U.S. patent application number 17/083048 was filed with the patent office on 2021-02-11 for feed mixing device and its use.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Sylvia BAUMANN, Jens HOFFMANN, Alexander JOCKWER, Christian KLINGER, Thomas TROEBS.
Application Number | 20210040528 17/083048 |
Document ID | / |
Family ID | 1000005178472 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040528 |
Kind Code |
A1 |
BAUMANN; Sylvia ; et
al. |
February 11, 2021 |
FEED MIXING DEVICE AND ITS USE
Abstract
Herein is reported a feed mixing device for adding feed
solutions with a non-physiologically pH value to a cell cultivation
vessel comprising a chamber for mixing the feed solutions prior to
their addition to the cell cultivation vessel as well as its use.
With the feed mixing device as reported herein feed components can
be provided in solution at a pH value at which they have good
solubility and/or good stability whereby the pH value can be
clearly different from the pH value of the cultivation medium, i.e.
different from the physiological pH value. This allows performing
the cultivation with more flexibility compared to a cultivation in
which the pH value of the feed solution is limited to a small range
around the pH value of the cultivation.
Inventors: |
BAUMANN; Sylvia;
(Benediktbeuern, DE) ; HOFFMANN; Jens; (Penzberg,
DE) ; JOCKWER; Alexander; (Eurasburg, DE) ;
KLINGER; Christian; (Benediktbeuern, DE) ; TROEBS;
Thomas; (Muenchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000005178472 |
Appl. No.: |
17/083048 |
Filed: |
October 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15713268 |
Sep 22, 2017 |
10837042 |
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17083048 |
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13909591 |
Jun 4, 2013 |
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15713268 |
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PCT/EP2011/071696 |
Dec 5, 2011 |
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13909591 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F 2215/044 20130101;
C12N 2500/60 20130101; C07K 16/244 20130101; C12P 21/005 20130101;
B01F 15/0479 20130101; C12M 29/26 20130101; C12N 5/00 20130101;
C07K 2317/14 20130101; C12M 41/26 20130101 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C07K 16/24 20060101 C07K016/24; C12M 1/00 20060101
C12M001/00; C12M 1/34 20060101 C12M001/34; C12N 5/00 20060101
C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 7, 2010 |
EP |
10194025.2 |
Claims
1-6. (canceled)
7. A cell cultivation apparatus comprising a) a cell cultivation
vessel, b) a gas supply, c) at least two liquid storage units for
feed solutions each with a non-physiological pH value, d) a sensor
for determining the pH value in the cell cultivation vessel, and e)
a device for adding a mixed feed solution to the cell cultivation
vessel, the device comprising separate inlets for the feed
solutions, a chamber for mixing the feed solutions to obtain the
mixed feed solution, and a single outlet for adding the mixed feed
solution to the cell cultivation vessel, whereby the ratio of the
volume of the chamber for mixing the feed solutions to the volume
of cultivation medium in the cultivation vessel is of from 0.8 ml/L
to 1.2 ml/L.
8. Use of a device according to claim or of an apparatus according
to claim 7 in a fed-batch or continuous cultivation of a cell.
9. A method for the production of a polypeptide comprising the
steps of: cultivating a cell comprising a nucleic acid encoding the
polypeptide in a fed-batch or continuous cultivation within a
cultivation vessel, wherein the cultivation vessel comprises a
device for adding at least two feed solutions, wherein the device
comprises separate inlets for the feed solutions, a chamber for
mixing the feed solutions, and a single outlet for adding the mixed
feed solution to the cell cultivation vessel; mixing at least two
feed solutions, wherein at least one feed solution is an acidic
feed solution and has a pH value of less than pH 6.5, and at least
one feed solution is an alkaline feed solution and has a pH value
of more than pH 7.5, in the chamber to produce a mixed feed
solution; adding the mixed feed solution to the cultivation vessel
containing the cultivation medium; further cultivating the cell
comprising the nucleic acid encoding the polypeptide in the
fed-batch or continuous cultivation within the cultivation vessel,
wherein the ratio of the volume of the mixed feed solution in the
chamber to the volume of the cultivation medium in the cultivation
vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the
cultivation medium in the cultivation vessel; and recovering the
polypeptide from the cultivation medium or the cells.
10. The method according to claim 9, wherein any acidic feed
solution has a pH value of pH 6.5 or lower, and/or any alkaline
feed solution has a pH value of pH 8.0 or higher, and the mixed
feed solutions have a pH value of from pH 7 to pH 7.5.
11. The method according to claim 9, wherein the mixing is
immediately prior to the addition to the cultivation vessel.
12. The method according to claim 9, wherein any acidic feed
solution has a pH value of pH 4.5, or lower, and/or any alkaline
feed solution has a pH value of pH 10.0 or higher.
