U.S. patent application number 13/572983 was filed with the patent office on 2012-12-20 for method for purifying protein.
This patent application is currently assigned to ASAHI KASEI CHEMICALS CORPORATION. Invention is credited to Naoyuki SHINOHARA, Hironobu SHIRATAKI.
Application Number | 20120322983 13/572983 |
Document ID | / |
Family ID | 40579333 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120322983 |
Kind Code |
A1 |
SHIRATAKI; Hironobu ; et
al. |
December 20, 2012 |
METHOD FOR PURIFYING PROTEIN
Abstract
The present invention provides a method for purifying a protein
to remove impurities from a mixture liquid containing a desired
protein and the impurities, comprising performing filtration using
a porous membrane having a graft chain on a pore surface and an
anion-exchange group fixed to the graft chain.
Inventors: |
SHIRATAKI; Hironobu; (Tokyo,
JP) ; SHINOHARA; Naoyuki; (Tokyo, JP) |
Assignee: |
ASAHI KASEI CHEMICALS
CORPORATION
Tokyo
JP
|
Family ID: |
40579333 |
Appl. No.: |
13/572983 |
Filed: |
August 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12681189 |
Apr 1, 2010 |
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PCT/JP2008/067540 |
Sep 26, 2008 |
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13572983 |
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Current U.S.
Class: |
530/387.3 ;
530/387.1; 530/388.1; 530/389.1; 530/413; 530/416 |
Current CPC
Class: |
B01D 69/08 20130101;
B01D 71/26 20130101; B01D 71/34 20130101; B01D 71/78 20130101; B01D
71/82 20130101; C07K 1/18 20130101 |
Class at
Publication: |
530/387.3 ;
530/416; 530/388.1; 530/389.1; 530/387.1; 530/413 |
International
Class: |
C07K 1/34 20060101
C07K001/34; C07K 1/36 20060101 C07K001/36; C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 26, 2007 |
JP |
2007-279406 |
Claims
1. A method for purifying a protein to remove impurities from a
mixture liquid containing a desired protein and the impurities,
comprising: performing filtration using a porous membrane having a
graft chain on a pore surface and an anion-exchange group fixed to
the graft chain.
2. The method for purifying the protein according to claim 1,
wherein the desired protein is one selected from the group
consisting of monoclonal antibodies, polyclonal antibodies,
humanized antibodies, human antibodies, and immunoglobulins.
3. The method for purifying the protein according to claim 1,
wherein the impurities are at least one selected from the group
consisting of a non-turbid component and a turbid component
dispersed in the mixture liquid.
4. The method for purifying the protein according to claim 3,
wherein the non-turbid component is at least one selected from the
group consisting of impurity proteins, HCP, DNA, viruses,
endotoxins, proteases, and bacteria dissolved in the mixture
liquid.
5. The method for purifying the protein according to claim 3,
wherein the turbid component dispersed in the mixture liquid is at
least one selected from the group consisting of cells and cell
debris.
6. The method for purifying the protein according to claim 1,
wherein a salt concentration of the mixture liquid is from 0.01 M
to 0.5 M (both inclusive).
7. The method for purifying the protein according to claim 6,
wherein the salt concentration of the mixture liquid is from 0.1 M
to 0.3 M (both inclusive).
8. The method for purifying the protein according to claim 1,
wherein: a base material of the porous membrane is polyethylene or
polyvinylidene fluoride, the graft chain is a polymer of glycidyl
methacrylate and has a graft rate of from 10% to 250% (both
inclusive), and the graft chain has 70% or more of epoxy groups
replaced with the anion-exchange groups.
9. The method for purifying the protein according to claim 8,
wherein the graft rate is from 10% to 150% (both inclusive).
10. The method for purifying the protein according to claim 8,
wherein the graft rate is from 10% to 90% (both inclusive).
11. The method for purifying the protein according to claim 8,
wherein the graft rate is from 30% to 60% (both inclusive).
12. The method for purifying the protein according to claim 1,
wherein the anion-exchange group is a diethylamino group and/or a
trimethylamino group.
13. The method for purifying the protein according to claim 1,
wherein the anion-exchange group is a diethylamino group.
14. The method for purifying the protein according to claim 1,
wherein the porous membrane has a maximum pore size of from 0.1
.mu.m to 0.8 .mu.m (both inclusive).
15. The method for purifying the protein according to claim 1,
wherein the mixture liquid is filtered using the porous membrane to
remove one or more impurities comprising a non-turbid
component.
16. The method for purifying the protein according to claim 1,
wherein the mixture liquid is an animal cell culture.
17. The method for purifying the protein according to claim 1,
wherein the porous membrane is a porous hollow fiber membrane.
18. The method for purifying a protein according to claim 1,
further comprising performing purification using affinity
chromatography.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 12/681,189, which is a National Stage of International
Patent Application No. PCT/JP2008/067540, filed Sep. 26, 2008, and
claims priority to Japanese Application No. 2007-279406, filed Oct.
26, 2007. The disclosures of application Ser. Nos. 12/681,189 and
PCT/JP2008/067540 are expressly incorporated by reference herein in
their entireties.
TECHNICAL FIELD
[0002] The present invention relates to a method for purifying a
protein. Specifically, the present invention relates to a method
for easily removing impurities from a mixture liquid containing a
desired protein and the impurities, represented by an animal cell
culture and efficiently purifying the desired protein.
BACKGROUND ART
[0003] In recent years, the practical large-scale purification of a
protein has represented an important challenge in the
biotechnology-based industry. Particularly in the field of
medicine, demand for antibody drugs is rapidly increasing; there is
a strong desire to establish technologies capable of efficiently
producing and purifying large amounts of a protein.
[0004] A protein is generally produced by cell culture using a cell
line derived from an animal. For practical use of a desired protein
(hereinafter sometimes referred to simply as "a target protein"),
particularly an antibody drug used as a medicine, it is necessary
to remove cell debris and the like providing turbid components and
a cell-derived dissolved protein and the like providing non-turbid
components from a cell culture, followed by purification to such a
degree as to give a composition sufficient for therapeutic
application in humans.
[0005] A typical operation of purifying a target protein from a
cell culture comprises first subjecting the cell culture to
centrifugation to precipitate and remove turbid components; then,
removing cell debris having a size of about 1 .mu.m or less
incapable of being completely removed by the centrifugation among
the turbid components by means of size filtering using a
microfiltration membrane; and further subjecting the filtrate to
sterilization filtration using a filtration membrane having a
maximum pore size of 0.22 .mu.m or less for sterilization to
provide a clear solution of the target protein (a harvest step).
When the clear solution containing the target protein is obtained,
the target protein is subsequently separated and purified using a
purification process employing a combination of a plurality of
chromatographic techniques including affinity chromatography (a
downstream step). Impurity proteins produced from cells in the
culture and dissolved in the culture are removed in the downstream
step. In such a conventional method by which the target protein and
the like are purified from the cell culture, the concentration of
the target protein in the culture is typically 1 g/L or less, and
the concentration of cell debris, dissolved impurity proteins and
the like contained in the culture is also comparable to the
concentration of the target protein. In the concentration range,
the purification process using the conventional harvest and
downstream steps is sufficiently useful in purifying the target
protein.
[0006] However, a rapid increase in demand for antibody drugs
allows the production of proteins providing the antibody drugs to
be large-scale-oriented; fast-paced advances in culture techniques
have recently increased the concentration of a target protein in a
cell culture, which is about to reach as high as 10 g/L. Such
fast-paced advances in culture techniques simultaneously mean the
result as which impurity proteins also increase, causing the
prediction of substantial loads applied to purification using a
conventional protein purification process.
[0007] Hence, as a technique for purifying large amounts of a
protein, for example, Patent Documents 1 and 2 each disclose a
protein adsorption membrane to which protein adsorption capability
is imparted by introducing ion-exchange groups into a porous
membrane, which can also be purchased.
[0008] As a usage example of a protein adsorption membrane, Patent
Document 3 also discloses a method for separating albumin from a
lymph fluid using two types of protein adsorption membranes, i.e. a
porous cellulose membrane into which anion-exchange groups are
introduced and a porous cellulose membrane into which
cation-exchange groups are introduced.
[0009] Further, Patent Document 4 discloses a method for separating
nucleic acid and endotoxin using a porous cellulose membrane into
which anion-exchange groups are introduced.
[0010] Sill further, Patent Documents 5 and 6 disclose protein
adsorption membranes in which cation-exchange groups and
anion-exchange groups, respectively are introduced into porous
polyether sulfone membranes.
[0011] Non-Patent Document 1 discloses a method for separating
nucleic acid and monoclonal antibody using a porous membrane
containing ion-exchange groups. [0012] Patent Document 1: U.S. Pat.
No. 5,547,575 [0013] Patent Document 2: U.S. Pat. No. 5,739,316
[0014] Patent Document 3: U.S. Pat. No. 6,001,947 [0015] Patent
Document 4: U.S. Pat. No. 6,235,892 [0016] Patent Document 5: U.S.
Pat. No. 6,783,937 [0017] Patent Document 6: U.S. Pat. No.
6,780,327 [0018] Non-Patent Document 1: Bioseparation 8:281-291,
1999
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0019] However, aggregated impurities are often observed to be
contained in a recovered solution when a cell culture containing a
high concentration of a target protein is subjected to
centrifugation, microfiltration and sterilization filtration to
make a clear fluid, which is then applied to a protein A affinity
column as an affinity chromatography column to selectively adsorb
the target protein, followed by eluting and recover the protein
with an acidic eluent. With increasing number of repeated uses of
the protein A affinity column, the amount of the aggregated
impurities tends to be increased while the amount of the recovered
target protein being decreased.
[0020] A larger amount of impurity proteins increases loads in the
affinity chromatography step as described above, and not only
requires a longer time of the purification step owing to the
washing of the column and the like, but also results in shorter
life of column beads used in the affinity chromatography,
particularly the life of the protein A affinity column. It is
undesirable for the protein A affinity column to have shorter life
because the column is expensive.
