U.S. patent application number 11/858844 was filed with the patent office on 2008-01-10 for animal protein-free media for cultivation of cells.
This patent application is currently assigned to Baxter Healthcare Corporation. Invention is credited to Friedrich Dorner, Leopold Grillberger, Wolfgang Mundt, Manfred Reiter.
Application Number | 20080009040 11/858844 |
Document ID | / |
Family ID | 35447241 |
Filed Date | 2008-01-10 |
United States Patent
Application |
20080009040 |
Kind Code |
A1 |
Grillberger; Leopold ; et
al. |
January 10, 2008 |
ANIMAL PROTEIN-FREE MEDIA FOR CULTIVATION OF CELLS
Abstract
The present invention relates to animal protein-free cell
culture media comprising polyamines and a plant- and/or
yeast-derived hydrolysate. The invention also relates to animal
protein-free culturing processes, wherein cells can be cultivated,
propagated and passaged without adding supplementary animal
proteins in the culture medium. These processes are useful in
cultivating cells, such as recombinant cells or cells infected with
a virus, and for producing biological products by cell culture
processes.
Inventors: |
Grillberger; Leopold;
(Vienna, AT) ; Reiter; Manfred; (Vienna, AT)
; Mundt; Wolfgang; (Vienna, AT) ; Dorner;
Friedrich; (Vienna, AT) |
Correspondence
Address: |
BAXTER HEALTHCARE CORPORATION
ONE BAXTER PARKWAY
MAIL STOP DF2-2E
DEERFIELD
IL
60015
US
|
Assignee: |
Baxter Healthcare
Corporation
Irvine
CA
|
Family ID: |
35447241 |
Appl. No.: |
11/858844 |
Filed: |
September 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10976399 |
Oct 29, 2004 |
|
|
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11858844 |
Sep 20, 2007 |
|
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Current U.S.
Class: |
435/70.1 |
Current CPC
Class: |
C12N 5/0682 20130101;
C12N 2500/46 20130101; C12N 2500/74 20130101; C12N 2500/92
20130101; C12N 5/0043 20130101; C12N 2500/76 20130101 |
Class at
Publication: |
435/070.1 |
International
Class: |
C12P 21/00 20060101
C12P021/00 |
Claims
1. A method for expressing a target protein, comprising the steps
of: a) providing a culture of cells that have been grown in an
animal protein-free cell culture medium comprising an animal
protein-free cell culture medium, comprising at least one polyamine
and at least one protein hydrolysate derived from the group
consisting of plants and yeast; b) introducing a nucleic acid
sequence comprising a sequence coding for the target protein into
the cells; c) selecting the cells carrying the nucleic acid
sequence; and d) selectively inducing the expression of the target
protein in the cells.
2. The method according to claim 1, wherein the cells are selected
from the group consisting of mammalian cells, insect cells, avian
cells, bacterial cells, and yeast cells.
3. The method according to claim 1 wherein the cell/target protein
combination is selected from the group consisting of CHO
cells/coagulation factor VIII, BHK cells/erythropoietin, Epstein
Barr virus transformed, immortalized human B cells/human
antibodies.
4. The method according to claim 1, wherein the cells are
cultivated by a method selected from the group consisting of
batch-cultivation, feed-batch-cultivation, perfusion cultivation,
and chemostat-cultivation.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to animal protein-free cell
culture media comprising a polyamine and a plant- and/or
yeast-derived hydrolysate. The invention also relates to animal
protein-free culturing processes, wherein cells can be cultivated,
propagated and passaged without adding supplementary animal
proteins in the culture medium. These processes are useful in
cultivating cells, such as recombinant cells or cells infected with
a virus, and for producing biological products by cell culture
processes.
BACKGROUND OF THE INVENTION
[0002] For cultivation of cells, particularly eukaryotic cells, and
more specifically mammalian cells, there is a constant need to use
special culture media that make available the growth nutrient
substances that are required for efficient growth of the cells and
for the production of the proteins or viruses that are desired. For
the efficient production of biological products, such as viruses or
recombinant proteins, it is important that an optimal cell density
is achieved as well as the protein expression itself is increased
to obtain maximal product yield.
[0003] Cell culture media formulations have been supplemented with
a range of additives, including undefined components like fetal
calf serum (FCS), several animal derived proteins and/or protein
hydrolysates of bovine origin.
[0004] In general, serum or serum-derived substances, such as
albumin, transferrin or insulin, may contain unwanted agents that
can contaminate the cell cultures and the biological products
obtained therefrom. Furthermore, human serum derived additives have
to be tested for all known viruses, including hepatitis and HIV,
that can be transmitted by serum. Moreover, bovine serum and
products derived therefrom bear the risk of BSE contamination. In
addition, all serum-derived products can be contaminated by unknown
constituents. In the case of serum or protein additives that are
derived from human or other animal sources in cell culture, there
are numerous problems (e.g. the varying quality in composition of
the different batches and the risk of contamination with
mycoplasma, viruses or BSE), particularly if the cells are used for
production of drugs or vaccines for human administration.
