U.S. patent application number 10/405794 was filed with the patent office on 2003-10-30 for medium for the protein-free and serum-free cultivation of cells.
Invention is credited to Dorner, Friedrich, Grillberger, Leopold, Mitterer, Artur, Mundt, Wolfgang, Reiter, Manfred.
Application Number | 20030203448 10/405794 |
Document ID | / |
Family ID | 3518214 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203448 |
Kind Code |
A1 |
Reiter, Manfred ; et
al. |
October 30, 2003 |
Medium for the protein-free and serum-free cultivation of cells
Abstract
A medium is described for the protein-free and serum-free
cultivation of cells, especially mammalian cells, whereby the
medium contains a proportion of soy hydrolysate.
Inventors: |
Reiter, Manfred; (Vienna,
AT) ; Mundt, Wolfgang; (Vienna, AT) ; Dorner,
Friedrich; (Vienna, AT) ; Grillberger, Leopold;
(Vienna, AT) ; Mitterer, Artur; (Orth/Donau,
AT) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
3518214 |
Appl. No.: |
10/405794 |
Filed: |
April 1, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10405794 |
Apr 1, 2003 |
|
|
|
09672240 |
Sep 28, 2000 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/325; 435/383 |
Current CPC
Class: |
C12N 2500/92 20130101;
C12N 2500/05 20130101; C07K 14/755 20130101; C12N 5/005 20130101;
C12N 5/0037 20130101; C12N 2500/60 20130101; C12N 5/0043 20130101;
C12P 21/00 20130101; C12N 5/0682 20130101; C12N 2500/76 20130101;
C12N 2500/32 20130101; C12N 2500/38 20130101; C12N 2500/46
20130101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/383 |
International
Class: |
C12P 021/02; C12N
005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 28, 1999 |
AT |
A 1659/99 |
Claims
1. A protein-free and serum-free medium for the cultivation of
cells, comprising soy hydrolysate wherein at least 40% of said soy
hydrolysate has a molecular weight of .ltoreq.500 daltons.
2. A medium in accordance with claim 1 wherein said medium contains
a quantity in excess of 10 wt % soy hydrolysate based on the total
dry weight of the medium.
3. A medium in accordance with claim 1, wherein said medium
contains ultrafiltered soy hydrolysate.
4. A medium in accordance with claim 1, wherein the soy hydrolysate
has an endotoxin content of <500 U/g.
5. A medium in accordance with claim 1, wherein at least 50% of the
soy hydrolysate has a molecular weight of .ltoreq.500 daltons.
6. A medium in accordance with claim 5, wherein at least 55% of the
soy hydrolysate has a molecular weight of .ltoreq.500 daltons.
7. A medium in accordance with claim 1, wherein said medium also
contains an amino acid.
8. A medium in accordance with claim 7, wherein said amino acid is
selected from the group consisting of L-asparagine, L-cysteine,
L-cystine, L-proline, L-tryptophan, L-glutamine and mixtures
thereof.
9. A medium in accordance with claim 1 wherein said medium also
contains auxiliary substances selected from the group consisting of
buffer substances, oxidation stabilizers, stabilizers to counteract
mechanical stress, and protease inhibitors.
10. A process for the cultivation of cells, comprising introducing
said cells into a medium in accordance with claim 1 and growing
said cells in said medium.
11. The process of claim 10, further comprising introducing
mammalian cells selected from the group consisting of CHO cells and
BHK cells.
12. A process for the production of a protein from cell culture
comprising: introducing cells into a medium in accordance with
claim 1, wherein said cells express a desired protein; growing said
cells in said medium and expressing said protein, thereby producing
a mixture of said cells and said protein in the medium; and
purifiying said protein from said mixture.
13. The process of claim 12, further comprising introducing cells
which produce a recombinant protein selected from the group
consisting of Factor II, Factor V, Factor VII, Factor VIII, Factor
IX, Factor X, Factor XI, Protein S, Protein C, activated forms of
these factors, and vWF.
14. A cell culture composition comprising mammalian cells and a
medium in accordance with claim 1.
15. A process for producing a cell culture medium according to
claim 1, comprising: obtaining a soy hydrolysate; ultrafiltering
said soy hydrolysate using an ultrafiltration process; purifying
said soy hydrolysate fraction using size exclusion chromatography;
and selecting the soy hydrolysate fractions consisting of soy
hydrolysate having a molecular weight .ltoreq.500 daltons.
Description
[0001] The present invention pertains to a medium for the
protein-free and serum-free cultivation of cells.
BACKGROUND OF THE INVENTION
[0002] The cultivation of cells, especially eukaryotic cells or
mammalian cells, constantly calls for the use of special culture
media that make available to the cells the nutrient substances and
growth substances that are required for efficient growth and for
the production of the proteins that are desired. As a rule, serum
or compounds that are derived from serum (e.g. bovine serum) are
used as a component of the medium in this regard.
