U.S. patent application number 14/190650 was filed with the patent office on 2014-08-28 for mixed-mode chromatography membranes.
This patent application is currently assigned to Natrix Separations Inc.. The applicant listed for this patent is Natrix Separations Inc.. Invention is credited to Charles H. Honeyman, Elena N. Komkova.
Application Number | 20140238935 14/190650 |
Document ID | / |
Family ID | 51387082 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140238935 |
Kind Code |
A1 |
Komkova; Elena N. ; et
al. |
August 28, 2014 |
Mixed-Mode Chromatography Membranes
Abstract
Described are composite materials and methods of using them for
mixed-mode chromatography. In certain embodiments, the composite
material comprises a support member, comprising a plurality of
pores extending through the support member; and a multi-functional
cross-linked gel. The multi-functional cross-linked gel possesses
at least two of the following functions or characteristics:
cationic, anionic, hydrophobic, hydrophilic, thiophilic, hydrogen
bond donating, hydrogen bond accepting, pi-pi bond donating, pi-pi
bond accepting, or metal chelating. The composite materials may be
used in the separation or purification of a biological molecule or
biological ion.
Inventors: |
Komkova; Elena N.;
(Hamilton, CA) ; Honeyman; Charles H.; (Toronto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Natrix Separations Inc. |
Burlington |
|
CA |
|
|
Assignee: |
Natrix Separations Inc.
Burlington
CA
|
Family ID: |
51387082 |
Appl. No.: |
14/190650 |
Filed: |
February 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61769330 |
Feb 26, 2013 |
|
|
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Current U.S.
Class: |
210/635 ;
156/272.2; 252/184; 502/402 |
Current CPC
Class: |
B01J 20/3285 20130101;
B01D 15/362 20130101; B01D 15/327 20130101; B01J 20/291 20130101;
B01J 47/12 20130101; C07K 1/165 20130101; B01J 43/00 20130101; B01D
15/3847 20130101; B01J 20/28033 20130101; B01J 20/286 20130101;
B01J 41/20 20130101; B01D 15/363 20130101; B01J 39/26 20130101;
B01D 15/36 20130101 |
Class at
Publication: |
210/635 ;
252/184; 502/402; 156/272.2 |
International
Class: |
B01J 20/32 20060101
B01J020/32; B01J 43/00 20060101 B01J043/00; B01J 20/291 20060101
B01J020/291; B01D 15/36 20060101 B01D015/36 |
Claims
1. A composite material, comprising: a support member, comprising a
plurality of pores extending through the support member; and a
cross-linked gel, wherein the cross-linked gel comprises a first
functionality and a second functionality; the first functionality
and the second functionality are cationic, anionic, hydrophobic,
hydrophilic, thiophilic, hydrogen bond donating, hydrogen bond
accepting, pi-pi bond donating, pi-pi bond accepting, or metal
chelating; and the first functionality is different from the second
functionality; wherein the cross-linked gel is located in the pores
of the support member.
2. The composite material of claim 1, wherein the cross-linked gel
is macroporous.
3. The composite material of claim 1, wherein the cross-linked gel
comprises a polymer derived from 2-(diethylamino)ethyl
methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate,
2-(methylthio)ethyl methacrylate, acrylamide,
N-acryloxysuccinimide, butyl acrylate or methacrylate,
N,N-diethylacrylamide, N,N-dimethylacrylamide,
2-(N,N-dimethylamino)ethyl acrylate or methacrylate,
N-[3-(N,N-dimethylamino)propyl]methacrylamide,
N,N-dimethylacrylamide, ethyl acrylate or methacrylate,
2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl
acrylate or methacrylate, ethylene glycol phenyl ether
methacrylate, methacrylamide, methacrylic anhydride, propyl
acrylate or methacrylate, N-isopropylacrylamide, styrene,
4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidinone (VP),
acrylamido-2-methyl-1-propanesulfonic acid, styrenesulfonic acid,
alginic acid, (3-acrylamidopropyl)trimethylammonium halide,
diallyldimethylammonium halide, 4-vinyl-N-methylpyridinium halide,
vinylbenzyl-N-trimethylammonium halide,
methacryloxyethyltrimethylammonium halide, 3-sulfopropyl
methacrylate, 2-(2-methoxy)ethyl acrylate or methacrylate,
hydroxyethyl acrylamide, N-(3-methoxypropyl acrylamide),
N-[tris(hydroxymethyl)methyl]acrylamide, N-phenyl acrylamide,
N-tert-butyl acrylamide, or diacetone acrylamide.
4. The composite material of claim 1, wherein the cross-linked gel
comprises a polymer derived from more than one monomer.
5. The composite material of claim 2, wherein the average pore
diameter of the macropores is about 25 nm to about 1500 nm.
6. The composite material of claim 1, wherein the composite
material is a membrane.
7. A method, comprising the step of: contacting at a first flow
rate a first fluid comprising a substance with a composite material
of claim 1, thereby adsorbing or absorbing a portion of the
substance onto the composite material.
8. The method of claim 7, wherein the first fluid further comprises
a fragmented antibody, aggregated antibodies, a host cell protein,
a polynucleotide, an endotoxin, or a virus.
9. The method of claim 7, wherein the substance is a biological
molecule or biological ion.
10. The method of claim 9, wherein the biological molecule or
biological ion is selected from the group consisting of albumins,
lysozyme, viruses, cells, .gamma.-globulins of human and animal
origins, immunoglobulins of human and animal origins, proteins of
recombinant and natural origins, polypeptides of synthetic and
natural origins, interleukin-2 and its receptor, enzymes,
monoclonal antibodies, trypsin and its inhibitor, cytochrome C,
myoglobin, myoglobulin, .alpha.-chymotrypsinogen, recombinant human
interleukin, recombinant fusion protein, nucleic acid derived
products, DNA of synthetic and natural origins, and RNA of
synthetic and natural origins.
11. The method of claim 1, wherein the first fluid is a clarified
cell culture supernatant.
12. A method, comprising the step of: contacting at a first flow
rate a first fluid comprising a substance and an unwanted material
with a composite material of claim 1, thereby adsorbing or
absorbing a portion of the unwanted material onto the composite
material.
13. The method of claim 12, wherein the unwanted material comprises
a fragmented antibody, aggregated antibodies, a host cell protein,
a polynucleotide, an endotoxin, or a virus.
14. The method of claim 12, wherein substantially all of the
unwanted material is adsorbed or absorbed onto the composite
material.
15. The method of claim 12, wherein the substance is a biological
molecule or biological ion.
16. The method of claim 15, wherein the biological molecule or
biological ion is selected from the group consisting of albumins,
lysozyme, viruses, cells, .gamma.-globulins of human and animal
origins, immunoglobulins of human and animal origins, proteins of
recombinant and natural origins, polypeptides of synthetic and
natural origins, interleukin-2 and its receptor, enzymes,
monoclonal antibodies, trypsin and its inhibitor, cytochrome C,
myoglobin, myoglobulin, .alpha.-chymotrypsinogen, recombinant human
interleukin, recombinant fusion protein, nucleic acid derived
products, DNA of synthetic and natural origins, and RNA of
synthetic and natural origins.
17. The method of claim 12, wherein the first fluid is a clarified
cell culture supernatant.
18. A method of making a composite material of claim 1, comprising
the steps of: combining a first monomer, a photoinitiator, a
cross-linking agent, and a solvent, thereby forming a monomeric
mixture; contacting a support member with the monomeric mixture,
thereby forming a modified support member; wherein the support
member comprises a plurality of pores extending through the support
member, and the average pore diameter of the pores is about 0.1 to
about 25 .mu.m; covering the modified support member with a
polymeric sheet, thereby forming a covered support member; and
irradiating the covered support member for a period of time,
thereby forming a composite material.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application Ser. Nos. 61/769,330, filed Feb. 26,
2013; the contents of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Mixed-mode chromatography (MMC), also known as multimodal
chromatography, refers to chromatographic method for separating one
analyte from another by utilizing more than one form of interaction
between the stationary phase and analytes. Importantly, the
secondary interactions in MMC must be strong enough to contribute
to retention of the analyte; this approach is distinct from
conventional single-mode chromatography.
[0003] MMC has many advantages over traditional single-mode
chromatography, and over other methods of separation. MMC exhibits
higher selectivity than single-mode chromatography. For example,
positive, negative and neutral substances could be separated by a
reversed phase (RP)/anion-cation exchange (ACE) column in a single
run. In addition, mixed-mode chromatographic media display a
remarkably higher loading capacity. Because MMC uses two forms of
interaction, one mixed-mode column can replace two or even more
single mode columns; so, MMC is economical and reduces waste.
[0004] The ability to combine and streamline separation methods can
enhance selectivity in a protein purification process. Unlike
affinity chromatography, where a known site on the protein is
targeted, mixed-mode ligands are not tailored to a known specific
site. Accordingly, screening mixed-mode media becomes a search for
sites on the target protein that provide useful affinity and
selectivity.
[0005] Monoclonal antibodies constitute the largest number of
protein-based therapeutic molecules in current use or in clinical
trials. Protein A chromatography is routinely employed as a first
capture step in industrial monoclonal antibody purification
processes due to its high selectivity, resulting in good overall
yields and purities. However, a major drawback of affinity
chromatography is its high price, which, especially in case of
therapeutic antibodies needed at high doses or for chronic
administration, can account for a significant component of the cost
of goods. In addition, leached protein A ligand from the affinity
matrix must be removed by further chromatography steps due to its
potential immunogenicity (Roque A. C. et al. Biotechnology Progress
20(2004), 639-654). In addition, traditional protein A
chromatography requires elution at low pH, which can result in
product aggregation or precipitation.
[0006] Monoclonal antibodies can be separated by MMC on multimodal
media exhibiting, for instance, ionic and hydrophobic
functionalities. These multimodal media offer a valuable
alternative to the classical affinity approach for the separation
of monoclonal antibodies. Due to the salt tolerability of the
hydrophobic component, the clarified cell culture supernatant can
be directly loaded on the matrix, resulting in an effective
capturing of the monoclonal antibody. However, due to the
multimodal nature of the resin, different types of interaction of
the ligand with a particular monoclonal antibody are possible,
requiring unique wash and elution conditions, differing from
traditional ion-exchange or hydrophobic interaction
chromatography.
[0007] There exists a need for MMC media and methods that display
enhanced selectivity, high flow velocity, and low back pressure,
are inexpensive, and allow for longer column lifetimes, reduced
process times, increased productivity, and operational flexibility
compared to affinity based methods.
BRIEF SUMMARY OF THE INVENTION
[0008] In certain embodiments, the invention relates to a composite
material, comprising:
[0009] a support member, comprising a plurality of pores extending
through the support member; and
[0010] a cross-linked gel, wherein the cross-linked gel comprises a
first functionality and a second functionality; the first
functionality and the second functionality are cationic, anionic,
hydrophobic, hydrophilic, thiophilic, hydrogen bond donating,
hydrogen bond accepting, pi-pi bond donating, pi-pi bond accepting,
or metal chelating; and the first functionality is different from
the second functionality,
[0011] wherein the cross-linked gel is located in the pores of the
support member.
[0012] In certain embodiments, the invention relates to a composite
material, comprising:
[0013] a support member, comprising a plurality of pores extending
through the support member; and
[0014] a cross-linked gel, wherein the cross-linked gel comprises a
first functionality and a second functionality; the first
functionality and the second functionality are strong cations, weak
cations, strong anions, weak anions, hydrophobic, hydrophilic,
thiophilic, hydrogen bond donating, hydrogen bond accepting, pi-pi
bond donating, pi-pi bond accepting, or metal chelating; and the
first functionality is different from the second functionality;
[0015] wherein the cross-linked gel is located in the pores of the
support member.
[0016] In certain embodiments, the invention relates to a method,
comprising the step of: [0017] contacting at a first flow rate a
first fluid comprising a substance with any one of the
aforementioned composite materials, thereby adsorbing or absorbing
a portion of the substance onto the composite material.
