U.S. patent application number 15/145618 was filed with the patent office on 2016-08-25 for cell culture processes.
The applicant listed for this patent is Baxalta GmbH, Baxalta Incorporated. Invention is credited to Virginie Charlot, Veronique Ducros, Andre Giovagnoli, Sylvain Roy, Yves-Olivier Stauffer.
Application Number | 20160244506 15/145618 |
Document ID | / |
Family ID | 40350258 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160244506 |
Kind Code |
A1 |
Giovagnoli; Andre ; et
al. |
August 25, 2016 |
CELL CULTURE PROCESSES
Abstract
Culturing heterologous protein-secreting mammalian cells, such
as CHO or BHK cells, at 35.1-36.5.degree. C. and/or at pH 7.15-7.20
and/or at a dissolved CO.sub.2 concentration of 10% or lower.
Preferred heterologous proteins are Factor VIII, ADAMTS-13, furin
or Factor VII.
Inventors: |
Giovagnoli; Andre;
(Villers-Le-Lac, FR) ; Roy; Sylvain; (Savagnier,
CH) ; Ducros; Veronique; (Champagne, CH) ;
Charlot; Virginie; (La Chaux-de-Fonds, CH) ;
Stauffer; Yves-Olivier; (Epalinges, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta GmbH |
Bannockburn
Glattpark (Opfikon) |
IL |
US
CH |
|
|
Family ID: |
40350258 |
Appl. No.: |
15/145618 |
Filed: |
May 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12341807 |
Dec 22, 2008 |
9359629 |
|
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15145618 |
|
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|
61009328 |
Dec 27, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/21075 20130101;
C07K 14/755 20130101; C12N 9/6437 20130101; C12P 21/02 20130101;
C12Y 304/24087 20130101; C12N 9/6489 20130101; C12P 21/00 20130101;
C12Y 304/21021 20130101; C12N 9/6454 20130101 |
International
Class: |
C07K 14/755 20060101
C07K014/755; C12P 21/00 20060101 C12P021/00; C12N 9/64 20060101
C12N009/64 |
Claims
1-51. (canceled)
52. A method comprising: expressing a heterologous protein in a
mammalian cell in continuous cell culture, wherein the continuous
cell culture has a dissolved carbon dioxide concentration of from
1% to 10% and is maintained at a cell culture density of from
1.4.times.10.sup.6 to 2.8.times.10.sup.6 cells/ml.
53. The method of claim 52, wherein the continuous cell culture has
a dissolved carbon dioxide concentration of from 4% to 9%.
54. The method of claim 52, wherein the continuous cell culture has
a dissolved carbon dioxide concentration of from 5.5% to 8.5%.
55. The method of claim 52, wherein the continuous cell culture is
maintained at a temperature of from 34.2.degree. C. to 36.9.degree.
C.
56. The method of claim 52, wherein the continuous cell culture is
maintained at a temperature of 36.+-.0.9.degree. C.
57. The method of claim 54, wherein the continuous cell culture is
maintained at a temperature of 36.+-.0.9.degree. C.
58. The method of claim 52, wherein the continuous cell culture is
maintained at a pH of from 7.10 to 7.25.
59. The method of claim 52, wherein the continuous cell culture is
maintained at a pH of 7.20.+-.0.05.
60. The method of claim 54, wherein the continuous cell culture is
maintained at a pH of 7.20.+-.0.05.
61. The method of claim 56, wherein the continuous cell culture is
maintained at a pH of 7.20.+-.0.05.
62. The method of claim 57, wherein the continuous cell culture is
maintained at a pH of 7.20.+-.0.05.
63. The method of claim 52, wherein the continuous cell culture is
buffered with bicarbonate.
64. The method of claim 52, wherein the dissolved CO.sub.2
concentration of the continuous cell culture is maintained by
sparging the continuous mammalian cell culture with air.
65. The method of claim 52, wherein the heterologous protein is a
blood protein.
66. The method of claim 52, wherein the heterologous protein is
Factor VIII.
67. The method of claim 66, wherein the Factor VIII is co-expressed
with von Willebrand Factor.
68. The method of claim 52, wherein the heterologous protein is
ADAMTS-13, Furin, or Factor VII.
69. The method of claim 52, wherein the continuous cell culture
comprises copper at a concentration of at least 5 parts per billion
(ppb).
70. The method of claim 52, wherein the continuous cell culture
comprises copper at a concentration of at least 7 parts per billion
(ppb).
71. The method of claim 52, wherein the mammalian cell is a rodent
cell.
72. The method of claim 52, wherein the mammalian cell is a CHO
cell.
73. The method of claim 52, wherein the mammalian cell is a BHK
cell.
74. The method of claim 52, wherein the continuous cell culture is
operated under chemostatic conditions.
75. The method of claim 52, wherein the continuous cell culture is
operated under turbostatic conditions.
Description
[0001] This application claims priority of U.S. Provisional
Application No. 61/009,328, filed Dec. 27, 2007, which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to processes for culturing mammalian
cells, particularly mammalian cells that secrete heterologous
and/or recombinant proteins and, more particularly, mammalian cells
that secrete blood proteins, such as blood clotting factor VIII
(hereinafter `Factor VIII`, or just `FVII`), ADAMTS-13, furin or
clotting factor VII (hereinafter `Factor VII`, or just `FVII`).
BACKGROUND OF THE INVENTION
[0003] Blood clotting Factor VIII is a trace plasma glycoprotein
that is found in mammals and is involved as a cofactor of IXa in
the activation of Factor X. An inherited deficiency of Factor VIII
results in the bleeding disorder haemophilia A, which can be
treated successfully with purified Factor VIII. The Factor VIII can
be extracted from blood plasma or can be produced by
recombinant-DNA-based techniques. In the plasma, it circulates as a
complex with von Willebrand Factor (vWF).
[0004] Recombinant Factor VIII (rFVIII) can be produced by Chinese
Hamster Ovary (CHO) cells transfected with a vector carrying a DNA
sequence encoding the Factor VIII molecule. In some cases,
recombinant Factor VIII is co-produced with recombinant von
Willebrand Factor (rvWF), which stabilises the Factor VIII. Such
co-production can involve the co-culturing of respective cell lines
that express FVIII and vWF, or the co-expression of the two
proteins in the same cell. See U.S. Pat. No. 5,250,421 (Genetics
Institute) and Kaufman et al (1989) Mol. Cell. Biol. 9,
1233-1242.
[0005] In a typical process for preparing recombinant Factor VIII,
cells are cultured in a medium and secrete Factor VIII into the
medium. Factor FVIII may then be purified from the medium,
optionally as a complex with vWF.
[0006] Recombinant Factor VIII is expensive to produce due to the
relatively low yields obtained in processes known in the art. The
yield per cell tends to be low compared to the yield that might be
obtained for other recombinant proteins. If the culture medium is
not supplemented with animal products, such as serum, the medium
may support only relatively low cell densities. This reduces the
yield per volume of medium. However, it is desirable not to
supplement the culture medium with animal products in order to
reduce the risk of contamination with viruses and other
transmissible agents. Animal-protein-free media for the production
of FVIII are known from U.S. Pat. No. 6,936,441 (Baxter AG), for
example.
[0007] The present invention provides processes for producing blood
proteins, including rFVIII, in which the yield is improved compared
to processes known in the art.
SUMMARY OF THE INVENTION
[0008] A first aspect of the invention provides a method of
culturing heterologous protein-secreting mammalian cells in a cell
culture supernatant wherein the cell culture supernatant is
maintained at a temperature that is set at X.+-.0.9.degree. C.
wherein X has a value of from 35.1 to 36.5, with the proviso that
the temperature is set at less than 37.degree. C.
[0009] A second aspect of the invention provides a method of
culturing heterologous protein-secreting mammalian cells in a cell
culture supernatant wherein the cell culture supernatant is
maintained at a pH that is set at X.+-.0.05 wherein X has a value
of from 7.15 to 7.20, with the proviso that the pH is set at
greater than 7.10.
[0010] A third aspect of the invention provides a method of
culturing heterologous protein-secreting mammalian cells in a cell
culture supernatant wherein the cell culture supernatant has a
CO.sub.2 concentration of 1-10%.
[0011] A fourth aspect of the invention provides a method of
continuous culture of FVIII-secreting mammalian cells in a vessel
comprising a cell culture supernatant wherein the density of the
cells in the cell culture supernatant is measured by an in-line
sensor and the influx of fresh medium into the vessel is
automatically controlled so as to maintain the density of the cells
in a desired range.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0012] In the process of the first aspect of the invention, the
cell culture supernatant in which the mammalian cells are cultured
is maintained at a temperature that is set at X.+-.0.9.degree. C.
wherein X has a value of from 35.1 to 36.5, with the proviso that
the temperature is set at less than 37.degree. C. In preferred
embodiments, the temperature is set at 36.+-.0.9.degree. C.,
preferably 36.+-.0.5.degree. C., more preferably 36.+-.0.2.degree.
C. and most preferably 36.degree. C.; or 35.1.+-.0.9.degree. C.,
preferably 35.1.+-.0.5.degree. C., more preferably
35.1.+-.0.2.degree. C. and most preferably 35.1.degree. C.; or
36.5.+-.0.9.degree. C., 36.5.+-.0.5.degree. C., more preferably
36.5.+-.0.2.degree. C. and most preferably 36.5.degree. C.
[0013] The "cell culture supernatant" is the medium in which the
mammalian cells are cultured. This medium is not to be confused
with feed medium that may be added to the culture, although feed
medium is also preferably added to the culture at the temperature
at which the cell culture supernatant is set. By "culture" we mean
the cell culture supernatant and the mammalian cells cultured
therein. Conventionally, mammalian cells are cultured at 37.degree.
C. Surprisingly, the applicant has found that culturing the
mammalian cells at a lower temperature, such as 36.degree. C.
increases the yield of recombinant protein.
[0014] By "culturing at" or "maintaining at" a temperature, we
refer to the temperature to which the process control systems are
set, in other words the intended, target, temperature. Clearly,
there will be small variations of the temperature of a culture over
time, and from location to location through the culture vessel.
Where we refer to "culturing at" or "maintaining at" a temperature
that is set at X.+-.Y .degree. C., we mean that the set point is at
a value of from X+Y .degree. C. to X-Y .degree. C. So, for example
where X is 36.0.+-.0.9.degree. C., the set-point is set at a value
of from 35.1 to 36.9. For each of the preferred values of X, the
set-point is at a value within the range X.+-.0.9.degree. C.,
.+-.0.8.degree. C., .+-.0.7.degree. C., .+-.0.6.degree. C.,
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., or .+-.0.1.degree. C. Narrower ranges are
preferred. A set-point of X is most preferred.
[0015] For any given set-point, slight variations in temperature
may occur. Typically, such variation may occur because heating and
cooling elements are only activated after the temperature has
deviated somewhat from the set-point. In that case, the set-point
is X(.+-.Y) and the heating or cooling element is activated when
the temperature varies by .+-.Z .degree. C., as appropriate.