[0021] In each of the methods disclosed in Patent Documents 1 to 4,
the separation operation is performed after passing a stock
solution used in the separation through a microfiltration membrane
in advance to remove particulate impurities. This is because the
protein adsorption membrane cannot remove particulate impurities
since the protein adsorption membrane has a maximum pore size of as
large as 3 .mu.m to 5 .mu.m.
[0022] Also in each of the methods disclosed in Patent Documents 5
and 6, the protein adsorption membrane cannot remove fine
particulate impurities since it has a maximum pore size of 0.8
.mu.m to 1.0 .mu.m.
[0023] In addition, a cell culture generally contains a salt; when
an ion-exchange membrane is used as disclosed in Non-Patent
Document 1, impurity proteins cannot be practically removed from
the cell culture because the amount of adsorption of proteins from
a solution containing 0.1 M or more of the salt is markedly
decreased.
[0024] As described above, the existing protein adsorption
membranes have maximum pore sizes of about 0.8 .mu.m or more and
decreased amounts of adsorption in the presence of a salt. Thus it
is not assumed that the adsorption membranes are used to
simultaneously perform clarification intended to remove fine
insoluble matter such as cell debris and adsorption of dissolved
impurity proteins; the adsorption membranes are not suitable for
the purpose.
[0025] Each of the protein adsorption membranes has a dynamic
adsorption volume of 30 mg/mL or less per membrane volume even
under conditions in which no salt is present in the solution; thus,
a large amount of the protein adsorption membrane will be required
to effectively adsorb and remove impurity proteins from the cell
culture. In addition, each of these existing protein adsorption
membranes is in the form of a flat membrane and therefore a large
amount of the protein adsorption membrane cannot be housed in a
compact size. As such, a large amount of the protein adsorption
membrane housed in a large-size container will be required and thus
cannot be said to be practical. The existing protein adsorption
membranes having anion-exchange groups are more unsuitable for
practical use because their amount of adsorption is markedly
decreased when a salt is present in the solution.
[0026] In view of such circumstances, an object of the present
invention is to provide a method for easily removing impurities
from a mixture liquid containing a target protein and the
impurities, represented by an animal cell culture and efficiently
purifying the target protein.
Means for Solving the Problems
[0027] As the result of intensive studies for solving the
above-described problems, the present inventors have found that the
use of a porous membrane having graft chains on the pore surface
and anion-exchange groups fixed to the graft chains is surprisingly
effective to solve the above problems.
[0028] Thus, the present invention provides a method for purifying
a protein and a porous hollow fiber membrane as described
below.
[1] A method for purifying a protein to remove impurities from a
mixture liquid containing a desired protein and the impurities,
comprising the step of:
[0029] performing filtration using a porous membrane having a graft
chain on a pore surface and an anion-exchange group fixed to the
graft chain.
[2] The method for purifying the protein according to item [1]
above, wherein the desired protein is one selected from the group
consisting of monoclonal antibodies, polyclonal antibodies,
humanized antibodies, human antibodies and immunoglobulins. [3] The
method for purifying the protein according to item [1] or [2]
above, wherein the impurities are at least one selected from the
group consisting of a non-turbid component and a turbid component
dispersed in the mixture liquid. [4] The method for purifying the
protein according to item [3] above, wherein the non-turbid
component is at least one selected from the group consisting of
impurity proteins, HCP, DNA, viruses, endotoxins, proteases and
bacteria dissolved in the mixture liquid. [5] The method for
purifying the protein according to item [3] or [4] above, wherein
the turbid component dispersed in the mixture liquid is at least
one selected from the group consisting of cells and cell debris.
[6] The method for purifying the protein according to any one of
items [1] to [5] above, wherein a salt concentration of the mixture
liquid is from 0.01 M to 0.5 M (both inclusive). [7] The method for
purifying the protein according to any one of items [1] to [5]
above, wherein a salt concentration of the mixture liquid is from
0.1 M to 0.3 M (both inclusive). [8] The method for purifying the
protein according to any one of items [1] to [7] above,
wherein:
[0030] a base material of the porous membrane is polyethylene or
polyvinylidene fluoride,
[0031] the graft chain is a polymer of glycidyl methacrylate and
has a graft rate of from 10% to 250% (both inclusive), and
[0032] the graft chain has 70% or more of epoxy groups replaced
with the anion-exchange groups.
[9] The method for purifying the protein according to item [8]
above, wherein the graft rate is from 10% to 150% (both inclusive).
[10] The method for purifying the protein according to item [8]
above, wherein the graft rate is from 10% to 90% (both inclusive).
[11] The method for purifying the protein according to item [8]
above, wherein the graft rate is from 30% to 60% (both inclusive).
[12] The method for purifying the protein according to any one of
items [1] to [11] above, wherein the anion-exchange group is a
diethylamino group and/or a trimethylamino group. [13] The method
for purifying the protein according to any one of items [1] to [12]
above, wherein the anion-exchange group is a diethylamino group.
[14] The method for purifying the protein according to any one of
items [1] to [13] above, wherein the porous membrane has a maximum
pore size of from 0.1 .mu.m to 0.8 .mu.m (both inclusive). [15] The
method for purifying the protein according to any one of items [1]
to [14], wherein the mixture liquid is filtered using the porous
membrane to remove one or more impurities comprising a non-turbid
component. [16] The method for purifying the protein according to
any one of items [1] to [15], wherein the mixture liquid is an
animal cell culture. [17] The method for purifying the protein
according to any one of items [1] to [16], wherein the porous
membrane is a porous hollow fiber membrane. [18] A porous hollow
fiber membrane used in the method for purifying the protein
according to item [17] above. [19] A module comprising the porous
hollow fiber membrane according to item [18] above. [20] A porous
hollow fiber membrane having a graft chain on a pore surface and an
anion-exchange group fixed to the graft chain, wherein:
[0033] a base material of the porous hollow fiber membrane is
polyethylene or polyvinylidene fluoride,
[0034] the graft chain is polymers of glycidyl methacrylate and has
a graft rate of from 10% to 250% (both inclusive), and
[0035] the graft chain has 70% or more of epoxy groups replaced
with the anion-exchange group.
[21] The porous hollow fiber membrane according to item [20] above,
wherein the graft rate is from 10% to 150% (both inclusive). [22]
The porous hollow fiber membrane according to item [20] above,
wherein the graft rate is from 10% to 90% (both inclusive). [23]
The porous hollow fiber membrane according to item [20] above,
wherein the graft rate is from 30% to 60% (both inclusive). [24]
The porous hollow fiber membrane according to any one of items [20]
to [23] above, wherein the porous membrane has a maximum pore size
of from 0.1 .mu.m to 0.8 .mu.m (both inclusive). [25] A module
comprising the porous hollow fiber membrane according to any one of
items [20] to [24] above. [26] The method for purifying a protein
according to any one of items [1] to [17] above, further comprising
the step of performing purification using affinity
chromatography.
Advantages of the Invention
[0036] In accordance with the method for purifying a protein
according to the present invention, the clarification of a cell
culture before an affinity chromatography step, carried out by the
three steps of centrifugation, microfiltration and sterilization
filtration can be easily performed through filtration using a
porous membrane having graft chains and anion-exchange groups fixed
to the graft chains.
[0037] In addition, removal of dissolved impurity proteins
incapable of being removed by conventional methods can be made
possible by filtration with the porous membrane having the fixed
anion-exchange groups. Further, it is made possible to greatly
reduce loads in the affinity chromatography step.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows results of the SDS-PAGE analysis of straightly
passed-through and adsorbed components in the filtration of a
mixture liquid of BSA and .gamma.-globulin using an anion-exchange
hollow fiber membrane module in Example 3;
[0039] FIG. 2 shows results of the SDS-PAGE analysis of the
purification of a .gamma.-globulin-containing cell culture by
filtration using various anion-exchange membranes in Examples 4 to
6 and Comparative Example 1;
[0040] FIG. 3 illustrates a schematic drawing of an estimation
apparatus for performing clarification, impurity protein removal
and sterilization by a single step using an anion-exchange hollow
fiber membrane module in Example 10;
[0041] FIG. 4 shows results of the SDS-PAGE analysis of straightly
passed-through and adsorbed components in the filtration using an
anion-exchange hollow fiber membrane module in Example 10; and
[0042] FIG. 5 illustrates a schematic drawing of an estimation
apparatus for performing impurity protein removal and sterilization
by 2 steps using an anion-exchange hollow fiber membrane module
after clarification with a microfiltration hollow fiber membrane
module in Example 11.
DESCRIPTION OF SYMBOLS
[0043] 1 Cell culture tank [0044] 2 Perister pump [0045] 3 Pressure
indicator (at entry side of module) [0046] 4 Anion-exchange hollow
fiber membrane module [0047] 5 Pressure indicator (at exit side of
module) [0048] 6 Flow control cock [0049] 7 Microfiltration hollow
fiber membrane module for clarification
BEST MODE FOR CARRYING OUT THE INVENTION
[0050] A best mode for carrying out the present invention
(hereinafter referred to as "the present embodiment") is described
below in detail. Note that the present invention is not intended to
be limited to the following embodiment, and various modifications
can be made within the scope of the gist of the invention.
[0051] The method for purifying a protein according to the present
embodiment is a method for purifying a protein to remove impurities
from a mixture liquid represented by an animal cell culture,
containing a desired protein useful as a medicine and the
impurities, comprising the step of performing filtration using a
porous membrane having a graft chain on a pore surface and an
anion-exchange group fixed to the graft chain.
[0052] The method for purifying a protein according to the present
embodiment is a method for purifying a protein which can easily
remove impurities contained in a mixture liquid to provide a clear
solution of a target protein.
[0053] Preferably, the method for purifying the protein according
to the present embodiment further comprises the step of purifying
the clear target protein solution obtained by the filtration step
using affinity chromatography.
[0054] Examples of the mixture liquid containing a desired protein
and impurities can include, but not limited to, an animal cell
culture.
[0055] The animal cell culture is not particularly limited provided
that it is a culture containing a target protein; however, examples
thereof can include a culture containing a recombinant protein
obtained by cell culture from host cells including Chinese hamster
ovary (CHO) cells.
[0056] Examples of the target protein can include antibodies used
as medicine; examples thereof can include monoclonal antibodies,
polyclonal antibodies, humanized antibodies, human antibodies and
immunoglobulins.