[0018] In certain embodiments, the invention relates to a method,
comprising the step of: [0019] contacting at a first flow rate a
first fluid comprising a substance and an unwanted material with
any one of the aforementioned composite materials, thereby
adsorbing or absorbing a portion of the unwanted material onto the
composite material.
[0020] In certain embodiments, the invention relates to a method of
making a composite material, comprising the steps of:
[0021] combining a first monomer, a photoinitiator, a cross-linking
agent, and a solvent, thereby forming a monomeric mixture;
[0022] contacting a support member with the monomeric mixture,
thereby forming a modified support member; wherein the support
member comprises a plurality of pores extending through the support
member, and the average pore diameter of the pores is about 0.1 to
about 25 .mu.m;
[0023] covering the modified support member with a polymeric sheet,
thereby forming a covered support member; and
[0024] irradiating the covered support member for a period of time,
thereby forming a composite material.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 depicts the performance of a mixed-mode membrane in a
hIgG bind-elute experiment.
[0026] FIG. 2 depicts the performance of a mixed-mode membrane in a
salt tolerance experiment investigating the effect of salt content
in binding buffer on hIgG binding capacity.
[0027] FIG. 3 depicts the performance of a mixed-mode membrane in
an experiment investigating the effect of pH on hIgG binding
capacity.
[0028] FIG. 4 depicts the performance of a mixed-mode membrane in
bind-elute mode to purify monoclonal antibodies.
[0029] FIG. 5 depicts SEC column analysis: mAbs feed solution.
[0030] FIG. 6 depicts SEC column analysis: purification of mAbs
post protein using mixed-mode membrane in bind-elute mode--eluent
B.
[0031] FIG. 7 depicts SEC column analysis: purification of mAbs
post protein A using mixed-mode membrane in bind-elute mode--eluent
C.
[0032] FIG. 8 tabulates performance results for multicycle
mixed-mode membranes.
[0033] FIG. 9 tabulates the dynamic binding capacity at 10%
breakthrough for various mixed-mode media.
[0034] FIG. 10 depicts selective separation of Cytochrome C (1) and
lysozyme (2) onto mixed-mode membrane prepared according to the
Example 11.
[0035] FIG. 11 depicts the effect of membrane exposure into 0.5 M
NaOH/0.1 M NaCl on membrane performance.
[0036] FIG. 12 depicts the removal of aggregates from hIgG using
anion-exchange mixed-mode membrane.
[0037] FIG. 13 depicts summary data on aggregates clearance in
elution fractions.
[0038] FIG. 14 depicts summary data on HCP/DNA clearance in elution
fractions.
[0039] FIG. 15 depicts the selective separation of myoglobin (1),
ribonuclease A (2) and lysozyme (3) onto mixed-mode membrane
prepared according to the Example 17.
[0040] FIG. 16 depicts the effect of the nature of the co-monomer
on membrane permeability.
[0041] FIG. 17 depicts the effect of the nature of the co-monomer
used on membrane performance.
[0042] FIG. 18 depicts the effect of the nature of the cross-linker
on membrane permeability.
[0043] FIG. 19 depicts the effect of the nature of the cross-linker
on membrane performance.
[0044] FIG. 20 depicts an ESEM micrograph of anion-exchange
mixed-mode membrane prepared according to Example 8.
[0045] FIG. 21 depicts an ESEM micrograph of cation-exchange
mixed-mode membrane prepared according to Example 11.
[0046] FIG. 22 depicts an ESEM micrograph of cation-exchange
mixed-mode membrane prepared according to Example 17.
[0047] FIG. 23 tabulates formulation components and concentrations
used to make mixed-mode strong cation-exchange membranes.
[0048] FIG. 24 tabulates performance characteristics of mixed-mode
strong cation-exchange membranes.
[0049] FIG. 25 tabulates formulation components and concentrations
used to make mixed-mode strong cation-exchange membranes.
[0050] FIG. 26 tabulates performance characteristics mixed-mode
strong cation-exchange membranes.
DETAILED DESCRIPTION OF THE INVENTION
Overview
[0051] In certain embodiments, the invention relates to the
preparation and use of membrane-based stationary phase mixed-mode
chromatographic supports that employ multiple chemical mechanisms
to adsorb or separate proteins or other solutes. In certain
embodiments, the invention relates to purification of a protein
from a mixture containing other materials, including fragmented or
aggregated antibodies, host cell proteins, DNA, endotoxins, and
viruses. Examples include, but are not limited to, chromatographic
supports that exploit combination of at least two or possibly more
of the following mechanisms: cation exchange, anion exchange,
hydrophobic interaction, hydrophilic interaction, thiophilic
interaction, hydrogen bonding, pi-pi bonding, and metal
affinity.
[0052] In certain embodiments, the composite materials of the
invention can be effectively used in both "bind-elute" and
"flow-through" modes. Importantly, since the individual
functionalities are included through the incorporation of
functional monomers, the relative amount of each functional group
can be easily and readily tuned for optimal performance
characteristics.
[0053] "Bind-elute mode" as it relates to invention herein, refers
to an operational approach to chromatography in which the buffer
conditions are established so that both a target protein and
undesired contaminants bind to the mixed mode chromatographic
support or composite material. Fractionation of target protein from
the other components is achieved subsequently by changing the
conditions such that the target protein and contaminants are eluted
separately from the support. In certain embodiments, a multimodal
cation-exchange membrane of the invention may be used in
"bind-elute mode" featuring high dynamic binding capacities at high
conductivity, high volume throughput and selectivity. In certain
embodiments, the eluent is reduced in aggregates of the target
protein by about 50% to about 99%. In certain embodiments, the
eluent is reduced in aggregates of the target protein by about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98%, or about 99%.
[0054] As it relates to the invention herein, the term
"flow-through mode" refers to an operational approach to
chromatography in which the buffer conditions are established so
that the intact target protein flows through the membrane upon
application while contaminants are selectively retained. In certain
embodiments, a multimodal anion-exchange membrane of the invention
may be used in "flow-through mode" in a post-protein A purification
process to remove key contaminants such as DNA, host cell proteins
(HCP), leached protein A, aggregates and viruses in a single
step.
Various Characteristics of Exemplary Composite Materials
[0055] Composition of the Gels
[0056] In certain embodiments, the cross-linked gels may be formed
through the in situ reaction of one or more polymerizable monomers
with one or more cross-linkers. In certain embodiments, the gels
may be formed through the reaction of one or more cross-linkable
polymers with one or more cross-linkers. In certain embodiments, a
cross-linked gel having macropores of a suitable size may be
formed.
[0057] The gel can be selected to comprise specific monomers having
specific functionality. Copolymers of these monomers can be
used.
[0058] In certain embodiments, the properties of the composite
materials may be tuned by adjusting the average pore diameter of
the macroporous gel. The size of the macropores is generally
dependent on the nature and concentration of the cross-linking
agent, the nature of the solvent or solvents in which the gel is
formed, the amount of any polymerization initiator or catalyst and,
if present, the nature and concentration of porogen. In certain
embodiments, the composite material may have a narrow pore-size
distribution.
[0059] Porous Support Member
[0060] In some embodiments, the porous support member contains
pores of average diameter between about 0.1 and about 25 .mu.m.
[0061] In some embodiments, the porous support member has a volume
porosity between about 40% and about 90%.
[0062] In certain embodiments, the porous support is flat.
[0063] In certain embodiments, the porous support is
disk-shaped.
[0064] Many porous substrates or membranes can be used as the
support member. In some embodiments, the porous support member is
made of polymeric material. In certain embodiments, the support may
be a polyolefin, which is available at low cost. In certain
embodiments, the polyolefin may be poly(ethylene), poly(propylene),
or poly(vinylidene difluoride). Extended polyolefin membranes made
by thermally induced phase separation (TIPS), or non-solvent
induced phase separation are mentioned. In certain embodiments, the
support member may be made from natural polymers, such as cellulose
or its derivatives. In certain embodiments, suitable supports
include polyethersulfone membranes, poly(tetrafluoroethylene)
membranes, nylon membranes, cellulose ester membranes, fiberglass,
or filter papers.
[0065] In certain embodiments, the porous support is composed of
woven or non-woven fibrous material, for example, a polyolefin such
as polypropylene. Such fibrous woven or non-woven support members
can have pore sizes larger than the TIPS support members, in some
instances up to about 75 .mu.m. The larger pores in the support
member permit formation of composite materials having larger
macropores in the macroporous gel. Non-polymeric support members
can also be used, such as ceramic-based supports. The porous
support member can take various shapes and sizes.
[0066] In some embodiments, the support member is in the form of a
membrane.
[0067] In some embodiments, the support member has a thickness from
about 10 to about 2000 .mu.m, from about 10 to about 1000 .mu.m, or
from about 10 to about 500 .mu.m.
[0068] In other embodiments, multiple porous support units can be
combined, for example, by stacking. In one embodiment, a stack of
porous support membranes, for example, from 2 to 10 membranes, can
be assembled before the gel is formed within the void of the porous
support. In another embodiment, single support member units are
used to form composite material membranes, which are then stacked
before use.
[0069] Relationship Between Gel and Support Member
[0070] The gel may be anchored within the support member. The term
"anchored" is intended to mean that the gel is held within the
pores of the support member, but the term is not necessarily
restricted to mean that the gel is chemically bound to the pores of
the support member. The gel can be held by the physical constraint
imposed upon it by enmeshing and intertwining with structural
elements of the support member, without actually being chemically
grafted to the support member, although in some embodiments, the
gel may be grafted to the surface of the pores of the support
member.
[0071] Because the macropores are present in the gel that occupies
the pores of the support member, the macropores of the gel must be
smaller than the pores of the support member. Consequently, the
flow characteristics and separation characteristics of the
composite material are dependent on the characteristics of the gel,
but are largely independent of the characteristics of the porous
support member, with the proviso that the size of the pores present
in the support member is greater than the size of the macropores of
the gel. The porosity of the composite material can be tailored by
filling the support member with a gel whose porosity is partially
or completely dictated by the nature and amounts of monomer or
polymer, cross-linking agent, reaction solvent, and porogen, if
used. As pores of the support member are filled with the same gel
material, a high degree of consistency is achieved in properties of
the composite material, and for a particular support member these
properties are determined partially, if not entirely, by the
properties of the gel. The net result is that the invention
provides control over macropore-size, permeability and surface area
of the composite materials.
[0072] The number of macropores in the composite material is not
primarily dictated by the number of pores in the support material.
The number of macropores in the composite material can be much
greater than the number of pores in the support member because the
macropores are smaller than the pores in the support member. As
mentioned above, the effect of the pore-size of the support
material on the pore-size of the macroporous gel is generally
negligible. An exception is found in those cases where the support
member has a large difference in pore-size and pore-size
distribution, and where a macroporous gel having very small
pore-sizes and a narrow range in pore-size distribution is sought.
In these cases, large variations in the pore-size distribution of
the support member are weakly reflected in the pore-size
distribution of the macroporous gel. In certain embodiments, a
support member with a narrow pore-size range may be used in these
situations.
[0073] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the composite
materials are relatively non-toxic.
[0074] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the composite
materials are tolerant to relatively wide ranges of salt
concentration in the contacting liquid.
Preparation of Composite Materials
[0075] In certain embodiments, the composite materials of the
invention may be prepared by single-step methods. In certain
embodiments, these methods may use water or other environmentally
benign solvents as the reaction solvent. In certain embodiments,
the methods may be rapid and, therefore, may lead to relatively
simple and low-cost manufacturing processes.
[0076] In certain embodiments, the composite materials of the
invention may be prepared by mixing more than one monomer, one or
more cross-linking agents, one or more initiators, and optionally
one or more porogens, in one or more suitable solvents. In certain
embodiments, the resulting mixture may be homogeneous. In certain
embodiments, the mixture may be heterogeneous. In certain
embodiments, the mixture may then be introduced into a suitable
porous support, where a gel forming reaction may take place.