Typically, the permissible degree of deviation of the temperature
from the set-point before heating or cooling elements are activated
may be programmed in the process control system. Temperature may be
controlled to the nearest .+-.0.5.degree. C., .+-.0.4.degree. C.,
.+-.0.3.degree. C., .+-.0.2.degree. C. or even .+-.0.1.degree. C.
by heating and cooling elements controlled by thermostats. Larger
differentials in temperature may also be programmed, such as
.+-.0.9.degree. C., .+-.0.8.degree. C., .+-.0.7.degree. C. or
.+-.0.6.degree. C. The temperature may also be controlled by
immersion of the culture vessel in a heating bath at a particular
temperature. Conceivably, there is no variation from the set-point
because the heating is applied continually. Another source of
variation arises due to measurement error in the temperature of the
cell culture supernatant. Typical thermometers used in cell culture
equipment may have a variability of .+-.0.3.degree. C. or
.+-.0.2.degree. C., or even .+-.0.1.degree. C.
[0016] Where the set-point is set at a value within the range
X.+-.Y .degree. C., and the tolerance of the temperature is .+-.Z
.degree. C. (i.e. a heater or cooler is activated when the
temperature deviates by .+-.Z .degree. C., as appropriate) this can
also be expressed as a set-point of (X-Y to X+Y) .+-.Z .degree. C.
For each possible value of X, all combinations of .+-.Y .degree. C.
and .+-.Z .degree. C., as indicated above, are envisaged, with the
proviso that the temperature is set at less than 37.degree. C.
[0017] In one preferred embodiment, the temperature is set at
36.+-.Y .degree. C. Preferably, the temperature is set at
(35.4-36.6) .+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree.
C. or .+-.0; or (35.5-36.5) .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (35.6-36.4)
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (35.7-36.3)
.+-.0.6.degree. C., .+-.0.5.degree. C., .+-.0.4.degree. C.,
.+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree. C. or
.+-.0; or (35.8-36.2) .+-.0.7.degree. C..+-.0.6.degree. C.,
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (35.9-36.1)
.+-.0.8.degree. C., .+-.0.7.degree. C..+-.0.6.degree. C.,
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or
36.+-.0.9.degree. C..+-.0.8.degree. C., .+-.0.7.degree.
C..+-.0.6.degree. C., .+-.0.5.degree. C., .+-.0.4.degree. C.,
.+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree. C. or
.+-.0.degree. C.
[0018] In another preferred embodiment, the temperature is set at
35.1.+-.Y .degree. C. Preferably, the temperature is set at
(34.5-35.7) .+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree.
C. or .+-.0; or (34.6-35.6) .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (34.7-35.5)
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (34.8-35.4)
.+-.0.6.degree. C., .+-.0.5.degree. C., .+-.0.4.degree. C.,
.+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree. C. or
.+-.0; or (34.9-35.3) .+-.0.7.degree. C..+-.0.6.degree. C.,
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or (35.0-35.2)
.+-.0.8.degree. C., .+-.0.7.degree. C..+-.0.6.degree. C.,
.+-.0.5.degree. C., .+-.0.4.degree. C., .+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or
35.1.+-.0.9.degree. C..+-.0.8.degree. C., .+-.0.7.degree.
C..+-.0.6.degree. C., .+-.0.5.degree. C., .+-.0.4.degree. C.,
.+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree. C. or
.+-.0.degree. C.
[0019] In another preferred embodiment, the temperature is set at
36.5.+-.Y .degree. C. Preferably, the temperature is set at
(36.1-36.9) .+-.0; or (36.2-36.8) .+-.0.1.degree. C. or .+-.0; or
(36.3-36.7) .+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0; or
(36.4-36.6) .+-.0.3.degree. C., .+-.0.2.degree. C., .+-.0.1.degree.
C. or .+-.0; or 36.5.+-.0.4.degree. C..+-.0.3.degree. C.,
.+-.0.2.degree. C., .+-.0.1.degree. C. or .+-.0.
[0020] In the process of the second aspect of the invention, the
cell culture supernatant is maintained at a pH that is set at
X.+-.0.05 wherein X has a value of from 7.15 to 7.20, with the
proviso that the pH is set at greater than 7.10. In preferred
embodiments, the pH is set at 7.20.+-.0.05, preferably
7.20.+-.0.03, more preferably 7.20.+-.0.01 and most preferably at
7.20; or 7.15.+-.0.05, preferably 7.15.+-.0.03, more preferably
7.15.+-.0.01 and most preferably at 7.15. In a conventional process
for producing a recombinant protein, the cell culture supernatant
is maintained at pH 7.1. Surprisingly, the applicant has found that
culturing the mammalian cells at a higher pH, such as pH 7.2
increases the yield of recombinant protein.
[0021] By "culturing at" or "maintaining at" a pH, we refer to the
pH to which the process control systems are set, in other words the
intended, target, pH. Where we refer to "culturing at" or
"maintaining at" a pH that is set at X.+-.Y, we mean that the set
point is at a value of from X+Y to X-Y. For each of the preferred
values of X, the set-point is at a value within the range
X.+-.0.05, .+-.0.04, .+-.0.03, .+-.0.02 or .+-.0.01. Narrower
ranges are preferred. A set-point of X is most preferred.
[0022] For any given set-point, slight variations in pH may occur.
Typically, such variation may occur because means which control pH,
for example by adding acid or base, or changing the sparge rate,
are only activated after the pH has deviated somewhat from the
set-point. Typically, the pH is controlled to the nearest .+-.0.05,
.+-.0.04, .+-.0.03, .+-.0.02 or .+-.0.01 units of pH.
[0023] Where the pH set-point is set at a value within the range
X.+-.Y, and the tolerance is .+-.Z, this can also be expressed as a
set-point of (X-Y to X+Y) .+-.Z. For each possible value of X, all
combinations of .+-.Y and .+-.Z, as indicated above, are envisaged,
with the proviso that the pH is set at greater than 7.10.
[0024] In one preferred embodiment, the pH is set at 7.20.+-.Y.
Preferably, the pH is set at (7.15-7.25) .+-.0; or (7.16-7.24)
.+-.0.1 or .+-.0; or (7.17-7.23) .+-.0.2, .+-.0.1 or .+-.0; or
(7.18-7.22) .+-.0.3, .+-.0.2, .+-.0.1 or .+-.0; or (7.19-7.21)
.+-.0.4, .+-.0.3, .+-.0.2, .+-.0.1 or .+-.0; or 7.20.+-.0.5,
.+-.0.4, .+-.0.3, .+-.0.2, .+-.0.1 or .+-.0.
[0025] In another preferred embodiment, the pH is set at 7.15.+-.Y.
Preferably, the pH is set at (7.11-7.19) .+-.0; or (7.12-7.18)
.+-.0.1 or .+-.0; or (7.13-7.17) .+-.0.2, .+-.0.1 or .+-.0; or
(7.14-7.16) .+-.0.3, .+-.0.2, .+-.0.1 or .+-.0; or 7.15.+-.0.4,
.+-.0.3, .+-.0.2, .+-.0.1 or .+-.0.
[0026] In the process of the third aspect of the invention, the
cell culture supernatant has a CO.sub.2 concentration of 1 to 10%,
for example 4.0-9.0%, 5.5-8.5% or about 6-8%. Conventionally,
CO.sub.2 concentration is higher than this due to the CO.sub.2
produced by the cells not being removed from the cell culture
supernatant. Surprisingly, the applicant has found that maintaining
the CO.sub.2 concentration at 10% or lower increases the yield of
recombinant protein. It helps the dCO.sub.2 to be kept low if the
feed medium is degassed (for example by bubbling air through it) as
well as the cell culture supernatant in the bioreactor being
sparged.
[0027] Preferably, the process of each of the first three aspects
of the invention is operated to include the particular feature
specified in relation to the process of one or more of the other
aspects of the invention. In other words, where the temperature is
maintained at X.+-.0.9.degree. C., wherein X has a value of from
35.1 to 36.5.degree. C., it is advantageous to also maintain the pH
at X.+-.0.05 wherein X has a value of from 7.15 to 7.20, and/or the
CO.sub.2 concentration at 10% or lower. Where the pH is maintained
at X.+-.0.05 wherein X has a value of from 7.15 to 7.20, it is
advantageous also to maintain the temperature at X.+-.0.9.degree.
C., wherein X has a value of from 35.1 to 36.5.degree. C., and/or
the CO.sub.2 concentration at 10% or lower. Where the CO.sub.2
concentration is maintained at 10% or lower, it is advantageous
also to maintain the pH at X.+-.0.05 wherein X has a value of from
7.15 to 7.20, and/or the temperature at X.+-.0.9.degree. C.,
wherein X has a value of from 35.1 to 36.5.degree. C.
[0028] Ways of monitoring the three defined parameters
(temperature, pH and CO.sub.2 concentration) are well known in this
art and generally rely on probes that are inserted into the
bioreactor, or included in loops through which the culture medium
is circulated, or inserted into extracted samples of culture
medium. A suitable in-line dCO.sub.2 sensor and its use are
described in Pattison et al (2000) Biotechnol. Prog. 16:769-774. A
suitable in-line pH sensor is Mettler Toledo InPro 3100/125/Pt100
(Mettler-Toledo Ingold, Inc., Bedford, Mass.). A suitable off-line
system for measuring dCO.sub.2, in addition to pH and pO.sub.2 is
the BioProfile pHOx (Nova Biomedical Corporation, Waltham Mass.).
In this system, dCO.sub.2 is measured by potentiometric electrodes
within the range 3-200 mmHg with an imprecision resolution of 5%.
pH may be measured in this system at a temperature of 37.degree.
C., which is close to the temperature of the cell culture
supernatant in the bioreactor. Ways of altering the specified
parameter in order to keep it at the predefined level are also well
known. For example, keeping the temperature constant usually
involves heating or cooling the bioreactor or the feed medium (if
it is a fed-batch or continuous process); keeping the pH constant
usually involves choosing and supplying enough of an appropriate
buffer (typically bicarbonate) and adding acid, such as
hydrochloric acid, or alkali, such as sodium hydroxide, sodium
bicarbonate or a mixture thereof, to the feed medium as necessary;
and keeping the CO.sub.2 concentration constant usually involves
adjusting the sparging rate (see further below), or regulating the
flow of CO.sub.2 in the head space. It is possible that the
calibration of an in-line pH probe may drift over time, such as
over periods of days or weeks, during which the cells are cultured.
In that event, it may be beneficial to reset the in-line probe by
using measurements obtained from a recently calibrated off-line
probe. A suitable off-line probe is the BioProfile pHOx (Nova
Biomedical Corporation, Waltham Mass.).
[0029] The inventors have found that increasing pH (e.g. by adding
NaOH) is not enough on its own to achieve the maximum benefit in
terms of the production of active protein. Instead, it is desirable
to reduce the CO.sub.2 concentration. Normally, one would keep the
other parameters of the process constant. However, the inventors
have found that it is advantageous to reduce the CO.sub.2
concentration but to allow the pH to rise from 7.1, for example to
7.15 or 7.2, preferably without adding NaOH.
