Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02381173 2002-02-04
The present invention relates to a stable recombinant cell clone which is
stable in a serum- and
protein-free medium for at least 40 generations, a biomass obtained by the
propagation of the
stable cell clone under serum- and protein-free culture conditions and a
method for the
preparation of recombinant proteins by means of the biomass. Moreover, the
invention relates to
a method for the preparation of stable recombinant cell clones. Furthermore,
the invention relates
to the preparation of a recombinant protein in a serum- and protein-free
synthetic minimal
medium.
The preparation of recombinant proteins, in particular biomedical products,
such as blood
factors, is becoming increasingly important. To allow optimal growth of
recombinant cells,
serum is usually added to the medium. Because of the high cost of the serum
and to prevent
possible contaminations by viral or molecular pathogens through the serum in
the culture
medium, a number of serum-free media have been developed, which, in
particular, should not
contain any additives of bovine or human origin. The use of such media in the
preparation
process allows not only a low risk of contamination of the prepared products
by viral and
molecular pathogen, but also a more simple purification of the expressed
proteins.
Recombinant cells are mostly cultivated* in the serum-containing medium until
a high
cell density, approximately equivalent to that of a "working cell bank," has
been reached, and
then, during the production phase, they are readapted to serum-free medium.
Miyaji et al. (1990, Cytotechnology 3:133-140) selected a serum-independent
cell clone
in a serum-free medium which contained insulin and transferrin. However, it
was shown that,
after 16 days, the viable count and the expression rate continuously
decreased. Miyaji et al.
(1990, Cytotechnology 4:173-180) attempted to improve the expression rate and
the productivity
of the recombinant cells by coamplification with a marker gene.
Yamauchi et al. (1992, Biosci. Biotechnol. Biochem. 56:600-604) established
serum-
independent recombinant CHO subclones by culturing serum dependent cells on
microtiter plate
as a monolayer for 3-4 weeks in a serum-free medium which contained human
serum albumin,
insulin and transfernn. Approximately 0.1 % of the cells were serum-
independent. A portion of
the subclones also grew in suspension culture in a serum-free medium, where
the cells, however,
formed aggregates and clumps. The doubling time of the cells was 1.5 days.
However, no
indications are provided on the stability of the serum-independent clones
obtained or on the long-
term culturing of these clones under serum-free conditions.
The media which allowed the maintenance of the metabolic activity and growth
of cells
during the cell free phase often contained additional substances, such as
growth factors, insulin
or transferrin, or adherence factors which replace the serum components.
[Editor's note: This is an unusual translation of "angiebien"]
CA 02381173 2002-02-04
In order to omit the addition of polypeptide factors, such as insulin or
transferrin, and to
allow protein-free culture conditions, various techniques have been developed.
For example,
specifically defined, complete protein-free media have been developed which
allow cell growth
even under protein-free conditions.
WO 97/05240 describes the preparation of recombinant proteins under protein-
free
conditions where the cells coexpress a growth factor in addition to the
desired protein.
JP 2696001 describes the use of protein-free media for the preparation of
factor VIII in
CHO cells with the addition of a nonionic surfactant or cyclodextrin to
improve the productivity
of the host cells. To increase the effectiveness of these additives, it has
been recommended, for
example, to add butyrate and lithium.
WO 96/26266 describes the culturing of cells in a medium which contains a
glutamine-containing protein hydrolysate, whose free amino acid content is
less than 15% of the
total protein weight and whose peptide has a molecular weight of less than 44
kd. A synthetic
minimal medium is used as base medium in the culturing media for cell
cultures, to which base
medium one also adds, besides protein hydrolysate, other additives, including
fetal calf serum,
gentamicin and mercaptoethanol. The use of this serum-containing medium for
the recombinant
preparation of blood factors is not mentioned.
US 5,393,668 describes special synthetic surfaces which allow the growth of
adherent
cells under protein-free conditions.
To stimulate the cell proliferation, CHO cells which overexpress human insulin
were
propagated on an artificial substrate to which the covalent insulin is bound
(Ito et al., 1996,
PNAS USA 93:3598-3601).