[0005] Therefore, many attempts have been made to provide efficient
host systems and cultivation conditions, which do not require serum
or other animal protein compounds. Simple serum free medium
typically includes basal medium, vitamins, amino acids organic or
inorganic salts, and optionally additional components to make the
medium nutritionally complex.
[0006] Soy hydrolysates are known to be useful for fermentation
processes and can enhance the growth of many fastidious organisms,
yeasts and fungi. WO 96/26266 describes that papaic digests of soy
meal are a source of carbohydrate and nitrogen and many of the
components can be used in tissue culture. Franek et al.
(Biotechnology Progress (2000) 16, 688-692) describe growth and
productivity promoting effects of defined soy hydrolysate peptide
fractions.
[0007] WO 96/15231 discloses serum-free medium composed of the
synthetic minimal essential medium and yeast extract for
propagation of vertebrate cells and virus production process. A
medium formulation composed of a basal cell culture medium
comprising a rice peptide and an extract of yeast and enzymatic
digest thereof, and/or a plant lipid for growth of animal cells is
disclosed in WO 98/15614. A medium comprising purified soy
hydrolysate for the cultivation of recombinant cells is disclosed
in WO 01/23527. WO 00/03000 discloses a medium that comprises a soy
hydrolysate and a yeast extract, but also requires the presence of
recombinant forms of animal proteins, such as growth factors.
[0008] EP-A-0 481 791 describes a biochemically defined culture
medium for culturing engineered CHO cells, which is free from
protein, lipid and carbohydrate isolated from an animal source,
further comprising a recombinant insulin or insulin analogue, 1% to
0.025% w/v papain digested soy peptone and putrescine. WO 98/08934
describes a serum-free eukaryotic cell culture comprising
hydrolyzed soy peptides (1-1000 mg/L), 0.01 to 1 mg/L putrescine
and a variety of animal-derived components, including albumin,
fetuin, various hormones and other proteins. In this context, it
should be also noted that putrescine is also known to be contained
in standard media like DMEM/Ham's F12 in a concentration of 0.08
mg/L.
[0009] However, the media known in the state of art are often
nutritionally insufficient and/or must be supplemented with
animal-derived protein supplements or recombinant versions of
proteins, such as insulin, insulin like growth factor or other
growth factors.
[0010] Therefore, a current need exists to increase the yield of
expressed recombinant protein or any other expression product, and
specific growth rate of cells, and to provide an optimal cell
culture medium completely free of animal proteins for production of
biological products, such as those used as pharmaceuticals or
vaccines in humans.
[0011] On the basis of soy peptone extracts (also designated as
"soy hydrolysates") media have been developed, which do not contain
animal proteins. However, the quality of commercially available
lots of soy hydrolysates varies extremely and as a result, there
are large variations in the production of recombinant proteins or
viral products (a variation of up to a factor of 3) as a function
of the lots of soy hydrolysates used ("lot-to-lot variation"). This
draw back affects the proliferation of the cells as well as the
protein expression of each cell.
[0012] Therefore, there is a need for an animal protein-free cell
culture medium which is completely free of animal proteins and
overcomes at least one of the above-mentioned problems.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide an animal
protein-free cell culture medium which does not contain any added
supplementary proteins derived from an animal source and/or
recombinant animal proteins, which allows efficient cell growth and
in particular protein production in a continuous quality with
respect to the amount of expression per cell. A further object of
the present invention is to provide a method for cultivating cells
and a method for efficient expression of recombinant proteins which
are free of animal proteins.
[0014] Another object of the present invention is to reduce plant
and/or yeast derived hydrolysate in order to overcome inhibitory
effects which would negatively impact the production yield of a
desired recombinant or viral product. Hydrolysates were
surprisingly found to be the cause of the lot-to-lot variations in
production.
[0015] The animal protein-free cell culture medium according to the
invention comprises at least one polyamine and a plant- and/or
yeast-derived hydrolysate, wherein the polyamine preferably
originates from a source other than the protein hydrolysate.
[0016] Surprisingly, the addition of at least one polyamine, in
particular the addition of putrescine, to the animal protein-free
cell culture medium provides the advantageous effect not only to
promote the cell growth but in particular to increase the protein
expression per cell and, in particular, recombinant protein
expression per cell.
[0017] Further, the animal protein-free medium according to the
present invention allows consistent cell growth and increased yield
of desired products, particularly of target proteins such as
recombinant proteins, independent of the quality or lot variations
of the protein hydrolysate, in particular of the vegetable
hydrolysates, in the animal protein-free cell culture medium. The
specific supplementation of cell culture media with polyamines and
a plant- and/or yeast-derived hydrolysate acts synergistically to
increase cell growth, cell specific productivity and final cell
density.