[0003] However, in the case of the use of serum or protein
additives that are derived from human or animal sources in cell
cultures, numerous problems exist, especially if the starting
material for the preparation of a medicinal agent that is to be
administered to humans is made available via the cell culture.
[0004] In the case of such serum preparations, therefore, the
composition and quality already vary from batch to batch just
because of the dissimilarity of the donor organisms for such
preparations. This represents a considerable problem, especially
for the standardization of cell production and in establishing
standard growth conditions for such cells. However, intensive and
constant quality control of the serum material that is used is
required in every case. However, this is extremely time-consuming
and cost intensive, especially in the case of such complex
compositions as serum.
[0005] Moreover, such complex preparations contain a plurality of
proteins that can act in a disruptive manner, especially within the
context of the purification process for the recombinant protein
that is to be recovered from the cell culture. This applies
particularly to those proteins that are homologous with or similar
to the protein that is to be recovered. Naturally, these problems
are especially acute in the case of the recombinant recovery of
serum proteins because the biogenic pendant in the medium that is
used (e.g., bovine protein) can be removed reliably within the
context of purification only via quite specific differential
purification (e.g., with antibodies that are directed specifically
only against the recombinant protein but not against the bovine
protein (Bjorck, L., J. Immunol., 1988, Vol. 140, pp. 1194-1197;
Nilson et al., J. Immunol Meth., 1993, 164, pp. 33-40).
[0006] Another issue in the use of serum or compounds which are
derived from serum in the culture medium is the possibility of
contamination by mycoplasma, viruses BSE agents, or
disease-inducing agents that are as yet unknown.
[0007] The addition of serum components in order to guarantee
adequate adhesion of the cells to their surfaces and to guarantee
adequate production of the desired substances from the cells has,
apart from a few exceptions, been previously regarded as
indispensable precisely for the cultivation of cells on solid
surfaces. Thus with the method that is described in WO 91/09935,
for example, it has been possible to achieve a process for the
serum-free and protein-free cultivation of the FSME virus/virus
antigen by means of the serum-free and protein-free cultivation of
surface-dependant permanent cells, preferably vero cells (see WO
96/15231). However these are not recombinant cells but, rather,
host cells that are used for the production of virus antigen in a
lytic process.
[0008] In contrast to this, the cells that are used preeminently
for a recombinant preparation, for example CHO cells, are capable
of adhering only to a limited extent. Thus, CHO cells that have
been bred by conventional methods bind to both smooth and porous
microcarriers only under serum-containing conditions (see U.S. Pat.
No. 4,973,616; Cytotechnology 9 (1992), 247-253). However, if such
cells are bred under serum-free conditions, they lose this property
and do not adhere to smooth carriers, or they become detached with
ease therefrom if other adhesion-promoting additions, such as e.g.,
fibronectin, insulin or transferrin, have not been provided in the
medium. However these are also proteins that are derived from
serum.
[0009] Alternatively to this, the cells can be bred using the
suspension culture technique as well as e.g., using the batch
process or using a continuous culture technique. Cultivation
preferably takes place using the chemostat process (Ozturk S. S. et
al., 1996, Abstr. Pap. Am. Chem. Soc., BIOT 164, Payne G. F. et
al., in "Large Scale Cell Culture Technology," 1987, ed. Lydersen
B. K., Hauser publishers; pp. 206-212). Kattinger H. et al
(Advances Mol. Cell. Biology, 1996, 15A, 193-207) describe the long
term cultivation of cells in protein-free medium, but these cells
must be cultivated on carriers and do not leave alternatives as
continuous culture techniques. It is stated that these cells only
show long term stability when adhered to the surface of carriers
because of reduced growth and, as a consequence, reduced demand for
growth factors.
[0010] In addition, attempts have been made on several occasions in
the prior art to adapt cells to a protein-free medium starting from
serum-containing conditions. However, in the case of such
adaptation, it has been found repeatedly that, compared to
serum-containing conditions, the yield of expressed protein and the
productivity of the recombinant cells are markedly reduced in the
protein-free medium following adaptation (Appl. Microbiol.
Biotechnol. 40 (1994), 691-658).
[0011] It has also been found that, in the case of a high cell
density, the production of recombinant proteins is considerably
restricted on occasions. During attempts to adapt the cells to
protein-free or serum-free media, instability with reduced growth
of the cells, which are used, is also found repeatedly so that
cells with reduced expression are produced, or even nonproducing
cells are produced, whereby these have a growth advantage, relative
to the producing cells, in protein-free and serum-free media, and
this leads to the fact that these overgrow the producing cells and
then, finally, the entire culture now generates very low product
yields.
SUMMARY OF THE INVENTION
[0012] The present invention has therefore an objective of
improving the possibilities for the protein-free and serum-free
cultivation of recombinant cells and of making agents and processes
available with which recombinant cells can be cultivated
efficiently in a serum-free or protein-free manner. Moreover, it
should then be possible not only to culture surface-dependent
cells, but also to use the suspension culture technique, whereby
instability in the productivity of the cells is required to be
repressed as much as possible.