[0077] In certain embodiments, suitable solvents for the
gel-forming reaction include 1,3-butanediol, di(propylene glycol)
propyl ether, N,N-dimethylacetamide, di(propylene glycol) dimethyl
ether, 1,2-propanediol, di(propylene glycol) methyl ether acetate
(DPMA), water, dioxane, dimethylsulfoxide (DMSO), dimethylformamide
(DMF), acetone, ethanol, N-methylpyrrolidone (NMP), tetrahydrofuran
(THF), ethyl acetate, acetonitrile, N-methylacetamide, propanol,
methanol, tri(ethylene glycol) dimethyl ether, tri(propylene
glycol) butyl ether, tri(propylene glycol) propyl ether, or
mixtures thereof. In certain embodiments, solvents that have a
higher boiling point may be used, as these solvents reduce
flammability and facilitate manufacture. In certain embodiments,
solvents that have a low toxicity may be used, so they may be
readily disposed of after use. An example of such a solvent is
dipropyleneglycol monomethyl ether (DPM).
[0078] In certain embodiments, a porogen may be added to the
reactant mixture, wherein porogens may be broadly described as
pore-generating additives. In certain embodiments, the porogen may
be selected from the group consisting of thermodynamically poor
solvents and extractable polymers, for example,
poly(ethyleneglycol), surfactants, and salts.
[0079] In some embodiments, components of the gel forming reaction
react spontaneously at room temperature to form the gel. In other
embodiments, the gel forming reaction must be initiated. In certain
embodiments, the gel forming reaction may be initiated by any known
method, for example, through thermal activation or UV radiation. In
certain embodiments, the reaction may be initiated by UV radiation
in the presence of a photoinitiator. In certain embodiments, the
photoinitiator may be selected from the group consisting of
2-hydroxy-1-[4-2(hydroxyethoxy)phenyl]-2-methyl-1-propanone
(Irgacure 2959), 2,2-dimethoxy-2-phenylacetophenone (DMPA),
benzophenone, benzoin and benzoin ethers, such as benzoin ethyl
ether and benzoin methyl ether, dialkoxyacetophenones,
hydroxyalkylphenones, and .alpha.-hydroxymethyl benzoin sulfonic
esters. Thermal activation may require the addition of a thermal
initiator. In certain embodiments, the thermal initiator may be
selected from the group consisting of
1,1'-azobis(cyclohexanecarbonitrile) (VAZO.RTM. catalyst 88),
azobis(isobutyronitrile) (AIBN), potassium persulfate, ammonium
persulfate, and benzoyl peroxide.
[0080] In certain embodiments, the gel-forming reaction may be
initiated by UV radiation. In certain embodiments, a photoinitiator
may be added to the reactants of the gel forming reaction, and the
support member containing the mixture of monomer, cross-linking
agent, and photoinitiator may be exposed to UV radiation at
wavelengths from about 250 nm to about 400 nm for a period of a few
seconds to a few hours. In certain embodiments, the support member
containing the mixture of monomer, cross-linking agent, and
photoinitiator may be exposed to UV radiation at about 350 nm for a
period of a few seconds to a few hours. In certain embodiments, the
support member containing the mixture of monomer, cross-linking
agent, and photoinitiator may be exposed to UV radiation at about
350 nm for about 10 minutes. In certain embodiments, visible
wavelength light may be used to initiate the polymerization. In
certain embodiments, the support member must have a low absorbance
at the wavelength used so that the energy may be transmitted
through the support member.
[0081] In certain embodiments, the rate at which polymerization is
carried out may have an effect on the size of the macropores
obtained in the macroporous gel. In certain embodiments, when the
concentration of cross-linker in a gel is increased to sufficient
concentration, the constituents of the gel begin to aggregate to
produce regions of high polymer density and regions with little or
no polymer, which latter regions are referred to as "macropores" in
the present specification. This mechanism is affected by the rate
of polymerization. In certain embodiments, the polymerization may
be carried out slowly, such as when a low light intensity in the
photopolymerization is used. In this instance, the aggregation of
the gel constituents has more time to take place, which leads to
larger pores in the gel. In certain embodiments, the polymerization
may be carried out at a high rate, such as when a high intensity
light source is used. In this instance, there may be less time
available for aggregation and smaller pores are produced.
[0082] In certain embodiments, once the composite materials are
prepared, they may be washed with various solvents to remove any
unreacted components and any polymer or oligomers that are not
anchored within the support. In certain embodiments, solvents
suitable for the washing of the composite material include water,
acetone, methanol, ethanol, propanol, and DMF.
Exemplary Uses of the Composite Materials
[0083] In certain embodiments, the invention relates to a method,
wherein a fluid is passed through the cross-linked gel of any one
of the aforementioned composite materials. By tailoring the
conditions for binding or fractionation, good selectivity can be
obtained.
[0084] In certain embodiments, the invention relates to a method of
separating biomolecules, such as proteins or immunoglobulins, from
solution. In certain embodiments, the invention relates to a method
of purifying biomolecules such as proteins or immunoglobulins. In
certain embodiments, the invention relates to a method of purifying
proteins or monoclonal antibodies with high selectivity. In certain
embodiments, the invention relates to a method, wherein the
biological molecule or biological ion retains its tertiary or
quaternary structure, which may be important in retaining
biological activity. In certain embodiments, biological molecules
or biological ions that may be separated or purified include
proteins such as albumins, e.g., bovine serum albumin, and
lysozyme. In certain embodiments, biological molecules or
biological ions that may be separated include .gamma.-globulins of
human and animal origins, immunoglobulins such as IgG, IgM, or IgE
of human and animal origins, proteins of recombinant and natural
origin including protein A, phytochrome, halophilic protease,
poly(3-hydroxybutyrate) depolymerase, aculaecin-A acylase,
polypeptides of synthetic and natural origin, interleukin-2 and its
receptor, enzymes such as phosphatase, dehydrogenase, ribonuclease
A, etc., monoclonal antibodies, fragments of antibodies, trypsin
and its inhibitor, albumins of varying origins, e.g.,
.alpha.-lactalbumin, human serum albumin, chicken egg albumin,
ovalbumin etc., cytochrome C, immunoglobulins, myoglobulin,
recombinant human interleukin, recombinant fusion protein, nucleic
acid derived products, DNA and RNA of synthetic and natural origin,
DNA plasmids, lectin, .alpha.-chymotrypsinogen, and natural
products including small molecules. In certain embodiments, the
invention relates to a method of recovering an antibody fragment
from variants, impurities, or contaminants associated therewith. In
certain embodiments, biomolecule separation or purification may
occur substantially in the cross-linked gel. In certain
embodiments, biomolecule separation or purification may occur
substantially in the macropores of the macroporous cross-linked
gel.
[0085] In certain embodiments, the invention relates to a method of
reversible adsorption of a substance. In certain embodiments, an
adsorbed substance may be released by changing the liquid that
flows through the gel. In certain embodiments, the uptake and
release of substances may be controlled by variations in the
composition of the cross-linked gel.
[0086] In certain embodiments, the invention relates to a method,
wherein the substance may be applied to the composite material from
a buffered solution.
[0087] In certain embodiments, the invention relates to a method,
wherein the substance may be eluted using varying concentrations of
aqueous salt solutions.
[0088] In certain embodiments, the invention relates to a method
that exhibits high binding capacities. In certain embodiments, the
invention relates to a method that exhibits binding capacities of
about 100 mg/mL.sub.membrane, about 110 mg/mL.sub.membrane, about
120 mg/mL.sub.membrane, about 130 mg/mL.sub.membrane, about 140
mg/mL.sub.membrane, about 150 mg/mL.sub.membrane, about 160
mg/mL.sub.membrane, about 170 mg/mL.sub.membrane, about 180
mg/mL.sub.membrane, about 190 mg/mL.sub.membrane, about 200
mg/mL.sub.membrane, about 210 mg/mL.sub.membrane, about 220
mg/mL.sub.membrane, about 230 mg/mL.sub.membrane, about 240
mg/mL.sub.membrane, about 250 mg/mL.sub.membrane, about 260
mg/mL.sub.membrane, about 270 mg/mL.sub.membrane, about 280
mg/mL.sub.membrane, about 290 mg/mL.sub.membrane, about 300
mg/mL.sub.membrane, about 310 mg/mL.sub.membrane, about 320
mg/mL.sub.membrane, about 330 mg/mL.sub.membrane, about 340
mg/mL.sub.membrane, about 350 mg/mL.sub.membrane, about 360
mg/mL.sub.membrane, about 370 mg/mL.sub.membrane, about 380
mg/mL.sub.membrane about 390 mg/mL.sub.membrane, or about 400
mg/mL.sub.membrane at 10% breakthrough.
[0089] In certain embodiments, methods of the invention result in
binding capacities higher than those reported with the use of
conventional MMC resins. In certain embodiments, the inventive
methods may be run at significantly higher flow rates, due to
convective flow, than the flow rates achieved in methods MMC
resins. In certain embodiments, the methods of the present
invention do not suffer from the problematic pressure drops
associated with methods using MMC resins.
[0090] In certain embodiments, the flow rate during binding (the
first flow rate) may be about 0.1 to about 10 mL/min. In certain
embodiments, the flow rate during elution (the second flow rate)
may be about 0.1 to about 10 mL/min. In certain embodiments, the
first flow rate or the second flow rate may be about 0.1 mL/min,
about 0.5 mL/min, about 1.0 mL/min, about 1.5 mL/min, about 2.0
mL/min, about 2.5 mL/min, about 3.0 mL/min, about 4.0 mL/min, about
4.5 mL/min, about 5.0 mL/min, about 5.5 mL/min, about 6.0 mL/min,
about 6.5 mL/min, about 7.0 mL/min, about 7.5 mL/min, about 8.0
mL/min, about 8.5 mL/min, about 9.0 mL/min, about 9.5 mL/min, or
about 10.0 mL/min. In certain embodiments, the first flow rate or
the second flow rate may be about 0.5 mL/min to about 5.0
mL/min.
[0091] The water flux, Q.sub.H2O (kg/m.sup.2 h), was calculated
using the following equation:
Q H 2 O = ( m 1 - m 2 ) A t ##EQU00001##
where m.sub.1 is the mass of water transferred through the membrane
at t.sub.1, m.sub.2 is the mass of water transferred through the
membrane at t.sub.2, A is the membrane cross-sectional area and t
is the elapsed time (t.sub.1-t.sub.2), where
t.sub.1>t.sub.2.
[0092] In certain embodiments, an additive may be added to the
eluting salt solution (the second fluid, or the third or later
fluid). In certain embodiments, the additive is added in a low
concentration (e.g., less than about 1 M, about 0.5 M, or about 0.2
M). In certain embodiments, the additive is a water-miscible
alcohol, a detergent, dimethyl sulfoxide, dimethyl formamide, or an
aqueous solution of a chaotropic salt. In certain embodiments, not
wishing to be bound by any particular theory, the additive may
decrease the surface tension of water, thus weakening the
hydrophobic interactions to give a subsequent dissociation of the
ligand-solute complex.
[0093] In certain embodiments, the mixed-mode media combines both
hydrophobic and ion-exchange characteristics, so that its
selectivity can be manipulated in order for the retention magnitude
of each retention mode may be adjusted by changing the mobile phase
ionic strength, pH, or organic solvent content. In certain
embodiments, the selectivity can be manipulated either concurrently
or individually.
[0094] In certain embodiments, changing pH is an effective elution
tool for protein elution without changing the conductivity of the
mobile phase.
[0095] In certain embodiments, the invention relates to a one-step
method of biomolecule purification. In certain embodiments, the
invention relates to a method of biomolecule separation that is
easier to scale-up, is less labor intensive, is faster, and has
lower capital costs than the commonly used conventional
packed-column chromatography techniques.
Pore Size Determination
[0096] SEM and ESEM
[0097] The average diameter of the macropores in the macroporous
cross-linked gel may be estimated by any one of many methods. One
method that may be employed is scanning electron microscopy (SEM).