[0030] Mammalian cell cultures need oxygen for the cells to grow.
Normally, this is provided by forcing oxygen into the culture
through injection ports. It is also necessary to remove the
CO.sub.2 that accumulates due to the respiration of the cells. This
is achieved by `sparging`, i.e., passing a gas through the
bioreactor in order to entrain and flush out the CO.sub.2.
Conventionally, this can also be done using oxygen. However, the
inventors have found that it is advantageous to use air instead. It
has been found that usually a conventional inert gas such as
nitrogen is less effective at sparging CO.sub.2 than using air.
Given that air is about 20% oxygen, one might have thought that
five times as much air would be used. However, this has been found
to be inadequate in large scale cultures, particularly in cultures
at 2500 L scale. In a 2500 L bioreactor, 7 to 10 times as much air,
preferably about 9 times as much air, is used. For example, under
standard conditions, the 2500 L bioreactor is sparged with O.sub.2
at a 10 .mu.m bubble size at a rate of 0.02 VVH (volume O.sub.2 per
volume of culture per hour). The same 2500 L bioreactor used
according to the method of the invention would be sparged with air
at a 10 .mu.m bubble size at a rate of 0.18 VVH.
[0031] Hence, the use of surprisingly high volumes of air has been
found to provide adequate oxygen supply and to remove the unwanted
CO.sub.2.
[0032] During production phase, it is preferred to remove CO.sub.2
by air sparging, as described above. This is especially the case
when using bioreactors of large capacity, in which the cell culture
supernatant would otherwise accumulate CO.sub.2 to deleteriously
high levels. However, at the beginning of culture, or in small
scale culture, such as at 1 L or 2.5 L scale, the head space may be
overlayed with CO.sub.2. Under such conditions, low levels of
dCO.sub.2 may still be achieved. Overlaying the headspace with
CO.sub.2 may also be used to reduce the pH to the set-point, if the
pH is too basic.
[0033] The cells may be any mammalian cell that can be cultured,
preferably in a manufacturing process (i.e. at least 1 litre), to
produce a desired protein such as FVIII. Examples include the
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);
human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension culture, Graham et al., J. Gen Virol., 36:59 [1977]);
baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR, such as the DUKX-B11 subclone (CHO, Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA, 77:4216 [1980]); mouse sertoli cells
(TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey kidney
cells (CV1 ATCC CCL 70); African green monkey kidney cells
(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HeLa,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor
(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y.
Acad. Sci., 383:44-68 [1982]); MRC 5 cells; FS4 cells; and the
human hepatoma line (Hep G2). Preferably, the cell line is a rodent
cell line, especially a hamster cell line such as CHO or BHK.
[0034] A preferred method of preparing stable CHO cell clones
expressing a recombinant protein is as follows. A DHFR deficient
CHO cell line DUKX-BII is transfected with a DHFR expression vector
to allow for expression of the relevant recombinant protein,
essentially as described in U.S. Pat. No. 5,250,421 (Kaufman et al,
Genetics Institute, Inc.) Transfection is selected for with
methotrexate. Amplification of the relevant region coding for
expression of the recombinant protein and DHFR gene is achieved by
propagation of the cells in increasing concentrations of
methotrexate. Where appropriate, CHO cell lines may be adapted for
growth in serum and/or protein free medium, essentially as
described in U.S. Pat. No. 6,100,061 (Reiter et al, Immuno
Aktiengesellschaft)
[0035] The basal medium chosen for culturing the host cell line is
not critical to the present invention and may be any one of, or
combination of, those known to the art which are suitable for
culturing mammalian cells. Media such as Dulbecco's Modified Eagle
Medium, Ham's F-12 Medium, Eagle's Minimal Essential Medium and
RPMI-1640 Medium and the like are commercially available. The
addition of growth factors such as recombinant insulin is
optional.
[0036] Historically, animal cells have been cultured in media
containing animal serum. However, such media are incompletely
defined and carry the risk of infection. Those in the art have
therefore devised "protein-free" media that are either completely
free of any protein or at least are free of any protein that is not
recombinantly produced. Due to the labile nature of Factor VIII,
the productivity of the engineered host cells is severely reduced
under protein-free conditions. Human serum albumin is commonly used
as a serum-free culture supplement for the production of
recombinant proteins. The albumin itself stabilizes the FVIII and
the impurities present in serum-derived albumin preparations may
also contribute to the stabilizing effect of albumin. Factors such
as lipoprotein have been identified as a replacement for human
serum albumin for the production of recombinant Factor VIII under
serum-free conditions.
[0037] Preferred media include those disclosed in U.S. Pat. No.
6,171,825 (Bayer, Inc) and U.S. Pat. No. 6,936,441 (Baxter AG).
[0038] The medium of U.S. Pat. No. 6,171,825 consists of modified
Dulbecco's Minimum Essential Medium and Ham's F-12 Medium (50:50,
by weight) supplemented with recombinant insulin, iron, a polyol,
copper and optionally other trace metals.
[0039] The insulin should be recombinant and can be obtained as
`Nucellin` insulin from Eli Lilly). It can be added at 0.1 to 20
.mu.g/ml (preferably 5-15 .mu.g/ml, or about 10 .mu.g/ml). The iron
is preferably in the form of Fe.sup.2+ ions, for example provided
as FeSO.sub.4.EDTA, and can be present at 5-100 .mu.M (preferably
about 50 .mu.m). Suitable polyols include non-ionic block
copolymers of poly(oxyethylene) and poly(oxypropylene) having
molecular weights ranging from about 1000 to about 16,000. A
particularly preferred polyol is Pluronic F-68 (BASF Wyandotte),
which has an average molecular weight of 8400 and consists of a
centre block of poly(oxypropylene) (20% by weight) and blocks of
poly(oxyethylene) at both ends. It is also available as Synperonic
F-68 from Unichema Chemie BV. Others include Pluronics F-61, F-71
and F-108. Copper (Cu.sup.2+) may be added in an amount equivalent
to 50-800 nM CuSO.sub.4, preferably 100-400 nM, conveniently about
250 nM. The inclusion of a panel of trace metals such as manganese,
molybdenum, silicon, lithium and chromium can lead to further
increases in Factor VIII production. BHK cells grow well in this
protein-free basal medium.
[0040] The medium of U.S. Pat. No. 6,936,441 is also based on a
50/50 mixture of DMEM and Ham's F12 but includes soybean peptone or
yeast extract at between 0.1 and 100 g/l, preferably between 1 and
5 g/l. As a particularly preferred embodiment, soybean extract,
e.g. soybean peptone, may be used. The molecular weight of the
soybean peptone can be less than 50 kD, preferably less than 10 kD.
The addition of ultrafiltered soybean peptone having an average
molecular weight of 350 Dalton has proven particularly advantageous
for the productivity of the recombinant cell lines. It is a soybean
isolate having a total nitrogen content of about 9.5% and a free
amino acid content of about 13%, or about 7-10%.
[0041] A particularly preferred medium has the following
composition: synthetic minimum medium (e.g. 50/50 DMEM/Ham's F12) 1
to 25 g/l; soybean peptone 0.5 to 50 g/l; L-glutamine 0.05 to 1
g/l; NaHCO.sub.3 0.1 to 10 g/l; ascorbic acid 0.0005 to 0.05 g/l;
ethanolamine 0.0005 to 0.05; and sodium selenite 1 to 15 .mu.g/l.
Optionally, a non-ionic surface-active agent such as a
polypropylene glycol (e.g. Pluronic F-61, Pluronic F-68, Pluronic
F-71 or Pluronic F-108) maybe added to the medium as a defoaming
agent. This agent is generally applied to protect the cells from
the negative effects of aeration ("sparging"), since without the
addition of a surface-active agent the rising and bursting air
bubbles may damage those cells that are at the surface of the air
bubbles.
[0042] The amount of non-ionic surface-active agent may range
between 0.05 and 10 g/l, preferably between 0.1 and 5 g/l.
Furthermore, the medium may also contain cyclodextrine or a
derivative thereof. Preferably, the serum- and protein-free medium
contains a protease inhibitor, such as a serine protease inhibitor,
which is suitable for tissue culture and which is of synthetic or
vegetable origin.
[0043] In another preferred embodiment the following amino acid
mixture is additionally added to the above-mentioned medium:
L-asparagine (0.001 to 1 g/l; preferably 0.01 to 0.05 g/l;
particularly preferably 0.015 to 0.03 g/1), L-cysteine (0.001 to 1
g/l; preferably 0.005 to 0.05 g/l; particularly preferably 0.01 to
0.03 WI), L-cysteine (0.001 to 1 g/l; preferably 0.01 to 0.05 g/l;
particularly preferably 0.015 to 0.03 g/l), L-proline (0.001 to 1.5
g/l; preferably 0.01 to 0.07 g/l; particularly preferably 0.02 to
0.05 g/l), L-tryptophan (0.001 to 1 g/l; preferably 0.01 to 0.05
g/l; particularly preferably 0.015 to 0.03 g/l) and L-glutamine
(0.05 to 10 g/l; preferably 0.1 to 1 g/l). These amino acids may be
added to the medium individually or in combination. The combined
addition of the amino acid mixture containing all of the
above-mentioned amino acids is particularly preferred.
[0044] In a particular embodiment a serum- and protein-free medium
is used additionally containing a combination of the
above-mentioned amino acid mixtures and purified, ultrafiltered
soybean peptone.
[0045] The medium of U.S. Pat. No. 6,936,441 is particularly well
suited to the culturing of CHO cells but may be used with other
cells as well.
[0046] A further suitable medium is the oligopeptide-free medium
disclosed in US 2007/0212770 (Grillberger et al; Baxter
International Inc., Baxter Healthcare S.A.)
[0047] Preferably, the culture medium is buffered by the use of
bicarbonate ions, typically supplied as sodium bicarbonate.
[0048] Suitably, the culture medium has an osmolality of between
210 and 650 mOsm, preferably 270 to 450 mOsm, more preferably 310
to 350 mOsm and most preferably 320 mOsm. Preferably, the
osmolality of the supernatant is maintained within one or more of
these ranges throughout the method of the invention.
[0049] The culture can be any conventional type of culture, such as
batch, fed-batch or continuous, but is preferably fed-batch or
continuous. Suitable continuous cultures included repeated batch,
chemostat, turbidostat or perfusion culture.
[0050] A batch culture starts with all the nutrients and cells that
are needed, and the culture proceeds to completion, i.e. until the
nutrients are exhausted or the culture is stopped for some
reason.
[0051] A fed-batch culture is a batch process in the sense that it
starts with the cells and nutrients but it is then fed with further
nutrients in a controlled way in order to limit the growth of the
cells. The fed-batch strategy is typically used in bio-industrial
processes to reach a high cell density in the bioreactor. The feed
solution is usually highly concentrated to avoid dilution of the
bioreactor. The controlled addition of the nutrient directly
affects the growth rate of the culture and allows one to avoid
overflow metabolism (formation of metabolic by-products) and oxygen
limitation (anaerobiosis). In most cases the growth-limiting
nutrient is glucose which is fed to the culture as a highly
concentrated glucose syrup (for example 600-850 g/l).