[0013] A further objective of the present invention additionally is
to efficiently increase the production of recombinant cells.
[0014] Finally, in accordance with the invention, the adaptation of
recombinant cells to serum-free and protein-free media is required
to be improved and configured more efficiently.
[0015] In accordance with the invention, these tasks are
accomplished by means of a medium for the protein-free and
serum-free cultivation of cells, especially mammalian cells,
characterized by the feature that the medium contains a proportion
of soy hydrolysate.
[0016] Surprisingly, it has been possible to show that the
objectives, which were defined above, can be achieved by
cultivating cells in a medium that contains soy hydrolysate,
without having to tolerate the disadvantages of serum-free
cultivation which are described in the prior art. In contrast to
other hydrolysates which are known in the prior art, such as for
example wheat hydrolysates, rice hydrolysates or yeast
hydrolysates, it has been found that only soy hydrolysate mediates
the properties in accordance with the invention and leads, for
example, to a significantly increased yield of the recombinant
target protein. When dealing with these terms, either the term soy
hydrolysate or the term soy peptone can be used whithout having
different meanings.
SUMMARY OF THE FIGURES
[0017] FIG. 1 shows the results of the cultivation of a rFVIII-CHO
cell clone in a 10-L perfusion bioreactor:
[0018] a) Factor VIII activity (milliunits/mL) and the perfusion
rate (1-5/day) over a period of 42 days;
[0019] b) volumetric productivity (units of Factor VIII/L/day) in
the perfusion bioreactor;
[0020] FIG. 2 shows a comparison of the Factor VIII productivity
(mU/mL) in the case of cultivation, using the batch process, of CHO
cells which express rFactor VIII, in various media. Mix 1 consists
of serum-free and protein-free medium without soy hydrolysate, but
containing an amino acid mixture as listed in table 4; Mix 2
consists of serum-free and protein-free medium containing soy
hydrolysate; Mix 3 consists of serum-free and protein-free medium
containing soy hydrolysate and an amino acid mixture as listed in
table 4 and Mix 4 consists of serum-free and protein-free medium
containing 2.5 g/l purified, ultrafiltrated soy hydrolysate and an
amino acid mixture as listed in table 4. For the purification of
the ultrafiltrated soy hydrolysate a Sephadex.RTM. column was
used.
[0021] FIG. 3 shows the Factor VIII productivity (U/L) in the case
of the continuous growth of CHO cells, which express rFactor VIII,
in a serum-free and protein-free medium after the start of the
addition of purified, ultrafiltered soy peptone, namely on the 6th
day of cultivation; and
[0022] FIG. 4 shows BHK cells expressing recombinant Factor II that
have been bred in a protein-free and serum-free medium that
contains soy hydrolysate.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The medium in accordance with the invention preferably
contains soy hydrolysate in a quantity of more than 10 wt % based
on the total dry weight of the medium. As a rule, the soy
hydrolysate in the medium is provided in a quantity of 4-40%.
[0024] The choice of specific soy hydrolysate is not critical in
accordance with the invention. A plurality of soy preparations,
which are to be found on the market, can be used in accordance with
the invention, e.g., peptones from soy flour, digested
enzymatically (e.g., by papain), with a pH value between 6.5 and
7.5 and a total nitrogen content between 9% and 9.7% and an ash
content between 8 and 15%. These are peptones from soybeans in the
form in which they are generally used for cell culture by the
expert in the field.
[0025] In accordance with a preferred form of embodiment, use is
made of a purified preparation of a soy hydrolysate or a crude
fraction thereof in the medium in accordance with the invention.
Impurities which could interfere with efficient cultivation are
preferably eliminated during this purification, or the precision of
the hydrolysate is improved, e.g., in regard to the molecular
weight.
[0026] In accordance with the invention, the provision of an
ultrafiltration step has proven to be especially valuable in
practice during this purification; because of this, the use of
ultrafiltered soy hydrolysate is especially preferred in the medium
in accordance with the invention.
[0027] Ultrafiltration can take place in accordance with the
process as described comprehensively in the prior art, e.g., with
use being made of membrane filters with a defined cut-off
limit.
[0028] The purification of the ultrafiltered soy peptone can take
place by means of gel chromatography, e.g., by means of Sephadex
chromatography, for example, with Sephadex G25 or Sephadex G10 or
equivalent materials, ion-exchange chromatography, affinity
chromatography, size exclusion chromatography or "reversed-phase"
chromatography. These are processes from the prior art with which
the expert in the field is familiar. Using this method, those
fractions can be selected which contain soy hydrolysate of defined
molecular weight, i.e. .ltoreq.1000 Dalton, preferably .ltoreq.500
Dalton, more preferably .ltoreq.350 Dalton. Therefore the invention
also comprises a process for producing a serum-free and
protein-free cell culture medium, comprising obtaining a soy
hydrolysate, ultrafiltering said soy hydrolysate using an
ultrafiltration process, purifying said soy hydrolysate fraction
using size exclusion chromatography and selecting the soy
hydrolysate fractions consisting of soy hydrolysate having a
molecular weight .ltoreq.1000 Dalton, preferably .ltoreq.500
Dalton, more preferably .ltoreq.350 Dalton.
