF8 cells were inoculated in
CellCubes with 21,500 cm2 - 85,000 cm2 of surface area. Inoculation density
was
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
maintained between 2 -6 x 104 cells/cm2 to provide growth in an exponential
phase.
Appropriate basal media supplemented with 11% FBS and 2-4 mM L-Glutamine was
used for inoculation. Perfusion was initiated on day 1 to remove waste
metabolites,
collect viral product and supplement cell culture with fresh nutrients.
Perfusion rates
were gradually increased to 5-7 volume exchanges per day after 7-8 days.
Perfusion
media was usually changed to appropriate media with low serum on day 4. Levels
of
dissolved oxygen and pH of the culture were controlled by using appropriate
mixture of
air, oxygen and CO2. DO was usually maintained at 50-80% of air saturation,
and pH
was controlled at physiological levels. Gradients of DO and pH and their
oscillations
were minimized by increasing recirculation rate in the CellCube. Samples were
collected daily for analysis of glucose, lactate, ammonia and lactate
dehyrodogenase
using Kodak Ektachem machine. Samples were collected daily for titer analysis
to
measure retroviral activity.
The titer of vector producing cell lines can be readily determined by a
variety of methods. For example, a titer assay can be performed utilizing the
transduction of expression (TOE) principle. Briefly, HT-1080 cells were plated
on day
1 in 6 well plates and transduced with retroviral vector on day 2.
Transduction was
conducted using several dilutions of retroviral vector samples. A standard
with known
amounts of retroviral vector titers was used to generate a standard curve.
Factor VIII
activity in the supernatant fluid was measured on day 5 using a Factor VIII
assay kit
from Chromogenix using the manufacturer's supplied instructions. Factor VIII
levels
measured in the supernatant is directly correlated to retroviral vector titers
using a
standard curve.
The following examples are offered by way of illustration, and not by
way of limitation.
16
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
EXAMPLES
EXAMPLE 1
INCREASE IN VECTOR TITER AND DECREASE IN CELL DEATH BY USING MS-162 MEDIA
Production of retroviral vector was conducted in T-flasks. In particular,
HA-II or HA-LB based vector producing cell lines were inoculated in T-75
flasks at 2-4
x 104 cells/cm2 in 15 ml of DMEM supplemented with 10% FBS and 4 mM L-
Glutamine. On day 4 or 5 when cells became confluent, media was replaced with
appropriate media supplemented with various serum amounts. Media was replaced
on a
daily basis by fresh media, and spent media was analyzed for retroviral vector
concentration.
Cells were grown in 10% DMEM until day 6 and media was switched to
MS-162 +10% FBS on day 6. For a controlled comparison, DMEM +10% FBS was
also used. Figure 1 shows retroviral vector titers on day 7 and 8 in these two
different
conditions. Titers in MS-162 + 10% FBS were 35-65% higher than DMEM +10% FBS
media. Cell death was also analyzed by comparing LDH levels in the
supernatant.
LDH is one of the enzymes that are released when cells lyse, and it has been
traditionally used in the bioprocess development environment to correlate to
the cell
death. Figure 2 shows significantly reduced LDH levels for MS-162 media
compared
to DMEM. This indicates that MS-162 media can prevent apoptosis of cells to
provide
a sustained level of viable cells for a prolonged time period.
EXAMPLE 2
INCREASE IN VECTOR TITER IN A CONTINUOUS PERFUSION CULTURE
BY USING MS-1~4 MEDIA COMBINED WITH REDUCED SERUM
Production of retroviral vector using HA-LB/CF8 VPCL was carned out
in 21,250 cm2 CellCubes (Pilot Scales) and average of a number of runs are
compared
for vector titers. DMEM media with 11 % serum usually results in a plateau by
day 7-8
followed by a decline in the vector titers (Figure 3). Vector titers for DMEM
17
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
supplemented with 11% or 2% serum is around 0.5-1.5 x 108 eq.cfu/ml for the
entire
production phase (day 4-13). MS-174 supplemented with 2% or 11% serum results
in
vector levels in the range of 1-3 x 10g eq.cfu/ml. MS-174 +11% plateaued on
day 10
and then dropped similar to DMEM +11% FBS levels. MS-174 +2% FBS, in contrast,
increased vector production further during last three days. This demonstrates
that MS-
174 + 2% FBS has superior titer capabilities compared to DMEM +2%, DMEM +11
and MS-174 +11% FBS.
EXAMPLE 3
INCREASE IN TOTAL VECTOR PRODUCTION IN A CONTINUOUS PERFUSION CULTURE
1O BY USING MS-174 MEDIA COMBINED WITH REDUCED SERUM
Production of retroviral vectors using HA-LB/CF8 VPCL was carried
out in 21,250 cm2 CellCubes and average of a number of runs are compared for
total
vector production per day. This measurement is a true indication of the yields
and
performance of various culture systems. Figure 4 shows total vector production
per day
for various culture systems. The total vector production is also set forth in
Table 1
below:
TABLE 1
Total Vector
Produced (Area Total Vector in
Under the Curvel Harvest
DMEM +2% FBS 6.25E+12 4.45E+12
DMEM + 11% FBS 1.19E+13 5.26E+12
MS-174 +2% FBS 1.66E+13 1.30E+13
MS-174 +11% FBS 1.33E+13 1.10E+13
Briefly, DMEM media with 11% serum showed a peak on day 9 at 2.0 x
10'2 eq.cfu/day followed by a three fold decline to level close to 7 x 10" eq.