Reiter et al. ( 1992, Cytotechnology 9:247-253) describe the immobilization of
r-CHO
cells which are cultured in the serum-containing medium at a high density on
supports and the
subsequent perfusion of the immobilized cells in the protein-free medium
during the production
phase, where a continuous release of protein into the cell supernatant was
observed. However,
the cells were perfused for less than 10 generations in the protein-free
medium.
The methods which are available to date for the successful preparation of an
industrial
"large-scale" cell culture under protein-free conditions have been described
for continuous cell
lines, particularly VERO cells (WO 96/15231). The cells were cultivated here
under serum- and
protein-free conditions from the original ampule to the industrial scale of
120(1 L. However, the
cells used are not recombinant cells, but host cells which are used for the
production of virus
antigen in a lytic process.
In contrast to adherent VERO cells, CHO cells, for example, are only dependent
to a
united degree on adhesion. CHO cells which are cultured by conventional
methods under serum-
containing conditions are capable of binding both to smooth and porous
microsupports (US '
4
CA 02381173 2002-02-04
4,978,616, Reiter et al., 1992, Cytotechnology 9:247-253). If CHO cells are
grown under serum-
free conditions, they lose this property and do not adhere to smooth supports,
such as, for
example, Cytodex 3, or they readily separate from them to the extent that no
adherence-
promoting additives, such as, for example, fibronectin, insulin or transferrin
have been added to
the medium. Because of the low adhesion of CHO cells to supports under semm-
free conditions,
the production of recombinant proteins is therefore usually carried out in
suspension culture. The
production process can here be run by a continuous or batch method. The
recombinant cell
culture is here cultivated in a bioreactor until an optimal cell density has
been reached; the
protein expression is optionally induced, and, for the harvest, the medium
which contains the
expressed proteins but also recombinant cells is drawn off at certain
intervals from the reaction
tank and thus removed from the production process. As a result of the
continuous loss of
biomass, the production efficiency in the bioreactor decreases, and it
increases only after the
slow addition of fresh medium, because the cells have to grow until the
desired cell density is
reached. Therefore, and in spite of the continuous process, there is always a
delay phase in which
the production rate decreases in this system. In addition, the capacities for
growth and production
are limited by the maximum achievable cell density in such a system.
In the adaptation of cells cultured under serum-containing conditions on
protein-free
medium, it was consistently observed that the yield of expressed protein and
the productivity of
recombinant CHO cells strongly decrease after adaptation in a protein-free
medium in
comparison to serum-containing conditions (Paterson et al., 1994, Appl.
Microbiol. Biotechnol.
40:691-658).
This is explained by an instability or reduced growth of the recombinant clone
as a result
of changes in the culture conditions. Because of the changed fermentation
conditions-and in
spite of the use of a stable original clone-a large portion of the cells is
always converted to cells
with reduced expression or to nonproducing cells, which, during the production
process,
overgrow product-producing cells, resulting in a fermentation culture which,
in the end, consists
of a large portion of nonproducing cells or of cells with low expression.
The result of this situation is that the maximum production capacity of the
fermentation
culture continuously decreases and the maximum product production is limited
to a certain
number of generations or cell passages.
Therefore there is a need for a system which allows continuous production over
as long a
time period as possible, in particular in the industrial production of
recombinant proteins under
senun- and protein-free conditions.
Moreover, it would be desirable to obtain recombinant cell clones which are
stable over
many generations in the production phase under protein-free conditions and
which expresses a
recombinant protein. Therefore the problem of the present invention is to
provide an efficient
CA 02381173 2002-02-04
method for the preparation of recombinant proteins under serum- and protein-
free culture and
production conditions.
6
An additional goal is to provide a stable recombinant cell clone.
According to the invention, the problem is solved by making available a
recombinant cell
clone which can be obtained from a cell culture, which cell clone is obtained
after the culturing
of the recombinant original cell clone on serum-containing medium and
readaptation of the cells
to a serum- and protein-free medium. Here, the cells are further cultured for
at least 40
generations in a serum- and protein-free medium under conditions equivalent to
the production
conditions.