[0029] An especially advantageous soy hydrolysate is characterized
by the feature that it has a free amino acids content of between
10.3 and 15.6% or, preferably, between 12 and 13.5%, a total
nitrogen content of between 7.6 and 11.4% or, preferably, between
8.7 and 9.5% and an endotoxin content of <500 U/g and whereby at
least 40% or, preferably, at least 50% or, especially preferably,
at least 55% thereof has a molecular weight of 200-500 daltons and
at least 10% or, preferably, 15% thereof has a molecular weight of
500-1000 daltons. Most preferably, at least 90% of the soy
hydrolysate is of a molecular weight of .ltoreq.500 Daltons. Such a
soy hydrolysate is especially well suited to the industrial
production of recombinant proteins since, because of its special
features, it can be standardized especially easily and it is usable
in routine processes.
[0030] In addition to soy hydrolysate, the medium in accordance
with the invention can also contain synthetic media in a way that
is known as such, such as e.g., DMEM/HAM's F12, Medium 199 or RPMI,
that are adequately known from the literature.
[0031] Moreover, the medium in accordance with the invention also
preferably contains amino acids, preferably those selected from the
group comprising L-asparagine, L-cysteine, L-cystine, L-proline,
L-tryptophan, L-glutamine, or mixtures thereof.
[0032] The following amino acids are also preferably added to the
medium in accordance with the invention: L-asparagine (in a
quantity of 0.001-1 g/L of medium, preferably 0.1-0.05 g/L,
especially preferably 0.015-0.03 g/L); L-cysteine (0.001-1 g/L,
preferably 0.005-0.05 g/L, especially preferably 0.01-0.03 g/L);
L-cystine (0.001-1 g/L, preferably 0.01-0.05 g/L, especially
preferably 0.015-0.03 g/L); L-proline (0.001-1.5 g/L, preferably
0.01-0.07 g/L, especially preferably 0.02-0.05 g/L); L-tryptophan
(0.001-1 g/L, preferably 0.01-0.05 g/L, especially preferably
0.015-0.03 g/L); and L-glutamine (0.05-1 g/L, preferably 0.1-1
g/L).
[0033] The amino acids designated above can be added to the medium
in accordance with the invention either individually or in
combination. The combined addition of an amino acid mixture, which
contains all of the above-mentioned amino acids, is especially
preferred.
[0034] A serum-free and protein-free medium is used in a special
form of embodiment, whereby this medium additionally contains a
combination of the above-mentioned amino acid mixture and purified
ultrafiltered soy peptone.
[0035] Surprisingly, for example, it has been found that in order
to inactivate viruses or other pathogens, the medium can be heated,
without negative effects, for approximately 5-20 min or,
preferably, 15 min at 70-95.degree. C. or, preferably,
85-95.degree. C.
[0036] In accordance with the invention, a known synthetic medium
can be used in combination with the soy hydrolysate. Conventional
synthetic media can contain inorganic salts, amino acids, vitamins,
a source of carbohydrates and water. For example, use can be made
of DMEM/HAM's F12 medium. The concentration of soy extract in the
medium can preferably be between 0.1 and 100 g/L, especially
preferably, 1 and 5 g/L. In accordance with an especially preferred
form of embodiment, soy peptone can be used which has been
standardized in regard to its molecular weight. The molecular
weight of the soy peptone preferably is less than 50 kD, especially
preferably less than 10 kD, most preferably, less than 1 kD.
[0037] The addition of ultrafiltered soy peptone has proven to be
especially advantageous for the productivity of the recombinant
cell lines, whereby the average molecular weight of the soy peptone
is 350 daltons (Quest Company). This is a soy isolate with a total
nitrogen content of approximately 9.5% and a free amino acid
content of approximately 13%.
[0038] The use of purified, ultrafiltered soy peptone with a
molecular weight of .ltoreq.1,000 daltons, preferably .ltoreq.500
daltons, especially preferably .ltoreq.350 daltons is especially
preferred.
[0039] The medium in accordance with the invention also preferably
contains auxiliary substances, such as e.g., buffer substances,
oxidation stabilizers, stabilizers to counteract mechanical stress,
or protease inhibitors.
[0040] Use is especially made of 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), NaHCO.sub.3 (0.1-10 g/L),
ascorbic acid (0.0005-0.05 g/L), ethanolamine (0.0005-0.05 g/L) and
Na selenite (1-15 .mu.g/L).
[0041] If required, a nonionic surfactant, such as, e.g.,
polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC
F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium
as a defoaming agent in accordance with the invention.
[0042] This agent is generally used in order to protect the cells
from the negative effects of aeration since, without an addition of
a surfactant, ascending and bursting air bubbles can lead to damage
of those cells that are located on the surface of these air bubbles
("sparging") (Murhammer and Goochee, 1990, Biotechnol. Prog.