13. The method according to claim 9, wherein from two to four
separate feed solutions are added to the cultivation medium whereof
at least one is an acidic feed solution and at least one is an
alkaline feed solution.
14. The method according to claim 9, characterized in that each of
the alkaline or acidic feed solutions comprises at least a compound
selected from amino acid, sugar, vitamin, trace element, lactate,
and growth factor.
15. A method for obtaining a polypeptide with a reduced G(0)
glycoform and/or increased G(1) glycoform comprising the steps of:
cultivating a cell comprising a nucleic acid encoding the
polypeptide in a fed-batch or continuous cultivation within a
cultivation vessel, wherein the cultivation vessel comprises a
device for adding at least two feed solutions, wherein the device
comprises separate inlets for the feed solutions, a chamber for
mixing the feed solutions, and a single outlet for adding the mixed
feed solution to the cell cultivation vessel; mixing at least two
feed solutions, wherein at least one feed solution is an acidic
feed solution and has a pH value of less than pH 6.5, and at least
one feed solution is an alkaline feed solution and has a pH value
of more than pH 7.5, in the chamber to produce a mixed feed
solution; adding the mixed feed solution to the cultivation vessel
containing the cultivation medium; further cultivating the cell
comprising the nucleic acid encoding the polypeptide in the
fed-batch or continuous cultivation within the cultivation vessel,
wherein the ratio of the volume of the mixed feed solution in the
chamber to the volume of the cultivation medium in the cultivation
vessel is from 0.8-1.2 ml chamber volume per 1 liter volume of the
cultivation medium in the cultivation vessel 0.8 ml/L to 1.2 ml/L;
and recovering the polypeptide from the cultivation medium or the
cells, wherein the feed solution mixture has a pH value upon
addition to the cultivation vessel of from pH 4.0 to pH 6.0.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/909,591, filed Jun. 4, 2013, which is a
continuation of International Application No. PCT/EP2011/071696
having an international filing date of Dec. 5, 2011, the entire
contents of each of which are incorporated herein by reference, and
which claims benefit under 35 U.S.C. .sctn. 119 to European Patent
Application No. 10194025.2 filed Dec. 7, 2010.
TECHNICAL FIELD
[0002] Herein is reported a feed mixing device that allows
simultaneous feeding of two or more feeding solutions with
non-physiological pH value to a cultivation medium whereby the pH
value of the combined feed solutions is adjusted, e.g. to a
physiological pH value, prior to the addition to the cultivation
medium within the feed mixing device.
BACKGROUND OF THE INVENTION
[0003] To increase product yield or cultivation time in the
cultivating of mammalian cells feeding solutions are added to the
cultivation medium to maintain the concentration of essential
medium components at or above a critical level.
[0004] Feeding of aqueous solutions with physical properties
different from the cultivation medium will affect physical
parameters of the cultivation medium like osmolality or pH value.
Due to poor solubility or required stabilization of different
components, the pH value of feeding solutions sometimes has to be
changed to non-physiological values.
[0005] Luan, Y. T., et al., report strategies to extend longevity
of hybridomas in culture and promote yield of monoclonal antibodies
(Biotechnol. Lett. 9 (1987) 691-696). Improved fermentation
processes for NSO cell lines expressing human antibodies and
glutamine synthetase is reported by Dempsey, J., et al.
(Biotechnol. Prog. 19 (2003) 175-178). Bibila, T. A., et al.,
report monoclonal antibody process development using medium
concentrates (Biotechnol. Prog. 10 (1994) 87-96).
[0006] In WO 2008/013809 cell culture methods are reported. Dry
powder cells and cell culture reagents and methods of production
thereof are reported in US 2006/0003448. In U.S. Pat. No. 5,081,036
a method and apparatus for cell culture is reported. Media
concentrate technology is reported in U.S. Pat. No. 5,681,748. In
U.S. Pat. No. 6,924,124 feeding strategies for cell culture are
reported.
[0007] In WO 2009/132616 a supply system is reported. A method and
apparatus for producing alcohol or sugar using a commercial-scale
bioreactor is reported in WO 2010/045168. In US 2003/0092652 a
protected one-vial formulation for nucleic acid molecules, methods
of making the same by in-line mixing, and related products and
methods are reported.
SUMMARY OF THE INVENTION
[0008] With a feed mixing device as reported herein feed components
can be provided e.g. in solutions with a pH value at which the
components have good solubility and/or good stability, whereby the
pH value can be/is different from the pH value of the cultivation
medium, i.e. different from a physiologically acceptable pH value
of pH 6.5 to pH 7.5, i.e. the solutions have independently of each
other a pH value of less than pH 6.5 or more than pH 7.5. This
allows performing a cultivation with more flexibility compared to a
cultivation in which e.g. the pH value of the feed solution is
limited to a small range around the pH value of the cultivation
medium.