[0052] Different strategies can be used to control the growth in a
fed-batch process. For example, any of dissolved oxygen tension
(DOT, pO2), oxygen uptake rate (OUR), glucose concentration,
lactate concentration, pH and ammonia concentration can be used to
monitor and control the culture growth by keeping that parameter
constant. In a continuous culture, nutrients are added and,
typically, medium is extracted in order to remove unwanted
by-products and maintain a steady state. Suitable continuous
culture methods are repeated batch culture, chemostat, turbidostat
and perfusion culture.
[0053] CHO cells, for example, may be cultured in a stirred tank or
an airlift tank that is perfused with a suitable medium at a
perfusion rate of from 2 to 10 volume exchanges per day and at an
oxygen concentration of between 40% and 60%, preferably about 50%.
Moreover, the cells may be cultured by means of the chemostat
method, using the preferred pH value given above, an oxygen
concentration of between 10% and 60% (preferably about 20%) and a
dilution rate D of 0.25 to 1.0, preferably about 0.5.
[0054] In a repeated batch culture, also known as serial
subculture, the cells are placed in a culture medium and grown to a
desired cell density. To avoid the onset of a decline phase and
cell death, the culture is diluted with complete growth medium
before the cells reach their maximum concentration. The amount and
frequency of dilution varies widely and depends on the growth
characteristics of the cell line and convenience of the culture
process. The process can be repeated as many times as required and,
unless cells and medium are discarded at subculture, the volume of
culture will increase stepwise as each dilution is made. The
increasing volume may be handled by having a reactor of sufficient
size to allow dilutions within the vessel or by dividing the
diluted culture into several vessels. The rationale of this type of
culture is to maintain the cells in an exponentially growing state.
Serial subculture is characterised in that the volume of culture is
always increasing stepwise, there can be multiple harvests, the
cells continue to grow and the process can continue for as long as
desired.
[0055] In the chemostat and turbidostat methods, the extracted
medium contains cells. Thus, the cells remaining in the cell
culture vessel must grow to maintain a steady state. In the
chemostat method, the growth rate is typically controlled by
controlling the dilution rate i.e. the rate at which fresh medium
is added. The cells are cultured at a sub-maximal growth rate,
which is achieved by restricting the dilution rate. The growth rate
is typically high. In contrast, in the turbidostat method, the
dilution rate is set to permit the maximum growth rate that the
cells can achieve at the given operating conditions, such as pH and
temperature.
[0056] In a perfusion culture, the extracted medium is depleted of
cells because most of the cells are retained in the culture vessel,
for example by being retained on a membrane through which the
extracted medium flows. However, typically such a membrane does not
retain 100% of cells, and so a proportion are removed when the
medium is extracted. It may not be crucial to operate perfusion
cultures at very high growth rates, as the majority of the cells
are retained in the culture vessel.
[0057] Continuous cultures, particularly repeated batch, chemostat
and turbidostat cultures, are typically operated at high growth
rates. According to common practice, it is typical to seek to
maintain growth rates at maximum, or close to maximum, in an effort
to obtain maximum volumetric productivity. Volumetric productivity
is measured in units of protein quantity or activity per volume of
culture per time interval. Higher cell growth equates to a higher
volume of culture being produced per day and so is conventionally
considered to reflect a higher volumetric productivity. The present
inventors have unexpectedly found that, in certain embodiments,
maximum volumetric productivity is not attained at the maximum
growth rate of the cell. As described in the Examples, a maximum
growth rate of a furin expressing CHO cell clone in chemostat
culture was observed at a temperature of 36.5.degree. C., but the
maximum volumetric productivity was observed at 35.1.degree. C.
Despite the lower harvest volumes obtained, arising from a lower
growth rate at the lower temperature, the amount of recombinant
protein produced was so much greater that the lower temperature
culture was, overall, the more productive.
[0058] Suitably, in any of the first, second or third aspects of
the invention, the cell culture supernatant is maintained at a
temperature that is set at a temperature which is lower than the
temperature at which maximum growth rate is observed by at least
0.5.degree. C., preferably at least 1.0.degree. C. In this
embodiment, it is preferred that the culture is a continuous
culture, particularly a repeated batch, chemostat or turbidostat
culture.
[0059] Mammalian cells such as CHO and BHK cells are generally
cultured as suspension cultures. That is to say, the cells are
suspended in the medium, rather than adhering to a solid support.
The cells may alternatively be immobilized on a carrier, in
particular on a microcarrier. Porous carriers, such as
Cytoline.RTM., Cytopore.RTM. or Cytodex.RTM., may be particularly
suitable.
[0060] The cell density is commonly monitored in cell cultures. In
principle, a high cell density would be considered to be desirable
since, provided that the productivity per cell is maintained, this
should lead to a higher productivity per bioreactor volume.
However, increasing the cell density can actually be harmful to the
cells, and the productivity per cell is reduced. There is therefore
a need to monitor cell density. To date, in mammalian cell culture
processes, this has been done by extracting samples of the culture
and analysing them under a microscope or using a cell counting
device such as the CASY TT device sold by Scharfe System GmbH,
Reutlingen, Germany. We have now found that it is advantageous to
analyse the cell density by means of a suitable probe introduced
into the bioreactor itself (or into a loop through which the medium
and suspended cells are passed and then returned to the
bioreactor). Such probes are available commercially from Aber
Instruments, for example the Biomass Monitor 220, 210 220 or 230.
The cells in the culture act as tiny capacitors under the influence
of an electric field, since the non-conducting cell membrane allows
a build-up of charge. The resulting capacitance can be measured; it
is dependent upon the cell type and is directly proportional to the
concentration of viable cells. A probe of 10 to 25 mm diameter uses
two electrodes to apply a radio frequency field to the biomass and
a second pair of electrodes to measure the resulting capacitance of
the polarized cells. Electronic processing of the resulting signal
produces an output which is an accurate measurement of the
concentration of viable cells. The system is insensitive to cells
with leaky membranes, the medium, gas bubbles and debris.
[0061] Typically, the cell density is from 1.0.times.10.sup.6 to
5.0.times.10.sup.6 cells/ml, suitably 1.0.times.10.sup.6 to
3.5.times.10.sup.6 cells/ml, suitably 1.4.times.10.sup.6 to
2.8.times.10.sup.6 cells/ml, preferably 1.6.times.10.sup.6 to
2.6.times.10.sup.6 cells/ml, most preferably 1.8.times.10.sup.6 to
2.4.times.10.sup.6 cells/ml. Increasing the concentration of cells
toward the higher end of the preferred ranges can improve
volumetric productivity. Nevertheless, ranges of cell density
including any of the above point values as lower or higher ends of
a range are envisaged.
[0062] The culture is typically carried out in a bioreactor, which
is usually a stainless steel, glass or plastic vessel of 1 (one) to
10000 (ten thousand) litres capacity, for example 5, 10, 50, 100,
1000, 2500, 5000 or 8000 litres. The vessel is usually rigid but
flexible plastic bags can be used, particularly for smaller
volumes. These are generally of the `single use` type.
[0063] The heterologous or recombinant protein produced by the
method of any of the first three aspects of the invention is
preferably a blood protein. By "blood protein" we include any
protein that is or may be present in the blood of a human or
animal, including proteins that are engineered for intravenous use.
Suitable blood proteins include serum albumin, coagulation factors
I, II, III, V, VII, VIII, IX, X, XI, XII and XIII, furin, von
Willebrand factor, tissue plasminogen activator, interleukins,
interferons, metalloproteases such as ADAMTS proteases (e.g.
ADAMTS-13), immunoglobulins such as IgG, IgM, IgA or IgE and
immunoglobulin fragments. Suitable antibody or immunoglobulin
fragments include Fab-like molecules (Better et al (1988) Science
240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038);
single-chain Fv (ScFv) molecules where the V.sub.H and V.sub.L
partner domains are linked via a flexible oligopeptide (Bird et al
(1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
USA 85, 5879) and single domain antibodies (dAbs) comprising
isolated V domains (Ward et al (1989) Nature 341, 544).
Immunoglobulins and their fragments may be "humanised". In other
words, variable domains of rodent origin may be fused to constant
domains of human origin such that the resultant antibody retains
the antigenic specificity of the rodent parent antibody (Morrison
et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
[0064] In a preferred embodiment, the cell culture is used to
produce Factor VIII, optionally together with von Willebrand Factor
(vWF). The vWF can be added separately to the culture medium and is
preferably recombinant. Alternatively, the vWF can be co-produced
by including vWF-secreting cells in the culture, as well as the
FVIII-secreting cells. Preferably, however, the FVIII and vWF are
co-expressed, i.e. each cell secretes both FVIII and vWF.
Recombinant vWF can be obtained as in Schlokat, et al. (1995),
"Large Scale Production of Recombinant von Willebrand Factor",
Thrombosis and Haemostasis 78, 1160 or U.S. Pat. No. 6,114,146
(Baxter AG). The latter patent also discloses cells that can be
used to co-produce vWF with FVIII-secreting cells. Cells that
co-express both proteins are disclosed in U.S. Pat. No. 5,250,421
(Genetics Institute) and Kaufman et al (1989) Mol. Cell. Biol. 9,
1233-1242.
[0065] The term Factor VIII is used herein to denote any
polypeptide or complex of polypeptides that has clotting factor
VIII activity. Activated Factor VIII functions as a cofactor in the
conversion of Factor X to Factor Xa by activated Factor IXa in the
presence of phospholipids and calcium ions. Conveniently, the
quantity of active Factor VIII can be estimated from the degree to
which it promotes conversion of Factor X to Factor Xa in a suitable
assay. In a typical assay, Factor Xa hydrolyses a specific
chromogenic substrate, thereby liberating a chromophore, the
quantity of which is determined spectrophotometrically.
Commercially available assay kits include Factor VIII Chromogenic
Assay kit (Dade Behring, Switzerland; U.S. Pat. No. 6,100,050); and
Coatest Factor VIII kit (Chromogenix, Sweden). Factor VIII
concentration in humans is defined as 1 IU/mL blood. The Coatest
Factor VIII kit can determine FVIII activity equivalent to at least
0.01 IU/ml blood. To be considered as a Factor VIII as defined
above, a polypeptide or complex of polypeptides must have at least
1% of the activity of native Factor VIII such that, when present in
blood at the same nanomolar concentration as native Factor VIII,
its activity is detectable by the Coatest Factor VIII assay.
[0066] A suitable FVIII for production by the method of the
invention is native, full length FVIII. Porcine FVIII may be
produced in accordance with the invention but the FVIII is more
preferably human. As an alternative to native FVIII, variants and
analogues can be produced. Many are known in this art, for example
the variants and deletion derivatives described in U.S. Pat. Nos.