[0057] Examples of the impurities can include a turbid component
dispersed in a mixture liquid and a non-turbid component.
[0058] Examples of the turbid and non-turbid components can include
impurities other than a target protein contained in the culture
obtained by performing animal cell culture in order to produce the
desired protein.
[0059] Examples of the turbid component dispersed in a mixture
liquid can include cells and cell debris; examples of the
non-turbid component can include impurity proteins, host cell
proteins (HCP), nucleic acids (DNA), viruses, endotoxins,
proteases, and bacteria dissolved in the mixture liquid.
[0060] The method for purifying the protein according to the
present embodiment preferably involves filtering the mixture liquid
using the porous membrane to remove one or more impurities
including the non-turbid component.
[0061] The non-turbid component can be removed by adsorption to the
porous membrane. Preferably, the turbid component is also removed
in removing one or more impurities including the non-turbid
component.
[0062] The isopotential point (pI) of an antibody as a target
protein ranges from about 6 to 8. A typical CHO animal cell culture
has a pH ranging from roughly 7 to 8 and contains about 1% by mass
(about 0.17 M) of a salt. The pH and salt concentration can vary
depending on the type of the animal cell culture.
[0063] In an aqueous solution, the amino and carboxyl groups of the
protein are present in ionized states: --NH.sub.3.sup.+ and
--COO.sup.-, respectively and the total charge of the protein in
the aqueous solution depends on the pH of the culture. The total
charge of the protein is zero at the pH of the isoelectric point
(pI). The protein is made in a negatively charged state at a pH of
more than pI, and in a positively charged state at a pH of less
than pI.
[0064] Because an animal cell culture has a pH of from 7 to 8, a
target protein is present in the culture in such a state that the
protein has a total charge of nearly zero or is slightly positively
charged; thus, the target protein is not substantially adsorbed by
an anion-exchange group.
[0065] In contrast, the impurity proteins, host cell proteins
(HCP), nucleic acids (DNA), endotoxins and the like dissolved in a
mixture liquid are present in a negatively charged state in an
animal cell culture even when dissolved in the culture because most
of them have a pH of 6 or lower. The impurity proteins and the like
dissolved in the mixture liquid have the property of being adsorbed
by an anion-exchange group under conditions in which a salt is
absent.
[0066] However, an animal cell culture typically contains a salt on
the order of about 1% by mass (0.17 M) in practice, allowing few
impurity proteins and the like to be adsorbed by an anion-exchange
group even at a pI of 6 or lower. Thus, impurities cannot be
directly removed from an animal cell culture even by use of a
conventional adsorption membrane having anion-exchange groups or
anion-exchange chromatography.
[0067] The method for purifying the protein according to the
present embodiment can be used to purify even an animal cell
culture having a salt concentration of from 0.01 M to 0.5 M (both
inclusive).
[0068] According to the present embodiment, the salt concentration
of the animal cell solution capable of being preferably purified is
from 0.1 M to 0.3 M (both inclusive).
[0069] According to the present embodiment, when an animal cell
culture is filtered using the porous membrane having the graft
chains on the pore surface and the anion-exchange groups fixed to
the graft chains, it has surprisingly been found that the impurity
proteins, host cell proteins (HCP), nucleic acids (DNA), endotoxins
and the like dissolved in the mixture liquid are adsorbed by the
anion-exchange groups of the porous membrane despite that a salt is
contained in the cell culture.
[0070] The porous membrane having graft chains on the pore surface
and anion-exchange groups fixed to the graft chains is used in the
filtration process to enable that adsorption of the impurity
proteins, host cell proteins, nucleic acids, endotoxins and the
like dissolved in a mixture liquid from an animal cell culture,
which is not typically achieved. Although the detailed reason is
unclear, the following is speculated.
[0071] In a common anion-exchange membrane and anion-exchange
chromatography, anion-exchange groups are generally fixed to the
surface of porous membrane pores or a resin and its pores. A
protein is adsorbed by surface anion-exchange groups. The
anion-exchange groups are fixed only to the surface of porous
membrane pores or a resin and its pores and thereby rest
two-dimensionally. As such, the protein is adsorbed in a
point-contact manner on the surface of porous membrane pores or a
resin and its pores; thus, the anion-exchange groups capable of
being responsible for the adsorption are small in number. Thus, the
presence of a salt in the solution significantly reduces the
adsorption of the proteins.
[0072] This is the common concept about an anion-exchange membrane;
based on this principle, the use of a salt solution is adopted as a
conventional method in eluting adsorbed proteins.
[0073] In other words, the anion-exchange membrane is thought to
adsorb no protein when a salt is present in the solution; this
concept has been ditto for an anion-exchange membrane having graft
chains.
[0074] In the porous membrane used in the present embodiment,
anion-exchange groups are fixed to graft chains present on the
surface of pores.
[0075] The present inventors have found the fact that the use of
the porous membrane of the present embodiment, i.e. the
anion-exchange membrane having anion-exchange groups fixed to graft
chains, does not cause a significant reduction in the adsorption of
a protein even in a solution containing a salt.
[0076] The adsorption of a protein is two-dimensional for a
conventional porous membrane in which anion-exchange groups rest
only on the surface of pores or a resin, whereas the fixation of
anion-exchange groups to graft chains allows the anion-exchange
groups to be three-dimensionally (sterically) placed. The fixation
of anion-exchange groups to graft chains is thought to increase the
number of anion-exchange groups responsible for adsorption because
graft chains adsorb onto a protein in such a manner that the
protein become entangled with the chains. The increased number of
the anion-exchange groups involved in the adsorption causes little
reduction in the adsorption of a protein even when a salt is
present in the solution, which probably achieves the removal of
dissolved impurities by adsorption even from a practical cell
culture.
[0077] The porous membrane having anion-exchange groups used in the
present embodiment refers to a porous membrane composed of a porous
body having graft chains fixed to the surface of the porous body as
a base material and its pores and anion-exchange groups chemically
or physically fixed to the graft chains.
[0078] The base material for the porous membrane is not
particularly limited; however, it is preferably composed of a
polyolefin polymer for retention of mechanical properties.
[0079] Examples of the polyolefin polymer can include homopolymers
of olefins such as ethylene, propylene, butylene and vinylidene
fluoride, copolymers of two or more of the olefins, or copolymers
of one or two or more of olefins and perhalogenated olefins.
[0080] Examples of the perhalogenated olefin can include
tetrafluoroethylene and/or chlorotrifluoroethylene.
[0081] Among these base materials, preferred is polyethylene or
polyvinylidene fluoride, more preferably polyethylene, in that they
are raw materials excellent particularly in mechanical strength and
providing a high adsorption volume.
[0082] Methods for introducing graft chains into the surface and
pores of a porous membrane and further fixing anion-exchange groups
to the graft chains include, for example, but not limited to, a
method as disclosed in Japanese Patent Laid-Open No. 02-132132.
[0083] According to the present embodiment, the graft rate refers
to the ratio (percentage) of the weight of the graft chains
introduced into the base material before the fixation of
anion-exchange groups to the graft chains, to the weight of the
base material.
[0084] The graft rate is preferably from 10% to 250% (both
inclusive), more preferably from 10% to 150% (both inclusive),
still more preferably from 10% to 90% (both inclusive), yet more
preferably from 30% to 60% (both inclusive).
[0085] The graft rate of 10% or more makes the adsorption volume of
a protein significantly high and is practical.
[0086] The graft rate of 250% or less provides practical
strength.
[0087] A salt solution is generally passed through the porous
membrane in eluting the adsorbed protein, which is characterized by
expanding the membrane volume; a higher graft rate results in a
higher expansion coefficient. The expansion coefficient of the
membrane volume due to the passage of the salt solution depends on
the structures of the porous membrane and graft chain; however, it
is about 2% or less for the graft rate of 60%, about 3% or less for
90%, and about 5% or less for 150%. A graft rate of 250% or less
can make the expansion coefficient 10% or less; thus, a porous
membrane having a graft rate of 250% or less is practically
preferable.
[0088] According to the present embodiment, the graft chain
introduced into the base material refers to a chain having a
chemical structure which is not removed even by washing with an
organic solvent such as dimethylformamide (DMF) after the
introduction reaction.
[0089] Examples of the graft chain can include polymers of glycidyl
methacrylate, vinyl acetate and hydroxypropyl acetate; however,
preferred is a polymer of glycidyl methacrylate or vinyl acetate,
more preferably a polymer of glycidyl methacrylate since
anion-exchange groups are easily introduced thereinto.
[0090] The anion-exchange group is not particularly limited
provided that it is an anion-exchange group capable of adsorbing an
impurity protein, DNA, HCP, a virus, an endotoxin or the like
dissolved in the mixture liquid; examples thereof can include a
diethylamino (DEA, Et.sub.2N--) group, a quaternary ammonium (Q,
R.sub.3N.sup.+--) group, a quaternary aminoethyl (QAE,
R.sub.3N.sup.+--(CH.sub.2).sub.2--) group, a diethylaminoethyl
(DEAE, Et.sub.2N--(CH.sub.2).sub.2--) group, and a
diethylaminopropyl (DEAP, Et.sub.2N--(CH.sub.2).sub.3--) group. R
is not particularly limited, and R's bonded to the same N may be
the same or different; R preferably represents a hydrocarbon group
such as an alkyl group, a phenyl group and an aralkyl group.
[0091] Examples of the quaternary ammonium group can include a
trimethylamino group (a trimethylammonium group introduced into a
graft chain, Me.sub.3N.sup.+--).
[0092] Preferred are DEA and Q, more preferably DEA since these
groups are easily fixed chemically to the graft chain introduced
into the porous membrane and provide a high adsorption volume.
[0093] The anion-exchange group can be fixed to the graft chain by
ring-opening the epoxy group of the glycidyl methacrylate polymer
forming the graft chain and adding an amine such as diethylamine
and an ammonium salt such as diethylammonium or
trimethylammonium.
[0094] Seventy percent or more, preferably 75% or more, more
preferably 80% or more by mole fraction of the epoxy groups of the
graft chain are preferably replaced with anion-exchange groups. The
amount of the replacing anion-exchange groups being within the
above range can make a porous membrane having a high dynamic
adsorption volume.