[0009] One aspect as reported herein is a device for adding at
least two solutions each with a non-physiological pH value to a
cell cultivation vessel comprising a chamber for mixing the
solutions prior to their addition to the cell cultivation
vessel.
[0010] In one embodiment the ratio of the volume of the chamber for
mixing the solutions to the volume of the cultivation medium in the
cultivation vessel is of from 0.8 ml/I to 1.2 ml/l. In one
embodiment the ratio is of from 0.9 ml/l to 1.1 ml/l. In one
embodiment the ratio is about 1 ml/l. In one embodiment the ratio
is 0.95 ml/l. In one embodiment the volume of the cultivation
medium is the volume of liquid at the start of the cultivation in
the cultivation vessel.
[0011] In one embodiment the at least two solutions each with a
non-physiological pH value are at least one acidic solution and at
least one alkaline solution. In one embodiment the at least two
solutions each with a non-physiological pH value have a pH value
independently of each other of less than pH 6.5 or more than pH
7.5. In one embodiment the at least two solutions each with a
non-physiological pH value have a pH value independently of each of
pH 0 to pH 6.49 or pH 7.51 to pH 14.
[0012] In one embodiment the pH value of the acidic and alkaline
solution differs by at least 0.5 pH units from the pH value of the
cultivation medium. In one embodiment the acidic solution has a pH
value of pH 6.5 or lower. In one embodiment the acidic solution has
a pH value of pH 4.0 or lower. In one embodiment the alkaline
solution has a pH value of pH 8.0 or higher. In one embodiment the
alkaline solution has a pH value of 10.0 or higher.
[0013] In one embodiment the device is for adding of from two to
four separate solutions with non-physiological pH value, whereof
optionally at least one is an acidic solution and at least one is
an alkaline solution.
[0014] In one embodiment each of the solutions is a feed solution
comprising at least one compound selected from amino acid, sugar,
vitamin, trace element, lactate, and growth factor.
[0015] In one embodiment the chamber for mixing the solutions is
separated from the cultivation vessel and comprises an outlet to
the inside of the cultivation vessel. In one embodiment the chamber
is outside of the cultivation vessel or inside the cultivation
vessel.
[0016] In one embodiment the chamber has a volume of from 0.1 ml to
50,000 ml. In one embodiment the chamber has a volume of from 0.25
ml to 30,000 ml. In one embodiment the chamber has a volume of from
0.5 ml to 1,000 ml.
[0017] In one embodiment the chamber has a volume of about 1.15 ml,
or about 8 ml, or about 80 ml, or about 200 ml, or about 400 ml, or
about 800 ml, or about 1.6 l, or about 4 l, or about 8 l, or about
16 l, or about 40 l.
[0018] In one embodiment the mixing is immediately prior to the
addition to the cultivation vessel.
[0019] In one embodiment the chamber for mixing the solutions
comprises an inlet with individual connectors for each of the
solutions.
[0020] In one embodiment the device is sterilizable.
[0021] Another aspect as reported herein is the use of a device as
reported herein in the fed-batch or continuous cultivation of
cells.
[0022] Also an aspect as reported herein is a cultivation vessel
comprising a device as reported herein.
[0023] An aspect as reported herein is a method for the production
of a polypeptide comprising the following steps: [0024] cultivating
a cell comprising a nucleic acid encoding the polypeptide in a
fed-batch or continuous cultivation in a cultivation vessel
comprising a device as reported herein whereby at least one acidic
feed solution and at least one alkaline feed solution are added
during the cultivating, and [0025] recovering the polypeptide from
the cultivation medium or the cells and thereby producing the
polypeptide.
[0026] In one embodiment the mixed feed solutions have a pH value
of from pH 4.5 to pH 9.5 upon addition to the cultivation vessel.
In one embodiment the mixed feed solutions have a pH value of from
pH 6.5 to pH 7.5 upon addition to the cultivation medium.
[0027] In one embodiment the cultivation vessel has a volume of
about 2 l, or 10 l, or 20 l, or 100 l, or 250 l, or 500 l, or 1,000
l, or 2,000 l, or 5,000 l, or 10,000 l, or 20,000 l, or 50,000
l.
[0028] In one embodiment the volume of the cultivation medium is
about 1.2 l, or about 8 l, or about 16 l, or about 80 l, or about
200 l, or about 400 l, or about 800 l, or about 1,600 l, or about
4,000 l, or about 8,000 l, or about 16,000 l, or about 40,000
l.
[0029] In one embodiment the device as reported herein is operated
at room temperature.