6:142-148).
[0043] The quantity of nonionic surfactant can be between 0.05 and
10 g/L. Preferably, the smallest possible amount is between 0.1 and
5 g/L. In addition, the medium in accordance with the invention can
also contain cyclodextrin or a derivative thereof.
[0044] The serum-free and protein-free medium of the present
invention preferably contains a protease inhibitor, such as a
serine protease inhibitor, which is suitable for tissue culture and
is of synthetic or plant origin.
[0045] Cells that have already been adapted are preferably used as
the cells for cultivation in the medium in accordance with the
invention, i.e., cells that have already adapted to growth in the
protein-free and serum-free media. It has been found that not only
can increased yields be achieved with such preadapted cells, but
their stability for serum-free and protein-free cultivation is also
clearly improved by the use of the medium in accordance with the
invention.
[0046] However, recombinant cell clones have proven to be
especially valuable in accordance with the invention, whereby these
are stable from the outset for at least 40 generations and,
preferably, at least 50 generations in serum-free and protein-free
media, and express recombinant products.
[0047] Such cell clones are obtainable from a cell culture that is
obtained following the cultivation of a recombinant original cell
clone on a serum-containing medium and readaptation of the cells to
a serum-free and protein-free medium.
[0048] The term "original cell clone" can be understood to mean a
recombinant cell clone transfectant that, after transfection of the
host cells with a recombinant nucleotide sequence, expresses a
recombinant product in a stable manner under laboratory conditions.
The original clone is bred in a serum-containing medium in order to
optimize its growth. In order to increase its productivity, the
original clone is bred, optionally in the presence of a selection
agent, with selection on the selection marker and/or amplification
marker. For large-scale industrial production, the original cell
clone is bred, under serum-containing conditions of cultivation, to
a high cell density and then it is readapted to a serum-free or
protein-free medium just prior to the production phase. Cultivation
preferably takes place without selection pressure in this case.
[0049] The cultivation of the recombinant original cell clone can
take place from the beginning in a serum-free and protein-free
medium; as a result, readaptation is no longer necessary. If
required, use can also be made of a selection agent in this case
and selection can take place on the selection marker and/or the
amplification marker. A process for this is described in EP 0 711
835, for example.
[0050] The cell culture that is obtained after readaptation to a
serum-free and protein-free medium is tested for those cell clones
of the cell population which produce products in a stable manner
under serum-free and protein-free conditions, optionally in the
absence of selection pressure. This can take place, for example, by
means of immunofluorescence with marked specific antibodies which
are directed against the recombinant polypeptide or protein. The
cells that are identified as product producers are isolated from
the cell culture and are re-bred under serum-free and protein-free
conditions that are preferably equivalent to production conditions.
The isolation of the cells can thereby take place by isolating the
cells and testing them for product producers.
[0051] The cell culture, containing the stable cells, can be tested
again for stable recombinant clones, and these are isolated from
the cell culture and subcloned. The stable recombinant cell clones
that are obtained under serum-free and protein-free conditions can
then be bred further under serum-free and protein-free
conditions.
[0052] The recombinant cell clones or the cell populations, which
are prepared in this way in the medium in accordance with the
invention, excel in particular by way of the feature that they are
stable for at least 40 generations, preferably for at least 50
generations and, in particular, for more than 60 generations, and
express a recombinant product.
[0053] An example of such a recombinant stable cell clone or cell
population has been filed, in accordance with the Budapest
convention, under number 98012206 with the ECACC (UK).
[0054] The cell culture, which is to be cultivated in accordance
with the invention, is preferably derived from a recombinant
mammalian cell. Recombinant mammalian cells can hereby be all those
cells that contain sequences which code for a recombinant
polypeptide or protein. All continuously growing cells, which grow
either adherently or nonadherently, are encompassed in this regard.
Recombinant CHO cells or BHK cells are especially preferred.
Recombinant polypeptides or proteins can be blood factors, growth
factors or other biomedically relevant products.
[0055] In accordance with the present invention, cell clones are
preferred 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 that are capable of
expressing these in a stable manner over several generations.
Recombinant CHO cells that 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, are especially preferred in
this regard.
[0056] 30 generations are required in order to start a master cell
bank. At least approximately 40 generations are required in order
to carry out an average batch culture on the 1000-L scale. Starting
out from an individual cell clone, it is possible with the medium
in accordance with the invention to prepare a "master cell bank"
(MCB) and a "working cell bank" (WCB) with approximately 8-10
generations, and hence a cell culture with up to 20-25 generations
under protein-free and serum-free conditions on the production
scale (production biomass) whereas, by contrast, some generations
become unstable after growth on a serum-free or protein-free medium
with previous cell clones and media and, as a result, a) a uniform
cell culture with product producers is not possible and b) stable
product productivity over an extended period of time is not
possible.
[0057] However, in accordance with the invention, it was even
possible, by contrast, to find increased product productivity even
in comparison to the original cell clone that had been cultivated
in a serum-containing medium.