SEM is a well-established method for determining pore sizes and
porosities in general, and for characterizing membranes in
particular. Reference is made to the book Basic Principles of
Membrane Technology by Marcel Mulder (.COPYRGT. 1996) ("Mulder"),
especially Chapter IV. Mulder provides an overview of methods for
characterizing membranes. For porous membranes, the first method
mentioned is electron microscopy. SEM is a very simple and useful
technique for characterising microfiltration membranes. A clear and
concise picture of the membrane can be obtained in terms of the top
layer, cross-section and bottom layer. In addition, the porosity
and pore size distribution can be estimated from the
photographs.
[0098] Environmental SEM (ESEM) is a technique that allows for the
non-destructive imaging of specimens that are wet, by allowing for
a gaseous environment in the specimen chamber. The environmental
secondary detector (ESD) requires a gas background to function and
operates at from about 3 torr to about 20 torr. These pressure
restraints limit the ability to vary humidity in the sample
chamber. For example, at 10 torr, the relative humidity at a
specific temperature is as follows:
TABLE-US-00001 Relative Humidity at 10 torr (%) T (.degree. C.)
About 80 About 16 About 70 About 18 About 60 About 20 About 40
About 24 About 20 About 40 About 10 About 50 About 2 About 70 About
1 About 100
This is a useful guide to relative humidity in the sample chamber
at different temperatures. In certain embodiments, the relative
humidity in the sample chamber during imaging is from about 1% to
about 99%. In certain embodiments, the relative humidity in the
sample chamber during imaging is about 1%, about 2%, about 3%,
about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, or
about 99%. In certain embodiments, the relative humidity in the
sample chamber during imaging is about 45%.
[0099] In certain embodiments, the microscope has nanometer
resolution and up to about 100,000.times. magnification.
[0100] In certain embodiments, the temperature in the sample
chamber during imaging is from about 4.degree. C. to about
95.degree. C. In certain embodiments, the temperature in the sample
chamber during imaging is about 4.degree. C., about 6.degree. C.,
about 8.degree. C., about 10.degree. C., about 12.degree. C., about
14.degree. C., about 16.degree. C., about 18.degree. C., about
20.degree. C., about 25.degree. C., about 30.degree. C., about
35.degree. C., about 40.degree. C., about 45.degree. C., about
50.degree. C., about 55.degree. C., about 60.degree. C., about
65.degree. C., about 70.degree. C., about 75.degree. C., about
80.degree. C., or about 85.degree. C. In certain embodiments, the
temperature in the sample chamber during imaging is about 5.degree.
C.
[0101] In certain embodiments, the pressure in the sample chamber
during imaging is from about 3 torr to about 20 torr. In certain
embodiments, the pressure in the sample chamber during imaging is
about 4 torr, about 6 torr, about 8 torr, about 10 torr, about 12
torr, about 14 torr, about 16 torr, about 18 torr, or about 20
torr. In certain embodiments, the pressure in the sample chamber
during imaging is about 3 torr.
[0102] In certain embodiments, the working distance from the source
of the electron beam to the sample is from about 6 mm to about 15
mm. In certain embodiments, the working distance from the source of
the electron beam to the sample is about 6 mm, about 7 mm, about 8
mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm,
about 14 mm, or about 15 mm. In certain embodiments, the working
distance from the source of the electron beam to the sample is
about 10 mm.
[0103] In certain embodiments, the voltage is from about 1 kV to
about 30 kV. In certain embodiments, the voltage is about 2 kV,
about 4 kV, about 6 kV, about 8 kV, about 10 kV, about 12 kV, about
14 kV, about 16 kV, about 18 kV, about 20 kV, about 22 kV, about 24
kV, about 26 kV, about 28 kV, or about 30 kV. In certain
embodiments, the voltage is about 20 kV.
[0104] In certain embodiments, the average pore diameter may be
measured by estimating the pore diameters in a representative
sample of images from the top or bottom of a composite material.
One of ordinary skill in the art will recognize and acknowledge
various experimental variables associated with obtaining an ESEM
image of a wetted membrane, and will be able to design an
experiment accordingly.
[0105] Capillary Flow Porometry
[0106] Capillary flow porometry is an analytical technique used to
measure the pore size(s) of porous materials. In this analytical
technique, a wetting liquid is used to fill the pores of a test
sample and the pressure of a non-reacting gas is used to displace
the liquid from the pores. The gas pressure and flow rate through
the sample is accurately measured and the pore diameters are
determined using the following equation:
D=4.times..gamma..times.cos .theta./P
D=pore diameter .gamma.=liquid surface tension .theta.=liquid
contact angle P=differential gas pressure
[0107] This equation shows that the pressure required to displace
liquid from the wetted sample is inversely related to the pore
size. Since this technique involves the flow of a liquid from the
pores of the test sample under pressure, it is useful for the
characterization of through pores (interconnected pores that allow
fluid flow from one side of the sample to the other). Other pore
types (closed and blind pores) are not detectable by this
method.
[0108] Capillary flow porometry detects the presence of a pore when
gas starts flowing through that pore. This occurs only when the gas
pressure is high enough to displace the liquid from the most
constricted part of the pore. Therefore, the pore diameter
calculated using this method is the diameter of the pore at the
most constricted part and each pore is detected as a single pore of
this constricted diameter. The largest pore diameter (called the
bubble point) is determined by the lowest gas pressure needed to
initiate flow through a wet sample and a mean pore diameter is
calculated from the mean flow pressure. In addition, both the
constricted pore diameter range and pore size distribution may be
determined using this technique.
[0109] This method may be performed on small membrane samples
(.about.2.5 cm diameter) that are immersed in a test fluid (e.g.
water, buffer, alcohol). The range of gas pressure applied can be
selected from 0 to 500 psi.
[0110] Other Methods of Determining Pore Diameter
[0111] Mulder describes other methods of characterizing the average
pore size of a porous membrane, including atomic force microscopy
(AFM) (page 164), permeability calculations (page 169), gas
adsorption-desorption (page 173), thermoporometry (page 176),
permporometry (page 179), and liquid displacement (page 181). The
teachings of Mulder, and the references cited therein, form part of
the common general knowledge in the field, and are hereby
incorporated by reference.
Exemplary Composite Materials
[0112] In certain embodiments, the invention relates to a composite
material, comprising:
[0113] a support member, comprising a plurality of pores extending
through the support member; and
[0114] a cross-linked gel, wherein the cross-linked gel comprises a
first functionality and a second functionality; the first
functionality and the second functionality are cationic, anionic,
hydrophobic, hydrophilic, thiophilic, hydrogen bond donating,
hydrogen bond accepting, pi-pi bond donating, pi-pi bond accepting,
or metal chelating; and the first functionality is different from
the second functionality,
[0115] wherein the cross-linked gel is located in the pores of the
support member.
[0116] In certain embodiments, the invention relates to a composite
material, comprising:
[0117] a support member, comprising a plurality of pores extending
through the support member; and
[0118] a cross-linked gel, wherein the cross-linked gel comprises a
first functionality and a second functionality; the first
functionality and the second functionality are strong cations, weak
cations, strong anions, weak anions, hydrophobic, hydrophilic,
thiophilic, hydrogen bond donating, hydrogen bond accepting, pi-pi
bond donating, pi-pi bond accepting, or metal chelating; and the
first functionality is different from the second functionality;
[0119] wherein the cross-linked gel is located in the pores of the
support member.
[0120] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the first
functionality is a strong cation; and the second functionality is a
weak cation.
[0121] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein more than two
functionalities are employed. In certain embodiments, the invention
relates to any one of the aforementioned composite materials,
wherein three, four, five, six, seven, eight, or nine different
functionalities are employed.
[0122] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the first
functionality or the second functionality is cationic. In certain
embodiments, the cationic functionality is a weak cation. In
certain embodiments, the cationic functionality is a strong cation.
In certain embodiments, the strong cation is an ammonium cation. In
certain embodiments, the first functionality or the second
functionality is a trialkylammonium radical. In certain
embodiments, the strong cation is a trimethylammonium radical. In
certain embodiments, the weak cation is cationic only at a certain
pH. In certain embodiments, the weak cation is a protonated
ammonium radical. In certain embodiments, the weak cation is a
protonated diialkylammonium radical. In certain embodiments, the
weak cation is a protonated dimethylammonium radical.
[0123] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the first
functionality or the second functionality is anionic. In certain
embodiments, the anionic functionality is a weak anion. In certain
embodiments, the cationic functionality is a strong anion. In
certain embodiments, the strong anion is a sulfonate anion. In
certain embodiments, the first functionality or the second
functionality is a sulfonate radical. In certain embodiments, the
first functionality or the second functionality is a sulfonate
radical.
[0124] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel is macroporous.
[0125] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cationic,
anionic, hydrophobic, hydrophilic, thiophilic, hydrogen bond
donating, hydrogen bond accepting, pi-pi bond donating, pi-pi bond
accepting, or metal chelating ability of the cross-linked gel is
determined under conditions suitable for binding (room temperature,
first fluid, see below).
[0126] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from 2-(diethylamino)ethyl
methacrylate, 2-aminoethyl methacrylate, 2-carboxyethyl acrylate,
2-(methylthio)ethyl methacrylate, acrylamide,
N-acryloxysuccinimide, butyl acrylate or methacrylate,
N,N-diethylacrylamide, N,N-dimethylacrylamide,
2-(N,N-dimethylamino)ethyl acrylate or methacrylate,
N-[3-(N,N-dimethylamino)propyl]methacrylamide,
N,N-dimethylacrylamide, ethyl acrylate or methacrylate,
2-ethylhexyl methacrylate, hydroxypropyl methacrylate, glycidyl
acrylate or methacrylate, ethylene glycol phenyl ether
methacrylate, methacrylamide, methacrylic anhydride, propyl
acrylate or methacrylate, N-isopropylacrylamide, styrene,
4-vinylpyridine, vinylsulfonic acid, N-vinyl-2-pyrrolidinone (VP),
acrylamido-2-methyl-1-propanesulfonic acid, styrenesulfonic acid,
alginic acid, (3-acrylamidopropyl)trimethylammonium halide,
diallyldimethylammonium halide, 4-vinyl-N-methylpyridinium halide,
vinylbenzyl-N-trimethylammonium halide,
methacryloxyethyltrimethylammonium halide, 3-sulfopropyl
methacrylate, 2-(2-methoxy)ethyl acrylate or methacrylate,
hydroxyethyl acrylamide, N-(3-methoxypropyl acrylamide),
N-[tris(hydroxymethyl)methyl]acrylamide, N-phenyl acrylamide,
N-tert-butyl acrylamide, or diacetone acrylamide.
[0127] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from more than one monomer.
[0128] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and 2-(methylthio)ethyl methacrylate. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the cross-linked gel comprises a
polymer derived from 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and 2-(methylthio)ethyl methacrylate in a molar
ratio of about 1:about 0.2:about 0.1:about 0.06. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the cross-linked gel comprises a polymer derived
from 2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, ethylene glycol phenyl ether methacrylate, and
2-(methylthio)ethyl methacrylate in a molar ratio of about 1:about
0.22:about 0.14:about 0.06.
[0129] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and hydroxypropyl methacrylate. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the cross-linked gel comprises a
polymer derived from 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and hydroxypropyl methacrylate in a molar ratio
of about 1:about 0.2:about 0.2:about 0.1. In certain embodiments,
the invention relates to any one of the aforementioned composite
materials, wherein the cross-linked gel comprises a polymer derived
from 2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, ethylene glycol phenyl ether methacrylate, and hydroxypropyl
methacrylate in a molar ratio of about 1: about 0.25:about
0.15:about 0.14.