[0030] Also an aspect as reported herein is a method for obtaining
a polypeptide with a reduced G(0) glycoform and/or increased G(1)
glycoform comprising the following steps: [0031] cultivating a cell
comprising a nucleic acid encoding the polypeptide in a fed-batch
or continuous cultivation in a cultivation vessel comprising a
device as reported herein whereby at least one acidic feed solution
and at least one alkaline feed solution are added during the
cultivating, and [0032] recovering the polypeptide from the
cultivation medium or the cells and thereby obtaining a polypeptide
with a reduced G(0) glycoform and/or increased G(1) glycoform,
wherein the mixed feed solutions have a pH value upon addition to
the cultivation vessel of from pH 4.0 to pH 6.0.
[0033] In one embodiment the polypeptide is an antibody or an
Fc-fusion polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
[0034] A widely used format for the production of therapeutic
polypeptides or biomass is the fed-batch fermentation. Cell
densities of mammalian cell cultures often exceed 100*10.sup.5
cells/ml in fed-batch fermentations resulting in challenges to
provide sufficient amounts of the required cultivation substrates
due to low solubility and/or impaired stability of certain
substances or substance classes.
[0035] Therefore a common approach is to use feed solutions with
non-physiological pH-values to dissolve or stabilize the required
amounts of these substances. For example, an adequate supply of the
amino acid tyrosine via a liquid feed solution is hardly to achieve
at pH values around pH 7 due to its low solubility. Solubility
however increases at high, non-physiological pH values in this
case, enabling feed strategies matching overall tyrosine
consumption in fed batch processes with feed solutions at
non-physiological pH values. Especially continuous feeding
strategies require stable feed solutions. Thus, the shelf-life of
the feed components must exceed at least the feeding period.
[0036] Furthermore, addition of feed solutions with
non-physiological pH values, i.e. alkaline or acidic pH values,
triggers a response of the pH control mechanism of the cultivation
device. This results in an increased and undesired addition of acid
or base to compensate pH changes induced by the addition of feed
solutions with a non-physiological pH value.
[0037] To avoid effects generated by the use of feed solutions with
a non-physiological pH value, such as those with high or low pH
values, a feed mixing device as reported herein can be used,
enabling continuous mixing of at least two feed solutions just
before addition into the cultivation vessel. By using the feed
mixing device as reported herein, feed components can be dissolved
at pH values at which these have good solubility and/or good
stability, whereby the pH value can be clearly different from the
pH value of the cultivation medium, i.e. different from a
physiological pH value. This allows more flexible pH values for a
feed solution, as the pH value is now adjusted directly prior to
the addition to the cultivation medium and cell suspension.
[0038] The device as reported herein is a feed mixing device useful
in the cultivation of cells during which compounds have to be
added, such as in a fed-batch cultivation or in a continuous
cultivation. With a feed mixing device as reported herein the
cultivating can be performed with at least one additional degree of
freedom and, thereby, with more flexibility. With the device as
reported herein it is possible to use feed solutions that have a
non-physiological pH value and/or a high compound concentration.
The non-physiological pH value can be required e.g. for stabilizing
pH-sensitive feed components. Prior to the addition to the
cultivation vessel and, therewith, to the cultivation medium any pH
value can be adjusted. This allows in a discontinuous or continuous
feeding process to add defined amounts of compounds. For example,
by the addition of defined amounts of ions a pre-defined osmolality
can be adjusted.
[0039] By the gained variability of the pH value of the feed
solutions that is possible by using a device as reported herein
feed solutions with any pH value, i.e. alkaline, neutral or acidic
solutions, and with any concentration of individual components can
be used. It is also possible to exert a pH gradient in the added
feed, whereby also essentially the same amount of substances can be
added compared to a conventional feeding strategies not using the
device as reported herein. Thus, even feed solutions can be used in
which the components due to their low solubility or impaired
stability have to be provided at extremely alkaline or acidic, i.e.
non-physiological, pH values.
[0040] The pH value of the mixed feed solution leaving the feed
mixing device and being added to the cultivation medium depends on
the volumetric mixing ratio of the individual feeds and on the
residence time within the mixing chamber and, therewith, on the
volume flow of the individual feed solutions.
[0041] In one embodiment the total volume flow into the cultivation
vessel through the feed mixing device is of from 1 to 1.5 g/h/l. In
one embodiment the volume flow is of from 1.15 g/h/l to 1.35 g/h/l.
In one embodiment the volume flow is about 1.25 g/h/l. The unit
g/h/l denotes mass of feed/cultivation time/cultivation volume. In
one embodiment the cultivation volume is the volume of liquid in
the cultivation vessel at the start of the cultivation.
[0042] By using the device as reported herein for the mixing of
feed solutions the viability of the cultivated cells can be
maintained for a longer period of time above a pre-defined level
and, therewith, allows for a longer overall cultivation time. At
the same time the lactate concentration and the glucose consumption
can be reduced.