[0018] Therefore, the animal protein free medium according to the
present invention is more favorable for recombinant protein
expression and cell growth rate compared to the media known in the
art. Furthermore, the animal protein-free medium according to the
present invention allows the reduction of the amount of protein
hydrolysate to be added to a given volume of the cell culture
medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a graph which compares (A) the volumetric
FVIII-CoA productivity (expressed in [U/L/D]=FVIII COA Units per L
reactor volume per day and (B) the specific growth rate (.mu.
expressed in [d.sup.-1]=1 per day) of GD8/6 cells as a function of
the media used for culture, which were supplemented with different
lots (K119-1, K138-1, M022963, M024423, M022453) of soy
hydrolysates (0.4% (w/v)).
[0020] FIG. 2 shows a table which compares the volumetric FVIII-CoA
productivity of GD8/6 cells grown in media with different soy
hydrolysate concentrations.
[0021] FIG. 3 shows a graph which compares the volumetric FVIII-CoA
productivity of GD8/6 cells as a function of the media used for
culture, which were supplemented with 5 different lots (K119-1,
K138-1, M022963, M024423, M022453) of soy hydrolysates (0.25%
(w/v)) (A) in the absence of putrescine and (B) in the presence of
1 mg/L putrescine.2HCl.
[0022] FIG. 4 shows a graph which compares the specific growth
rates of GD8/6 cells as a function of the media used for culture,
which were supplemented with 5 different lots (K119-1, K138-1,
M022963, M024423, M022453) of soy hydrolysates (0.25% (w/v)) (A) in
the absence of putrescine and (B) in the presence of 1 mg/L
putrescine.2HCl.
[0023] FIG. 5 shows a table which compares the volumetric FVIII-CoA
productivity (QP [U/L/D]) and the specific growth rate (.mu.[d-1])
of GD8/6 cells and their standard deviation grown in media with 5
different selected lots (K119-1, K138-1, M022963, M024423, M022453)
of soy hydrolysates (0.4% (w/v) or 0.25% (w/v)) with the same soy
hydrolysates (0.25% (w/v)) with and without putrescine.2HCl at 1
mg/L.
[0024] FIG. 6 shows a table which describes the average putrescine
concentrations found in soy hydrolysates (0.4% (w/v) in cell
culture medium) from different manufacturers.
[0025] FIG. 7 shows a table which compares the effect of soy
hydrolysate (0.4% (w/v)) and soy hydrolysate (0.25% (w/v))+1.8 mg/L
putrescine.2HCl on the volumetric productivity (QP expressed in [mg
IgG1/L reactor volume/day] and cell specific productivity (qp
[.mu.g IgG1/10E06 Cells/d]) in ARH77 cells secreting a monoclonal
antibody.
[0026] FIG. 8 shows a graph which compares the effect of soy
hydrolysate (0.25% (w/v)) and soy hydrolysate (0.25% (w/v))+1 mg/L
putrescine (1.8 mg/L putrescine.2HCl) on the cell specific
erythropoeitin (EPO)-productivity of recombinant BHK cells (EPO
production (Units)/glucose consumption (g).
[0027] FIG. 9 shows a table comparing the effect of putrescine,
ornithine and spermine over a wider concentration range (0-18 mg/L)
on the specific growth (.mu. absolute, .mu. relative) and the cell
specific productivity (Qp absolute, Qp relative) of GD8/6 cells
cultivated in BAV-medium containing 0.0% soy hydrolysate and no
amines, or BAV-medium containing a reduced soy hydrolysate
concentration of 0.25% supplemented with polyamines in the
concentration range indicated above. BAV-SP 0.25%=BAV medium
containing 0.25% soy hydrolysate; BAV-SP 0.4%=BAV medium containing
0.4% soy hydrolysate; BAV w/o soy no polyamines=BAV medium
containing neither soy hydrolysate nor polyamines.
DETAILED DESCRIPTION OF THE INVENTION
[0028] One aspect of the invention relates to an animal
protein-free cell culture medium comprising at least one polyamine
and a plant- and/or yeast-derived hydrolysate, in a concentration
sufficiently reduced in order to avoid potential inhibitory effects
of the hydrolysate.
[0029] The term "polyamine" refers to any of a group of organic
compounds composed of carbon, nitrogen, and hydrogen, and
containing two or more amino groups. For example, the term
encompasses molecules selected from the group consisting of
cadaverine, putrescine, spermidine, spermine, agmatine, and
ornithine.
[0030] Unless stated differently, concentration values indicated
throughout this document refer to the free base form of the
component(s).