[0095] The maximum pore size of the porous membrane is preferably
from 0.1 .mu.m to 0.8 .mu.m (both inclusive), more preferably from
0.1 .mu.m to 0.6 .mu.m (both inclusive), still more preferably from
0.2 .mu.m to 0.5 .mu.m (both inclusive) to cut a turbid component
and bacteria and yet to obtain a high flux.
[0096] According to the present embodiment, the maximum pore size
of the porous membrane refers to a value as determined by a bubble
point method as given in Examples.
[0097] The dynamic adsorption volume for proteins from a salt-free
solution, which the porous membrane containing anion-exchange
groups has is preferably 30 mg/mL or more, more preferably 50 mg/mL
or more, still more preferably 70 mg/mL or more to effectively
reduce loads on an affinity chromatography column in the
purification step using affinity chromatography.
[0098] The dynamic adsorption volume when the buffer contains 0.1
mol/L of a salt is preferably 10 mg/mL or more, more preferably 20
mg/mL or more, still more preferably 30 mg/mL or more.
[0099] The amount of impurities adsorbed to an ion-exchange
membrane is proportional to the volume of the ion-exchange
membrane. Thus, the larger dynamic adsorption volume of the
ion-exchange membrane can decrease the size of a module used for
purifying a protein.
[0100] According to the present embodiment, the dynamic adsorption
volume refers to the mass (in mg/mL) of a protein adsorbed by a
porous membrane before breakthrough per volume of the porous
membrane. Bovine serum albumin (BSA) is used as a model protein for
estimating the adsorption volume; BSA dissolved in a 20 mM Tris-HCl
(pH 8.0) buffer can be used to estimate the dynamic adsorption
volume by a method as described in the following Examples.
[0101] The form of the porous membrane is not particularly limited
provided that it is a porous body; examples thereof can include a
flat membrane, nonwoven fabric, hollow fiber membrane, monolith,
capillary, disk or cylinder form.
[0102] The porous membrane is preferably a hollow fiber membrane in
view of ease of production, scalability, membrane packing
properties in modular molding, and the like.
[0103] According to the present embodiment, the porous hollow fiber
membrane having anion-exchange groups can be used in the harvest
step before the downstream step employing affinity chromatography
to filter an animal cell culture containing a target protein to
provide a clear solution of the target protein. The porous hollow
fiber membrane having anion-exchange groups can be used to remove
impurity proteins, HCP, DNA, viruses and the like as non-turbid
components, present in a dissolved state in the culture in addition
to cells, cell debris and the like present as turbid components and
yet remove bacteria to provide a clear target protein solution
which is made free of impurities and sterilized.
[0104] For the purpose of obtaining a clear target protein solution
from the cell culture, the porous hollow fiber membrane having
anion-exchange groups is preferably housed in a module.
[0105] The porous hollow fiber membrane module is a module housing
a porous hollow fiber membrane composed of a porous hollow fiber in
which anion-exchange groups are chemically or physically fixed to
the surface of the fiber and the surface of its pores. Using the
porous hollow fiber membrane module, an animal cell culture can be
passed therethrough to remove cells, cell debris and the like
present as turbid components and dissolved impurity proteins, HCP,
DNA, bacteria and the like present as non-turbid components from
the culture. The cells, cell debris and the like present in the
culture as turbid components can be removed by size filtering since
they are large in size and thus cannot be passed through the pores
of the porous hollow fiber. Impurity proteins and the like present
dissolved in the culture can be removed by adsorption to the
anion-exchange groups fixed to the surface of the porous hollow
fiber and the surface of its pores.
[0106] According to the present embodiment, to remove impurities
such as turbid and non-turbid components in an animal cell culture,
a cross-flow filtration method is preferably used which involves
passing a pressurized culture through the inside of a porous hollow
fiber to pass the culture out to the outside of the porous hollow
fiber.
[0107] The culture can be passed to the inside of the porous hollow
fiber by cross-flow to inhibit the sedimentation of turbid material
on the inside surface in passing the culture out to the outside of
the porous hollow fiber to suppress a considerable reduction in the
flux.
[0108] In the cross-flow filtration, the linear velocity of the
liquid in flowing through the porous hollow fiber is preferably
0.05 m/s to 5.0 m/s, more preferably 0.1 m/s to 2.0 m/s.
[0109] According to the present embodiment, the linear velocity is
the velocity of a liquid passing through the inner section of the
porous hollow fiber, and is expressed by: (the volume of the liquid
flowing through the porous hollow fiber per second)/(the inner
sectional area of the porous hollow fiber).
[0110] The filtering liquid passage pressure from the inside of the
porous hollow fiber to the outside thereof is preferably from 0.01
MPa to 0.5 MPa, more preferably from 0.05 MPa to 0.2 MPa.
[0111] According to the present embodiment, preferred methods for
obtaining a clear solution of a target protein from an animal cell
culture in a short time also include a method which involves
subjecting the animal cell culture only to clarification treatment
and then performing a filtration step using the porous hollow fiber
membrane having anion-exchange groups. The animal cell culture can
be subjected only to clarification treatment in advance before
filtration using the porous hollow fiber membrane having
anion-exchange groups to provide a high flux in the filtration
step, enabling the filtration step employing the porous hollow
fiber membrane to be performed by normal flow filtration.
[0112] The clarification treatment is preferably performed in
advance because the filtration using the porous hollow fiber
membrane having anion-exchange groups removes only non-turbid
components such as residual cell debris, dissolved impurity
proteins; HCP, DNA, and bacteria.
[0113] Non-limited examples of the clarification treatment of an
animal cell culture can include a method using centrifugation,
normal flow filtration with a depth filter such as a diatomaceous
earth filter, or cross-flow filtration with a microfiltration
membrane.
[0114] The liquid subjected to clarification treatment can be
directly fed to filtration using the porous hollow fiber membrane
having anion-exchange groups; thus, the cross-flow filtration with
a microfiltration membrane is preferable, and cross-flow filtration
with a microfiltration hollow fiber membrane module is more
preferable.
[0115] The maximum pore size of the porous hollow fiber in the
microfiltration hollow fiber membrane module used for clarification
treatment is preferably from 0.1 .mu.m to 0.8 .mu.m, more
preferably from 0.2 .mu.m to 0.6 .mu.m.
[0116] A smaller maximum pore size provides no sufficient flux, and
a larger maximum pore size tends to cause plugging in subsequent
normal flow filtration through the hollow fiber membrane module
comprising the porous hollow fibers having anion-exchange groups
according to the present embodiment, resulting in increased passage
pressure.
[0117] In performing the clarification treatment by cross-flow
filtration using the microfiltration hollow fiber membrane module,
the linear velocity of the animal cell culture when flowing on the
inner surface of the porous hollow fiber is preferably from 0.05
m/s to 5.0 m/s, more preferably from 0.1 m/s to 2.0 m/s.
[0118] The filtering liquid passage pressure from the inside of the
porous hollow fiber to the outside thereof is preferably from 0.01
MPa to 0.5 MPa, more preferably from 0.05 MPa to 0.2 MPa.
[0119] Preferably, the method for purifying the protein according
to the present embodiment further comprises the step of performing
purification using affinity chromatography.
[0120] The purification using affinity chromatography can be
carried out by a heretofore known method and can be performed using
a protein A affinity column.
[0121] In this process, the clarified solution containing a target
protein obtained by filtration using the porous membrane having
anion-exchange groups is first applied to the protein A affinity
column for the selective adsorption of the target protein.
[0122] The dissolved impurity proteins contained in the clarified
solution are removed by outflow without adsorption to the protein A
affinity column. Next, the column is washed with a buffer having
the same pH as that of the clarified solution to remove impurities
left in the column, and the adsorbed target protein can be then
eluted and recovered using an acidic eluent to provide a solution
of a purified target protein from which most of the impurity
proteins have been further removed.
[0123] Even with impurity proteins being dissolved in a solution to
be applied containing the target protein, the target protein can be
purified using the protein A affinity column.
[0124] In the step of performing purification by affinity
chromatography, a large amount of dissolved impurity proteins
results in the presence of aggregated impurities in the recovered
solution from the column, or reduces the life of the column because
loads are applied to the column. Before application to the column,
dissolved impurity proteins are preferably removed as much as
possible in advance using the porous hollow fiber membrane having
anion-exchange groups; thus, it is preferred to filter them using
the porous membrane used in the present embodiment.
[0125] Examples of the affinity chromatography column used in the
present embodiment can include columns having protein A, heparin,
Con A, Red (Procion Red HE-3B), Blue (Cibacron Blue 3GA), lysine,
arginine, benzamidine, and the like as ligands; when the target
protein is an antibody, the protein A affinity chromatography
column is often used.
[0126] The method for applying the clarified solution containing a
target protein obtained by filtration using the porous membrane
having anion-exchange groups to the affinity chromatography column
for adsorption can be carried out by a method which involves, for
example, supplying the clarified solution to the column after
equilibrating using a pump or hydrostatic pressure.
[0127] The washing of the affinity chromatography column to which
the target protein is adsorbed is not particularly limited provided
that it uses a buffer having the same pH as that of the clarified
solution containing the target protein; examples of the buffer can
include a buffer such as a sodium-phosphate buffer.
[0128] The washing with the buffer can be carried out by passing a
buffer having a volume of on the order of 2 to 10 times the column
volume through the column.
[0129] The recovery of the target protein from the affinity
chromatography column to which the target protein is adsorbed is
not particularly limited provided that it uses a buffer of an
acidic solution as an elution buffer; examples of the buffer can
include acidic solutions such as a pH 3 to 4 citrate-NAOH
buffer.
[0130] The recovery of the target protein using the acidic solution
can be carried out by feeding the elution buffer at a volume of 2
to 10 times the column volume through the column.
[0131] The previously observed generation of aggregated impurities
occurring in the recovery of a protein with an acidic solution can
be suppressed by filtering an animal cell culture containing a
target protein using the porous membrane having anion-exchange
groups to provide a clear solution of the target protein and then
purifying the clear solution of the target protein by affinity
chromatography to recover the target protein. A mixture liquid
containing a target protein can be filtered using the porous
membrane employed in the present embodiment to reduce loads on the
affinity column as well as to recover a highly purified target
protein.