[0130] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, and ethylene glycol
phenyl ether methacrylate. In certain embodiments, the invention
relates to any one of the aforementioned composite materials,
wherein the cross-linked gel comprises a polymer derived from
2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, and ethylene glycol phenyl ether methacrylate in a molar
ratio of about 1:about 0.3:about 0.1. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials, wherein the cross-linked gel comprises a polymer derived
from 2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, and ethylene glycol phenyl ether methacrylate in a molar
ratio of about 1:about 0.26:about 0.15.
[0131] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from
vinylbenzyl-N-trimethylammonium halide. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials, wherein the cross-linked gel comprises a polymer derived
from vinylbenzyl-N-trimethylammonium chloride.
[0132] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from 2-(diethylamino)ethyl
methacrylate, (ar-vinylbenzyl)trimethylammonium chloride, ethylene
glycol phenyl ether methacrylate, and 2-aminoethyl methacrylate. In
certain embodiments, the invention relates to any one of the
aforementioned composite materials, wherein the cross-linked gel
comprises a polymer derived from 2-(diethylamino)ethyl
methacrylate, (ar-vinylbenzyl)trimethylammonium chloride, ethylene
glycol phenyl ether methacrylate, and 2-aminoethyl methacrylate in
a molar ratio of about 1:about 0.4:about 0.5:about 0.1. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the cross-linked gel comprises a
polymer derived from 2-(diethylamino)ethyl methacrylate,
(ar-vinylbenzyl)trimethylammonium chloride, ethylene glycol phenyl
ether methacrylate, and 2-aminoethyl methacrylate in a molar ratio
of about 1:about 0.36:about 0.52:about 0.1.
[0133] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide, and N-phenylacrylamide. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the cross-linked gel comprises a
polymer derived from 2-acrylamido-2-methyl-1-propanesulfonic acid
sodium salt, N-isopropyl acrylamide, and N-phenylacrylamide in a
molar ratio of about 1:about 0.2:about 0.1. In certain embodiments,
the invention relates to any one of the aforementioned composite
materials, wherein the cross-linked gel comprises a polymer derived
from 2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, and ethylene glycol phenyl ether methacrylate in a molar
ratio of about 1:about 0.18:about 0.1.
[0134] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide (NIPAM) (or
N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm) or
N-(3-methoxypropyl) acrylamide (MPAAm) or N,N'-dimethylacrylamide
(DMAAm)) and N-phenylacrylamide. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials, wherein the cross-linked gel comprises a polymer derived
from 2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide (NIPAM) (or
N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm) or
N-(3-methoxypropyl) acrylamide (MPAAm) or N,N'-dimethylacrylamide
(DMAAm)) and N-phenylacrylamide in a molar ratio of about 1:about
0.2:about 0.1. In certain embodiments, the invention relates to any
one of the aforementioned composite materials, wherein the
cross-linked gel comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide (NIPAM) (or
N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm) or
N-(3-methoxypropyl) acrylamide (MPAAm) or N,N'-dimethylacrylamide
(DMAAm)) and N-phenylacrylamide in a molar ratio of about 1:about
0.18:about 0.1
[0135] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linked
gel comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide, and ethylene glycol phenyl ether
methacrylate. In certain embodiments, the invention relates to any
one of the aforementioned composite materials, wherein the
cross-linked gel comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide, and ethylene glycol phenyl ether
methacrylate in a molar ratio of about 1:about 0.1:about 0.2. In
certain embodiments, the invention relates to any one of the
aforementioned composite materials, wherein the cross-linked gel
comprises a polymer derived from
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide, and ethylene glycol phenyl ether
methacrylate in a molar ratio of about 1:about 0.1:about 0.15.
[0136] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is selected from the group consisting of glycerol
1,3-diglycerolate diacrylate, glycerol dimethacrylate,
3-(acryloyloxy)-2-hydroxypropyl methacrylate, glycerol propoxylate
triacrylate, bisacrylamidoacetic acid,
2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,
2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate and
dimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanediol
diacrylate and dimethacrylate, 1,10-dodecanediol diacrylate and
dimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,
2,2-dimethylpropanediol diacrylate and dimethacrylate,
dipentaerythritol pentaacrylate, dipropylene glycol diacrylate and
dimethacrylate, N,N-dodecamethylenebisacrylamide, divinylbenzene,
glycerol trimethacrylate, glycerol tris(acryloxypropyl)ether,
N,N'-hexamethylenebisacrylamide, N,N'-octamethylenebisacrylamide,
1,5-pentanediol diacrylate and dimethacrylate,
1,3-phenylenediacrylate, poly(ethylene glycol) diacrylate and
dimethacrylate, poly(propylene) diacrylate and dimethacrylate,
triethylene glycol diacrylate and dimethacrylate, triethylene
glycol divinyl ether, tripropylene glycol diacrylate or
dimethacrylate, diallyl diglycol carbonate, poly(ethylene glycol)
divinyl ether, N,N'-dimethacryloylpiperazine, divinyl glycol,
ethylene glycol diacrylate, ethylene glycol dimethacrylate,
N,N'-methylenebisacrylamide, 1,1,1-trimethylolethane
trimethacrylate, 1,1,1-trimethylolpropane triacrylate,
1,1,1-trimethylolpropane trimethacrylate (TRIM-M), vinyl acrylate,
1,6-hexanediol diacrylate and dimethacrylate, 1,3-butylene glycol
diacrylate and dimethacrylate, alkoxylated cyclohexane dimethanol
diacrylate, alkoxylated hexanediol diacrylate, alkoxylated
neopentyl glycol diacrylate, aromatic dimethacrylate, caprolactone
modified neopentylglycol hydroxypivalate diacrylate, cyclohexane
dimethanol diacrylate and dimethacrylate, ethoxylated bisphenol
diacrylate and dimethacrylate, neopentyl glycol diacrylate and
dimethacrylate, ethoxylated trimethylolpropane triacrylate,
propoxylated trimethylolpropane triacrylate, propoxylated glyceryl
triacrylate, pentaerythritol triacrylate, tris (2-hydroxy
ethyl)isocyanurate triacrylate, di-trimethylolpropane
tetraacrylate, dipentaerythritol pentaacrylate, ethoxylated
pentaerythritol tetraacrylate, pentaacrylate ester, pentaerythritol
tetraacrylate, caprolactone modified dipentaerythritol
hexaacrylate, N,N'-methylenebisacrylamide, diethylene glycol
diacrylate and dimethacrylate, trimethylolpropane triacrylate,
ethylene glycol diacrylate and dimethacrylate, tetra(ethylene
glycol) diacrylate, 1,6-hexanediol diacrylate, divinylbenzene,
1,3-butanediol dimethacrylate, poly(ethylene glycol) diacrylate,
1,3,5-triacryloylhexahydro-1,3,5-triazine, trimethylolpropane
diallyl ether, 2,4,6-triallyloxy-1,3,5-triazine,
1,3,5-triallyl-1,3,5-triazine-2,4,6-(1H,3H,5H)-trione,
N,N'-hexamethylenebis(methacrylamide), and glyoxal
bis(diallylacetal).
[0137] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is glycerol 1,3-diglycerolate diacrylate.
[0138] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent comprises glycerol dimethacrylate or
3-(acryloyloxy)-2-hydroxypropyl methacrylate. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the cross-linking agent comprises
glycerol dimethacrylate and 3-(acryloyloxy)-2-hydroxypropyl
methacrylate. In certain embodiments, the invention relates to any
one of the aforementioned composite materials, wherein the
cross-linking agent comprises glycerol dimethacrylate and
3-(acryloyloxy)-2-hydroxypropyl methacrylate in a molar ratio of
about 1:about 0.9.
[0139] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is glycerol propoxylate triacrylate.
[0140] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is 1,1,1-trimethylolpropane triacrylate or
1,1,1-trimethylolpropane trimethacrylate.
[0141] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is N,N-methylenebisacrylamide.
[0142] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is ethylene glycol dimethacrylate.
[0143] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is N,N'-hexamethylenebis(methacrylamide).
[0144] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is 1,3,5-triacryloylhexahydro-1,3,5-triazine.
[0145] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is 2,4,6-triallyloxy-1,3,5-triazine.
[0146] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is 1,3,5-triallyl-1,3,5-triazine-2,4,6-(1H,3H,5H)-trione.
[0147] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is N,N'-hexamethylenebis(methacrylamide).
[0148] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the cross-linking
agent is glyoxal bis(diallylacetal).
[0149] In certain embodiments, the invention relates to any one of
the aforementioned composite materials wherein the cross-linked gel
comprises macropores; the macroporous cross-linked gel has a volume
porosity of about 30% to about 80%; and the macropores have an
average pore diameter of about 10 nm to about 3000 nm.
[0150] In certain embodiments, the invention relates to any one of
the aforementioned composite materials wherein the cross-linked gel
comprises macropores; the macroporous cross-linked gel has a volume
porosity of about 40% to about 70%. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials wherein the cross-linked gel comprises macropores; the
macroporous cross-linked gel has a volume porosity of about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, or about
70%.
[0151] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the average pore
diameter of the macropores is about 25 nm to about 1500 nm.
[0152] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the average pore
diameter of the macropores is about 50 nm to about 1000 nm. In
certain embodiments, the invention relates to any one of the
aforementioned composite materials, wherein the average pore
diameter of the macropores is about 50 nm, about 100 nm, about 150
nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about
400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm,
about 650 nm, or about 700 nm.
[0153] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the average pore
diameter of the macropores is from about 300 nm to about 400
nm.
[0154] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the composite
material is a membrane.
[0155] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
has a void volume; and the void volume of the support member is
substantially filled with the macroporous cross-linked gel.
[0156] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
comprises a polymer; the support member is about 10 .mu.m to about
500 .mu.m thick; the pores of the support member have an average
pore diameter of about 0.1 .mu.m to about 25 .mu.m; and the support
member has a volume porosity of about 40% to about 90%.
[0157] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the thickness of
the support member is about 10 .mu.m to about 1000 .mu.m. In
certain embodiments, the invention relates to any one of the
aforementioned composite materials, wherein the thickness of the
support member is about 10 .mu.m to about 500 .mu.m. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the thickness of the support member is
about 30 .mu.m to about 300 .mu.m. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials, wherein the thickness of the support member is about 30
.mu.m, about 50 .mu.m, about 100 .mu.m, about 150 .mu.m, about 200
.mu.m, about 250 .mu.m, or about 300 .mu.m.
[0158] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the pores of the
support member have an average pore diameter of about 0.1 .mu.m to
about 25 .mu.m. In certain embodiments, the invention relates to
any one of the aforementioned composite materials, wherein the
pores of the support member have an average pore diameter of about
0.5 .mu.m to about 15 .mu.m. In certain embodiments, the invention
relates to any one of the aforementioned composite materials,
wherein the pores of the support member have an average pore
diameter of about 0.5 .mu.m, about 1 .mu.m, about 2 .mu.m, about 3
.mu.m, about 4 .mu.m, about 5 .mu.m, about 6 .mu.m, about 7 .mu.m,
about 8 .mu.m, about 9 .mu.m, about 10 .mu.m, about 11 .mu.m, about
12 .mu.m, about 13 .mu.m, about 14 .mu.m, or about 15 .mu.m.
[0159] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
has a volume porosity of about 40% to about 90%. In certain
embodiments, the invention relates to any one of the aforementioned
composite materials, wherein the support member has a volume
porosity of about 50% to about 80%. In certain embodiments, the
invention relates to any one of the aforementioned composite
materials, wherein the support member has a volume porosity of
about 50%, about 60%, about 70%, or about 80%.
[0160] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
comprises a polyolefin.
[0161] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
comprises a polymeric material selected from the group consisting
of polysulfones, polyethersulfones, polyphenyleneoxides,
polycarbonates, polyesters, cellulose and cellulose
derivatives.
[0162] In certain embodiments, the invention relates to any one of
the aforementioned composite materials, wherein the support member
comprises a fibrous woven or non-woven fabric comprising a polymer;
the support member is from about 10 .mu.m to about 2000 .mu.m
thick; the pores of the support member have an average pore
diameter of from about 0.1 .mu.m to about 25 .mu.m; and the support
member has a volume porosity of about 40% to about 90%.