[0058] In accordance with a further aspect, the present invention
also pertains to a process for the cultivation of cells, especially
mammalian cells, that is characterized by the feature that these
cells are introduced into a medium in accordance with the invention
and then are cultured in this medium.
[0059] Thus the present invention also pertains to the use of the
medium in accordance with the invention for the cultivation of
recombinant cells, preferably eukaryotic cells and, especially,
mammalian cells. The subject of the present invention, accordingly,
is also a cell culture that comprises the medium in accordance with
the invention and cells, preferably eukaryotic cells, and
especially mammalian cells.
[0060] The present invention further includes a process for the
production of a desired protein (especially a recombinant protein)
from cell culture comprising introducing cells which express such
desired protein into a medium of the present invention; growing
said cells in said medium and expressing said protein, thereby
producing a mixture of said cells and said protein in the medium;
and purifiying said protein from said mixture. In this way
recombinant proteins such as Factor II, Factor V, Factor VII,
Factor VIII, Factor IX, Factor X, Factor XI, Protein S, Protein C,
activated forms of these factors, and vWF can be produced.
[0061] The invention will be elucidated in more detail by means of
the following examples below, as well as by the figures in the
drawings, but it is not to be limited thereto.
EXAMPLES
Example 1
Stability of rvWF-CHO Cells After Switching from a Serum-containing
Medium to a Serum-free and Protein-free Medium
[0062] CHO-dhfr cells were plasmid phAct-rvWF and pSV-dhfr
co-transfected, and vWF-expressing clones were subcloned as
described by Fischer et al. (1994, FEBS Letters 351:345-348). A
working cell bank (WCB) was started from the subclones, which
expressed rvWF in a stable manner, under serum-containing
conditions but in the absence of MTX, and the cells were
immobilized on a porous microcarrier (Cytopore) under
serum-containing conditions. Switching the cells to a serum-free
and protein-free medium took place after a cell density of
2.times.10.sup.7 cells/mL of the matrix had been reached. The cells
were cultured further for several generations under serum-free and
protein-free conditions. The cells were tested in a serum-free and
protein-free medium at various points in time by means of
immunofluorescence with labelled anti-vWF antibodies. The
evaluation of the stability of the cells took place using the
working cell bank prior to switching the medium, after 10
generations and after 60 generations in the serum-free and
protein-free medium. Whereas the working cell bank still exhibited
100% rvWF producers, the proportion of rvWF producers declined to
approximately 50% after 10 generations in the serum-free and
protein-free medium. After 60 generations, more than 95% of the
cells were identified as nonproducers.
Example 2
Cloning of Stable Recombinant CHO Clones
[0063] A dilution series was prepared from the cell culture
containing rvWF-CHO cells in accordance with Example 1 (this stable
cell clone that was designated r-vWF-CHO F7 was filed, in
accordance with the Budapest convention, with the ECACC (European
Collection of Cell Cultures), Salisbury, Wiltshire SP4 OJG, UK, on
Jan. 22, 1998, and acquired the deposition number 98012206) which
had been cultured for 60 generations in a serum-free and
protein-free medium and 0.1 cells were seeded out in each well of a
microtiter plate. The cells were cultivated for approximately 3
weeks in DMEM/HAM's F12 without serum additions or protein
additions and without selection pressure, and the cells were tested
via immunofluorescence with labelled anti-vWF antibodies. A cell
clone, which had been identified as positive, was used as the
starting clone for the preparation of a seed cell bank. A master
cell bank (MCB) was started from the seed cell bank in a serum-free
and protein-free medium and individual ampules were put away and
frozen for the further preparation of a working cell bank. A
working cell bank was prepared in a serum-free and protein-free
medium from an individual ampule. The cells were immobilized on
porous microcarriers and cultivated further for several generations
under serum-free and protein-free conditions. The cells were tested
for productivity at various points in time in a serum-free and
protein-free medium by means of immunofluorescence with labelled
anti-vWF antibodies. The evaluation of the stability of the cells
took place at the working cell bank stage and after 10 and 60
generations in a serum-free and protein-free medium. Approximately
100% of the cells were identified as positive stable clones, which
express rvWF, at the working cell bank stage, after 10 generations,
and 60 generations.
Example 3
Cell Specific Productivity of the Recombinant Cell Clones
[0064] A defined number of cells was removed at defined stages
during the cultivation of the recombinant cells, and these were
incubated for 24 h with fresh medium. The rvWF: Risto-CoF-activity
was determined in the supernatant liquors of the cell cultures.
Table 1 shows that, in the case of the stable recombinant cell
clones in accordance with the invention, the cell-specific
productivity was stable even after 60 generations in a serum-free
and protein-free medium and it had even increased in comparison to
the original clone that had been cultivated in a serum-containing
medium.