5,422,260, 4,749,780, 4,868,112, 4,877,614 and 5,171,844. The term
"deletion derivative of recombinant Factor VIII" is defined as one
or more polypeptide chains having Factor VIII activity, derived
from full-length Factor VIII polypeptide by deleting one or more
amino acids. Preferably, the said deletion derivative is devoid of
most of the B-domain, but retains parts of the amino-terminal and
carboxy-terminal sequences of the B-domain that are essential for
in vivo proteolytic processing of the primary translation product
into two polypeptide chains. The production of such a Factor VIII
deletion derivative, identified as "r-VIII SQ", is described in WO
91/09122. The term "r-VIII SQ" is defined as a polypeptide chain
derived from full-length Factor VIII and lacking amino acids 743
through 1636. Further FVIII variants lacking all or part of the B
domain are described in U.S. Pat. No. 6,358,703.
[0067] Suitable vectors for transforming CHO and 293S cells are
disclosed in U.S. Pat. No. 5,854,021. BHK cells expressing FVIII
may be prepared as disclosed in Wood et al (1984) Nature 312,
330-337 or obtained from the ATCC as culture CRL-8544. CHO cells
expressing B-domain-deleted variants of FVIII are described in Lind
et al (1995) Eur. J. Biochem. 232, 19-27 and in U.S. Pat. No.
5,661,008. Three such cell types were deposited with Deutsche
Sammlung von Mikroorganismen and Zellkulturen as DSM 6415, DSM
6417, and DSM 6416.
[0068] When FVIII and vWF are co-produced, the complex between them
may be purified by centrifuging the medium to remove the cells and
then exposing the resulting liquid to an immobilised solid support
containing an antibody for either FVIII or vWF, or a peptide that
will specifically bind FVIII or vWF, under conditions that will not
cause the complex to dissociate. Suitable methods are taught in
U.S. Pat. No. 6,307,032 (Baxter AG) and U.S. Pat. No. 5,200,510
(ZymoGenetics).
[0069] The FVIII (optionally in complex with vWF) may then be
formulated and used in known ways. For example, FVIII, having been
produced in a culture free of animal proteins, is preferably
formulated in a protein-free composition, as disclosed for example
in U.S. Pat. No. 6,586,573 (Baxter International), WO 94/07510 or
U.S. Pat. No. 6,599,724, and used to treat patients with
haemophilia A.
[0070] Where the method of the invention is used to produce FVIII,
it is preferred that the cell culture supernatant is maintained at
a temperature that is set at 36.+-.0.9.degree. C., preferably
36.+-.0.5.degree. C., more preferably 36.+-.0.2.degree. C. and most
preferably 36.degree. C. and/or the pH is set at 7.20.+-.0.05,
preferably 7.20.+-.0.03, more preferably 7.20.+-.0.01, most
preferably 7.20; and/or the cell culture supernatant has a
dissolved CO.sub.2 concentration of 1 to 10%, preferably 4.0 to
9.0%, more preferably 5.5 to 8.5%. Preferably, at least two of
these parameters are within the preferred limits, namely
temperature and pH, temperature and dCO.sub.2 or pH and dCO.sub.2.
Most preferably, all three parameters are operated within the
preferred limits.
[0071] As shown in the Examples, it is advantageous to include
copper in the cell culture supernatant when the invention is used
to produce FVIII. Typically, cells are cultured in a cell culture
supernatant comprising 4 ppb Cu.sup.2+. Advantageously, the
concentration of concentration of Cu.sup.2+ in the cell culture
supernatant is at least 5 ppb, and preferably at least 7, 10, 15 or
25 ppb.
[0072] In an alternative preferred embodiment, the cell culture is
used to produce ADAMTS-13.
[0073] ADAMTS-13, also known as von Willebrand factor cleaving
protease (VWF-cp) is a member of the metalloprotease family. It has
the ability to metabolize large VWF multimers to smaller forms, by
cleaving the peptide bond between residues Tyr-842 and Met-843 of
VWF. This metalloprotease is activated by Ca.sup.2/Ba.sup.2, and is
not inhibited by inhibitors of serine or cysteine proteases.
Deficient von Willebrand factor (VWF) degradation has been
associated with thrombotic thrombocytopenic purpura (TTP). In
hereditary TTP, ADAMTS-13 is absent or functionally defective,
whereas in the nonfamilial, acquired form of TTP, an autoantibody
inhibiting ADAMTS-13 activity is found transiently in most
patients.
[0074] The cloning and expression of the human ADAMTS-13 gene are
described in Plaimauer et al, 2002, Blood. 15; 100(10):3626-32. The
cloning and expression of the human ADAMTS-13 gene, together with
the complete sequence of the cDNA are also disclosed in US
2005/0266528 A1 (Laemmle et al). A suitable ADAMTS-13 for
production by the method of the invention is native, full length
ADAMTS-13, preferably human ADAMTS-13. As an alternative to native
FVIII, variants and analogues can be produced.
[0075] The term ADAMTS-13 is used herein to denote any polypeptide
or complex of polypeptides that has ADAMTS-13 activity,
particularly the ability to cleave the peptide bond between
residues Tyr-842 and Met-843 of VWF. Conveniently, the quantity of
active ADAMTS-13 may be determined by functional assays, such as
functional assays employing modified von Willebrand factor peptides
as substrate for ADAMTS-13 (Tripodi et al J Thromb Haemost. 2008
September; 6(9):1534-41). A preferred method of determining r-hu
ADAMTS13 activity is disclosed in Gerritsen et al. Assay of von
Willebrand factor (vWF)-cleaving protease based on decreased
collagen binding affinity of degraded vWF: a tool for the diagnosis
of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999;
82:1386-1389. In this assay, 1 U corresponds to the level of
ADAMTS-13 activity in pooled normal human plasma. To be considered
as a ADAMTS-13 as defined above, a polypeptide or complex of
polypeptides must have at least 1% of the activity of native
ADAMTS-13. The quantity of ADAMTS-13 may also be determined by
measurement of ADAMTS-13 antigen, for example using the ELISA
method disclosed in Rieger et al, 2006, Thromb Haemost. 2006
95(2):212-20.
[0076] Proteolytically active recombinant ADAMTS-13 may be prepared
by expression in mammalian cell cultures, as described in Plaimauer
et al, 2002, supra and US 2005/0266528 A1. Methods of recombinant
culture of ADAMTS-13 expressing cells are disclosed in Plaimauer B,
Scheiflinger F. Semin Hematol. 2004 January; 41(1):24-33. Preferred
cell types for the expression of ADAMTS-13 include HEK-293 cells
and CHO cells.
[0077] US 2005/0266528 A1 and Zheng et al, 2001, Blood,
98:1662-1666 disclose methods of purifying ADAMTS-13. Purified
ADAMTS-13 may be formulated according to conventional methods and
used therapeutically, for example to treat TTP.
[0078] Where the method of the invention is used to produce
ADAMTS-13, it is preferred that the cell culture supernatant is
maintained at a temperature that is set at 36.0.+-.0.9.degree. C.,
preferably 36.0.+-.0.5.degree. C., more preferably
36.0.+-.0.2.degree. C. and most preferably 36.0.degree. C. and/or
the pH is set at 7.15.+-.0.05, preferably 7.15.+-.0.03, more
preferably 7.15.+-.0.01, most preferably 7.15; and/or the cell
culture supernatant has a dissolved CO.sub.2 concentration of 1 to
10%, preferably 4.0 to 9.0%, more preferably 5.5 to 8.5%.
Preferably, at least two of these parameters are within the
preferred limits, namely temperature and pH, temperature and
dCO.sub.2 or pH and dCO.sub.2. Most preferably, all three
parameters are operated within the preferred limits.
[0079] In an alternative preferred embodiment, the cell culture is
used to produce furin.
[0080] Furin, also termed PACE (paired basic amino acid cleaving
enzyme), belongs to the group of the subtilisin-like serine
proteases, which play an important role in the cleavage of
proproteins, especially in secretory synthesis (Van de Ven et al.,
Crit. Rev. Oncogen., 4:115-136, 1993). It is a calcium-dependent
serine endoprotease structurally arranged into several domains,
namely a signal peptide, propeptide, catalytic domain, homo-B or
P-domain, the C-terminally located cysteine-rich domain,
transmembrane domain and cytoplasmic tail. The protease cleavage
site comprises a recognition sequence which is characterized by the
amino acid sequence Arg-X-Lys/Arg-Arg. The protease furin cleaves
proproteins specifically after this consensus sequence (Hosaka et
al., 1991, J. Biol. Chem. 266:12127-12130).
[0081] Intact furin is incorporated into the membrane system of the
Golgi apparatus and there it is functionally active (Bresnahan et
al, J Cell Biol. 1990; 111:2851-9). Upon transit of the newly
synthesized furin precursor from the endoplasmic reticulum to the
Golgi compartment, the propeptide is autocatalytically removed in a
two step processing event (Anderson et al, EMBO J. 1997; 16:
1508-18). Furin also cycles between the trans-Golgi network and the
cell surface via endosomal vesicles, thereby processing both
precursor proteins during their transport through the constitutive
secretory pathway as well as molecules entering the endocytic
pathway. The cellular distribution of furin to the processing
compartments is directed by defined structural features within its
cytoplasmic tail (Teuchert et al, J Biol Chem. 1999;
274:8199-07).
[0082] Since an overexpression of the native furin protease
negatively affects the growth of continuously growing cell
cultures, solutions have been sought to reduce the toxic influence
of furin on the cells. The C-terminal domains have been found to be
dispensable for the functional activity of furin and a truncated
form of the over-expressed native furin of 75-80 kD could be
detected in the cell supernatant as secreted protein (Wise et al,
PNAS. 1990; 87:9378-82). This naturally secreted truncated furin is
also known as "shed furin" (Vidricaire et al, Biochem Biophys Res
Comm. 1993; 195:1011-8; Plaimauer et al, Biochem J. 2001;
354:689-95) and is cleaved N-terminally of the transmembrane
portion (Vey et al, J Cell Biol. 1994; 127: 1829-42).
[0083] Furin proteins truncated by genetic engineering, in which
the encoding part of the transmembrane and cytoplasmatic domains
has been deleted have been described for example for amino acids
.DELTA.714-794 (Leduc et al, J Biol Chem. 1992; 267: 14304-8;
Molloy et al, J Biol Chem. 1992; 267:16396-402) and for amino acids
.DELTA.716-794 ("Sol-PACE", Wasley et al., J Biol Chem. 1993;
268:8458-65; Rehemtulla and Kaufman, Blood. 1992; 79:2349-55) and
for amino acids .DELTA.705-794 (Hatsuzawa et al, J Biol Chem. 1992;
267:16094-9). Furin mutants additionally comprising a deletion of
the cystein-rich region have also been described (Hatsuzawa et al,
J Biochem. 1992; 101:296-301; Creemers et al, J Biol Chem. 1993;
268:21826-34).
[0084] WO 2008/141824 (Baxter International Inc., Baxter Healthcare
S.A.) discloses a truncated human furin lacking amino acids 578 to
794, i.e. .DELTA.578-794.