The cell clone according to the invention therefore forms a population of
cells which, in a
predominant portion, can be cultured for at least 40 generations in a stable
manner in the serum-
and protein-free medium. Here, it is preferred that more than 80%, in
particular more than 99%,
of the cell population according to the invention or the cell clone according
to the invention is
stable for at least 40 generations.
Here it is preferred for the culturing of the cells to be carried out without
selection for the
selection marker and/or amplification gene, for example, in the absence of MTX
in the case of
CHO-dhfr cells.
In the context of the invention, the term original cell clone denotes a
recombinant cell
clone transfectant, which, after transfection of host cells with a recombinant
nucleotide sequence
expresses recombinant product in a stable manner under laboratory conditions.
The original
clone is cultured for growth optimization in the serum-containing medium. To
increase the
productivity, the original clone is optionally cultivated in the presence of a
selection agent and
with selection for the selection marker and/or amplification marker. For
industrial production,
the original cell clone is cultivated under serum-containing culturing
conditions until a high cell
density has been reached, and it is adapted to serum and/or protein-free
medium shortly before
the production phase. Here, the culturing is preferably carried out without
selective pressure.
It was found that under these conditions a large portion, more than 95%, of
the cells in
such a cell culture which has been readapted to serum- and protein-free medium
is converted into
non-product-producing cells. By means of immunofluorescence with product-
specific antibodies,
it was possible to show that, as a function of the generation time of the
cells in serum- and
protein-free medium, the number of non-producing cells in a culture increases
and overgrows the
product-producing cells, resulting in a decrease in the production of the
culture.
The cell culture which is obtained after readaptation to serum- and protein-
free medium
is tested for the cell clone of the cell population which produces stable
products under serum-
and protein-free conditions, optionally in the absence of selective pressure.
This can be achieved,
for example, by immunofluorescence with specifically labeled antibodies made
against the a
CA 02381173 2002-02-04
recombinant polypeptide or protein. The cells which have been identified as
product-producing
cells are isolated from the cell culture, and again propagated under serum-
and protein-free
conditions, which are preferably equivalent to the production conditions. The
isolation of the
cells can here be achieved by isolation of the cells and testing for product-
producing cells.
Optionally, the cell culture which contains the stable cells is again tested
for stable recombinant
clones, which are then isolated from the cell culture and cloned. The stable
recombinant cell
clones obtained under serum- and protein-free conditions are then further
propagated under
serum- and protein-free conditions.
The recombinant cell clone according to the invention is characterized, in
particular, in
that it is stable in serum-free and protein-free medium for at least 40,
preferably at least 50, and
particularly advantageously more than b0, generations, and expresses a
recombinant protein.
Here, this stability appears without benefit of aids such as matrices or solid
surfaces, for
example, as supports. Furthermore, according to the invention it is not
required to carry out the
culturing using high cell densities.
According to a special aspect of the invention, the stable recombinant cell
clone is in an
isolated form. Starting from the stable cell clones, a cell culture is
obtained under serum- and
protein-free conditions by propagation of the stable cells.
The stable recombinant cell clone according to the invention is preferably
derived from a
recombinant mammalian cell. Here, the recombinant mammalian cells can be any
cells which
contain sequences coding for a recombinant polypeptide or protein. This
definition comprises all
continuously growing cells, both adherent and non-adherent. It is particularly
preferred to use
recombinant CHO cells or BHK cells. The recombinant polypeptides or proteins
can be blood
factors, growth factors and other biomedically relevant products.
According to the present invention, it is preferred to use stable recombinant
cell clones
which contain the coding sequence for a recombinant blood factor such as
factor II, factor V,
factor VII, factor VIII, factor IX, factor X, factor XI, protein S, protein C,
an activated form of
one of these factors, or vWF, and which are capable of expressing these blood
factors under
stable conditions over several generations. Here, it is preferred to use CHO
cells which express
vWF or a polypeptide with vWF activity, factor VIII or a polypeptide with VIII
activity, vWF
and factor VIII, factor IX or factor II.
The cell clone which was selected under serum- and protein-free conditions
according to
the invention is characterized, in particular, in that it is stable for at
least SO [sic; 40J, preferably
at least 50, generations, and particularly advantageously for more than 60
generations in serum-
and protein-free medium.