1 TABLE 1 Cell specific Cell specific Cell specific productivity
productivity productivity of the after 10 after 60 working cells
generations generations in mU in mU in mU Cell rvWF/10.sup.6
rvWF/10.sup.6 rvWF/10.sup.6 Clone cells/day cells/day cells/day
rvWF-CHO 55 30 <10 #808.68 original cell clone r-vWF-CHO 62 65
60 F7*) stable clone *)filed on January 22, 1998 (ECACC (European
Collection of Cell Cultures, Salisbury, Wiltshire SP4 OJG, UK);
deposition number 98012206)
Example 4
Composition of a Synthetic Serum-free and Protein-free Medium
[0065]
2TABLE 2 Preferred quantity (according to our knowledge at the time
of the patent Component g/L application) in g/L Synthetic minimal
1-100 11.00-12.00 medium (DMEM/HAM's F12) Soy peptone 0.5-50 2.5
L-glutamine 0.05-1 0.36 Ascorbic acid 0.0005-0.0 5 0.0035
NaHCO.sub.3 0.1-10 2.00 Ethanolamine 0.0005-0.05 0.0015 Na selenite
1-15 .mu.g/l 8.6 .mu.g/l Optionally: 0.01-10 0.25 Synperonic
F68
Example 5
Cultivation of rFVIII-CHO Cells in a Protein-free and Serum-free
Minimal Medium
[0066] A cell culture containing rFVIII-CHO cells was cultivated in
a 10-L stirred tank with perfusion. A medium in accordance with
Example 4 was used in this case. The cells were thereby immobilized
on a porous microcarrier (Cytopore, Pharmacia) and then cultivated
for at least 6 weeks. The perfusion rate was 4 volume changes per
day; the pH was 6.9-7.2; the O.sub.2 concentration was
approximately 20-50% and the temperature was 37.degree. C.
[0067] FIG. 1 shows the results of the cultivation of a rFVIII-CHO
cell clone in a 10 L perfusion bioreactor.
[0068] a) Factor VIII activity (milliunits/mL) and perfusion rate
(1-5/day) over a period of 42 days.
[0069] b) Volumetric productivity (units of Factor VIII/L/day) in
the perfusion bioreactor.
3TABLE 3 Cell specific Immunofluorescence Days of productivity (%
FVIII cultivation (mU/10.sup.6 cells/day) positive cells) 15 702
n.a. 21 1125 n.a. 25 951 >95% 35 691 >95% 42 910 n.a.
[0070] Table 3 shows the stability and specific productivity of the
rFVIII-expressing cells. In order to obtain these results, samples
were taken after 15, 21, 28, 35 and 42 days and then centrifuged at
300 g and resuspended in fresh serum-free and protein-free medium.
The Factor VIII concentration in the supernatant liquors of the
cell cultures and the cell count was determined after a further 24
h. The specific FVIII productivity was calculated from these
data.
[0071] A stable average productivity of 888 milliunits/10.sup.6
cells/day was achieved. This stable productivity was also confirmed
by immunofluorescence with labelled anti-FVIII antibodies after 15,
21, 28, 35 and 42 days in a serum-free and protein-free medium.
Example 6
Comparison of the Productivity of Recombinant FVIII-CHO Cells in a
Protein-free and Serum-free Medium Containing Further Medium
Components
[0072] A cell culture containing rFVIII-CHO cells was cultivated
batchwise. In this case, use was made of a medium in accordance
with Example 4 to which the following amino acids had been
added:
4 TABLE 4 Preferred quantity (according to our knowledge at the
time of the patent Amino acid mg/l application) in mg/L
L-Asparagine 1-100 20 L-Cysteine.HCl.H.sub.2O 1-100 15 L-Cystine
1-100 20 L-Proline 1-150 35 L-Glutamine 50-1000 240
[0073] The cells were bred at 37.degree. C. and pH 6.9-7.2. The
cells were bred using the batch process over periods of 24-72
h.
[0074] The productivity of the recombinant FVIII-CHO cells was
measured in the following medium compositions:
[0075] Mix 1: comprising a serum-free and protein-free medium
without soy peptone and additionally containing an amino acid
mixture in accordance with the table designated above.
[0076] Mix 2: comprising a serum-free and protein-free medium
containing soy peptone.
[0077] Mix 3: comprising a serum-free and protein-free medium
containing soy peptone and additionally containing an amino acid
mixture in accordance with the table designated above.
[0078] Mix 4: comprising a serum-free and protein-free medium, and
additionally containing an amino acid mixture in accordance with
the table designated above and 2.5 g/L of purified, ultrafiltered
soy peptone. The purification of the ultrafiltered soy peptone took
place chromatographically over a Sephadex column.
Example 7
Cultivation of Recombinant FVIII-CHO Cells in a Protein-free and
Serum-free Medium Using the Chemostat Culture Method
[0079] A cell culture containing rFVIII-CHO cells was cultivated in
a 10-L stirred bioreactor tank. In this case, use was made of a
medium in accordance with Example 4, without soy peptone,
containing an amino acid mixture in accordance with Example 6. The
cells were bred at 37.degree. C. and pH 6.9-7.2; the oxygen
concentration was 20-50% air saturation. Samples were taken every
24 h in order to determine the Factor VIII titer and the cell
concentration in the supernatant liquor of the culture. The total
cell concentration was constant from the 2nd day to the 14th day.