[0031] In a preferred embodiment of the animal protein-free cell
culture medium the concentration of the polyamine is present in a
concentration ranging from about 0.5 mg/L to about 30 mg/L, more
preferably from about 0.5 mg/L to about 20 mg/L, even more
preferably from about 0.5 mg/L to about 10 mg/L, more preferably
from about 2 mg/L to about 8 mg/L, most preferably from about 2 to
about 5 mg/L in the medium.
[0032] In a preferred embodiment the total concentration of the
plant- and/or yeast-derived protein hydrolysate in the animal
protein-free cell culture medium is about 0.05% to about 5% (w/v),
more preferably about 0.05% to about 2% (w/v), more preferably
about 0.05% to about 1% (w/v), more preferably about 0.05% to about
0.5% (w/v), most preferably about 0.05% to about 0.25% (w/v); i.e.
if the medium contains a plant- and a yeast derived protein
hydrolysate, the total concentration is calculated by the summing
up the concentration values of each of the protein hydrolysate
components contained in the medium.
[0033] The term "animal protein free cell culture medium" according
to the invention refers to a medium that does not contain proteins
and/or protein components from higher multicellular non-plant
eukaryotes. Typical proteins that are avoided are those found in
serum and serum-derived substances, such as albumin, transferrin,
insulin and other growth factors. The animal protein free cell
culture medium is also free of any purified animal derived products
and recombinant animal derived products as well as protein digests
and extracts thereof or lipid extracts or purified components
thereof. Animal proteins and protein components are to be
distinguished from non-animal proteins, small peptides and
oligopeptides obtainable from plants (usually 10-30 amino acids in
length), such as soy bean, and lower eukaryotes, such as yeast
which may be included into the animal protein free cell culture
medium according to the invention.
[0034] The animal protein free culture medium according to the
invention may be based on any basal medium such as DMEM, Ham's F12,
Medium 199, McCoy or RPMI generally known to the skilled worker.
The basal medium may comprise a number of ingredients, including
amino acids, vitamins, organic and inorganic salts, and sources of
carbohydrate, each ingredient being present in an amount which
supports the cultivation of a cell which is generally known to the
person skilled in the art. The medium may contain auxiliary
substances, such as buffer substances like sodium bicarbonate,
antioxydants, stabilisers to counteract mechanical stress, or
protease inhibitors. If required, a non-ionic surfactant such as
mixtures of polyethylene glycols and polypropylene glycols (e.g.
Pluronic F68.RTM., SERVA) can be added as a defoaming agent.
[0035] The polyamine employed for the animal protein free culture
medium according to the invention may be selected from the group
consisting of cadaverine, putrescine, spermidine, spermine,
agmatine, ornithine, and combinations thereof. Most preferably, the
animal protein free culture medium contains ornithine, putrescine
and spermine.
[0036] In an preferred embodiment of the animal protein free
culture medium the polyamine controls DNA- and RNA-synthesis, cell
proliferation, cell differentiation, membrane stabilization, and/or
antioxidative DNA-protection. Putrescine, spermidine, spermine, and
ornithine are examples of polyamines which exhibit these functions.
Another example of a polyamine is cadaverine.
[0037] In another preferred embodiment of the animal protein-free
cell culture medium according to the invention the polyamine
originates from a source other than the protein hydrolysate.
[0038] In a further preferred embodiment of the animal protein-free
cell culture medium the polyamine is present in a concentration
ranging from about 0.5 to about 30 mg/L, more preferably from about
0.5 mg/L to about 20 mg/L, even more preferably from about 0.5 mg/L
to about 10 mg/L, more preferably from about 2 mg/L to about 8
mg/L, most preferably from about 2 to about 5 mg/L in the medium,
and the plant- and/or yeast-derived protein hydrolysate is present
in the medium in a concentration ranging from about 0.05% to about
5% (w/v), more preferably about 0.05% to about 2% (w/v), more
preferably about 0.05% to about 1% (w/v), more preferably about
0.05% to about 0.5% (w/v), most preferably about 0.05% to about
0.25% (w/v).
[0039] The plant-derived protein hydrolysate used for the animal
protein-free cell culture medium according to the invention is
preferably selected from the group consisting of a cereal
hydrolysate and/or a soy hydrolysate. The soy hydrolysate may be a
highly purified soy hydrolysate, a purified soy hydrolysate or
crude soy hydrolysate.
[0040] The term "hydrolysate" includes any enzymatic digest of a
vegetable or yeast extract. The "hydrolysate" can be further
enzymatically digested, for example by papain, and/or formed by
autolysis, thermolysis and/or plasmolysis. Hydrolysates to be used
according to the present invention are also commercially available,
such as HyPep 1510.RTM., Hy-Soy.RTM., Hy-Yeast 412.RTM. and
Hi-Yeast 444.RTM., from sources such as Quest International,
Norwich, N.Y., OrganoTechnie, S.A. France, Deutsche Hefewerke GmbH,
Germany, or DMV Intl. Delhi, N.Y. Sources of yeast extracts and soy
hydrolysates are also disclosed in WO 98/15614, WO 00/03000, WO
01/23527 and U.S. Pat. No. 5,741,705.