[0085] The term "furin" is used herein to denote any polypeptide or
complex of polypeptides that has furin proteolytic activity.
[0086] The evaluation of proteolytic activity of a furin, truncated
furin or furin derivative can be performed by any suitable test,
for example by using fluorogenic substrates which are comprised of
a dibasic cleavage site for which furin is specific (Schlokat et
al, Biotechnol Appl Biochem. 1996; 24:257-67). With said assay 1
Unit is defined as the amount of furin that will release 1 pmol of
7-Amino-4-methylcoumarin (AMC) from the fluorogenic substrate
Boc-Arg-Val-Arg-Arg-AMC in 1 minute at 30.degree. C. The limit of
quantification for this test is typically 0.625 U/mL. Alternatively
the proteolytic activity can also be measured by incubating furin
with pro-proteins, for example pro-rvWF, for a sufficient time. The
degree of pro-rvWF processing can be analyzed for example by
Western blotting. The quantity of furin antigen can be measured by
an ELISA test. A suitable ELISA test is the human furin DuoSet
available from R&D systems, MN (cat. no. DY1503) in which mouse
anti-human furin is used as a capture antibody, and biotinylated
goat anti-human furin is used as a detection antibody.
[0087] A suitable furin for production by the method of the
invention is native, full length furin, preferably human furin. As
an alternative to native furin, variants and analogues can be
produced, including those described above.
[0088] Suitable vectors for transforming mammalian cells,
particularly CHO cells, with furin or variants of furin are
described in WO 2008/141824 (Baxter International Inc., Baxter
Healthcare S.A.), together with methods for purifying the furin so
produced. WO 91/06314 (Holland Biotechnology) describes furin
expression vectors, a method of expressing furin in mammalian
cells, particularly COS-1 cells, and the purification of
recombinantly produced furin. WO 92/09698 (Genetics Institute and
Chiron Corp) describes the expression of furin in CHO cells, either
alone, or in combination with vWF or Factor IX.
[0089] Pro-rVWF is processed to its mature form during cell culture
by endogenously produced furin, which is expressed at relatively
low levels in many cell types (Wise et al, 1990, PNAS
87:9378-9382). Pro-rVWF processing can be made more efficient by
coexpressing heterologous furin with the Pro-rVWF. Alternatively,
WO 2008/141824 suggests that a purified furin may be suitable for
use as a reagent to promote processing of rVWF.
[0090] Where the method of the invention is used to produce furin,
it is preferred that the cell culture supernatant is maintained at
a temperature that is set at 35.1.+-.0.9.degree. C., preferably
35.1.+-.0.5.degree. C., more preferably 35.1.+-.0.2.degree. C. and
most preferably 35.1.degree. C. and/or the pH is set at
7.15.+-.0.05, preferably 7.15.+-.0.03, more preferably
7.15.+-.0.01, most preferably 7.15; and/or the cell culture
supernatant has a dissolved CO.sub.2 concentration of 1 to 10%,
preferably 4.0 to 9.0%, more preferably 5.5 to 8.5%. Preferably, at
least two of these parameters are within the preferred limits,
namely temperature and pH, temperature and dCO.sub.2 or pH and
dCO.sub.2. Most preferably, all three parameters are operated
within the preferred limits.
[0091] In an alternative preferred embodiment, the cell culture is
used to produce Factor VII.
[0092] "Factor VII polypeptide" encompasses wild-type Factor VII
(i.e. a polypeptide having the amino acid sequence disclosed in
U.S. Pat. No. 4,784,950), as well as variants of Factor VII
exhibiting substantially the same or improved biological activity
relative to wild-type Factor VII, and Factor VII variants having
substantially modified or reduced biological activity relative to
wild-type Factor VII. The term "Factor VII" is intended to
encompass Factor VII polypeptides in their uncleaved (zymogen)
form, as well as those that have been proteolytically processed to
yield their respective bioactive forms, which may be designated
Factor VIIa. Typically, Factor VII is cleaved between residues 152
and 153 to yield Factor VIIa. The term "Factor VII polypeptide"
also encompasses polypeptides, including variants, in which the
Factor VIIa biological activity has been substantially modified or
somewhat reduced relative to the activity of wild-type Factor VIIa.
These polypeptides include, without limitation, Factor VII or
Factor VIIa into which specific amino acid sequence alterations
have been introduced that modify or disrupt the bioactivity of the
polypeptide.
[0093] The biological activity of Factor VIIa in blood clotting
derives from its ability to (i) bind to Tissue Factor (TF) and (ii)
catalyze the proteolytic cleavage of Factor IX or Factor X to
produce activated Factor IX or X (Factor IXa or Xa, respectively).
Biological activity of Factor VII polypeptides ("Factor VII
biological activity") may be quantified by measuring the ability of
a preparation to promote blood clotting using Factor VII-deficient
plasma and thromboplastin, as described, e.g., in U.S. Pat. No.
5,997,864 or WO 92/15686. In this assay, biological activity is
expressed as the reduction in clotting time relative to a control
sample and is converted to "Factor VII units" by comparison with a
pooled human serum standard containing 1 unit/mL Factor VII
activity. Alternatively, Factor VIIa biological activity may be
quantified by (i) measuring the ability of Factor VIIa (or the
Factor VII polypeptide) to produce activated Factor X (Factor Xa)
in a system comprising TF embedded in a lipid membrane and Factor X
(Persson et al., J. Biol. Chem. 272:19919-19924, 1997); (ii)
measuring Factor X hydrolysis in an aqueous system; (iii) measuring
the physical binding of Factor VIIa (or the Factor VII polypeptide)
to TF using an instrument based on surface plasmon resonance
(Persson, FEBS Letts. 413:359-363, 1997); (iv) measuring in vitro
hydrolysis of a synthetic substrate by Factor VIIa (or a Factor VII
polypeptide); or (v) measuring generation of thrombin in a
TF-independent in vitro system. Alternatively, FVII antigen may be
determined by ELISA. A suitable ELISA is the AssayMax Human Factor
VII (FVII) ELISA Kit available from Assay Pro (St Charles, Mo.)
Cat. no EF1007-1, which uses a monoclonal anti-human FVII as
capture antibody, and a biotinylated polyclonal anti-human FVII as
detection antibody.
[0094] A preferred in vitro proteolysis assay for native
(wild-type) factor VIIa and/or factor VIIa variant is carried out
in a microtiter plate (MaxiSorp, Nunc, Denmark), as described in US
2007/0219135 (Novo Nordisk HealthCare A/G). Factor VIIa (10 nM) and
factor X (0.8 microM) in 100 microL 50 mM Hepes, pH 7.4, containing
0.1 M NaCl, 5 mM CaCl.sub.2 and 1 mg/ml bovine serum albumin, are
incubated for 15 min. Factor X cleavage is then stopped by the
addition of 50 microL 50 mM Hepes, pH 7.4, containing 0.1 M NaCl,
20 mM EDTA and 1 mg/ml bovine serum albumin. The amount of factor
Xa generated is measured by addition of the chromogenic substrate
Z-D-Arg-Gly-Arg-p-nitroanilide (S-2765, Chromogenix, Sweden), final
concentration 0.5 mM. The absorbance at 405 nm is measured
continuously in a SpectraMax.TM. 340 plate. The absorbance
developed during 10 minutes, after subtraction of the absorbance in
a blank well containing no FVIIa, may be used to calculate the
ratio between the proteolytic activities of variant and wild-type
factor VIIa:
Ratio=(A.sub.405 nm factor VIIa variant)/(A.sub.405 nm factor VIIa
wild-type).
[0095] In a variation of this assay, FVII is determined. A
thromboplastin is included. FVII in the sample forms a complex with
Ca2+ ions and tissue factor which generates small amounts of FXa.
The FXa activates FVII to FVIIa.
[0096] A commercially available FVII activity assay is the HEMOCLOT
FVII reagent kit, available from Aniara (Mason, Ohio) Cat. no.
ACK081K in which clotting triggered by a calcium thromboplastin is
measured.
[0097] Factor VII variants having substantially the same or
improved biological activity relative to wild-type Factor VIIa
encompass those that exhibit at least about 25%, preferably at
least about 50%, more preferably at least about 75% and most
preferably at least about 90% of the specific activity of Factor
VIIa that has been produced in the same cell type, when tested in
one or more of a clotting assay, proteolysis assay, or TF binding
assay as described above. Factor VII variants having substantially
reduced biological activity relative to wild-type Factor VIIa are
those that exhibit less than about 25%, preferably less than about
10%, more preferably less than about 5% and most preferably less
than about 1% of the specific activity of wild-type Factor VIIa
that has been produced in the same cell type when tested in one or
more of a clotting assay, proteolysis assay, or TF binding assay as
described above. Factor VII variants having a substantially
modified biological activity relative to wild-type Factor VII
include, without limitation, Factor VII variants that exhibit
TF-independent Factor X proteolytic activity and those that bind TF
but do not cleave Factor X. Variants of Factor VII, whether
exhibiting substantially he same or better bioactivity than
wild-type Factor VII, or, alternatively, exhibiting substantially
modified or reduced bioactivity relative to wild-type Factor VII,
include, without limitation, polypeptides having an amino acid
sequence that differs from the sequence of wild-type Factor VII by
insertion, deletion, or substitution of one or more amino
acids.
[0098] Non-limiting examples of Factor VII variants having
substantially the same biological activity as wild-type Factor VII
include S52A-FVIIa, S60A-FVIIa (Lino et al., Arch. Biochem.
Biophys. 352: 182-192, 1998); Factor VIIa variants exhibiting
increased proteolytic stability as disclosed in U.S. Pat. No.
5,580,560; Factor VIIa that has been proteolytically cleaved
between residues 290 and 291 or between residues 315 and 316
(Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995); oxidized
forms of Factor VIIa (Komfelt et al., Arch. Biochem. Biophys.
363:43-54, 1999); Factor VII variants as disclosed in
PCT/DK02/00189; and Factor VII variants exhibiting increased
proteolytic stability as disclosed in WO 02/38162 (Scripps Research
Institute); Factor VII variants having a modified GIa-domain and
exhibiting an enhanced membrane binding as disclosed in WO 99/20767
(University of Minnesota); and Factor VII variants as disclosed in
WO 01/58935 (Maxygen ApS).
[0099] Non-limiting examples of Factor VII variants having
increased biological activity compared to wild-type Factor VIIa
include Factor VII variants as disclosed in WO 01/83725, WO
02/22776, WO 02/077218, WO 03/27147, WO 03/37932; WO 02/38162
(Scripps Research Institute); and Factor VIIa variants with
enhanced activity as disclosed in JP 2001061479
(Chemo-Sero-Therapeutic Res Inst.). Non-limiting examples of Factor
VII variants having substantially reduced or modified biological
activity relative to wild-type Factor VII include RI 52E-FVIIa
(Wild-goose et al., Biochem 29:3413-3420, 1990), S344A-FVIIa
(Kazama et al, J. Biol. Chem. 270:66-72, 1995), FFR-FVIIa (Hoist et
al, Eur. J. Vase. Endovasc. Surg. 15:515-520, 1998), and Factor
VIIa lacking the Gla domain, (Nicolaisen et al, FEBS Letts.