Exemplary Methods
[0163] In certain embodiments, the invention relates to a method,
comprising the step of: [0164] contacting at a first flow rate a
first fluid comprising a substance with any one of the
aforementioned composite materials, thereby adsorbing or absorbing
a portion of the substance onto the composite material.
[0165] In certain embodiments, the first fluid further comprises a
fragmented antibody, aggregated antibodies, a host cell protein, a
polynucleotide, an endotoxin, or a virus.
[0166] In certain embodiments, the invention relates to any one of
the aforementioned methods,
[0167] wherein the fluid flow path of the first fluid is
substantially through the macropores of the composite material.
[0168] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of: [0169]
contacting at a second flow rate a second fluid with the substance
adsorbed or absorbed onto the composite material, thereby releasing
a first portion of the substance from the composite material.
[0170] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the fluid flow path of the
second fluid is substantially through the macropores of the
composite material.
[0171] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of: [0172]
contacting at a third flow rate a third fluid with the substance
adsorbed or absorbed onto the composite material, thereby releasing
a second portion of the substance from the composite material.
[0173] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the substance is a biological
molecule or biological ion.
[0174] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the biological molecule or
biological ion is selected from the group consisting of albumins,
lysozyme, viruses, cells, .gamma.-globulins of human and animal
origins, immunoglobulins of human and animal origins, proteins of
recombinant and natural origins, polypeptides of synthetic and
natural origins, interleukin-2 and its receptor, enzymes,
monoclonal antibodies, trypsin and its inhibitor, cytochrome C,
myoglobin, myoglobulin, .alpha.-chymotrypsinogen, recombinant human
interleukin, recombinant fusion protein, nucleic acid derived
products, DNA of synthetic and natural origins, and RNA of
synthetic and natural origins.
[0175] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the biological molecule or
biological ion is lysozyme, hIgG, myoglobin, human serum albumin,
soy trypsin inhibitor, transferring, enolase, ovalbumin,
ribonuclease, egg trypsin inhibitor, cytochrome c, Annexin V, or
.alpha.-chymotrypsinogen.
[0176] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid is a buffer. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the concentration of the buffer in
the first fluid is about 20 mM, about 30 mM, about 40 mM, about 50
mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85
mM, about 90 mM, about 95 mM, about 0.1M, about 0.11M, about 0.12
M, about 0.13 M, about 0.14 M, about 0.15 M, about 0.16 M, about
0.17 M, about 0.18 M, about 0.19 M or about 0.2 M. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the pH of the first fluid is about 2, about 2.5,
about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about
6, about 7, about 8, or about 9.
[0177] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid comprises
sodium acetate. In certain embodiments, the invention relates to
any one of the aforementioned methods, wherein the first fluid
comprises sodium citrate. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the first
fluid comprises sodium phosphate.
[0178] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid comprises a
salt. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the salt is selected from the
group consisting of glycine-HCl, NaCl, and NH.sub.4Cl. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the first fluid comprises sodium chloride. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the first fluid comprises sodium
chloride in a concentration of about 10 mM to about 600 mM. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the first fluid comprises sodium
chloride in a concentration of about 50 mM, about 75 mM, about 100
mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about
225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM,
about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450
mM, about 475 mM, about 500 mM, or about 525 mM.
[0179] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid is a clarified
cell culture supernatant.
[0180] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the concentration of the
substance in the first fluid is about 0.2 mg/mL to about 10 mg/mL.
In certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the concentration of the substance
in the first fluid is about 0.2 mg/mL, about 0.4 mg/mL, about 0.6
mg/mL, about 0.8 mg/mL, about 1 mg/mL, about 2 mg/mL, about 3
mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL,
about 8 mg/mL, about mg/mL, or about 10 mg/mL.
[0181] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first flow rate is up to
about 50 bed volumes/min. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the first
flow rate is about 5 bed volumes/min, about 10 bed volumes/min,
about 20 bed volumes/min, about 30 bed volumes/min, about 40 bed
volumes/min, or about 50 bed volumes/min.
[0182] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first flow rate is about
0.5 mL/min to about 2 mL/min. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the first
flow rate is about 0.5 mL/min, about 0.6 mL/min, about 0.7 mL/min,
about 0.8 mL/min, about 0.9 mL/min, about 1 mL/min, about 1.1
mL/min, about 1.2 mL/min, about 1.3 mL/min, about 1.4 mL/min, about
1.5 mL/min, about 1.6 mL/min, about 1.7 mL/min, or about 1.8
mL/min.
[0183] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the second fluid is a buffer.
In certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the second fluid comprises
2-(N-morpholino)ethanesulfonic acid. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the second fluid comprises 2-(N-morpholino)ethanesulfonic acid in a
concentration of about 50 mM to about 150 mM. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the second fluid comprises
2-(N-morpholino)ethanesulfonic acid in about 50 mM, about 60 mM,
about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM,
about 120 mM, about 130 mM, about 140 mM, or about 150 mM.
[0184] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the pH of the second fluid is
about 4 to about 8. In certain embodiments, the invention relates
to any one of the aforementioned methods, wherein the pH of the
second fluid is about 5, about 5.2, about 5.4, about 5.5, about
5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.2, or about
6.4.
[0185] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the second fluid comprises a
salt. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the salt is selected from the
group consisting of glycine-HCl, NaCl, and NH.sub.4Cl. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the salt concentration in the second fluid is
about 70 mM to about 200 mM. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the salt
concentration is about 70 mM, about 80 mM, about 90 mM, about 100
mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about
130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM,
about 160 mM, about 170 mM, about 180 mM, about 190 mM, or about
200 mM.
[0186] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the third fluid is a buffer. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the third fluid comprises
2-amino-2-hydroxymethyl-propane-1,3-diol/HCl (TRIS/HCl). In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the third fluid comprises
2-amino-2-hydroxymethyl-propane-1,3-diol/HCl (TRIS/HCl) in a
concentration of about 15 mM to about 40 mM. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the third fluid comprises
2-amino-2-hydroxymethyl-propane-1,3-diol/HCl (TRIS/HCl) in about 15
mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40
mM.
[0187] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the pH of the third fluid is
about 7 to about 9. In certain embodiments, the invention relates
to any one of the aforementioned methods, wherein the pH of the
third fluid is about 7, about 7.2, about 7.4, about 7.6, about 7.8,
about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.6,
about 8.8, or about 9.
[0188] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the third fluid comprises a
salt. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the salt is selected from the
group consisting of glycine-HCl, NaCl, and NH.sub.4Cl. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the salt concentration in the second fluid is
about 125 mM to about 400 mM. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the salt
concentration is about 125 mM, about 150 mM, about 175 mM, about
200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM,
about 325 mM, about 350 mM, about 375 mM, or about 400 mM.
[0189] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the steps of:
[0190] cleaning the composite material; and
[0191] repeating the above-mentioned steps.
[0192] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein substantially all of the
substance is adsorbed or absorbed onto the composite material.
[0193] In certain embodiments, the invention relates to a method,
comprising the step of: [0194] contacting at a first flow rate a
first fluid comprising a substance and an unwanted material with
any one of the aforementioned composite materials, thereby
adsorbing or absorbing a portion of the unwanted material onto the
composite material.
[0195] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the unwanted material comprises
a fragmented antibody, aggregated antibodies, a host cell protein,
a polynucleotide, an endotoxin, or a virus.
[0196] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein substantially all of the
unwanted material is adsorbed or absorbed onto the composite
material.
[0197] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the fluid flow path of the
first fluid is substantially through the macropores of the
composite material.
[0198] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the substance is a biological
molecule or biological ion.
[0199] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the biological molecule or
biological ion is selected from the group consisting of albumins,
lysozyme, viruses, cells, .gamma.-globulins of human and animal
origins, immunoglobulins of human and animal origins, proteins of
recombinant and natural origins, polypeptides of synthetic and
natural origins, interleukin-2 and its receptor, enzymes,
monoclonal antibodies, trypsin and its inhibitor, cytochrome C,
myoglobin, myoglobulin, .alpha.-chymotrypsinogen, recombinant human
interleukin, recombinant fusion protein, nucleic acid derived
products, DNA of synthetic and natural origins, and RNA of
synthetic and natural origins.
[0200] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the biological molecule or
biological ion is lysozyme, hIgG, myoglobin, human serum albumin,
soy trypsin inhibitor, transferring, enolase, ovalbumin,
ribonuclease, egg trypsin inhibitor, cytochrome c, Annexin V, or
.alpha.-chymotrypsinogen.
[0201] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid is a buffer. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the concentration of the buffer in
the first fluid is about 20 mM, about 30 mM, about 40 mM, about 50
mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85
mM, about 90 mM, about 95 mM, about 0.1 M, about 0.11 M, about 0.12
M, about 0.13 M, about 0.14 M, about 0.15 M, about 0.16 M, about
0.17 M, about 0.18 M, about 0.19 M or about 0.2 M.
[0202] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid comprises
sodium acetate. In certain embodiments, the invention relates to
any one of the aforementioned methods, wherein the first fluid
comprises sodium citrate. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the first
fluid comprises sodium phosphate.
[0203] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid comprises a
salt. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the salt is selected from the
group consisting of glycine-HCl, NaCl, and NH.sub.4Cl. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the first fluid comprises sodium chloride. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the first fluid comprises sodium
chloride in a concentration of about 10 mM to about 600 mM. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the first fluid comprises sodium
chloride in a concentration of about 50 mM, about 75 mM, about 100
mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about
225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM,
about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450
mM, about 475 mM, about 500 mM, or about 525 mM.
[0204] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first flow rate is about
0.5 mL/min to about 2 mL/min. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the first
flow rate is about 0.5 mL/min, about 0.6 mL/min, about 0.7 mL/min,
about 0.8 mL/min, about 0.9 mL/min, about 1 mL/min, about 1.1
mL/min, about 1.2 mL/min, about 1.3 mL/min, about 1.4 mL/min, about
1.5 mL/min, about 1.6 mL/min, about 1.7 mL/min, or about 1.8
mL/min.
[0205] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the first fluid is a clarified
cell culture supernatant.
[0206] In certain embodiments, the invention relates to a method of
making a composite material, comprising the steps of:
[0207] combining a first monomer, a photoinitiator, a cross-linking
agent, and a solvent, thereby forming a monomeric mixture;
[0208] contacting a support member with the monomeric mixture,
thereby forming a modified support member; wherein the support
member comprises a plurality of pores extending through the support
member, and the average pore diameter of the pores is about 0.1 to
about 25 .mu.m;
[0209] covering the modified support member with a polymeric sheet,
thereby forming a covered support member; and
[0210] irradiating the covered support member for a period of time,
thereby forming a composite material.
[0211] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomeric mixture comprises
a plurality of different monomers. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the monomeric mixture comprises a first monomer and a second
monomer. In certain embodiments, the invention relates to any one
of the aforementioned methods, wherein the monomeric mixture
further comprises a third, fourth, fifth, sixth, seventh, eighth,
or ninth monomer.
[0212] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of washing
the composite material with a second solvent, thereby forming a
washed composite material. In certain embodiments, the second
solvent is water.
[0213] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of
contacting the composite material or the washed composite material
with a salt solution.
[0214] In certain embodiments, the salt solution comprises sodium.
In certain embodiments, the salt solution comprises sodium
hydroxide. In certain embodiments, the salt solution comprises
sodium hydroxide in a concentration of about 0.05 N to about 0.15
N. In certain embodiments, the salt solution comprises sodium
hydroxide at about 0.06 N, about 0.07 N, about 0.08 N, about 0.09
N, about 0.1 N, about 0.11 N, about 0.12 N, about 0.13 N, or about
0.14 N.
[0215] In certain embodiments, the salt solution comprises sodium
chloride. In certain embodiments, the salt solution comprises
sodium chloride in a concentration of about 0.05 N to about 0.5 N.