Ultrafiltered soy peptone was added to the medium starting from the
6th day. The Factor VIII productivity, is illustrated in 3; the
measurements took place by means of a CHROMOGENIX CoA FVIII:C/4
system. Immunofluorescence was carried out with labelled anti-FVIII
antibodies. It can be seen from the data that a distinct increase
in Factor VIII productivity and hence an increase in the volumetric
productivity of the bioreactor system, occurred as a result of the
addition of soy peptone, whereby this did not lead to a distinct
increase in cell growth. The absence of soy peptone in the
continuous culture leads to a distinct decline in Factor VIII
productivity after a few days, whereas the addition of soy peptone
leads, as a consequence, to an almost 10-fold increase in
productivity. However, since this addition does not increase the
cell count, it is hereby clearly shown that ultrafiltered soy
peptone leads, as a consequence, to a distinct increase in
productivity which is independent of cell growth.
Example 8
Comparison of the Growth Rate and the Productivity of Recombinant
FVIII-CHO Cells in a Protein-free and Serum-free Medium Containing
Different Hydrolysates
[0080] A rFVIII-CHO cell culture was cultivated batchwise. In this
case, use was made of a serum-free and protein-free medium as
described in Example 4 to which different hydrolysates (from soy,
yeast, rice and wheat) had been added. A serum-free and
protein-free medium, to which no hydrolysate had been added, was
used as the control.
[0081] The initial cell density was 0.6.times.10.sup.5 and
0.4.times.10.sup.6, respectively. The cells were cultured at
37.degree. C. using the batch process at pH 6.9-7.2.
[0082] Table 5: shows the results of the cultivation experiments
with rFVIII-CHO cells in a serum-free and protein-free medium to
which soy hydrolysate (ultrafiltered) and yeast hydrolysate had
been added. The initial cell density was 0.6.times.10.sup.5 cells.
A serum-free and protein-free medium without hydrolysate additions
was used as the control.
5 TABLE 5 FVIII Final cell clotting density FVIII titer activity
Hydrolysate (.times.10.sup.6/mL) (mU/mL) (mU/mL) Soy 3.6 485 508
Yeast 3.3 226 230
[0083] Table 6: shows the results of the cultivation experiments
with rFVIII-CHO cells in a serum-free and protein-free medium to
which soy hydrolysate (ultrafiltered), rice hydrolysate and wheat
hydrolysate had been added. The initial cell density was
0.6.times.10.sup.5 cells. A serum-free and protein-free medium
without hydrolysate additions was used as the control.
6 TABLE 6 Final cell density FVIII titer vWF - Antigen Hydroysate
(.times.10.sup.6/mL) (mU/mL) (.mu.g/L) Soy 3.1 1142 6.7 Rice 3.0
419 3.2 Wheat 3.4 522 3.9 Control 3.0 406 3.1
[0084] Table 7 shows the results of the cultivation experiments
with rFVIII-CHO cells in a serum-free and protein-free medium to
which soy hydrolysate (ultrafiltered) and wheat hydrolysate had
been added. The initial cell density amounted to 0.4.times.10.sup.6
cells.
7TABLE 7 Final cell FVIII FVIII VWF- density titer Antigen Antigen
Hydrolysate (.times.10.sup.6/mL) (mU/mL) (.mu.g/mL) (.mu.g/mL) Soy
1.6 1427 166 17.2 Wheat 1.0 1120 92 1.9
Example 9
Cultivation of BHK Cells in a Protein-free and Serum-free Medium
Containing Soy Hydrolysate
[0085] BHK-21 (ATCC CCL 10) cells were co-transfected three times
with the following plasmids by means of a CaPO.sub.4 procedure: 25
.mu.g of the plasmid pSV-FII (Fischer, B. et al., J. Biol. Chem.,
1996, Vol. 271, pp. 23737-23742) which contains the human Factor II
(prothrombin)-cDNA under the control of a SV40 promotor (SV40 early
gene promoter); 4 .mu.g of the plasmid pSV-DHFR for methotrexate
resistance and 1 .mu.g of the plasmid pUCSV-neo (Schlokat, U. et
al., Biotech. Appl. Biochem., 1996, Vol. 24, pp. 257-267) which
mediates G418/neomycin resistance. Stable cell clones were selected
by means of cultivation in a medium, which contained 500 .mu.g/mL
of G418, by increasing the methotrexate concentration in a stepwise
manner up to a concentration of 3 .mu.M.
[0086] The clones that were obtained in this way were subcloned and
adapted to a protein-free and serum-free medium. Cultivation took
place using the suspension culture technique.
[0087] The results can be seen in Table 6; the BHK cells, which
were bred in the protein-free and serum-free medium containing soy
hydrolysate, exhibited a high and stable rate of production of
recombinant Factor II.
* * * * *