[0043] The general course of the pH value of a cell cultivation is
shown in FIG. 1. After inoculation (FIG. 1, "1") the pH value of
the cultivation decreases and approaches the lower margin of the
pre-defined pH range (FIG. 1, "2"). This is due to the formation of
lactate and the accumulation of carbon dioxide in the cultivation
medium. An engaged pH control mechanism ensures that the pH value
is maintained at the lower margin of the pre-set pH range by the
addition of base. The base addition is continued until the cell
metabolism changes and the lactate in the cultivation medium is
re-metabolized and/or the accumulated carbon dioxide is removed
(FIG. 1, "3"). Afterwards the pH value increases until it reaches
the upper margin of the pre-set pH range (FIG. 1, "4"). The pH
control mechanism maintains the pH value at this upper margin value
by the addition of acid.
[0044] An alkaline feed solution can maintain the pH value of the
cultivation medium above the lower margin of a pre-set pH range
e.g. without engagement of the pH control mechanism. Thus, the
change of the pH value in the cultivation vessel can be
counteracted by changing the ratio of the individual volume flow
rates of the two or more feed solutions added to the cultivation
medium using the feed mixing device as reported herein. Thus, with
the device as reported herein a pH control mechanism or at least
the time of engagement thereof (and likewise the added amounts of
acid and base) might be obsolete or can be reduced,
respectively.
[0045] By using an alkaline feed solution and an acidic feed
solution with individual feed rates and the feed mixing device as
reported herein the pH value of the combined feed solutions can be
adjusted to any target value. By varying the individual feed rates
and/or the feed solutions during the course of the cultivation the
pH value of the combined feed solutions can be changed during the
cultivation. Thus, an adaptation and/or a control of the pH value
depending on the metabolism of the cultivated cells is possible.
The variable pH value of the combined feed solutions can be used to
support or replace other means for adjusting the cultivation pH
value.
[0046] By using the feed mixing device as reported herein two or
more feed solutions can be combined continuously. The feed
solutions may comprise any compound. For example, compounds, such
as vitamins, can be stabilized at (high) non-physiological pH
values. The pH value is adjusted prior to the addition to the
cultivation medium with the feed mixing device as reported herein
to a pH value in the physiological range. Thus, the time at which
the fed compound is kept at a stability impairing pH value is
reduced.
[0047] By using one or more alkaline solutions and one or more
acidic solutions a variable pH value adjustment is possible. With
the device as reported herein the pH value in the cultivation
medium can be controlled online by adjusting the ratio and the
individual flow rates of the feed solutions (FIG. 1, "5").
[0048] By using feed solutions with different properties a time
dependent adjustment of e.g. the ion concentration, the pH value,
the osmolality, compound ratios, the viscosity, the surface
tension, the conductivity, the wettability, the specific
resistance, and/or the density is possible.
[0049] By using online sensors it is possible to combined different
feed solutions with different physical and/or chemical properties
with the device as reported herein. Therewith different parameters
can be changed according to a pre-set schedule.
[0050] By using a device as reported herein it is possible to used
solids, dispersion and hardly mixable compounds/solutions as feed
solution. The chemical and/or physical properties of the feed
solutions can be different.
[0051] By using a feed mixing device as reported herein a pH
gradient of the added mixed feed solution during a cultivation can
be performed.
[0052] The individual feeds can independently of each other be a
dispersion, an emulsion, or a solution. Solutions can be
transported to the feed mixing device by using conventional pumps
or any other known mechanical or flow-mechanical method. If a feed
is no true solution the feed can, e.g., be added by using a
mechanical transportation method.
[0053] The scale of the feed mixing device is variable and, thus,
the device can be used with any type and size of cultivation
device, such as e.g. with a cultivation vessel (stirred tank),
chip, or flow pipe reactor.
[0054] When using a device as reported herein almost no precipitate
is formed in the mixing chamber although the pH value of the feed
solution is profoundly changed prior to the addition to the
cultivation medium upon mixing the individual feed solutions.
[0055] By using a device as reported herein and thereby adjusting
the separate feeds to a combined (acidic) feed the G(0) glycoform
of a produced polypeptide, especially of a produced immunoglobulin,
can be reduced compared to a cultivation with a single alkaline
feed and without employing the device as reported herein. Likewise
the G(1) glycoform can be increased by using a device as reported
herein and adjusting the separates feeds to a mixed acidic feed
compared to a cultivation with a single alkaline feed and without
employing the device as reported herein.
[0056] By adjusting the cell culture conditions the content of host
cell protein in the cultivation medium prior to downstream
processing can be changed. This should be possible by using
specific feed solutions. Thus, with an adjusted feed strategy
comprising pH adjusted feed solutions with a defined concentration
of protons, i.e. a defined pH value, an identical or at least
similar amount of feed components could be added and concurrently
the amount of base or acid required in order to correct the pH
value of the cultivation medium can be reduced and also
concurrently the host cell protein content in the cultivation
supernatant could be reduced.