EXAMPLES
[0132] The present embodiment is more specifically described below
based on Examples and Comparative Examples. However, the present
embodiment is not intended to be limited only to these Examples.
Estimation and measurement methods used in the present embodiment
are as follows.
[0133] (1) Bubble Point Method
[0134] The bubble point method was used to measure the maximum pore
size of a porous hollow fiber. One end of a porous hollow fiber 8
cm in length was blocked, and the other end was connected to a
nitrogen gas supply line via a pressure indicator. Nitrogen gas was
supplied in the connected state to replace the inside of the line
with nitrogen, followed by immersing the porous hollow fiber in
ethanol. At this time, the immersion was carried out in a state
only slightly pressurized with nitrogen to avoid backflow of
ethanol into the line. The pressure of nitrogen gas was slowly
increased in a state in which the porous hollow fiber was immersed,
and the pressure (p) at which bubbles of nitrogen gas started to
stably occur from the porous hollow fiber was recorded.
[0135] The maximum pore size of the porous hollow fiber was
calculated by
d=C.gamma./p (I)
wherein d is the maximum pore size and .gamma. is the surface
tension of the interface between ethanol and air.
[0136] Here, C is a constant. Because the immersion fluid is
ethanol, C=0.632 (kg/cm); the maximum pore size d (.mu.m) was
determined by substituting p (kg/cm.sup.2) into the above
equation.
[0137] (2) Bacteria Challenge Test
[0138] A bacteria challenge test was carried out to determine the
sterilization performance of minimodules as prepared in Examples 1,
2 and 6.
[0139] A 100 ppm sodium hypochlorite aqueous solution (200 mL) was
fed to, and simultaneously passed through, the inside of the module
in the manner of cross-flow for sterilization, and then extra pure
water (500 mL) was similarly fed while passing therethrough for
washing. Using Pseudomonas dimunuta as an indicator bacterium for a
pore size of 0.22 .mu.m, 200 mL of an aqueous solution containing a
concentration of 10.sup.6 cells/mL of the indicator bacteria was
fed to, and simultaneously passed through, the inside thereof also
in the manner of cross-flow. The amount of Pseudomonas dimunuta
contained in the filtrate was measured, demonstrating that it was
10 cells/100 mL or less and the LRV (logarithmic reduction value)
of the 0.22 .mu.m indicator bacterium for the module was 7 or less.
From this, it was determined that this module enabled almost
complete sterilization.
[0140] (3) Preparation of Model Fluid for Animal Cell Culture
Containing Target Protein
[0141] A serum-free CHO cell culture containing no antibody protein
was provided which had a salt concentration of about 0.9% by mass
(0.15 M), a protein concentration of about 1 g/L and a cell density
of 3.0.times.10.sup.7/mL, and .gamma.-globulin (from SIGMA) as a
target protein was added thereto to a concentration of 1 g/L to
prepare a model fluid for an animal cell culture containing the
target protein and being not clarified.
[0142] In this model fluid, .gamma.-globulin is a target protein,
and all proteins derived from the cell culture containing a turbid
component are impurity proteins.
[0143] (4) Analysis of Protein by SDS-PAGE
[0144] SDS-PAGE was used to analyze proteins in the culture having
passed through each of the minimodules prepared in Examples 1, 2
and 6. The filtrate (10 .mu.L) to be used for analysis was mixed
with an equal amount of a sample treatment solution (Tris SDS
sample treatment solution or Tris SDS.beta. ME sample treatment
solution from Daiichi Pure Chemical Co., Ltd.), which was
heat-treated at 100.degree. C. for 5 minutes. The resultant sample
was applied to a gel plate for electrophoresis (Multigel II Mini
from Daiichi Pure Chemical Co., Ltd.) in an amount of 10 .mu.L per
well using a micropipette, which was inserted into an
electrophoresis tank (EasySeparator.TM. from Wako Pure Chemical
Industries Ltd.) filled with a phoresis buffer (SDS-Tris-glycin
phoresis buffer from Daiichi Pure Chemical Co., Ltd. was used after
1/10 dilution). Phoresis was performed at a constant current of 30
mA for one hour to separate proteins in the filtrate. The gel plate
after phoresis was stained using a staining reagent (InstantBlue
from Funakoshi Corporation or 2D-Silver Staining Reagent-II from
Daiichi Pure Chemical Co., Ltd.) to confirm protein bands.
[0145] (5) Quantification of HCP
[0146] The fluid to be estimated was applied to a 96-well plate in
CHO Host Cell Protein ELISA Kit from Cygnus Technologies Inc. to
quantify HCP as an impurity using Ultrospec Visible Plate Reader
II96 from GE Healthcare Bioscience Corporation as a plate
reader.
[0147] (6) Quantification of DNA
[0148] The fluid to be estimated was treated using Quant-iT.TM.
dsDNA HS Assay Kit from Invitrogen Corporation, followed by
quantifying DNA as an impurity employing Qubit.TM. fluorometer.
[0149] (7) Adsorption Recovery of Target Protein by Protein a
Affinity Column
[0150] Clear cultures obtained by passage through the minimodules
prepared in Examples 1, 2 and 6 were each used as a sample fluid to
estimate the adsorption recovery of a target protein therefrom by a
protein A affinity column. The protein A affinity column (HiTrap
Protein A HP 1 mL from GE Healthcare Bioscience Corporation) was
connected to a commercially available chromatography system
(AKTAexplorer100 from GE Healthcare Bioscience Corporation) for
fluid-feeding. The buffer for equilibrating the column and washing
after sample application used was 20 mM sodium-phosphate (pH 7.0),
and the buffer for eluting the adsorbed target protein used was 0.1
M citric acid-NaOH (pH 3.0). In the adsorption estimation, the
following five steps were carried out as one cycle to estimate the
adsorption amount of a target protein: 1) pass 10 mL of the buffer
for equilibration through the column; 2) pass a clear fluid
containing the target protein through the column to adsorb the
target protein thereto; 3) pass 10 mL of a buffer to the column to
wash impurity proteins therein; 4) pass 10 mL of the elution buffer
therethrough to elute and recover the adsorbed target protein; and
5) pass 10 mL of the buffer through the column to rewash it. At
this time, the passage was performed at a flow rate of 1.0
mL/minute in each step. These cycles were continuously performed in
the repeated estimation of the adsorption recovery of the target
protein. The recovered solution was diluted by 1/10 and measured
for ultraviolet absorbance at 280 nm to determine the amount of
adsorption from the calibration curve obtained by ultraviolet
absorbance measurements at known concentrations in advance.
[0151] (8) Microfiltration Hollow Fiber Membrane Module for
Clarification
[0152] A minimodule for clarification was prepared by bundling 11
polysulfonate microfiltration hollow fibers having an outer
diameter of 2.0 mm, an inner diameter of 1.4 mm and a maximum pore
size of 0.4 .mu.m and fixing both ends thereof to a
polycarbonate-made module case using an epoxy potting agent so that
the hollow parts of the hollow fibers were not blocked. The
resultant minimodule had an inner diameter of 0.9 cm, a length of
about 8 cm, and an effective membrane area of the hollow fiber
inner surface in the module of 39 cm.sup.2.
[0153] (9) Measurement of Dynamic Adsorption Volume
[0154] A BSA solution in which BSA was dissolved at a concentration
of 1 g/L in a 20 mmol/L Tris-HCl (pH 8.0) buffer was used to pass
the BSA solution through the estimation module until the start of
its breakthrough. Here, from the concentration (Q) of a BSA
solution, the volume (V.sub.B) of the BSA solution passed through
by the time the breakthrough of the estimation module occurred, and
the volume (V.sub.B) of an ion-exchange membrane according to each
Example in the estimation module, the dynamic adsorption volume was
calculated based on the equation (II) below:
A=Q.times.V.sub.B/V.sub.M (II)
[0155] The volume of an ion-exchange membrane is the volume of that
from which the hollow portion thereof is subtracted. The
breakthrough refers to a time point at which the concentration of
BSA in the filtrate has exceeded 0.1 g/L, i.e. 10% of the
concentration of the BSA solution supplied. The solution was passed
from the inside of the hollow ion-exchange membrane toward the
outside thereof in the estimation module. The dynamic adsorption
volumes of the anion-exchange membranes prepared in Examples 1, 2
and 6 were 70 mg/mL, 35 mg/mL and 75 mg/mL, respectively, as
measured by this method.
Example 1
[0156] (i) Introduction of Graft Chains into Porous Hollow Fiber
Membrane
[0157] In an airtight container was placed porous polyethylene
hollow fibers having an outer diameter of 3.0 mm, an inner diameter
of 2.0 mm and a maximum pore size measured by the bubble point
method described in the above (1) of 0.3 .mu.m, and the air in the
container was replaced with nitrogen. Then, the hollow fibers were
irradiated with 200 kGy of .gamma.-ray to generate radical while
cooling the container from the outside thereof with dry ice. The
resultant radical-containing porous polyethylene hollow fibers were
placed in a glass reaction tube, and the oxygen in the reaction
tube was removed by depressurization to 200 Pa or less. A reaction
solution containing 3 parts by volume of glycidyl methacrylate
(GMA) and 97 parts by volume of methanol, adjusted at 40.degree.
C., was injected thereinto in an amount of 20 parts by mass to the
hollow fibers and then allowed to stand in a closed state for 12
minutes to subject to graft polymerization reaction to introduce
graft chains into the porous hollow fibers.
[0158] The mixed solution was bubbled with nitrogen in advance to
replace the oxygen in the mixed solution with nitrogen.
[0159] After graft polymerization reaction, the reaction solution
in the reaction tube was discarded. The hollow fibers were then
washed by placing dimethylsulfoxide in the reaction tube to remove
the residual glycidyl methacrylate, its oligomers and graft chains
not fixed to the porous hollow fiber membranes.
[0160] After discarding the wash solution, washing was further
carried out twice by placing dimethylsulfoxide therein. Using
methanol, washing was similarly performed three times. When the
hollow fibers after washing were dried and weighed, the weight of
the porous hollow fiber membranes was 138% of that before
introduction of graft chains and the graft rate defined as the
ratio of the graft chain weight to the base material weight was
38%.