[0041] The hydrolysates are preferably purified from crude
fraction, because impurities could interfere with efficient
cultivation. Purification can be carried out by ultrafiltration or
Sephadex chromatography, for example with Sephadex 25 or Sephadex
G10 or equivalent materials, ion exchange chromatography, affinity
chromatography, size exclusion chromatography or
reverse-phase-chromatography. The fractions may contain
hydrolysates of defined molecular weight, preferably up to about
1000 Dalton, more preferably up to about 500 Dalton, most
preferably up to about 350 Dalton. At least about 90% of the
hydrolysate has preferably a molecular weight of up to about 1000
Dalton. The average molecular weight of the hydrolysates lies
preferably between about 220 and about 375 Daltons. The pH value of
the hydrolysate should be in the range of from about 6.5 to about
7.5. The total nitrogen content is preferably between about 5 and
about 15%, and the ash content is preferably up to about 20%. The
free amino acid content is preferably between about 5% and about
30%. The endotoxin content is preferably less than about 500
U/g.
[0042] The invention also provides a method of using at least one
polyamine for addition to an animal protein-free cell culture
medium containing a plant- and/or yeast-derived protein
hydrolysate, for increasing the protein expression yield in the
cultured cells. According to a preferred embodiment of the
invention, the polyamine is present in the culture medium in a
total concentration ranging from about 0.5 to about 30 mg/L, more
preferably from about 0.5 mg/L to about 20 mg/L, even more
preferably from about 0.5 mg/L to about 10 mg/L, more preferably
from about 2 mg/L to about 8 mg/L, most preferably from about 2 to
about 5 mg/L in the medium. Preferably, the polyamine is selected
from the group consisting of cadaverine, putrescine, spermidine,
spermine, agmatine, ornithine, and combinations thereof.
Preferably, the plant- and/or yeast-derived protein hydrolysate is
present in the medium in a concentration ranging from about 0.05%
to about 5% (w/v), more preferably about 0.05% to about 2% (w/v),
more preferably about 0.05% to about 1% (w/v), more preferably
about 0.05% to about 0.5% (w/v), most preferably about 0.05% to
about 0.25% (w/v).
[0043] The present invention further relates to a method for
cultivating cells, comprising the steps of: [0044] (a) providing an
animal protein-free cell culture medium according to the invention,
and [0045] (b) propagating the cells in the medium to form a cell
culture.
[0046] In a preferred embodiment the animal protein-free cell
culture medium comprises at least one polyamine and a plant- and/or
yeast-derived hydrolysate. Preferably the polyamine originates from
a source other than the protein hydrolysate.
[0047] The present invention is not limited to any type of cells.
In a preferred embodiment of the invention the cells used are for
example mammalian cells, insect cells, avian cells, bacterial
cells, yeast cells. The cells may be for example stem cells or
recombinant cells transformed with a vector for recombinant gene
expression, or cells transfected with a virus for producing viral
products. The cells may also be for example cells producing a
protein of interest without recombinant transformation, e.g. a
B-cell producing an antibody, which may be transformed into an
immortalized status e.g. by viral infection like Epstein Barr Virus
infection. The cells may also be for example primary cells, e.g.
chicken embryo cells, or primary cell lines. Preferred are cells
that are used for in vitro virus production. In a preferred
embodiment the cells may be BSC cells, LLC-MK cells, CV-1 cells,
COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF
cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK
cells, MDOK cells, BHK-21 cells, CHO cells, NS-1 cells, MRC-5
cells, WI-38 cells, BHK cells, 293 cells, RK cells, and chicken
embryo cells.
[0048] The cells used according to the present invention may be
cultivated by a method selected from the group of
batch-cultivation, feed-batch-cultivation, perfusion cultivation
and chemostate-cultivation all of which are generally known in the
field.
[0049] The present invention further relates to a method for
expressing a target protein such as a heterologous or autologous
protein or a recombinant protein, comprising the steps of: [0050]
a) providing a culture of cells that have been grown in an animal
protein-free cell culture medium according to the invention; and
[0051] b) introducing a nucleic acid sequence comprising a sequence
coding for the target protein into the cells; [0052] c) selecting
the cells carrying the nucleic acid sequence; and [0053] d)
selectively inducing the expression of the target protein in the
cells.
[0054] In a preferred embodiment the animal protein-free cell
culture medium comprises at least one polyamine and a plant- and/or
yeast-derived hydrolysate. Preferably, the polyamine originates
from a source other than the protein hydrolysate.