[0057] Thus, the feed mixing device as reported herein is used in
one embodiment for reducing the host cell protein content in a cell
cultivation supernatant.
[0058] The following examples and figures are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
DESCRIPTION OF THE FIGURES
[0059] FIG. 1 Scheme of the general course of the pH value of a
cell cultivation.
[0060] FIG. 2 Course of the viable cell density: open: single feed
(feed 1); filled: separate feeds (feed 2 and feed 3); circle: with
potassium chloride; square: with sodium chloride.
[0061] FIG. 3 Course of the viability: open: single feed (feed 1);
filled: separate feeds (feed 2 and feed 3); circle: with potassium
chloride; square: with sodium chloride. It can be seen that the
viability by using the mixing device as reported herein remains
longer at a value more than 90%.
[0062] FIG. 4 Course of the pH value: open: single feed (feed 1);
filled: separate feeds (feed 2 and feed 3); circle: with potassium
chloride; square: with sodium chloride. Course is comparable until
the start of the feeding (after 72 hours).
[0063] FIG. 5 Course of glucose consumption: open: single feed
(feed 1); filled: separate feeds (feed 2 and feed 3); circle: with
potassium chloride; square: with sodium chloride. The glucose
consumption is reduced when a mixing device as reported herein is
used.
[0064] FIG. 6 Course of lactate formation: open: single feed (feed
1); filled: separate feeds (feed 2 and feed 3); circle: with
potassium chloride; square: with sodium chloride. Lactate formation
and onset of re-metabolism is improved upon using a mixing device
as reported herein.
[0065] FIG. 7 Course of glutamine consumption: open: single feed
(feed 1); filled: separate feeds (feed 2 and feed 3); circle: with
potassium chloride; square: with sodium chloride.
[0066] FIG. 8 Course of ammonia accumulation: open: single feed
(feed 1); filled: separate feeds (feed 2 and feed 3); circle: with
potassium chloride; square: with sodium chloride.
[0067] FIG. 9 Acidic peak fraction: left: use of a device as
reported herein; dark right: without a device as reported
herein.
[0068] FIG. 10 Dependency of the required amount of added base
(after 14 days of cultivation) on the pH value of the feed
solution. Circle: without a device as reported herein; square: with
a device as reported herein.
[0069] FIG. 11 Dependency of lactate formation (after 14 days of
cultivation) on the pH value of the feed solution. Circle: without
a device as reported herein; square: with a device as reported
herein.
[0070] FIG. 12 Dependency of the glutamine concentration (after 14
days of cultivation) in the cultivation medium on the pH value of
the feed solution. Circle: without a device as reported herein;
square: with a device as reported herein.
[0071] FIG. 13 Dependency of the osmolality in the cultivation
medium (after 14 days of cultivation) on the pH value of the feed
solution. Circle: without a device as reported herein; square: with
a device as reported herein.
[0072] FIG. 14 Dependency of the dissolved carbon dioxide (after 14
days of cultivation) on the pH value of the feed solution. Circle:
without a device as reported herein; square: with a device as
reported herein.
[0073] FIG. 15 Dependency of viable cell density (after 14 days of
cultivation) on the pH value of the feed solution. Circle: without
a device as reported herein; square: with a device as reported
herein.
[0074] FIG. 16 Dependency of the G(0) fraction on the pH value of
the feed solution.
[0075] FIG. 17 Dependency of the G(1) fraction on the pH value of
the feed solution.
[0076] FIG. 18+19 Exemplary schemes of the device as reported
herein.
[0077] FIG. 20 Dependency of the host cell protein content on the
pH value of the feed solution.
EXAMPLES
Materials
Antibody
[0078] An exemplary antibody used in the method and examples as
reported herein is an anti-IL17 antibody as reported in WO
2010/034443 (incorporated herein by reference).
Feed Solutions
[0079] Feed 1: This solution comprises all feed components (amino
acids and pyruvate) at a pH value of about 9.5. [0080] Feed 2: This
solution comprises at a double concentration the components soluble
at an acid pH value of about pH 1.5. [0081] Feed 3: This solution
comprises at a double concentration the components soluble at a
basic pH value of about pH 10.
[0082] In this setup it is ensured that the concentration as well
as the number of components as well as the volume of the added feed
1 is the same as after the combination of feed 2 and feed 3. The pH
value of feed 1 is about pH 9.5 and the pH value of the combined
feeds 2 and 3 is about pH 7.2.
Feed Mixing Device Dimensions
[0083] Volume of the chamber for mixing the feed solutions: 1.146
ml
[0084] Volume of the cultivation medium at the start of a
cultivation with a 2 l-cultivation vessel: 1.21
[0085] Feed flow through the feed mixing device: 36 g/d
Example 1
[0086] Four 2 l-cultivation vessels (Sartorius Biostat B-DCU Quad,
Sartorius, Goettingen, Germany) have been inoculated in parallel
with an inoculum solution pre-cultivated in the same shaker flask.