[0161] This is equivalent to the ratio of the number of moles of
the introduced GMA (molecular weight: 142) to the number of moles
of CH.sub.2 groups (molecular weight: 14) as the skeleton unit of
the base material polyethylene being 3.75% as calculated by the
following equation (III).
Mole Number % of Introduced GMA=(Graft Rate/142)/(100/14).times.100
(III)
[0162] The ratio of the number of moles of the polyethylene
skeleton unit CH.sub.2 group in the porous hollow fiber membrane
after graft reaction and the number of moles of ester groups (COO
groups) characteristic of GMA constituting the graft chain was
measured by the solid NMR method.
[0163] Using 0.5 g of the powdered sample obtained by freezing and
grinding the hollow fibers after graft reaction, the measurement
was carried out at room temperature under conditions of a waiting
time of 100 s and an integration of 1,000 times by the quantitative
determination mode of the High Power Decoupling (HPDEC) method
employing DSX400 from Bruker Biospin Corp. and the nuclide
.sup.13C.
[0164] The ratio of the peak area corresponding to the ester group
and the peak area corresponding to the CH.sub.2 group in the
resultant NMR spectrum is equivalent to the ratio of the numbers of
moles of GMA and CH.sub.2 groups; thus, when the ratio of the
number of moles of the introduced GMA to the number of moles of
CH.sub.2 groups was calculated from the measurement results, it was
found to be 3.8%. This is equivalent to a graft rate of 38.5%; it
was shown that the graft rate was obtained by measuring the sample
after graft reaction by the solid NMR method.
[0165] (ii) Fixation of Anion-Exchange Group (Tertiary Amino Group)
to Graft Chain
[0166] A dried hollow fiber into which graft chains were introduced
was swollen by immersion in methanol for 10 minutes or more and
then immersed in purified water for replacement with water. The
hollow fiber after graft reaction (20 parts by mass) was placed in
a glass reaction tube containing a reaction solution containing a
mixed solution of 50 parts by volume of diethylamine and 50 parts
by volume of purified water, and adjusted to 30.degree. C. The
porous hollow fiber into which graft chains were introduced was
inserted thereinto and allowed to stand for 210 minutes to replace
the epoxy groups of the graft chains with diethylamino groups to
provide a porous hollow fiber membrane having diethylamino groups
as anion-exchange groups.
[0167] The resultant porous hollow fiber membrane has an outer
diameter of 3.3 mm and an inner diameter of 2.1 mm, and 80% of the
epoxy groups of the graft chain in the porous hollow fiber membrane
were replaced with diethylamino groups.
[0168] A replacement ratio T was calculated by the following
equation (IV), assuming that T is the ratio of the number (N.sub.1)
of moles of epoxy groups replaced with diethylamino groups to the
number (N.sub.2) of moles of epoxy groups.
T=100.times.N.sub.1/N.sub.2=100.times.{(w.sub.2-w.sub.1)/M.sub.1}/{w.sub-
.1(dg/(dg+100))/M.sub.2} (IV)
[0169] In this equation, M.sub.1 is the molecular weight of
diethylammonium (73.14); w.sub.1 is the weight of the porous hollow
fiber membrane after graft polymerization reaction; w.sub.2 is the
weight of the porous hollow fiber membrane after replacement
reaction with the dimethylamino group; dg is the graft rate; and
M.sub.2 is the molecular weight of GMA (142).
[0170] In the same way as described above using the solid NMR
method, the ratio of the number of moles of ester groups
characteristic of GMA to the number of moles of CH.sub.2 groups as
polyethylene skeleton units in the porous hollow fiber membrane
into which diethylamino groups were introduced was measured. As a
result, it was found to be 3.75%. This was equivalent to the graft
rate of 38%. From this result, it was determined that there was no
change in the graft rate due to the introduction of diethylamino
groups.
[0171] (iii) Preparation of Anion-Exchange Membrane Module
[0172] A hollow fiber module for the anion-exchange membrane was
prepared by bundling three porous hollow fibers having diethylamino
groups as anion-exchange groups and fixing both ends thereof to a
polysulfonate-made module case using an epoxy potting agent so that
the hollow parts of the porous hollow fibers were not blocked.
[0173] The resultant module had an inner diameter of 0.9 cm, a
length of about 3.3 cm, a module inner volume of about 2 mL, an
effective volume of the porous hollow fibers in the module of 0.85
mL, and a volume of only porous hollow fiber membranes from which
hollow parts were subtracted of 0.54 mL.
Example 2
[0174] (iv) Fixation of Anion-Exchange Groups (Quaternary Amino
Groups) to Graft Chain
[0175] In the same way as in Example 1, a dried hollow fiber into
which graft chains were introduced was swollen by immersion in
methanol for 10 minutes or more, and then immersed in purified
water for replacement with water. A mixed solution containing 50
parts by volume of purified water and 50 parts by volume of
dimethylsulfoxide was provided, and trimethylammonium chloride was
added to a concentration of 0.5M to the mixed solution, which was
then mixed to provide a uniform reaction solution. The reaction
solution was placed in a glass reaction tube in an amount of 20
parts by mass to the hollow fiber after graft reaction, and
adjusted to 60.degree. C. The porous hollow fiber into which graft
chains were introduced was then inserted thereinto and allowed to
stand for 200 minutes to replace the epoxy groups of the graft
chains with trimethylamino groups to provide a porous hollow fiber
membrane having trimethylamino groups as anion-exchange groups.
[0176] The resultant porous hollow fiber membrane had an outer
diameter of 3.2 mm and an inner diameter of 2.1 mm, and 80% of the
epoxy groups of the graft chain were replaced with trimethylamino
groups. The replacement ratio was calculated in the same way as for
the diethylamino group by substituting the molecular weight of
trimethylammonium chloride (95.57) into M.sub.1 in the above
equation (IV).
[0177] (v) Preparation of Anion-Exchange Membrane Module
[0178] A porous hollow fiber membrane module having trimethylamino
groups as anion-exchange groups was also prepared in the same way
as in Example 1. A module was obtained which has an effective
volume of the porous hollow fibers of 0.75 mL and a volume of only
porous hollow fiber membranes from which hollow parts were
subtracted of 0.46 mL.
Example 3
[0179] NaCl was added to 20 mM Tris-HCl (pH 8.0) to a concentration
of 0.17 M to prepare a metal salt-containing buffer. In this buffer
dissolved were BSA (pI: 5.6) and .gamma.-globulin at a
concentration of 1 g/L each to prepare a mixed solution of
proteins. This mixed solution of proteins was passed through the
anion-exchange membrane module having diethylamino groups prepared
in Example 1 at a flow rate of 2 mL/minute, and the filtrate was
collected in the form of 5 mL each of fractions. SDS-PAGE was used
to analyze proteins in the fractions having passed through the
module. The filtrate (10 .mu.L) to be used for the analysis was
mixed with an equal amount of a sample treatment solution (Tris SDS
sample treatment solution from Daiichi Pure Chemical Co., Ltd.),
which was then heat-treated at 100.degree. C. for 5 minutes. The
resultant sample was applied to a gel plate for electrophoresis
(Multigel II Mini from Daiichi Pure Chemical Co., Ltd.) in an
amount of 10 .mu.L per well using a micropipette, which was
inserted into an electrophoresis tank (EasySeparator.TM. from Wako
Pure Chemical Industries Ltd.) filled with a phoresis buffer
(SDS-Tris-glycin phoresis buffer from Daiichi Pure Chemical Co.,
Ltd. was used after 1/10 dilution) to perform phoresis at a
constant current of 30 mA for one hour to separate the proteins in
the filtrate. The gel plate after phoresis was stained using a
staining reagent (InstantBlue from Funakoshi Corporation).
[0180] The results obtained are shown in FIG. 1. Lane 1 is BSA;
lane 2 is .gamma.-globulin alone; lane 3 is a mixture liquid of BSA
and .gamma.-globulin; lanes 5 to 12 are fractions of the filtrate;
and lane 13 is an eluate of adsorbate. There was the almost
exclusive presence of .gamma.-globulin in the filtrate reaching up
to 20 mL (lanes 5 to 8); BSA was totally adsorbed. After
adsorption, elution was carried out using a salt solution in which
1 M NaCl was dissolved in a buffer. There was the exclusive
presence of BSA in the eluate (lane 13): only BSA was selectively
adsorbed to the module and .gamma.-globulin was non-adsorbed. The
results shown in FIG. 1 indicated that the method of the present
embodiment is effective for separating a target protein from a
salt-containing solution.
Example 4
[0181] A serum-free CHO cell culture containing 0.5 g/L of
.gamma.-globulin as a target protein was subjected to dead end
filtration using a microfiltration hollow fiber membrane module to
provide a clarified supernatant. This supernatant fluid (54 mL
(equivalent to 100 times the hollow fiber membrane volume)) was
passed at a flow rate of 2 mL/minute through the anion-exchange
membrane module having diethylamino groups prepared in Example 1 to
collect all the filtrate. SDS-PAGE was used to estimate proteins in
the fractions having passed through the module. The filtrate (10
.mu.L) to be used for the analysis was mixed with an equal amount
of a sample treatment solution (Tris SDS.beta. ME sample treatment
solution from Daiichi Pure Chemical Co., Ltd.), which was then
heat-treated under reduction at 100.degree. C. for 5 minutes. The
resultant sample was applied to a gel plate for electrophoresis
(Multigel II Mini from Daiichi Pure Chemical Co., Ltd.) in an
amount of 10 .mu.L per well using a micropipette, which was
inserted into an electrophoresis tank (EasySeparator.TM. from Wako
Pure Chemical Industries Ltd.) filled with a phoresis buffer
(SDS-Tris-glycin phoresis buffer from Daiichi Pure Chemical Co.,
Ltd. was used after 1/10 dilution). Phoresis was performed at a
constant current of 30 mA for one hour to separate proteins in the
filtrate.
[0182] The gel plate after phoresis was stained using a staining
reagent (2D-Silver Staining Reagent-II from Daiichi Pure Chemical
Co., Ltd.).