In certain embodiments, the salt solution comprises sodium chloride
in about 0.06 N, about 0.07 N, about 0.08 N, about 0.09 N, about
0.1 N, about 0.11 N, about 0.12 N, about 0.13 N, about 0.14 N,
about 0.15 N, about 0.18 N, about 0.2 N, about 0.22 N, about 0.24
N, about 0.26 N, about 0.28 N, about 0.3 N, about 0.32 N, about
0.34 N, about 0.36 N, about 0.38 N, about 0.4 N, about 0.42 N,
about 0.44 N, about 0.46 N, about 0.48 N, or about 0.5 N.
[0216] In certain embodiments, the salt solution comprises sodium
hydroxide and sodium chloride.
[0217] In certain embodiments, the invention relates to any one of
the aforementioned methods, further comprising the step of removing
any excess monomeric mixture from the covered support member.
[0218] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
2-(diethylamino)ethyl methacrylate, 2-aminoethyl methacrylate,
2-carboxyethyl acrylate, 2-(methylthio)ethyl methacrylate,
acrylamide, N-acryloxysuccinimide, butyl acrylate or methacrylate,
N,N-diethylacrylamide, N,N-dimethylacrylamide,
2-(N,N-dimethylamino)ethyl acrylate or methacrylate,
N-[3-(N,N-dimethylamino)propyl]methacrylamide,
N,N-dimethylacrylamide, n-dodecyl acrylate, n-dodecyl methacrylate,
ethyl acrylate or methacrylate, 2-ethylhexyl methacrylate,
hydroxypropyl methacrylate, glycidyl acrylate or methacrylate,
ethylene glycol phenyl ether methacrylate, methacrylamide,
methacrylic anhydride, propyl acrylate or methacrylate,
N-isopropylacrylamide, styrene, 4-vinylpyridine, vinylsulfonic
acid, N-vinyl-2-pyrrolidinone (VP),
acrylamido-2-methyl-1-propanesulfonic acid, styrenesulfonic acid,
alginic acid, (3-acrylamidopropyl)trimethylammonium halide,
diallyldimethylammonium halide, 4-vinyl-N-methylpyridinium halide,
vinylbenzyl-N-trimethylammonium halide,
methacryloxyethyltrimethylammonium halide, 3-sulfopropyl
methacrylate, 2-(2-methoxy)ethyl acrylate or methacrylate,
hydroxyethyl acrylamide, N-(3-methoxypropyl acrylamide),
N-[tris(hydroxymethyl)methyl]acrylamide, N-phenyl acrylamide,
N-tert-butyl acrylamide, or diacetone acrylamide.
[0219] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
more than one monomer. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the
monomer mixture further comprises a second monomer.
[0220] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, ethylene glycol phenyl ether methacrylate, and
2-(methylthio)ethyl methacrylate. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the monomer mixture comprises 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and 2-(methylthio)ethyl methacrylate in a molar
ratio of about 1:about 0.2:about 0.1:about 0.06. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the monomer mixture comprises 2-carboxyethyl
acrylate, acrylamido-2-methyl-1-propanesulfonic acid, ethylene
glycol phenyl ether methacrylate, and 2-(methylthio)ethyl
methacrylate in a molar ratio of about 1:about 0.22:about
0.14:about 0.06.
[0221] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, ethylene glycol phenyl ether methacrylate, and hydroxypropyl
methacrylate. In certain embodiments, the invention relates to any
one of the aforementioned methods, wherein the monomer mixture
comprises 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and hydroxypropyl methacrylate in a molar ratio
of about 1:about 0.2:about 0.2:about 0.1. In certain embodiments,
the invention relates to any one of the aforementioned methods,
wherein the monomer mixture comprises 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, ethylene glycol phenyl
ether methacrylate, and hydroxypropyl methacrylate in a molar ratio
of about 1: about 0.25:about 0.15:about 0.14.
[0222] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
2-carboxyethyl acrylate, acrylamido-2-methyl-1-propanesulfonic
acid, and ethylene glycol phenyl ether methacrylate. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the monomer mixture comprises 2-carboxyethyl
acrylate, acrylamido-2-methyl-1-propanesulfonic acid, and ethylene
glycol phenyl ether methacrylate in a molar ratio of about 1:about
0.3:about 0.1. In certain embodiments, the invention relates to any
one of the aforementioned methods, wherein the monomer mixture
comprises 2-carboxyethyl acrylate,
acrylamido-2-methyl-1-propanesulfonic acid, and ethylene glycol
phenyl ether methacrylate in a molar ratio of about 1:about
0.26:about 0.15.
[0223] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
vinylbenzyl-N-trimethylammonium halide. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the monomer mixture comprises vinylbenzyl-N-trimethylammonium
chloride.
[0224] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomer mixture comprises
2-(diethylamino)ethyl methacrylate,
(ar-vinylbenzyl)trimethylammonium chloride, ethylene glycol phenyl
ether methacrylate, and 2-aminoethyl methacrylate. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the monomer mixture comprises
2-(diethylamino)ethyl methacrylate,
(ar-vinylbenzyl)trimethylammonium chloride, ethylene glycol phenyl
ether methacrylate, and 2-aminoethyl methacrylate in a molar ratio
of about 1:about 0.4:about 0.5:about 0.1. In certain embodiments,
the invention relates to any one of the aforementioned methods,
wherein the monomer mixture comprises 2-(diethylamino)ethyl
methacrylate, (ar-vinylbenzyl)trimethylammonium chloride, ethylene
glycol phenyl ether methacrylate, and 2-aminoethyl methacrylate in
a molar ratio of about 1:about 0.36:about 0.52:about 0.1.
[0225] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomers are present in the
solvent in about 6% to about 38% (w/w), collectively.
[0226] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the monomers are present in the
solvent in an amount of about 6%, about 7%, about 8%, about 9%,
about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,
about 16%, about 17%, about 18%, about 19%, about 20%, about 21%,
about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,
about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,
about 34%, about 35%, about 36%, about 37%, or about 38% (w/w),
collectively.
[0227] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the photoinitiator is present
in the monomeric mixture in an amount of about 0.4% (w/w) to about
2.5% (w/w) relative to the total weight of monomer.
[0228] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the photoinitiator is present
in the monomeric mixture in about 0.6%, about 0.8%, about 1.0%,
about 1.2%, or about 1.4% (w/w) relative to the total weight of
monomer.
[0229] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the photoinitiator is selected
from the group consisting of
1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one,
2,2-dimethoxy-2-phenylacetophenone, benzophenone, benzoin and
benzoin ethers, dialkoxyacetophenones, hydroxyalkylphenones, and
.alpha.-hydroxymethyl benzoin sulfonic esters.
[0230] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
1,3-butanediol, di(propylene glycol) propyl ether,
N,N-dimethylacetamide, di(propylene glycol) dimethyl ether,
1,2-propanediol, di(propylene glycol) methyl ether acetate (DPMA),
water, dioxane, dimethylsulfoxide (DMSO), dimethylformamide (DMF),
acetone, ethanol, N-methylpyrrolidone (NMP), tetrahydrofuran (THF),
ethyl acetate, acetonitrile, N-methylacetamide, propanol,
tri(propylene glycol) propyl ether, or methanol.
[0231] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
N,N-dimethylacetamide, di(propylene glycol) dimethyl ether,
1,2-propanediol, and water.
[0232] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
N,N-dimethylacetamide. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the
solvent comprises N,N-dimethylacetamide in an amount of about 15%
to about 40% by weight. In certain embodiments, the invention
relates to any one of the aforementioned methods, wherein the
solvent comprises N,N-dimethylacetamide in about 20%, about 21%,
about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,
about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,
about 34%, or about 35% by weight.
[0233] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
di(propylene glycol) dimethyl ether. In certain embodiments, the
invention relates to any one of the aforementioned methods, wherein
the solvent comprises di(propylene glycol) dimethyl ether in an
amount of about 30% to about 90% by weight. In certain embodiments,
the invention relates to any one of the aforementioned methods,
wherein the solvent comprises di(propylene glycol) dimethyl ether
in about 40%, about 42%, about 44%, about 46%, about 48%, about
50%, about 52%, about 54%, about 56%, about 58%, about 60%, about
62%, about 64%, about 66%, about 68%, about 70%, about 72%, about
74%, about 76%, about 78%, or about 80% by weight.
[0234] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
1,2-propanediol. In certain embodiments, the invention relates to
any one of the aforementioned methods, wherein the solvent
comprises 1,2-propanediol in an amount of about 3% to about 75% by
weight. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
1,2-propanediol in about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,
about 60%, about 65%, about 70%, or about 75% by weight.
[0235] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises water. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the solvent comprises water in an
amount of about 2% to about 9% by weight. In certain embodiments,
the invention relates to any one of the aforementioned methods,
wherein the solvent comprises water in about 2%, about 3%, about
4%, about 5%, about 6%, about 7%, about 8%, or about 9% by
weight.
[0236] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
di(propylene glycol) methyl ether acetate. In certain embodiments,
the invention relates to any one of the aforementioned methods,
wherein the solvent comprises di(propylene glycol) methyl ether
acetate in an amount of about 24% to about 72% by weight. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the solvent comprises di(propylene
glycol) methyl ether acetate in about 30%, about 32%, about 34%,
about 36%, about 38%, about 40%, about 42%, about 44%, about 46%,
about 48%, about 50%, about 52%, about 54%, about 56%, about 58%,
about 60%, about 62%, about 64%, or about 66% by weight.
[0237] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
1,3-butanediol. In certain embodiments, the invention relates to
any one of the aforementioned methods, wherein the solvent
comprises 1,3-butanediol in an amount of about 35% to about 95% by
weight. In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the solvent comprises
1,3-butanediol in about 40%, about 45%, about 50%, about 55%, about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, or
about 90% by weight.
[0238] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
present in the solvent in about 4% to about 25% (w/w).
[0239] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
present in the solvent in an amount of about 4%, about 5%, about
6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,
about 13%, about 14%, about 15%, about 16%, about 17%, about 18%,
about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,
or about 25% (w/w).
[0240] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
selected from the group consisting of glycerol 1,3-diglycerolate
diacrylate, glycerol dimethacrylate,
3-(acryloyloxy)-2-hydroxypropyl methacrylate, glycerol propoxylate
triacrylate, bisacrylamidoacetic acid,
2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,
2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate and
dimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanediol
diacrylate and dimethacrylate, 1,10-dodecanediol diacrylate and
dimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,
2,2-dimethylpropanediol diacrylate and dimethacrylate,
dipentaerythritol pentaacrylate, dipropylene glycol diacrylate and
dimethacrylate, N,N-dodecamethylenebisacrylamide, divinylbenzene,
glycerol trimethacrylate, glycerol tris(acryloxypropyl)ether,
N,N'-hexamethylenebisacrylamide, N,N'-octamethylenebisacrylamide,
1,5-pentanediol diacrylate and dimethacrylate,
1,3-phenylenediacrylate, poly(ethylene glycol) diacrylate and
dimethacrylate, poly(propylene) diacrylate and dimethacrylate,
triethylene glycol diacrylate and dimethacrylate, triethylene
glycol divinyl ether, tripropylene glycol diacrylate or
dimethacrylate, diallyl diglycol carbonate, poly(ethylene glycol)
divinyl ether, N,N'-dimethacryloylpiperazine, divinyl glycol,
ethylene glycol diacrylate, ethylene glycol dimethacrylate,
N,N'-methylenebisacrylamide, 1,1,1-trimethylolethane
trimethacrylate, 1,1,1-trimethylolpropane triacrylate,
1,1,1-trimethylolpropane trimethacrylate (TRIM-M), vinyl acrylate,
1,6-hexanediol diacrylate and dimethacrylate, 1,3-butylene glycol
diacrylate and dimethacrylate, alkoxylated cyclohexane dimethanol
diacrylate, alkoxylated hexanediol diacrylate, alkoxylated
neopentyl glycol diacrylate, aromatic dimethacrylate, caprolactone
modified neopentylglycol hydroxypivalate diacrylate, cyclohexane
dimethanol diacrylate and dimethacrylate, ethoxylated bisphenol
diacrylate and dimethacrylate, neopentyl glycol diacrylate and
dimethacrylate, ethoxylated trimethylolpropane triacrylate,
propoxylated trimethylolpropane triacrylate, propoxylated glyceryl
triacrylate, pentaerythritol triacrylate, tris (2-hydroxy
ethyl)isocyanurate triacrylate, di-trimethylolpropane
tetraacrylate, dipentaerythritol pentaacrylate, ethoxylated
pentaerythritol tetraacrylate, pentaacrylate ester, pentaerythritol
tetraacrylate, caprolactone modified dipentaerythritol
hexaacrylate, N,N'-methylenebisacrylamide, diethylene glycol
diacrylate and dimethacrylate, trimethylolpropane triacrylate,
ethylene glycol diacrylate and dimethacrylate, tetra(ethylene
glycol) diacrylate, 1,6-hexanediol diacrylate, divinylbenzene,
poly(ethylene glycol) diacrylate,
1,3,5-triacryloylhexahydro-1,3,5-triazine, trimethylolpropane
diallyl ether, 2,4,6-triallyloxy-1,3,5-triazine,
1,3,5-triallyl-1,3,5-triazine-2,4,6-(1H,3H,5H)-trione,
N,N'-hexamethylenebis(methacrylamide), and glyoxal
bis(diallylacetal).