In order to develop a master cell bank, 30 generations are needed. To carry
out an
average batch culture on the 1000 L scale, at least 40 generations are
required. Thus, for the first
7
CA 02381173 2002-02-04
time it is possible to prepare from an individual clone a "master cell bank"
(MCB), a "working
cell bank" (WCB) with approximately 8-10 generations and thus to prepare a
cell culture on the
production scale (production biomass) with up to 20-25 generations under these
conditions,
because the cell clones available to date became unstable after a few
generations of growth on
serum- or protein-free medium, resulting in the inability to obtain a) a
uniform cell culture with
product-producing cells, and b) stable product productivity over a longer time
period.
The cell clone according to the invention is thus stable for at least 40
generations under
production conditions in serum- and protein-free medium. The methods which
have been
described to date provided only a generation number of less than 10
generations with product
productivity under protein-free conditions (Reiter et al., 1992, supra).
As stability criterion, a minimal number of at least 40 generations,
preferably more than
50, and particularly advantageously more than 60, generations are used in the
production
process, during which stable expression of the proteins occurs and the cell
morphology and
phenotype do not change and no tumorogenic characteristics are present.
Unexpectedly, it was found that the cell clone according to the invention,
under serum-
8
and protein-free conditions, presents an increased productivity even in
comparison to the original
cell clone which was cultured in serum-containing medium.
According to another aspect, the present invention makes available a cell
culture which
contains at least 90%, preferably more than 95%, and particularly more
advantageously more
than 98%, stable recombinant cells which, under serum- and protein-free
conditions, are stable
for at least 40 generations, in particular at least 50 generations, and
express recombinant product.
In the context of the present invention, a cell culture denotes a master cell
bank (MCB), a
working cell bank (WCB-working cell bank) or a production biomass in an
industrial production
bioreactor.
According to the invention, the cell culture is obtained, in particular, by
culturing a stable
recombinant cell clone of the type mentioned above under serum- and protein-
free conditions.
The cell culture according to the invention can here be obtained by
propagation of the
isolated stable cell clone from the individual clone, that is the seed cells,
to the MCB, the WCB
or a biomass on the production scale in the bioreactor under serum- and
protein-free conditions,
preferably without selective pressure on the selection and/or marker gene. In
particular, it has
been shown that the recombinant cells in a cell culture obtained from the
stable recombinant
clone according to the invention are stable for at least 40 generations under
serum- and protein-
free conditions.
The cell culture made available according to the present invention, which is
prepared
from a serum and protein-independent stable cell clone, under most protein-
free culturing and
production conditions presents at least 90%, preferably at least 95%,
particularly advantageously
CA 02381173 2002-02-04
9
at least 98%, stable recombinant cells. The term "stable recombinant cells"
here denotes, in
particular, recombinant mammalian cells which are derived from the stable cell
clone. It is here
preferred to use recombinant CHO cells, preferably CHO-dhfi cells, CHO-K 1
cells, and BHK
cells which express a blood factor, preferably recombinant vWF, factor VIII,
factor VIII and
vWF, factor IX or factor II.
The cell culture according to the invention can contain the stable recombinant
cells as
suspension culture. The cells can also be immobilized on a support, in
particular a microsupport,
where porous microsupports are particularly preferred. It was found that
porous supports such as,
for example, Cytoline~ or Cytopore~, are particularly suitable.
According to another aspect, the present invention represents a method for the
industrial
production of a recombinant product under serum- and protein-free conditions,
using the stable
cell clone made available according to the invention. The method here
comprises the steps of
preparation of an isolated, stable recombinant cell clone of the above
described type for the
preparation of a cell culture. Here the propagation of the isolated stable
cell clone occurs under
serum- and protein-free conditions from the stable individual cell clone to
the cell culture. In
particular, the subculturing of the stable cell clone also occurs under
protein-free conditions, in
particular without the addition of a protease such as, for example, trypsin.