[0183] The results obtained are shown in FIG. 2. Lane 2 is
.gamma.-globulin alone; lane 3 is a serum-free CHO cell culture
supernatant alone; lanes 4 and 9 are serum-free CHO cell culture
supernatants containing .gamma.-globulin; and lane 10 is the
filtrate for this estimation. It was shown that passage through the
anion-exchange membrane module removed much impurities in the
salt-containing cell culture to provide a purified target protein.
The concentrations of HCP and DNA as typical impurities were 346
.mu.g/mL and 7,200 ng/mL, respectively in the cell culture
supernatant before passage through the anion-exchange membrane
module and considerably decreased to 39 .mu.g/mL and 52 ng/mL,
respectively in the filtrate, indicating that this method was
excellent in the property of removing these protein impurities
present in a dissolved state.
Example 5
[0184] Using the anion-exchange membrane module containing
trimethylamino groups prepared in Example 2, in the same way as in
Example 4, the same supernatant of the serum-free CHO cell culture
containing 0.5 g/L of .gamma.-globulin as a target protein was
passed therethrough in an amount of 46 mL equivalent to 100 times
the membrane volume to remove impurities. Estimation using SDS-PAGE
was performed in the same way as in Example 4. The results are
shown as in Example 4 in FIG. 2. Lane 12 is the filtrate for this
estimation. It was shown that passage through the anion-exchange
membrane module removed much impurities in the salt-containing cell
culture to provide a purified target protein. The concentrations of
HCP and DNA as typical impurities were 346 .mu.g/mL and 7,200
ng/mL, respectively in the cell culture supernatant before passage
through the anion-exchange membrane module and considerably
decreased to 73.8 .mu.g/mL and 32.8 ng/mL, respectively in the
filtrate, indicating that this method was excellent in the property
of removing these protein impurities present in a dissolved
state.
Example 6
[0185] The introduction of graft chains and the fixation of
diethylamino groups to the graft chains were carried out in the
same way as in Example 1 except for the composition of the reaction
solution containing 10 parts by volume of glycidyl methacrylate and
90 parts by volume of methanol to provide a porous hollow fiber
membrane containing diethylamino groups as anion-exchange groups
and having a graft rate of 140%. As the result of calculation using
the equation (IV), 93% of the epoxy groups of the graft chain were
replaced by diethylamino groups. The resultant porous hollow fiber
membrane had an outer diameter of 4.0 mm and an inner diameter of
2.5 mm. This porous hollow fiber membrane was used to prepare a
hollow fiber module having an effective volume of the porous hollow
fibers of 1.53 mL and a volume of only porous hollow fiber
membranes from which hollow parts were subtracted of 0.92 mL in the
same way as in Example 1.
[0186] Lane 11 in FIG. 2 shows the result of the estimation
performed in the same way as in Example 4 using the hollow fiber
module. It was shown that passage through the module removed many
impurities in the salt-containing cell culture to provide a
purified target protein. The concentrations of HCP and DNA as
typical impurities were 346 .mu.g/mL and 7,200 ng/mL, respectively
in the cell culture supernatant before passage through the
anion-exchange membrane module and considerably decreased to 42.6
.mu.g/mL and 32.8 ng/mL, respectively in the filtrate, indicating
that this method is excellent in the property of removing these
impurities present in a dissolved state.
Comparative Example 1
[0187] Using 3 types of commercially available membranes having
anion-exchange groups, i.e. SartobindQ MA75 (membrane volume: 2.06
mL) from Sartorius AG, MustangQ Acrodisc (membrane volume: 0.18 mL)
from Pall Corporation, and BioCap 25 Filter 90ZA (membrane volume:
5 mL or more) from Cuno Incorporated, a supernatant of a serum-free
CHO cell culture containing 0.5 g/L of .gamma.-globulin as a target
protein was passed therethrough in volumes equivalent to 100 times
the respective membrane volumes in the same way as in Example 4 to
remove impurities. Estimation using SDS-PAGE was performed in the
same way as in Example 4. The results are shown as in Example 4 in
FIG. 2. Lane 6 was a filtrate of SartobindQ MA75; lane 7 was a
filtrate of MustangQ Acrodisc'; and lane 8 was a filtrate of BioCap
25 Filter 90ZA. These results showed that the use of the
commercially available membranes having anion-exchange groups did
not allow the effective removal of impurities from the
salt-containing cell culture. The concentrations of HCP and DNA in
the cell culture supernatant before passage through the
anion-exchange membrane module were 346 .mu.g/mL and 7,200 ng/mL,
respectively, whereas those after the passage were 269 .mu.g/mL and
492 ng/mL, respectively, for SartobindQ MA75, 248 .mu.g/mL and
3,916 ng/mL, respectively, for MustangQ Acrodisc, and 310 .mu.g/mL
and 7,600 ng/mL, respectively, for BioCap 25 Filter 90ZA. These
results indicated that this method was inferior in the property of
removing impurities to the method of the present embodiment.
Example 7
[0188] Protein A Purification of Solution Having Passed Through DEA
Anion-Exchange Membrane
[0189] The solution having passed through a hollow fiber membrane
module having diethylamino groups obtained in Example 4 was used as
a cell culture from which dissolved impurities were removed to
purify .gamma.-globulin therefrom by adsorption and recovery using
a protein A affinity column. According to the method described in
the above (7), 30 mL of the filtrate of Example 4 was passed
through the protein A affinity column to adsorb .gamma.-globulin,
followed by purification and recovery using an elution buffer. The
recovered solution was diluted by 1/10 and measured for ultraviolet
absorption intensity to estimate the recovery amount. As a result,
14.2 mg of .gamma.-globulin was contained in the recovered
solution; the recovery rate was found to be 95%. The concentrations
of HCP and DNA contained in the recovered solution were 0.162
.mu.g/mL and 11.8 ng/mL, respectively; thus, the method of the
present embodiment was shown to have a high recovery rate and be
excellent in the property of removing impurities.
Example 8
[0190] Protein A Purification of Solution Having Passed Through TMA
Anion-Exchange Membrane
[0191] The solution having passed through a hollow fiber membrane
module having trimethylamino groups obtained in Example 5 was used
as a cell culture from which dissolved impurities were removed to
purify .gamma.-globulin therefrom by adsorption and recovery using
a protein A affinity column as described in Example 7. According to
the method described in the above (7), 30 mL of the filtrate of
Example 5 was passed through the protein A affinity column to
adsorb .gamma.-globulin, followed by purification and recovery
using an elution buffer. The recovered solution was diluted by 1/10
and measured for ultraviolet absorption intensity to estimate the
recovery amount. As a result, 13.8 mg of .gamma.-globulin was
contained in the recovered solution; the recovery rate was found to
be 92%. The concentrations of HCP and DNA contained in the
recovered solution were 0.289 .mu.g/mL and 19.1 ng/mL,
respectively; thus, the method of the present embodiment was shown
to have a high recovery rate and be excellent in the property of
removing impurities.
Example 9
[0192] Protein A Purification of Solution Having Passed Through DEA
Anion-Exchange Membrane (2)
[0193] The solution having passed through a hollow fiber membrane
module having diethylamino groups obtained in Example 6 was used as
a cell culture from which dissolved impurities were removed to
purify 7-globulin therefrom by adsorption and recovery using a
protein A affinity column. According to the method described in the
above (7), 30 mL of the filtrate of Example 6 was passed through
the protein A affinity column to adsorb .gamma.-globulin, followed
by purification and recovery using an elution buffer. The recovered
solution was diluted by 1/10 and measured for ultraviolet
absorption intensity to estimate the recovery amount. As a result,
13.9 mg of .gamma.-globulin was contained in the recovered
solution; the recovery rate was found to be 93%. The concentrations
of HCP and DNA contained in the recovered solution were 0.186
.mu.g/mL and 13.8 ng/mL, respectively; thus, the method of the
present embodiment was shown to have a high recovery rate and be
excellent in the property of removing impurities.
Comparative Example 2
[0194] Purification was estimated as described in Example 7, using
the cell culture containing 0.5 mg/mL of .gamma.-globulin,
subjected only to clarification with a microfiltration membrane,
obtained in Example 4 as a cell culture from which dissolved
impurities were not removed. The solution recovered from the
protein A affinity column was diluted by 1/10 and measured for
ultraviolet absorption intensity to estimate the recovery amount.
As a result, 14.5 mg of .gamma.-globulin was contained in the
recovered solution; the recovery rate was found to be 97%. The
concentrations of HCP and DNA contained in the recovered solution
were 2.93 .mu.g/mL and 63.2 ng/mL, respectively. The rate of
recovery of the target protein was high, but much impurities were
contained in the recovered solution in comparison to the case where
the impurity removal was not performed using the hollow fiber
membrane module having anion-exchange groups according to the
present embodiment.
Comparative Example 3
[0195] The solutions having passed through commercially available
anion-exchange membranes obtained in Comparative Example 1 were
used to purify .gamma.-globulin therefrom by adsorption and
recovery using a protein A affinity column as described in Example
7. The solutions recovered from the protein A affinity column were
diluted by 1/10 and measured for ultraviolet absorption intensity
to estimate the rate of recovery from the solutions having passed
through the respective anion-exchange membranes. As a result, the
recovery rate was high for each of the membranes, i.e. 97% for
SartobindQ MA75, 93% for MustangQ Acrodisc, and 95% for BioCap 25
Filter 90ZA. The concentrations of HCP and DNA in the recovered
solutions were 3.83 .mu.g/mL and 32.6 ng/mL, respectively for
SartobindQ MA, 2.54 .mu.g/mL and 46 ng/mL, respectively for
MustangQ Acrodisc, and 3.27 .mu.g/mL and 88.2 ng/mL, respectively
for BioCap 25 Filter 90ZA. Thus, this method was inferior in the
property of removing impurities to the method of the present
embodiment and only provided almost the same purification degree as
for Comparative Example 2 in which no passage treatment using an
anion-exchange membrane was carried out.
[0196] The concentrations of HCP and DNA obtained in Examples 4 to
9 and Comparative Examples 1 to 3 were shown in Table 1. The
results of Table 1 showed that the excellent property of removing
impurities was obtained in each of Examples 4 to 9.