[0055] The nucleic acid sequence comprising a sequence coding for
the target protein may be a vector. The vector may be a virus or a
plasmid. The sequence coding for a target protein may be a specific
gene or a biological functional part thereof. In a preferred
embodiment the target protein is at least a biologically active
part of a blood coagulation factor such as the Factor VIII or at
least a biologically active part of a protein involved in the
production of red blood cells and angiogenesis such as
erythropoeitin, or a monoclonal antibody.
[0056] Preferably, the nucleic acid further comprises other
sequences suitable for controlled expression of a target protein
such as promotor sequences, as enhancers, TATA boxes, transcription
initiation sites, polylinkers, restriction sites, poly-A-sequences,
protein processing sequences, selection markers, and the like which
are generally known to the person skilled in the art.
[0057] Most preferred are the following cell lines transformed with
a recombinant vector for the expression of the respective products:
CHO cells for the production of recombinant coagulation factor
VIII, BHK cells for the production of recombinant erythropoietin,
Epstein Barr virus transformed, immortalized human B cells for the
production of human antibodies.
[0058] The present invention further relates to a method for
producing a virus or part of a virus, comprising the steps of:
[0059] a) providing a culture of cells that have been grown in an
animal protein-free cell culture medium according to the invention;
and [0060] b) infecting the cells with a virus; [0061] c) selecting
the virus-infected cells; and [0062] d) incubating the cells to
propagate the virus.
[0063] In a preferred embodiment the animal protein-free cell
culture medium comprises at least one polyamine and a plant- and/or
yeast-derived hydrolysate. More preferably, the polyamine
originates from a source other than the protein hydrolysate.
[0064] The virus used in the method according to the invention may
be any pathogenic virus, mammalian, preferably human virus, such as
a vaccinia or attenuated vaccinia virus, e.g. for smallpox
vaccines, coronavirus, preferably SARS virus, e.g. for production
of SARS vaccines, orthomyoxyvirus, preferably influenza virus, e.g.
for production of influenza vaccines, paramyxovirus, retrovirus,
influenza A or B virus, Ross River virus, flavivirus, preferably
West Nile virus or FSME virus (i.e. tick borne encephalitis virus),
e.g. for the production of the respective vaccines, picornavirus,
arena virus, herpesvirus, poxvirus or adenovirus.
[0065] The virus may be a wild-type-virus, an attenuated virus, a
reassortant virus, or a recombinant virus or combinations thereof,
e.g. attenuated and recombinant. In addition, instead of actual
virions being used to infect cells with a virus, an infectious
nucleic acid clone may be used. Split virions may also be used.
[0066] The method for expressing a protein or producing a virus may
be used for producing immunogenic compositions comprising a virus
or a virus antigen.
[0067] The cells used for the method for producing a virus may be
selected from the group consisting of mammalian cells, insect
cells, avian cells, bacterial cells, and yeast cells. Preferably,
the cells are cultivated by a method selected from the group
consisting of batch-cultivation, feed-batch-cultivation, perfusion
cultivation and chemostat-cultivation.
[0068] Preferred combinations of cells with viruses for producing a
virus or part of a virus are Vero cell/attenuated vaccinia, Vero
cell/Vaccinia, Vero cell/Hepatitis A, Vero cell/Influenza Virus,
Vero cell/West Nile Virus, Vero cell/SARS Virus, chicken embryo
cells/FSME virus.
[0069] The present invention further relates to a method of using
the animal protein-free cell culture medium according to the
invention for culturing cells expressing a target protein.
[0070] The present invention will now be further illustrated in the
following examples, without being limited thereto.
EXAMPLES
Example 1 (BAV-medium)
[0071] Animal protein free medium was prepared with basal
DMEM/HAM's F12 (1:1) medium supplemented with inorganic salts,
amino acids, vitamins and other components (Life technologies,
32500 Powder). Also added were L-glutamine (600 mg/L), ascorbic
acid (20 .mu.M), ethanol amine (25 .mu.M), Synperonic.RTM. (SERVA)
(0.25 g/L), sodium selenite (50 nM). Additionally, essential amino
acids were supplemented to the cell culture medium. Further,
varying concentrations of soy hydrolysate (Quest Technologies, NY
or DMV Intl., NY) in the range of 0.0-1.0% and varying
concentrations of polyamines (0-10 mg/L) were added (FIG. 1-9)
Example 2
[0072] Cell cultures of recombinant mammalian cells (e.g. CHO-cells
stably expressing Factor VIII=GD8/6-cells) were grown in suspension
in a chemostat culture in 10 l bioreactors. The culture conditions
of 37.degree. C., oxygen saturation 20% and pH 7.0 to 7.1 were kept
constant. The cultures were supplied with a constant feed of
BAV-medium as defined in Example 1 additionally supplemented with
soy hydrolysates in the range of 0.1-1.0% and/or addition of
putrescine.2HCl in the range of 0-1 mg/L (cf. FIG. 1-5).