317:245-249, 1993).
[0100] Following production of FVII, the polypeptide may be
purified from the medium Purification of Factor VII polypeptides
may involve, e.g., affinity chromatography on an anti-Factor VII
antibody column (see, e.g., Wakabayashi et al., J. Biol. Chem.
261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988) and
activation to by proteolytic cleavage, using Factor XIIa or other
proteases having trypsin-like specificity, such as, e.g., Factor
IXa, kallikrein, Factor Xa, and thrombin. See, e.g., Osterud et
al., Biochem. 11:2853 (1972); Thomas, U.S. Pat. No. 4,456,591; and
Hedner et al., J. Clin. Invest. 71:1836 (1983). Alternatively,
Factor VII may be activated by passing it through an ion-exchange
chromatography column, such as Mono Q.RTM. (Pharmacia) or the
like.
[0101] Factor VII or activated Factor VII may be formulated and
used it known ways. For example, it may be used in treatment of
bleeding in hemophiliacs.
[0102] Where the method of the invention is used to produce Factor
VII, it is preferred that the cell culture supernatant is
maintained at a temperature that is set at 36.5.+-.0.9.degree. C.,
preferably 36.5.+-.0.5.degree. C., more preferably
36.5.+-.0.2.degree. C. and most preferably 36.5.degree. C. and/or
the pH is set at 7.20.+-.0.05, preferably 7.20.+-.0.03, more
preferably 7.29.+-.0.01, most preferably 7.29; and/or the cell
culture supernatant has a dissolved CO.sub.2 concentration of 1 to
10%, preferably 4.0 to 9.0%, more preferably 5.5 to 8.5%.
Preferably, at least two of these parameters are within the
preferred limits, namely temperature and pH, temperature and
dCO.sub.2 or pH and dCO.sub.2. Most preferably, all three
parameters are operated within the preferred limits.
[0103] The present invention will be further illustrated in the
following examples, without any limitation thereto.
Example 1
Basic Cell Culture
FVIII Production
[0104] Typical cultures are established in bioreactors, using a
subclone of the 10A106 CHO cell line transformed to co-express
Factor VIII and von Willebrand Factor described in Kaufman et al
(1989) Mol. Cell. Biol. 9:1233-1242 and U.S. Pat. No. 5,250,421.
The particular subclone was obtained by adaptation to a standard
medium that does not contain animal-derived products, and
subcloning in a microplate.
[0105] The standard culture medium is:
TABLE-US-00001 DMEM/Ham's F12 50/50 11.76 g/kg to which was added:
L-glutamine 0.6 g/kg Ethanolamine 1.53 mg/kg Synperonic F68 0.25
g/kg NaHCO.sub.3 2 g/kg Soya peptone 4 g/kg
CuSO.sub.4.cndot.5H.sub.2O 17.02 mg/kg
[0106] The basal DM EM/Ham's F12 50/50 medium contains 1.3 mg/kg
Cu.sup.2+ sufficient for the complete medium to contain 4.3 ppb
Cu.sup.2+.
ADAMTS-13 Production
[0107] CHO DUKX-B11 cells were transfected using the calcium
phosphate coprecipitation method, to introduce the ADAMTS-13 gene.
Cells were cultured under neomycin selection conditions and were
selected after methotrexate and G418 treatment. After serum-free
adaptation, cells were subcloned and subclone 640-2 was chosen as
production clone.
[0108] The standard culture medium is a serum-, insulin- and
oligopeptide-free medium based on the medium disclosed in US
2007/0212770 (Grillberger et al; Baxter International Inc., Baxter
Healthcare S.A.)
Furin Production
[0109] CHO DUKX-B11 cells were transfected using the calcium
phosphate coprecipitation method, to introduce the furin gene.
Cells were selected with DHFR medium without hypoxanthine,
thymidine and glycine. Production clone 488-3 was identified by
subcloning and selection in medium containing 100 nM methotrexate,
followed by serum-free adaptation.
[0110] The standard culture medium is a serum-, insulin- and
oligopeptide-free medium based on the medium disclosed in US
2007/0212770 (Grillberger et al; Baxter International Inc., Baxter
Healthcare S.A.)
[0111] FVII Production
[0112] CHO DUKX-B11 cells were transfected with a bicistronic
vector to allow coexpression of FVII and VKORC (vitamin K epoxide
reductase complex). Gene expression is driven by the CMV promoter,
and an internal ribosomal entry site (IRES) is located between the
FVII gene and the VKORC gene Cells were transfected using the
calcium phosphate precipitation method. The selection medium
contained 200 .mu.g/ml hygromycin B. Cells were subcloned under
serum-free conditions and a high expressing subclone 1 E9 was
chosen as production clone. 1E9 was selected as having advantageous
properties with regard to growth, productivity and stability under
continuous culture conditions. Stability was evaluated for a period
of two months in chemostat mode.
[0113] The standard culture medium is based on the culture medium
disclosed in U.S. Pat. No. 6,936,441 (Baxter AG), and contains,
inter alia, 2.5 g/L soy peptone and 5 mg/L insulin (Nucellin.RTM.;
Eli Lilly or Novolin.RTM., Novo Nordisk).
Standard Processes Used for FVIII Production
5 L Continuous Culture
[0114] Medium is pre-conditioned for several hours in a CO.sub.2
incubator (5-15% CO.sub.2) at 37.degree. C. To establish the
culture, at least one 1 ml vial (10.sup.7 CHO cells/ml) is
defrosted and the cells diluted in 60 ml pre-conditioned medium in
Roux flasks (200 ml), and cultured in the CO.sub.2 incubator at
37.degree. C. After about 3 days, the 60 ml culture is added to 140
ml of fresh medium in 1 roller bottle (1.8 L). The roller bottle is
sparged with 15% CO.sub.2 and cultured at 37.degree. C. with
rotation. After two days, the cells are split 1/3 and cultured in 2
roller bottles in fresh medium (200 ml medium+100 ml culture=300 ml
per roller bottle). After two or three further days, the cells are
again split 1/3 and cultured in a total of 6 roller bottles in
fresh medium as described above. Once the required cell density of
approximately 1.times.10.sup.6 cells/ml is reached, the 1800 ml
inoculum is inoculated in 3.2 L medium that had been preconditioned
as described above, and cultured in the 5 L bioreactor. Under
standard conditions, the 5 L bioreactor is run at pH 7.2, a cell
density of 1.4.times.10.sup.6 cells/ml and a temperature of
37.degree. C. Under standard conditions, the culture is sparged
with O.sub.2 having a 10 .mu.m bubble size at a rate of 0.25 VVH
(volume of O.sub.2 per volume of culture per hour).
Inoculum Build-Up in the 40 L Bioreactor
[0115] An inoculum pool is obtained essentially as described above
in relation to the 5 L continuous culture. However, the pool is
approximately 5 L, and is obtained from 18 rather than 6 roller
bottles. The BR-40 bioreactor is cleaned and sterilized before the
operation, and approximately 8 L of medium is transferred to BR-40
prior to inoculation. The inoculum pool of approximately 5 L is
transferred from the pooling tank to the bioreactor via a transfer
line to reach a total culture volume of approximately 13 L. Once
the cell density reaches .gtoreq.9.times.10.sup.5 cells/mL, the
culture is diluted (1:3) with media. After 1 to 3 more days, the
cell concentration again reaches .gtoreq.9.times.10.sup.5 cells/mL,
and the transfer of the inoculum to the 320 L bio reactor is
carried out.
Expansion in the 320 L Bioreactor
[0116] The BR-320 bioreactor is cleaned and sterilized before the
operation, and approximately 80 L of medium is transferred to
BR-320 prior to inoculation. The inoculum of approximately 40 L is
transferred from the BR-40 bioreactor to the BR-320 bioreactor via
a transfer line to reach an initial culture volume of approximately
120 L. Once the cell density reaches .gtoreq.9.times.10.sup.5
cells/mL, the culture is diluted (1:3) with medium. After 3 to 6
days (total time), the cell concentration again reaches
.gtoreq.9.times.10.sup.5 cells/mL, and the transfer of the inoculum
to the 2500 L bioreactor is carried out.
2500 L Bioreactor
Build-up
[0117] The inoculum of approximately 320 L is transferred from the
BR-320 to the BR1 bioreactor via a transfer line that already
contained approximately 630 L of medium to reach an initial culture
volume of approximately 950 L. Once the cell density reaches
.gtoreq.9.times.10.sup.5 cells/mL, the culture is diluted
(.about.1:3) with medium to a final volume of approximately 2500 L.
After 4 to 7 days (total time), the cell concentration again
reaches .gtoreq.9.times.10.sup.5 cells/mL, and approximately 1150 L
of the inoculum is transferred from the BR1 to the BR2 bioreactor
that contains approximately 1350 L of medium. After transfer,
approximately 1150 L of medium is added to bioreactor BR1, to reach
a final culture volume of approximately 2500 L.
Chemostat
[0118] The `chemostat` culture mode is started as soon as the cell
concentration in each bioreactor reaches .gtoreq.1.2.times.10.sup.6
cells/mL. Approximately 1250 L of medium per day is added in a
continuous mode to each bioreactor. The cell concentration is
between 9.times.10.sup.5-1.6.times.10.sup.6 cells/mL in each 2500 L
bioreactor. Multiple harvests of approximately 1250
L/day/bioreactor are stored in sterile bags at 2-8.degree. C. The
culture is maintained for about 50-57 days in the chemostat mode.
Under standard conditions, the pH is set to 7.2, the temperature is
set to 37.degree. C. and the culture is sparged with O.sub.2 having
a 10 .mu.m bubble size at a rate of 0.02 VVH (volume of O.sub.2 per
volume of culture per hour).
[0119] Similar cultivation methods are also applicable to the
culture of CHO cells expressing ADAMTS-13, furin or FVII.
Example 2
Effects of Changing Various Parameters on FVIII Productivity
[0120] FVIII productivity of the FVIII- and vWF-expressing CHO cell
clone described in Example 1 was determined under various culture
conditions.
[0121] In separate experiments, the pH, cell density and
temperature were varied in a 5 L scale continuous culture. In each
case, the control experiment used pH 7.1, a cell density of
1.4.times.10.sup.6 cells/ml and 37.degree. C.
[0122] When the cell density was increased, the volumetric FVIII
productivity (IU per litre per day), relative to the value for a
cell density of 1.2.times.10.sup.6 cells/ml, increased as
follows:
TABLE-US-00002 Percentage Productivity Cell density increase in
increase (.times.10.sup.6 cells/ml) cell density (%) 1.2 -- -- 1.4
17 43 1.6 33 51 1.8 50 65 2.0 67 73 2.2 83 74 2.4 100 76 2.6 117
49
[0123] Hence, a substantial increase in productivity could be
achieved by increasing cell density. This could not have been
predicted, since increasing cell density can reduce the
productivity per cell. Moreover, at certain cell densities, the
increase in productivity was found to be greater than the increase
in cell density, which is even more surprising.