TABLE-US-00001 TABLE 1 Solution recovered Filtrate from protein A
column Treatment HCP DNA HCP DNA method (.mu.g/mL) (ng/mL)
(.mu.g/mL) (ng/mL) Examples Diethylamino 39 52 0.162 11.8 4 and 7
group module Examples Trimethyl- 73.8 32.8 0.289 19.1 5 and 8 amino
group module Examples Diethylamino 42.6 32.8 0.186 13.8 6 and 9
group module Comparative Untreated 346 7200 2.93 63.2 Example 2
Comparative SartobindQ 269 492 3.83 32.6 Examples MustangQ 248 3916
2.54 46 1 and 3 BioCap 90ZA 310 7600 3.27 88.2
Example 10
[0197] The cross-flow filtration estimation apparatus shown in FIG.
3 was set up; 300 mL of the cell culture containing a target
protein prepared in the above (3) was placed in a cell culture tank
1 and, using a perister pump 2, the culture was passed at a linear
velocity of 0.5 m/s through a hollow fiber membrane module
comprising porous hollow fiber membranes having diethylamino groups
as anion-exchange groups as prepared in Example 1 (hereinafter
referred to as an anion-exchange hollow fiber module) 4 for
cross-flow filtration. Here, internal pressure type filtration was
adopted by which the filtrate moved from the inside of the porous
hollow fiber toward the outside thereof. The passage pressure was
regulated by a flow control cock 6 so that the average of the
pressures measured with a pressure indicator 3 (at the entry side
of the module) and a pressure indicator 5 (at the exit side of the
module) was 0.1 MPa, and 30 mL each of the solutions having passed
through the module were collected so as to provide a total amount
of 210 mL, followed by final collection of 40 mL of the solution
and thereby the cross-flow filtration was performed until the total
amount of the filtrate reached 250 mL. During the filtration, the
average flux was 21 L/m.sup.2/hr (21 LMH). All of the filtrates
collected were found to be clear by visual observation.
[0198] FIG. 4 shows the results of the SDS-PAGE of the filtrates
and eluates performed according to the method described in the
above (4). For all of the filtrates collected, the target protein
straightly passed therethrough into the filtrate at the same
concentration as that of the stock solution, and it was also shown
that impurity proteins were evidently adsorbed by the
anion-exchange hollow fiber membrane module. The anion-exchange
hollow fiber membrane module after cross-flow filtration estimation
was back washed with the buffer of 20 mM Tris-HCl (pH 8.0) to
remove a turbid component deposited inside the porous hollow fiber,
followed by eluting the adsorbed components using a buffer solution
containing 1 M NaCl to analyze the eluate by SDS-PAGE. It was shown
that a large amount of impurity proteins were adsorbed to the
anion-exchange hollow fiber membrane module, while the target
protein was not adsorbed to the module.
[0199] All of the clear cultures obtained by the passage placed in
the same container to make a uniform solution, which was then
passed through an unused protein A affinity column to estimate the
amount of the target protein adsorbed to the protein A affinity
column according to the method described in the above (7). Here,
the amount of the culture passed through the column was 10 mL. In
the estimation, the cycle of adsorption estimation described in the
above (7) was repeated 10 times to measure the change of the
adsorption amount. As a result, the adsorption amount in the first
cycle was 9.8 mg and the adsorption amount in the tenth cycle was
9.8 mg, showing no decrease in the adsorption amount during the 10
times repetition. When the eluates were visually observed, the
eluates were found to be clear in all of the estimations and no
aggregates were identified. After the end of estimation, when the
protein A affinity column was disassembled to observe inside beads
under an optical microscope, no adhesion of impurities to the beads
was seen. From these results, it was determined that the
clarification of a cell culture and the removal of dissolved
impurity proteins can be simultaneously carried out using the
anion-exchange hollow fiber membrane module, which can greatly
reduce loads on a protein A affinity column.
Comparative Example 4
[0200] Porous polyethylene hollow fibers comprising no
anion-exchange groups, having an inner diameter of 3.0 mm and an
outer diameter of 2.0 mm were used to prepare a minimodule by the
same method as for the anion-exchange hollow fiber membrane module
described in Example 1. Using this minimodule, a filtrate of a cell
culture containing a target protein was collected in the same way
as in Example 3. The resultant filtrate was clear by visual
observation; however, when it was analyzed by SDS-PAGE according to
the method described in the above (4), it was determined that an
extremely large amount of impurity proteins were dissolved therein
compared to the filtrate of Example 10. In addition, when the
minimodule after estimation was back washed with a buffer as
described in Example 10 and then eluted with a 1 M NaCl buffer
solution, no impurity protein or target protein was shown to be
adsorbed to the minimodule.
[0201] The clear culture obtained by the passage was passed through
an unused protein A affinity column as described in Example 10 to
estimate the adsorption amount of the target protein. When the
estimation was repeated 10 times without exchanging the column to
measure the change of the adsorption amount, the adsorption amount
in the first estimation was 9.8 mg; however, the adsorption amount
decreased with increasing number of repeats, resulting in the
adsorption amount in the tenth estimation being 9.4 mg. When the
eluate was visually observed, white turbidity, although slight, due
to aggregates was identified. After the end of estimation, when the
protein A affinity column was disassembled to observe inside beads
under an optical microscope, adhesion of aggregated impurities to
the beads was seen. This showed that the removal of dissolved
impurity proteins was important for the reduction of loads on the
protein A affinity column, demonstrating the effect of the porous
hollow fiber membrane having anion-exchange groups.
Example 11
[0202] The cross-flow filtration estimation apparatus shown in FIG.
5 was set up; 300 mL of the cell culture containing a target
protein prepared in the above (3) was placed in a cell culture tank
1 and, using a perister pump 2, the culture was passed in
cross-flow at a linear velocity of 0.5 m/s through the
microfiltration hollow fiber membrane module 7 for clarification
described in the above (8) for filtration. The passage pressure was
regulated by a flow control cock 6 so that the average of the
pressures measured with a pressure indicator 3 (at the entry side
of the module) and a pressure indicator 5 (at the exit side of the
module) was 0.1 MPa. The solution having passed through the
microfiltration hollow fiber membrane module 7 for clarification
was directly fed to the same anion-exchange hollow fiber membrane
module 4 as that of Example 10 to collect 30 mL each of the
filtrates obtained by normal flow filtration. The time taken until
the total filtrate amount reached 250 mL was 160 minutes, and the
average flux was 120 L/m.sup.2/hr (120 LMH). All of the filtrates
collected were found to be clear by visual observation.
[0203] When the filtrates collected according to the method
described in the above (4) were analyzed using SDS-PAGE, for all of
the filtrates collected, the target protein had straightly passed
therethrough into the filtrate at the same concentration as that of
the stock solution, and it was also shown that impurity proteins
had been evidently adsorbed by the anion-exchange hollow fiber
membrane module.
Example 12
[0204] Using the microfiltration hollow fiber membrane module
described in the above (8), the animal cell culture containing a
target protein prepared in the above (3) was filtered to provide
300 mL of a clarified (clear) solution. After completely blocking
the flow control cock 6 in the estimation apparatus of FIG. 3, the
resultant clear solution was placed in the cell culture tank 1 of
FIG. 3 and, using the perister pump 2, fed at a feed flow rate of
10 mL/minute therefrom to the same anion-exchange hollow fiber
membrane module 4 as that of Example 10 to collect 30 mL each of
the filtrates obtained by normal flow filtration. The time taken
until the total filtrate amount reached 250 mL was 25 minutes, and
the average flux was 816 L/m.sup.2/hr (816 LMH). All of the
filtrates collected were found to be clear by visual observation.
From these results, it was determined that even the culture
containing dissolved impurity proteins could be subjected to
microfiltration before performing the filtration thereof using the
anion-exchange hollow fiber membrane module to remove the impurity
proteins at an extremely high processing speed.
[0205] When the filtrates collected according to the method
described in the above (4) were analyzed by SDS-PAGE, for all of
the filtrates collected, the target protein had straightly passed
therethrough into the filtrate at the same concentration as that of
the stock solution, and it was also shown that impurity proteins
had been evidently adsorbed by the anion-exchange hollow fiber
membrane module.
[0206] As described above, dissolved impurity proteins were capable
of being markedly removed in Examples 3 to 12 in which filtration
was performed using the anion-exchange hollow fiber membrane
modules comprising porous hollow fiber membranes having
anion-exchange groups prepared in Examples 1, 2 and 6 compared to
in Comparative Examples 1, 3 and 4 in which the polyethylene hollow
fiber membrane modules given no anion-exchange groups were used, or
to in Comparative Example 2 in which no anion-exchange hollow fiber
membrane module was used; the solutions of the target protein
having a higher purity were also capable of being obtained when
purification was performed using affinity chromatography.
Particularly in Example 10, even when the solution obtained by
performing the filtration was repeatedly applied to the protein A
affinity column, the purifying capability of the column was not
reduced, the adhesion of impurities to the column was prevented,
and loads on the column were sufficiently diminished.
[0207] In addition, filtration was performed using the
microfiltration hollow fiber membrane module before filtration with
the anion-exchange hollow fiber membrane module to enable the
filtration step employing the anion-exchange hollow fiber module to
be performed at an extremely high processing speed. Filtration
using the anion-exchange hollow fiber module could also be
performed by normal flow filtration to markedly remove dissolved
impurity proteins.
[0208] The present application is based on Japanese Patent
Application No. 2007-279406 filed Oct. 26, 2007, the content of
which is incorporated herein by reference.
INDUSTRIAL APPLICABILITY
[0209] The use of the method for purifying the protein according to
the present embodiment enables the clarification of a cell culture
before an affinity chromatography step, conventionally carried out
by the three steps of centrifugation, microfiltration and
sterilization filtration to be completed by the single step of
filtration using a porous hollow fiber membrane having
anion-exchange groups. As a result, reduction in cost is made
possible by simplifying the process and contracting necessary
equipment. Loads in the step of affinity chromatography can also be
considerably reduced to more highly purify a desired protein as
well as to achieve reduced cost of the purification.
* * * * *