As a result, it is
guaranteed that at no time during the preparation of a cell culture used in
the production of a
recombinant product that contamination occurs which may under certain
circumstances be
caused by the addition of serum and protein-containing additives of human or
animal origin to
the cell culture. Thus, for the first time a method is described which allows
the preparation,
starting from the initial clone and via the preparation of a working cell
bank, a production
biomass, and the subsequent production of recombinant protein under serum- and
protein-free
conditions.
The preparation of the recombinant products with the cell culture according to
the
invention, which contains more than 90%, preferably more than 95%, and
particularly
advantageously more than 98%, of stable product-producing cells, can be
carried out as
suspension culture or with cells immobilized with a support. The process here
can be carried out
in batch or continuous mode, or by a perfusion technique with serum- and
protein-free medium.
The recombinant proteins expressed are then obtained from the cell culture
supernatant,
then purified with known methods of the state of the art and further
processed.
As serum- and protein-free medium one can use any known synthetic medium.
Conventional synthetic minimal media can contain inorganic salts, amino acids,
vitamins and a
carbohydrate source and water. For example, it can be a DMEMIHAM F 12 medium.
The content
of soy and yeast extract can be 0.1-100 g/L, particularly advantageously 1-5
g,~L. In a particularly
CA 02381173 2002-02-04
preferred embodiment one can use soy extract, for example, soy peptone. The
molecular weight
of the soy peptone is less than 50 kd, preferably less than 10 kd.
It is particularly preferred~to use a medium with the following composition:
synthetic
minimal medium (1-25 g/L), soy peptone (0.5-50 g/L), L-glutamine (0.05-1 g/L),
NaHC03
(0.1-10 g/L), ascorbic acid (0.0005-0.05 g/L), ethanolamine (0.0005-0.05 g/L).
Na selenite
(0.0001-0.01 g/L). A nonionic surfactant such as, for example, polypropylene
glycol
(PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC
F108) as defoaming agent can optionally be added to the medium.
This agent is generally used to protect the cells from the negative effects of
aeration,
because, without the addition of a surfactant, the ascending bursting air
bubbles can lead to
damage to those cells located on the surface of these air bubbles ("sparging")
(Murhammer and
Goochee, 1990, Biotechnol. Prog. 6:142-148).
The quantity of nonionic. surfactant can here be 0.05-10 g/L, however it is
particularly
preferred to use as small a quantity as possible of 0.1-5 g/L. Moreover, the
medium can also
contain cyclodextrin or a derivative thereof.
The addition of the nonionic surfactant or of cyclodextrin is, however, not
essential to the
invention. It is preferred for the serum- and protein-free medium to contain a
protease inhibitor,
such as, for example, serine protease inhibitors which are suitable for use in
tissue culture and of
synthetic or plant origin.
The parameters for the culturing of the cells, such as 02 concentration, rate
of perfusion
or change in medium, pH, temperature and culturing technique are here
dependent on the
individual cell types used and they can be determined in an easy manner by a
person skilled in
the art. For example, the culturing of CHO cells can be carried out in a
stirred vessel and
perfusion with protein-free medium can occur at a perfusion rate of 1-10
volume changes/day, a
pH of 7.0-7.8, preferably at pH 7.4, an 02 concentration of 40-60%, preferably
50%, and a
temperature of 34-38°C, preferably 37°C.
According to an additional aspect, the present invention makes available a
method for the
obtention of a stable recombinant cell clone, comprising the steps of
- propagation of a recombinant original clone up to the cell culture in serum-
containing
medium, preferably without selective pressure,
- culturing the cells under serum- and protein-free conditions which are
preferably
equivalent to production conditions,
- testing the cell culture for product-producing cells under serum- and
protein-free
conditions,
CA 02381173 2002-02-04
- cloning the stable recombinant cell clone under serum- and protein-free
conditions,
where the cloning can be carried out by generally known techniques such as
isolation of the cells
by dilution and growing of the individual clones,
- propagation of the isolated cell clones under serum- and protein-free
conditions, and,
- optionally, testing the cell culture for product-producing-cells.
Here only those recombinant cell clones should be considered stable which
express stable
recombinant protein in a protein-free medium for at least 10, preferably at
least 20, and
particularly advantageously at least 50 generations.