Two cultivation vessels comprised the device as reported herein
whereas the other two cultivation vessels comprised two individual
conventional feeding devices with a Luer fitting but without a
mixing chamber.
[0087] All cultivations were performed with a constant aeration
rate, constant temperature and constant agitation speed over the
entire cultivation. All feed rates are calculated based on the
start working volume and are given in feed volume per fermentation
starting volume per day.
[0088] The feed in the cultivation vessels comprising the feed
mixing device was started after 72 hours cultivation time with feed
2 and feed 3 as continuous feed with the same volume flow. The
feeds were combined in the feed mixing device and added to the
cultivation medium after mixing.
[0089] The feed in the cultivation vessels comprising the
conventional feeding device was started after 72 hours cultivation
time with feed 1 at a volume flow twice that of the corresponding
cultivations with the feed mixing device.
[0090] Thus, the added volume as well as the added amount of all
feed components is identical in all four cultivations (see Tables 1
and 2).
TABLE-US-00001 TABLE 1 parameter feed solution 1 feed solution 2
feed solution 3 use of device as no yes yes reported herein pH
value alkaline alkaline acidic volume flow 100% 50% 50%
concentration 1x 2x 2x feed rate 0.03 1/d 0.015 1/d 0.015 1/d total
feed volume 100% 50% 50%
TABLE-US-00002 TABLE 2 cultivation 1 cultivation 2 cultivation 3
cultivation 4 feed 1 feed 1 feed 2 + feed 3 feed 2 + feed 3 +sodium
+potassium +sodium +potassium chloride chloride chloride chloride
feed pH feed pH mixed feed pH mixed feed value 9.5 value 9.5 valu
7.2 pH value 7 indicates data missing or illegible when filed
[0091] The course during the cultivation of the viable cell density
is shown in FIG. 2, the course of the cell viability is shown in
FIG. 3, the course of the pH value in the cultivation medium is
shown in FIG. 4, and the course of the glucose consumption is shown
in FIG. 5. FIG. 6 shows the course of the lactate concentration
during the cultivation. The course of glutamine concentration
during the cultivation is shown in FIG. 7. FIG. 8 shows the course
of the ammonia concentration during the cultivation. The amount of
the acidic peak of the produced immunoglobulin is shown in FIG.
9.
[0092] As can be seen from the figures the viability can be
maintained above 90% for an extended period of time by using the
device as reported herein. The course of the pH value is comparable
for the first 72 hours, i.e. prior to the start of the feeding.
Thereafter the pH value of the cultivations employing the feed
mixing device is below that of the other cultivations. The glucose
consumption is reduced in the cultivations employing the device as
reported herein. The maximum lactate concentration during the
course of the cultivation with the feed mixing device is lower
compared to the maximum lactate concentration of the cultivation
without the feed mixing device.
Example 2
[0093] In this example only the influence of the pH value of the
feed solutions on different parameters of the cultivation, such as
base consumption, lactate formation, growth kinetic or product
formation is analyzed. All other parameters were kept
comparable.
[0094] The feed solution were composed in such a way that only the
pH value after the mixing is different but all other parameters,
such as the added amount of feed components, feed volume, or
osmolality, are comparable. Therefore the feed solutions were not
added at a constant feed rate but added by a gravimetric feeding
controller.
[0095] The pO.sub.2 value was adjusted to a value of 35% air
saturation and determined with a pO.sub.2 probe (Mettler-Toledo
InPro 6820). The pO.sub.2 probe was calibrated at process
conditions after 72 hours of gassing in based on the mean value
determined with a certified gas analytics (GA4, Dasgip). The
aeration during the cultivation was kept at a constant rate of 75
ml/min of a mixture of nitrogen, air, pure oxygen and carbon
dioxide. The fraction of carbon dioxide in the total gas flow was
constant at 7 vol % of the total gas flow and was changed solely
due to increased demand of the pH control.
[0096] The pH value of the cultivation medium was adjusted with a 1
mol/l sodium carbonate solution as base and carbon dioxide as acid
to a set point of 7.0+/-0.05 pH units. The required carbon dioxide
was added to the total carbon dioxide flow of 75 ml/min. The pH
probe (Mettler Toledo 405-DPAS-SC-K8S/200) was calibrated with
reference buffer solution of pH values 7.0 and 4.01 and after
equilibration of the cultivation medium for at least 72 hours under
process conditions as mean value of a blood gas analyzer
(Bioprofile, PHOx BGA).
[0097] The cultivation was performed at a constant stirrer speed of
about 230 rpm. A mixer a dish stirrer was used. The power input was
about 80 W/m.sup.3. The same power input was used in the
pre-cultivation to ensure comparability and avoid a rapid change in
the conditions.