[0124] pH was increased to 7.2 by altering the sparge parameters.
In the control vessel at pH 7.1, the culture was sparged with
O.sub.2 having a 10 .mu.m bubble size at a rate of 0.25 VVH (volume
of O.sub.2 per volume of culture per hour). In the test vessel at
pH 7.2 the culture was sparged with air having a 10 .mu.m bubble
size at a rate of 1.25 VVH (volume of air per volume of culture per
hour). By increasing the pH from 7.1 to 7.2, the productivity could
be increased by about 16%.
[0125] By lowering the temperature from 37.degree. C. to 36.degree.
C., the productivity increased by about 22%.
Example 3
Influence of Cell Density and Copper Concentration on FVIII
Productivity
[0126] FVIII productivity of the FVIII- and vWF-expressing CHO cell
clone described in Example 1 was determined under various culture
conditions.
[0127] A control culture was operated at pH 7.1, 37.degree. C., 4
ppb Cu.sup.2+ and a cell density of 1.4.times.10.sup.6
cells/ml.
[0128] Comparative cultures were run at pH 7.2 and 36.degree. C. In
one culture the cell density was increased to 1.6.times.10.sup.6
cells/ml and in another it was increased to 2.0.times.10.sup.6
cells/ml. In a third culture, the cell density was
2.0.times.10.sup.6 cells/ml and the copper concentration was
increased from 4 ppb to 6 ppb.
[0129] Results: by reducing the temperature, increasing the pH and
increasing the cell density to 1.6.times.10.sup.6 cells/ml (an
increase of 14%), the FVIII productivity increased by 41-50%. A
further increase in cell density to 2.0.times.10.sup.6 cells/ml
(43%) gave an increase in productivity (compared to the control
culture) of 39-77%. When the copper in a 2.0.times.10.sup.6
cells/ml culture was increased to 6 ppb, the productivity (compared
to the control culture) was increased by 48-98%.
[0130] Hence, by raising the pH slightly, decreasing the
temperature slightly, increasing the cell density by only 43% and
increasing the copper concentration by 50% the FVIII productivity
can be almost doubled.
Example 4
Influence of Cell Density and Copper Concentration on vWF
Productivity
[0131] Example 3 was repeated but the volumetric vWF productivity
was measured. At 1.6.times.10.sup.6 cells/ml and 4 ppb copper
(36.degree. C., pH7.2) the productivity was 124% of the control. At
2.0.times.10.sup.6 cells/ml and 6 ppb copper the productivity was
182%.
[0132] Hence, again substantial increases in productivity can be
achieved by making seemingly small changes in the process
parameters.
Example 5
Influence of Cell Density, pH and dCO.sub.2
[0133] FVIII productivity of the FVIII- and vWF-expressing CHO cell
clone described in Example 1 was determined under various culture
conditions.
[0134] In this experiment, the cell density was increased from
1.41.times.10.sup.6 cells/ml to 2.03.times.10.sup.6 cells/ml, the
pH was increased from 7.1 to 7.2 and the dCO.sub.2 concentration
was reduced from 9.5% to 6.2%.
[0135] The volumetric FVIII productivity (IU per litre per day)
increased by 98% and the specific FVIII productivity (IU per
million cells per day) increased by 36%.
Example 6
Influence of pH and Temperature on Furin Production
[0136] Furin expressing CHO cells were cultivated in 2.5 L
bioreactors in chemostatic mode. The cell density was maintained at
an average of between 1.52.times.10.sup.6 and 1.78.times.10.sup.6
cells/ml for individual cultures over 5 cultivation days. Dissolved
oxygen was controlled in all experiments at a set-point of 20% air
saturation. Dissolved CO.sub.2 concentration was maintained between
5%-6% by overlaying the headspace of the bioreactors with
CO.sub.2.
[0137] By means of the "design of experiments method", different
temperatures were combined with different pH values to ascertain
the conditions which result in maximum volumetric productivity of
furin. Five temperatures were combined with three pH values
according to the "Doehlert Matrix", resulting in seven combinations
of temperature and pH as follows:
TABLE-US-00003 Fermentation lot Temp (.degree. C.) pH 1 35.1 7.20 2
35.8 7.10 3 35.8 7.30 4 36.5 7.20 5 36.5 7.20 6 37.2 7.10 7 37.2
7.30 8 37.9 7.20
[0138] The combination of 36.5.degree. C. and pH 7.20 was chosen as
the center point, which was applied to two fermentation lots (4 and
5 in the above table).
[0139] The data, including volumetric and specific productivity,
and growth rate, were analyzed statistically with the Response
Surface Methodology (RSM), using the "Minitab" software.
[0140] Temperature, but not pH, significantly influenced growth
rate, with a maximum for the growth rate occurring at 36.5.degree.
C. By decreasing the temperature from 37.degree. C. to 35.1.degree.
C., the volumetric productivity could be raised by approximately
2.7-fold. A similar trend was seen for specific productivity. This
is a surprising result, as it might have been expected that the
maximum volumetric productivity would be observed at the
temperature at which growth rate was maximal. The influence of the
pH for specific and volumetric productivity was minor in the
investigated range of 7.20+/-0.1. A slightly higher productivity
was observed in the lower pH range of 7.15+/-0.05 (or between 7.10
and 7.20), therefore pH 7.15 was selected as set-point for furin
production.
Example 7
Influence of dCO.sub.2 on Furin Production
[0141] Two fermentation runs were carried out in parallel in
chemostat mode in 2.5 L bioreactors, one run with a CO.sub.2
concentration of approximately 7.5% and the other with a CO.sub.2
concentration of approximately 12%. The CO.sub.2 concentration was
adjusted by varying the CO.sub.2 fraction in the head space flow.
The fermentations were carried out at 37.degree. C., at a pH of
7.15 and with a pO.sub.2 of 20%. The cell count was approximately
1.07.times.10.sup.6 cells/ml over 12 days in the high CO.sub.2
culture, and 1.49.times.10.sup.6 cells/ml in the low CO.sub.2
culture.
[0142] Reducing the CO.sub.2 concentration from 12% to 7.5% had the
effect of increasing the volumetric productivity by approximately
2.78-fold and the specific productivity by 2-fold. Cell growth rate
was also higher in the low CO.sub.2 culture.
Example 8
Influence of pH and Temperature on FVII Production
[0143] FVII expressing CHO cells were cultivated in 2.5 L
bioreactors in chemostatic mode, where the cell density was
maintained at an average of about 2.5.times.10.sup.6 cells/ml
(between 2.times.10.sup.6 and 3.times.10.sup.6 cells/ml) for
individual cultures over 4 cultivation weeks. Dissolved oxygen was
controlled in all experiments at a setpoint of 20% air saturation.
Dissolved CO.sub.2 concentration was maintained between 4%-7% by
overlaying the headspace of the bioreactors with CO.sub.2.
[0144] By means of the "design of experiments method", different
temperatures were combined with different pH values to ascertain
the conditions which result in maximum volumetric productivity of
FVII. Three temperatures were combined with three pH values
according to the "Doehlert Matrix", resulting in five combinations
of temperature and pH as follows:
TABLE-US-00004 Fermentation lot Temp (.degree. C.) pH 1 36.0 7.15 2
36.0 7.25 3 36.5 7.20 4 37.0 7.15 5 37.0 7.25
[0145] The mean maximum volumetric kinetic productivity was
achieved at 36.5.degree. C. and a pH setpoint of 7.20. There was a
positive interaction of the parameters, such that the result of
optimising both parameters was greater than the combined effects of
optimising each parameter individually.
Example 9
Influence of dCO.sub.2 on FVII Production
[0146] The effect of four different CO.sub.2 concentrations (5.0,
6.3, 7.6 and 8.9%) on FVII productivity was tested in small scale
continuous culture.
[0147] Cells were cultivated until chemostat day eight, and then
transferred into 2.5 L Rushton Bioreactors and cultivated under
continuous conditions at pH 7.20 and 36.5.degree. C. for almost
four weeks. Data from only the last three weeks were analysed due
to the necessary equilibration to the different CO.sub.2
concentrations during the first week. CO.sub.2 levels were
controlled by off line measurement and addition of CO.sub.2 into
the headspace of the bioreactors.
[0148] The recorded cell densities varied from 2.36.times.10.sup.6
cells/ml at a CO.sub.2 level of 8.9% to 2.87.times.10.sup.6
cells/ml at 5.0%. The growth rates for the same range are 0.42
d.sup.-1 and 0.49 d.sup.-1. Increased specific growth rates at low
CO.sub.2 level correlated with increased specific productivities.
The compound effects of CO.sub.2 on growth rate and specific
productivity resulted in a substantial effect on volumetric
productivity.
[0149] A decrease in the CO.sub.2 concentration from 8.9% to 5%
increases the specific growth rate by 17%, the specific
productivity by 10% and the volumetric kinetic productivity by
35%.
Example 10
Influence of Temperature and pH on ADAMTS-13 Production
[0150] Transfected CHO cells expressing recombinant ADAMTS-13 were
cultivated in chemostat cultures in 1.5 L bioreactors.
[0151] In a first experiment, pH and temperature were set to
different setpoints in the range of 36.degree. C. to 38.degree. C.
and pH 7.10 to 7.30. Samples from the steady state were analyzed
for cell count and ADAMTS-13 expression by ELISA, and dilution
rates from the chemostat cultures were measured to calculate the
growth rate and volumetric ADAMTS-13 expression. Once the optimum
was found to be at the outer range of the design space, a second
experiment was set up with a temperature range from 35.degree. to
37.degree. C. and pH 7.05 to 7.15, and data were analyzed from the
steady state. Cell densities ranged from 1.17-1.71.times.10.sup.6
cells/ml. CO.sub.2 was controlled by overlaying the headspace with
CO.sub.2 to reach a dissolved CO.sub.2 concentration of 4-6%.
[0152] Data from both experiments were normalized and analyzed
using statistical software Minitab.
[0153] Specific growth rate was found to have its optimum at pH
7.13 and 36.0.degree. C. using a quadratic model for pH and
temperature. The effect of temperature on growth rate was weak.
[0154] Volumetric productivity was found to have its optimum at pH
7.15 and 36.0.degree. C. Despite the weak effect of temperature on
growth rate, there was a relatively strong effect of temperature on
volumetric productivity.
[0155] Assuming a constant temperature of 37.degree. C., the effect
of raising pH from 7.10 to 7.15 was to increase volumetric
productivity by 10%. Assuming a constant pH of 7.10, the effect of
decreasing temperature from 37.degree. C. to 36.degree. C. was to
increase volumetric productivity by 14%. The overall effect of
changing conditions from pH 7.10 and temperature of 37.degree. C.
to pH 7.15 and temperature of 36.degree. C. was to increase
volumetric productivity by 24%.
[0156] The contents of all references cited herein are included by
reference.
* * * * *