According to another aspect, the invention makes available a method for the
obtention of
a stable recombinant cell clone, comprising the steps of
- propagation of a nonrecombinant initial cell or cell line under serum- and
protein-free
conditions and cloning a stable nonrecombinant cell clone under serum- and
protein-free
conditions,
- transfecting the stable cell clone with a recombinant nucleic acid and
isolation of stable
recombinant cell clones,
- culturing the stable cell clone transfectants in a serum- and protein-free
medium under
conditions which are optionally equivalent to production conditions,
- testing the stable recombinant cells for production and product stability.
The invention is described with reference to the following examples, without
being
limited to the examples.
Brief description of the figures
Figure 1: shows the microscopic view of a working cell bank of an original
clone at the
time of the readaptation from serum-containing medium to serum- and protein-
free medium (A),
after 10 generations in serum- and protein-free medium (B), and after 60
generations in serum-
and protein-free medium (C).
Figure 2: shows the microscopic view of a cell culture starting from a stable
recombinant
cell clone under serum- and protein-free conditions at the stage of the
working cell bank (A),
after 10 generations (B) and after 60 generations (C).
Figure 3: shows the results of culturing an rFVIII CHO cell clone in a 10 L
perfusion
bioreactor.
a) FVIII activity (mU/mL) and perfusion rate (1-S/day) over a time period of
42 days.
b) Volumetric productivity (units factor VIII/l/day) in the perfusion
bioreactor.
CA 02381173 2002-02-04
12
Examples:
Example 1: Stability of rvWF CHO cells after readaptation from serum-
containing medium to
serum- and protein-free medium
CHO-dhfr cells cotransfected with plasmid phAct-rvWF and pSV-dhfr, and vWF
expressing clones, as described in Fischer et al. (1994, FEBS Letters 351:345-
348), are
subcloned. From the subclones which expressed rvWF in a stable manner, a
working cell bank
(WCB) was prepared under serum-containing conditions but in the absence of
MTX, and the
cells were immobilized under serum-containing conditions on a porous
microsupport
(Cytopore~). After a cell density of 2 x 10' cells/mL support matrix was
reached, the conversion
of the cells to serum- and protein-free medium was carried out. The cells were
further cultured
for several generations under serum- and protein-free conditions. By means of
immunofluorescence with labeled anti-vWF antibodies, the cells were tested at
various times in
the serum- and protein-free medium. The evaluation of the stability of the
cells was carried out
on the working cell bank before the change of medium, after 10 and 60
generations in serum-
and protein-free medium. While the working cell bank still presented 100% rvWF
producing
cells (Figure 1 A), the proportion of rvWF producing cells decreased to
approximately 50% after
generations in serum- and protein-free medium (Figure 1 B). After 60
generations more than
95% of the cells were identified as nonproducing cells (Figure 1 C).
Example 2: Cloning of stable recombinant CHO clones
From the rVWF CHO cell-containing cell culture according to Example 1, which
had
been cultured for 60 generations in serum- and protein-free medium (Figure 1
C), a dilution was
prepared, and in each case 0.1 cell/well was inoculated in a microtiter plate.
The cells were
cultured in DMEM/HAM F 12 without serum or protein additives and without
selective pressure
for approximately 3 weeks, and the cells were tested by means of
immunofluorescence with
labeled anti-vWF antibodies. A cell clone which had been identified as
positive was used as
starting clone for the preparation of a seed cell bank. From the seed cell
bank a master cell bank
(MCB) was prepared in serum- and protein-free medium, and individual ampules
were frozen
and stored for the later preparation of a working cell bank. Starting from an
individual ampule, a
working cell bank was prepared in serum- and protein-free medium. The cells
were immobilized
on porous microsupports and continued to be cultured for several generations
under serum- and
protein-free conditions. By means of immunofluorescence with labeled anti-vWF
antibodies, the
cells were tested at different times in serum- and protein-free medium for
productivity. The
evaluation of the stability of the cells was carried out at the stage of the
working cell bank and
after 10 and 60 generations in serum- and protein-free medium. At the stage of
the working cell
CA 02381173 2002-02-04
13
bank (Figure 2A), and after 10 (Figure 2B) and 60 generations (Figure 2C),
approximately 100%
of the cells were identified as positive stable recombinant clones which
express rvWF.