cfu/day by
day 12. DMEM supplemented with 2% FBS resulted in reduced levels of vector
production in the range 5-8 x 10" eq.cfu/day. MS-174 + 2% or 11% FBS resulted
in a
2-3 fold higher total vector production with levels in the range of 1.0 - 2.0
x 10'Z
eq.cfu/day during days 5-10 of culture. This is a significant improvement over
DMEM
18
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
+2% orl l% FBS cultures. During last three days, MS-174 +11% shows a 3 fold
drop
in vector production levels, while MS-174+2% shows further increases in vector
production rates. This was an unexpected result since based on DMEM results we
would have expected MS-174+11% to perform superior to MS-174+2% FBS.
EXAMPLE 4
COMBINING MS-174 MEDIA WITH REDUCED SERUM CONDITIONS CAN REDUCE APOPTOSIS
Growth of HA-LB/CF8 cells were carned out in 21,250 cmz CellCubes
and cell death is compared by plating LDH produced per day in these cultures.
When
cell lyses, it releases LDH in the surrounding media, which is directly
proportional to
the number of dead cells. Higher amount of cell death is very undesirable as
it
increases impurity levels and decreases duration of manufacturing runs. Figure
5 shows
LDH/day, which is an indication of the cell death/day during CellCube
cultures. All
data points represent averages of multiple runs. Standard media conditions
(DMEM +
11% FBS) indicates a 4-6 fold higher cell death compared to MS-174 + 2% FBS.
This
graph also shows MS-174 +2% FBS has 2-3 fold lower cell death compared to DMEM
+2% FBS cultures. More importantly, cell death levels during entire MS-174 +2%
FBS
culture is lower than day 9 of DMEM culture supplemented with either 2% or 11
FBS, indicating that cell death has been delayed significantly. This
demonstrates that
using this method it is possible to significantly increase duration of vector
production
phase.
This graph also shows limited effect of rational media design on
delaying apoptosis. A comparison of DMEM and MS-174 cultures, both
supplemented
with 11% FBS, indicate a 3-day delay in cell apoptosis in MS-174 media
culture.
However, the number of cells dying during last two days of culture is similar
in both
cultures. Reducing serum levels in these cultures provides a much superior
culture
conditions showing a delay of 7 days using MS-174+2% FBS culture.
19
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
EXAMPLE 5
HIGHER TITER AT MANUFACTURING SCALE USING MS-174 MEDIA
COMBINED WITH REDUCED SERUM
Growth of HA-LB/CF8 cells was carried out in 85,000 cm2 CellCubes
and two manufacturing scale runs are compared with respect to vector titers.
Results
are shown in Figures 6 and 7. Briefly, these graphs show 3-10 fold higher
titers in MS-
174 +2% FBS media compared to DMEM + 2% FBS media. Titers in DMEM are in
the range of 3-6 x 10' eq.cfu/ml, while MS-174 shows titers in the range of
1.5-4.0 x
107 eq.cfu/ml. This provides a significant improvement in vector quality and
reduces
manufacturing costs considerably.
EXAMPLE 6
HIGHER TOTAL VECTOR PRODUCTION RATE AT MANUFACTURING SCALE
USING MS-174 MEDIA COMBINED WITH REDUCED SERUM
Growth of HA-LB/CF8 cells was carried out in 85,000 cm2 CellCubes
and two manufacturing scale runs are compared for vector production rate. This
graph
also shows a 2-3 times higher vector production levels in MS-174 media
initially, and
5-10 fold higher levels after day 10. This represents a very significant
improvement in
product yields while considerably reducing cost of manufacturing.
EXAMPLE 7
2O REDUCTION IN CELL APOPTOSIS IN MANUFACTURING SCALE CULTURES
USING MS-174 MEDIA WITH REDUCED SERUM
Growth of HA-LB/CF8 cells was carned out in 85,000 cm2 CellCubes
and two manufacturing scale runs are compared for cell death. Results are
shown in
Figure 8, briefly, this graph shows similar cell death levels during 11% FBS
cultures
(day 1-4), followed by a significant delay in apoptosis for MS-174 media
supplemented
with 2% FBS. The levels of cell death in MS-174 media during entire production
phase
(day S-13) are lower than DMEM. This demonstrates that it is possible to delay
apoptosis by changing cell environmental conditions at bioproduction scales.
CA 02389057 2002-04-26
WO 01/32838 PCT/US00/29549
EXAMPLE 8
INCREASE IN VECTOR PRODUCTION FOR HA-LB/EPO VECTOR IN A BATCH CULTURE
HA-LB/EPO producer cell line for retroviral vector was grown as
indicated in the Materials and Methods section. Figure 9 shows average titer
values for
multiple DMEM and MS-162 runs in T-flasks, both supplemented with 10% FBS. MS-
162 media with 10% FBS results in a 60% increase in viral vector titers in the
culture
supernatant.
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
various modifications may be made without deviating from the spirit and scope
of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
21