[0073] Small scale experiments with GD8/6 cells in suspension
culture were carried in Techne spinner flasks at 200 ml working
volume in batch refeed mode at 37.degree. C., without pH and pO2
control. The cultures were supplied with BAV-medium as defined in
Example 1 without supplementation of soy hydrolysate and
polyamines, or supplemented with soy hydrolysate in the range of
0.1-0.4% and/or putrescine.2HCl, ornithine.HCl, spermine.4HCl in
the range of 0-18 mg/L (equivalent to 0-10 mg/L of the polyamine
without .HCl (cf. FIG. 9).
Example 3 (cf. FIG. 1 to 5 7, and 9)
[0074] Cell counts from suspension cells or immobilized cells were
determined either by counting with a CASY.RTM. cell counter as
described by Scharfe et al., (Biotechnologie in LaborPraxis 10:
1096-1103 (1988)) or by citric acid extraction and flourescent
staining of the nuclei followed by counting with a
NucleoCounter.RTM. (Chemometec, DK). The specific growth rate
(.mu.) is calculated from the increase of the cell densities
(X.sub.t) and/or the dilution rate (D) of the steady state of
chemostat cultures of suspensions cells over a certain time
interval (t): .mu.=D+ln(Xt/X0)/t
Example 4
[0075] The activity of Factor VIII (FVIII) (cf. FIGS. 1 to 5 and 9)
was measured by a chromogenic assay (Chromogenic, Sweden). The
activity of erythropoeitin (cf. FIG. 8) and the monoclonal antibody
titer (cf. FIG. 7) were measured by ELISA test systems.
[0076] The volumetric productivity is calculated from the amount of
activity units or antigen titers yielded per liter reactor volume
per day (U/L/d or mg/L/d) in the respective production systems.
[0077] The cell specific productivity is defined as the specific
amount of produced protein (U or .mu.g) per number of cells per day
(cf. FIGS. 7 and 9) or as the specific amount of produced protein
(U) produced per amount of D-glucose consumed by the cells (cf.
FIG. 8).
Example 5
[0078] GD8/6 cells were supplied with BAV-medium containing 0.4%
(w/v) of different soy hydrolysate lots. The volumetric
FVIII-productivity varied from about 600 to 1800 U/L/d and the
specific growth rates varied of from 0.35 to 0.52 .mu.[d-1] between
the different lots (cf. FIG. 1). This indicates that the soy
hydrolysate lots at the 0.4% concentration does not allow
consistent growth of the GD8/6 cells, possibly due to inhibitory
substances affecting the specific growth rate (.mu.) which are
contained in the soy hydrolysates.
Example 6
[0079] GD8/6 cells were supplied with BAV-medium containing
different concentrations of soy hydrolysate lot M022257 (in the
range of 0.15-1.0% w/v). The volumetric FVIII-productivity varied
of from 500 to 1.100 U/L/d and reached an optimum productivity of
1.100 U/L/d at a soy hydrolysate concentration of 0.4% (w/v) (cf.
FIG. 2).
Example 7
[0080] GD8/6 cells were supplied with BAV-medium containing 0.25%
(w/v) of the same 5 different soy hydrolysate lots as described in
Example 5 (FIGS. 3A and 4A) and 0.25% (w/v) soy hydrolysate of the
same soy hydrolysate lots additionally supplemented with 1 mg/L
putrescine.2 HCl (FIGS. 3B and 4B), respectively. The volumetric
FVIII-productivity varied of from 1700 U/L/d to 500 U/L/d in the
cells grown BAV-SP medium containing 0.25% (w/v) soy hydrolysate of
different soy hydrolysate lots (FIG. 3A). The specific growth rate
varied of from 0.58 to 0.24 .mu.[d-1], indicating that the
reduction of the soy hydrolysate concentration does not lead to an
improved or more consistent growth rate of the cells (FIG. 4A).
[0081] In contrast, only minor variations of the volumetric
FVIII-productivity (FIG. 3B) and specific growth rates (FIG. 4B)
between the same soy hydrolysate lots are observed in the cells
grown BAV-medium containing 0.25% (w/v) soy hydrolysate when
supplemented with 1 mg/L putrescine.2HCl. The addition of 1 mg/L
putrescine.2HCl approximately compensates the reduction of this
polyamine by the reduction of soy hydrolysate concentration from
0.4% (w/v) to 0.25% (w/v). From this it can be concluded that not
the concentration of the polyamine itself, but the addition of the
polyamine in combination with the reduction of the soy hydrolysate
concentrations leads to a reduction of inhibitory substances which
reduce growth and productivity (see Example 5). Furthermore, the
addition of putrescine also leads to an over proportional increased
volumetric productivity of FVIII due to an increase of the cell
specific FVIII productivity (FIG. 5).