Example 3: Cell specific productivity of the recombinant cell clones
From the defined stage during the culturing of recombinant cells, a defined
cell number
was removed and incubated with fresh medium for 24 h. The rvWF:Risto-CoF
[ristocetin
cofactor] activity was determined in the cell culture supernatants. Table 1
shows that the
cell-specific productivity in the stable recombinant cell clones according to
the invention was
stable even after 60 generations in serum- and protein-free medium, and it was
elevated even in
comparison with the original clone which was cultured in serum-containing
medium.
Table 1
Cell clone Cell specific Cell specific Cell specific
-
productivity of productivity afterproductivity after
the 10 60
working cells, generations, mU generations, mU
mU
rvWF1106 cells/dayrvWF/106 cells/dayrvWF/106 cells/day
rvWf CHO *808.68SS 30 - <10
original cell
clone
r-vWF-CHO F7 62 65 60
*)
stable clone
*) deposited in accordance with the Budapest Treatise of January 22, 1998
(ECAC (European
Collection of Cell Cultures, Salisbury, Wiltshire SP4 OJG, UK), deposit No.
98012206)
Example 4: Culturing of rFVIII CHO cells in protein and serum-free minimal
medium
An rFVIII CHO cell-containing cell culture was cultured in a 10 L stirred tank
and
perfused. Thereby a serum- and protein-free medium was used. The cells were
here immobilized
on a porous microsupport (Cytopore~, Pharmacia) and cultured for at least 6
weeks. The
perfusion rate was 4 volume changes/day, the pH was 6.9-7.2, the 02
concentration
approximately 20-50%, and the temperature 37°C.
Figure 3 shows the results of culturing an rFVIII CHO cell clone in a 10 L
perfusion
bioreactor.
a) FVIII activity (rnU/mL) and perfusion rate (1-S/day) over a time period of
42 days.
b) Volumetric productivity (units factor VIII/L/day) in the perfusion
bioreactvr.
CA 02381173 2002-02-04
Table 2
14
Culturing days Cell specific productivity Immunofluorescence
(mU/106 cells/day) (% FVIII positive cells)
15 702 n.a.
21 1125 n.a.
28 951 >95%
35 691 >95%
42 970 n.a.
Table 2 shows the stability and specific productivity of the rFVIII expressing
cells. For
these results, samples were removed after 15, 21, 28, 35 and 42 days,
centrifuged at 300 G and
resuspended in fresh serum- and protein-free medium. After an additional 24 h,
the factor VIII
concentration in the cell culture supernatants and the cell count were
determined. From these
data, the specific FVIII productivity was calculated.
A stable average productivity of 888 mU/106 cells/day was reached. This stable
productivity was also confirmed by immunofluorescence with labeled anti-FVIII
antibodies after
15, 21, 28, 35 and 42 days in serum- and protein-free medium.
Claims
1. Recombinant cell clone, characterized in that it is stable in a serum- and
protein-free
medium for at least 40, preferably 50, generations and expresses recombinant
product.
2. Cell clone according to Claim 1 or 2, characterized in that it is in an
isolated form.
3. Cell clone according to one of Claims 1-3 [sic], characterized in that it
is derived from
a recombinant mammalian cell.
4. Cell clone according to Claim 3, characterized in that the mammalian cell
is a
recombinant CHO cell or a BHK cell.
5. Cell clone according to one of Claims 1-4, characterized in that it
contains sequences
coding for a recombinant polypeptide or protein.
6. Cell clone according to Claim 5, characterized in that the recombinant
protein is a
blood factor chosen from the group consisting of factor II, factor V, factor
VII, factor VIII, factor
IX, factor X, factor XI, protein S, protein C, or an activated form of one of
the factors, or vWF.
7. Cell clone according to one of Claims 1-6, characterized in that the cell
is a
recombinant CHO cell which expresses von Willebrand's factor.