[0098] An anti-foam solution was added based on the foam formation.
No anti-foam probe was employed. The anti-foam amount required by
the cultivation vessel with the highest foam formation was also
added to the other cultivation vessels. As anti-foam agent 1%
medical grade Dow was used.
[0099] The cultivation medium was a chemically defined medium. For
each fermentation one liter of medium was used. The inoculation
volume was 200 ml. Thus, the cultivation was performed with a
starting volume of 1,200 ml.
[0100] As cell line a CHO cell transfected with a nucleic acid
encoding an anti-IL17 antibody was used. The cell density in the
200 ml inoculation volume was adjusted to ensure a cell density of
about 3.5.times.10.sup.5 cells/ml in the cultivation. The power
input in the inoculation cultivation was kept at the same value as
the thereafter following main cultivation. The inoculation
cultivation was performed at about 36.5.degree. C., 7% CO.sub.2,
and a relative humidity of 85%.
[0101] After transfer of the inoculation medium to the main
cultivation samples were withdrawn on a daily basis. The main
cultivation was performed as a fed-batch cultivation, wherein the
feeding was started approximately 72 hours after start of the main
cultivation.
[0102] Five different cultivations were carried out simultaneously.
The parameters thereof are given in Table 3.
TABLE-US-00003 TABLE 3 culti- culti- culti- culti- culti- parameter
vation 1 vation 2 vation 3 vation 4 vation 5 use of device yes yes
no yes yes reported herei feed 1 alkaline alkaline complete
alkaline alkaline feed feed 2 acidic acidic -- acidic acidic pH set
of feed 9.5 7.0 9.5 4.5 4.5 change of pH no no no Yes, change no
set of feed t pH set of 9.5 after pH value dropped to lower pH ban
border indicates data missing or illegible when filed
Results:
[0103] By using feed solution the pH value of the cultivation
medium is directly affected. Also affected but only indirectly is
the amount of acid and/or base that has to be added during the
cultivation by the pH control mechanisms.
[0104] In FIGS. 10 to 17 the influence of the pH value of the feed
solution on the parameters amount of added base, amount of added
acid, resulting pCO.sub.2 in the cultivation medium after a
cultivation time of 14 days is depicted.
[0105] As can be seen from FIG. 10 the amount of added base is
dependent on the pH value of the feed solution.
[0106] As can be seen from FIG. 11 the amount of lactate in the
cultivation medium after a cultivation of 14 days is dependent on
the pH value of the feed solution whereby the use of a feed
solution of a pH value of 9.5 results in the highest amount of
lactate. By using a device as reported herein the overall amount of
lactate is reduced by about 30% compared to a combined feed.
[0107] As can be seen from FIG. 12 the amount of glutamine in the
cultivation medium after a cultivation time of 14 days is dependent
on the pH value of the feed solution.
[0108] As can be seen from FIG. 13 the osmolality in the
cultivation medium after a cultivation time of 14 days is depending
on the pH value of the feed solution.
[0109] In FIG. 14 the pCO.sub.2 value in the cultivation medium
after a cultivation time of 14 days is shown. It can be seen that
the pCO.sub.2 is dependent on the pH value of the feed solution,
whereby the highest pCO.sub.2 value was obtained with alkaline feed
solutions, either as single feed or as mixed feed using the device
as reported herein. It can be seen that by using a feed of a lower
pH value the pCO.sub.2 value after a cultivation time of 14 days
can be dramatically reduced avoiding unphysiologically high
pCO.sub.2 values in the cultivation medium.
[0110] In FIG. 15 the viable cell density after a cultivation time
of 14 days is shown. It can be seen that the viable cell density of
the cultivations is at or above a value of 90%.
[0111] In FIG. 16 the amount of the G(0) glycoform depending on the
pH value of the feed solution is shown. It can be seen that with a
neutral and an alkaline feed solution comparable amounts of the
G(0) glycoform were obtained. With an acidic feed solution the
amount of the G(0) glycoform was reduced.
[0112] In FIG. 17 the amount of the G(1) glycoform depending on the
pH value of the feed solution is shown. It can be seen that with a
neutral and alkaline feed solution comparable amounts of the G(1)
glycoform were obtained. With an acidic feed solution the amount of
the G(l) glycoform was increased.
Example 3
Behavior of the Mixed Feeds
[0113] An alkaline feed solution of a pH value of 11.3 and an
acidic feed solution of a pH value of about 1.0 were combined to
obtain a target pH value of about pH 6.5. The mixing of the
individual solutions was performed at room temperature and at
4.degree. C. by combining 10 ml of each feed solution of the
respective temperature.
[0114] After an incubation time of 110 min. a slight precipitate
was observed for the feeds mixed and incubated at room temperature.
In the feeds mixed and incubated at 4.degree. C. a precipitate was
formed already shortly after the mixing was performed.
* * * * *