[0082] Thus addition of putrescine to animal protein-free cell
culture media not only promotes protein expression rate of cultured
cells but it also reduces the amount of plant hydrolysate to be
included into the culture media in order to obtain the same cell
growth. As a result, culture media become less affected by the
lot-by-lot variation of quality of plant hydrolysate and thus an
overall improvement of the cell culture conditions is achieved.
Example 8
[0083] FIG. 5 comprises the statistical analysis of the Examples
shown in FIGS. 1, 2 and 4: GD8/6 cells were supplied with
BAV-medium containing 0.4% (w/v) of soy hydrolysate or 0.25% (w/v)
soy hydrolysate or 0.25% (w/v) soy hydrolysate and 1 mg/L
putrescine.2HCl. Standard deviations are calculated based on five
selected lots of soy hydrolysates (K119-1, K138-1, M022963,
M024423, M022453). The volumetric and cell specific
FVIII-productivity and the specific growth rate with 0.25% (w/v)
soy hydrolysate was lower than with 0.4% (w/v) soy hydrolysate,
which confirms the optimum depicted in FIG. 2. However, the
volumetric and cell specific FVIII-productivity and the specific
growth rate increases in cell culture medium containing 0.25% (w/v)
soy hydrolysate+1 mg/L putrescine.2HCl. Further, the standard
deviation calculated from five different lots of soy hydrolysates
is significantly reduced (cf. FIG. 5 [QP [U/L/D]=volumetric
productivity; qp [mU/106 cells/day]=cell specific
productivity).
Example 9
[0084] Examples 7 and 8 show that putrescine is an active compound
supporting cell growth and, more specifically protein expression.
Therefore, the concentration of putrescine from different soy
hydrolysate lots from 2 different suppliers (Quest and DMV) were
quantitatively analysed by a HPLC method and evaluated
statistically. The concentration in the cell culture media prepared
with soy hydrolysate from both suppliers was approximately 2.3 mg/L
putrescine, when soy hydrolysate was added to the medium in a
concentration of 0.4% (w/v) (cf. FIG. 6).
Example 10
[0085] ARH77 cells (human lymphoblastoid cell line stably
expressing hIgG) were grown in a perfusion culture after
immobilization on macroporous microcarriers in a 80 L stirred tank
bioreactor at 37.degree. C., pH 7.0-7.2 and pO2 20-80% air
saturation, supplied with BAV medium containing 0.4% (w/v) of soy
hydrolysate or 0.25% (w/v) soy hydrolysate+1.8 mg/L
putrescine.2HCl. Arithmetic means and standard deviations were
calculated from data points representing the steady states for the
respective medium formulations. The volumetric hIgG-volumetric
productivity/cell specific productivity in BAV-medium supplemented
with 0.4% (w/v) soy hydrolysate was lower than in BAV-medium
supplemented with 0.25% (w/v) soy hydrolysate+1.8 mg/L
putrescine.2HCl. This experiment indicates that the medium
composition according to the present invention is capable to
promote also the expression of monoclonal antibodies from a
transformed cell line. Further, the specific medium composition can
also be used in perfusion cultures (cf. FIG. 7).
Example 11
[0086] Recombinant BHK cells were grown to confluence in 5% (v/v)
fetal calf serum containing medium. The cells were washed with
protein-free medium and incubated for 3 days in BAV medium
supplemented with 0.25% (w/v) soy hydrolysate or 0.25% (w/v) soy
hydrolysate+1.8 mg/L putrescine.2HCl (FIG. 8). Since no cell
counting in this experiment was performed, the glucose consumption
rate (g/L) was measured over three days to prove equivalent biomass
in the culture system. The EPO-activity (mU/ml) was correlated with
the glucose consumption rate (g/L) over three days. The addition of
putrescine gives a 16% increase in EPO productivity compared to
BAV-medium merely supplemented with 0.25% (w/v) soy peptone. This
experiment also indicates that the medium composition according to
the present invention is capable to promote the expression of
different recombinant proteins.
Example 12
[0087] To prove the specific effect of putrescine, ornithine and
spermine over a wider concentration range (0-18 mg/L equivalent to
0-10 mg/L of the polyamine without --.HCl) an experiment was
carried where the GD8/6 cells were incubated in Techne spinner
flasks at 1-1.5 E06 cells/ml in BAV-medium containing 0.25% and
0.4% soy hydrolysate without polyamines, and BAV-medium containing
the reduced soy hydrolysate concentration of 0.25% with the
polyamines in the above mentioned concentration range. All three
polyamines in the investigated concentration range resulted in a
significant increase of cell specific productivity (expressed in
mU/106 cells/day) compared to the unsupplemented medium formulation
with 0.25% soy hydrolysate, or the increased concentration of 0.4%.
The increase of the cell specific productivity is clearly not
correlating with an increased specific growth rate, which confirms
the specific effect on the expression rate of recombinant FVIII of
the GD8/6 cells (FIG. 9).
* * * * *