[0241] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
glycerol 1,3-diglycerolate diacrylate.
[0242] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent
comprises glycerol dimethacrylate or
3-(acryloyloxy)-2-hydroxypropyl methacrylate. In certain
embodiments, the invention relates to any one of the aforementioned
methods, wherein the cross-linking agent comprises glycerol
dimethacrylate and 3-(acryloyloxy)-2-hydroxypropyl methacrylate. In
certain embodiments, the invention relates to any one of the
aforementioned methods, wherein the cross-linking agent comprises
glycerol dimethacrylate and 3-(acryloyloxy)-2-hydroxypropyl
methacrylate in a molar ratio of about 1:about 0.9.
[0243] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
glycerol propoxylate triacrylate.
[0244] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
1,1,1-trimethylolpropane triacrylate or 1,1,1-trimethylolpropane
trimethacrylate.
[0245] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
ethylene glycol dimethacrylate.
[0246] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the cross-linking agent is
N,N'-methylenebisacrylamide.
[0247] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the covered support member is
irradiated at about 350 nm.
[0248] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the period of time is about 1
minute, about 5 minutes, about 10 minutes, about 15 minutes, about
20 minutes, about 30 minutes, about 45 minutes, or about 1
hour.
[0249] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the composite material
comprises macropores.
[0250] In certain embodiments, the invention relates to any one of
the aforementioned methods, wherein the average pore diameter of
the macropores is less than the average pore diameter of the
pores.
EXEMPLIFICATION
[0251] The following examples are provided to illustrate the
invention. It will be understood, however, that the specific
details given in each example have been selected for purpose of
illustration and are not to be construed as limiting the scope of
the invention. Generally, the experiments were conducted under
similar conditions unless noted.
Example 1
[0252] This example illustrates a method of preparing a
cation-exchange material of the present invention with multi-modal
functionality.
[0253] A 20 wt % solution was prepared by dissolving 2-carboxyethyl
acrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid, ethylene
glycol phenyl ether methacrylate, 2-(methylthio) ethyl methacrylate
in a molar ratio of 1:0.22:0.14:0.06, respectively, in a solvent
mixture containing 27.0 wt % N,N'-dimethylacetamide, 61.0 wt %
di(propylene glycol)dimethyl ether, 7.15 wt % 1,2-propanediol and
4.85 wt % water. Glycerol 1,3-diglycerolate diacrylate was used as
a cross-linking agent to achieve cross-linking density of 8%
(mol/mol). The photo-initiator Irgacure 2959 was added in the
amount of 1 wt % with respect to the mass of the monomers.
[0254] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the following general
procedure. A weighed support member was placed on a poly(ethylene)
(PE) sheet and a monomer or polymer solution was applied the
sample. The sample was subsequently covered with another PE sheet
and a rubber roller was run over the sandwich to remove excess
solution. In situ gel formation in the sample was induced by
polymerization initiated by irradiation with the wavelength of 350
nm for the period of 10 minutes. The resulting composite material
was thoroughly washed with RO and then placed in a solution
containing 0.1 N sodium hydroxide and 0.1 N sodium chloride to
transfer the membrane into Na.sup.+-form. Thereafter membrane was
washed with RO water and dried at room temperature.
[0255] Membranes were characterized in terms of solute flux, hIgG
and lysozyme binding capacity, hIgG and lysozyme recovery.
[0256] Solute flux measurements through the composite materials
were carried out after the samples had been wetted with RO water.
As a standard procedure, a sample in the form of a disk of diameter
7.8 cm was mounted on a sintered grid of 3-5 mm thickness and
assembled into a cell supplied with compressed nitrogen at a
controlled pressure. The cell was filled with 85 mM sodium acetate
buffer containing 250 mM NaCl, pH 4.5 and pressure of 100 kPa was
applied. The buffer solution that passed through the composite
material in a specified time was collected in a pre-weighed
container and weighed. All experiments were carried out at room
temperature and at atmospheric pressure at the permeate outlet.
Each measurement was repeated three or more times to achieve a
reproducibility of .+-.5%.
[0257] Protein adsorption experiments were carried out by the
following procedure. In adsorption step, a composite material
sample in a form of a single membrane disk of diameter 25 mm was
installed into Natrix membrane holder. Waters 600E HPLC system was
used for carrying out the membrane chromatographic studies. The
cell and the membrane sample were primed by passing 85 mM sodium
acetate buffer containing 250 mM sodium chloride, pH 4.5 and
conductivity 30 mS/cm (buffer A). The UV absorbance (at 280 nm) of
the effluent stream from the Natrix membrane holder and the system
pressure were continuously recorded. Protein was dissolved in
buffer A to prepare 0.5 mg/mL solution. Buffer A was referred as a
binding buffer. The elution buffer, containing 25 mM TRIS/HCl, 250
mM NaCl, pH 8.2, was referred as buffer B. All buffers and protein
solutions were filtered through a polyethersulfone (PES)
microporous membrane with pore size of 0.2 .mu.m (Nalgene).
[0258] In chromatographic experiments, buffer A was passed through
the membrane until a stable UV absorbance baseline was established.
The method developed for bind-elute experiment included following
steps: in the first step the membrane was preconditioning with
binding buffer A; in the second step, the protein dissolved in a
buffer A passed through the membrane to reach 20-30% breakthrough;
in the third step the membrane was washed with buffer A and in a
final step protein was eluted from the membrane using a buffer B
which passed through the membrane. Chromatographic experiments were
performed at a flow rate of 1 mL/min.
[0259] Protein recovery was calculated as the amount of the
desorbed substance compared to the amount of the substance applied
to the membrane in the adsorption/binding step.
[0260] The composite material produced by this method had a solute
flux of 2,950 kg/m.sup.2 h at applied pressure of 100 kPa. Dynamic
binding capacity at 10% breakthrough was 220.1 mg/mL for hIgG and
135.0 mg/mL for lysozyme. The recovery of hIgG and lysozyme
exceeded 95%.
[0261] FIG. 1 shows the hIgG bind-elute curve obtained in described
above experiment.
Example 2
[0262] This example illustrates a method of preparing a
cation-exchange material of the present invention with multi-modal
functionality
[0263] A 23 wt % solution was prepared by dissolving 2-carboxyethyl
acrylate, 2-acrylamido-2-methyl-1-propanesulfonic acid, ethylene
glycol phenyl ether methacrylate, hydroxypropyl methacrylate in a
molar ratio of 1:0.25:0.15:0.14, respectively, in a solvent mixture
containing 26.3 wt % N,N'-dimethylacetamide, 59.6 wt % di(propylene
glycol)dimethyl ether, 7.3 wt % 1,2-propanediol and 6.8 wt % water.
Glycerol dimethacrylate (GDA) and 3-(acryloyloxy)-2-hydroxypropyl
methacrylate (AHM) were used as cross-linking agents to achieve
cross-linking density of 10.6% (mol/mol). Cross-linking agents GDA
and AHM were added in a molar ratio of 1:0.9, respectively. The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0264] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and then placed in a solution containing 0.1 N sodium
hydroxide and 0.1 N sodium chloride to transfer the membrane into
Na.sup.+-form. Thereafter membrane was washed with RO water and
dried at room temperature.
[0265] Membrane thus obtained was characterized in terms of solute
flux, hIgG binding capacity and hIgG recovery as described in
Example 1.
[0266] The composite material produced by this method showed solute
flux of 4,150.00 kg/m.sup.2 h and hIgG binding capacity of 175.8
mg/mL at 10% breakthrough. The recovery of hIgG exceeded 95%.
Example 3
[0267] This example illustrates a method of preparing a
cation-exchange material of the present invention with multi-modal
functionality
[0268] A 20.6 wt % solution was prepared by dissolving
2-carboxyethyl acrylate, 2-acrylamido-2-methyl-1-propanesulfonic
acid, ethylene glycol phenyl ether methacrylate in a molar ratio of
1:0.26:0.15, respectively, in a solvent mixture containing 27.0 wt
% N,N'-dimethylacetamide, 60.0 wt % di(propylene glycol)dimethyl
ether, 6.5 wt % 1,2-propanediol and 6.5 wt % water. Glycerol
propoxylate (1PO/OH) triacrylate was used as cross-linking agents
to achieve cross-linking density of 7.7% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0269] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and then placed in a solution containing 0.1 N sodium
hydroxide and 0.1 N sodium chloride to transfer the membrane into
Na.sup.+-form. Thereafter membrane was washed with RO water and
dried at room temperature.
[0270] Membrane thus obtained was characterized in terms of solute
flux, hIgG binding capacity and hIgG recovery as described in
Example 1.
[0271] The composite material produced by this method showed solute
flux of 2,740.00 kg/m.sup.2 h and hIgG binding capacity of 144
mg/mL at 10% breakthrough. The recovery of hIgG exceeded 95%.
Example 4
[0272] This example illustrates effect of ionic strength on hIgG
binding capacity of a cation-exchange material of the present
invention with multi-modal functionality
[0273] One of the most important features of mixed mode media is a
salt tolerance. Due to dual functionality of mixed-mode media,
containing hydrophobic and ionic elements, increasing ionic
strength will disrupt ionic bonds but the increasing salt
concentration will promote hydrophobic adsorption leading to the
salt independent performance.
[0274] Multimodal cation-exchange membrane prepared as described in
Example 1 was used to examine an effect of ionic strength on hIgG
binding capacity. 85 mM sodium acetate buffer containing sodium
chloride ranging from 0 to 400 mM, pH 4.5 was used as a binding
buffer. hIgG was dissolved in binding buffer with various ionic
strength to prepare 0.5 mg/mL solution. Binding experiments were
performed as described in Example 1. FIG. 2 illustrates hIgG
dynamic binding capacity at 10% breakthrough as a function of salt
content in a binding buffer. Natrix weak-cation exchange membrane
(Natrix C) and Natrix strong cation exchange membrane (Natrix S)
were also examined in the salt tolerance study (FIG. 2). As can be
seen from FIG. 2, incorporating various functionalities in the
mixed-mode membrane allows not only maintain salt-tolerance
performance as also seen in the case of weak-cation exchange
membrane (Natrix C), but enhance binding capacity as well. The
multimodal membrane of the present invention can be successfully
used in a much larger operating range in terms of conductivity of
starting material than traditional cation exchangers. Secondly,
mixed-mode membrane can be employed for direct load of clarified
feed stocks, without prior dilution to reduce the conductivity of
starting material.
Example 5
[0275] This example illustrates effect of pH on hIgG binding
capacity of a cation-exchange material of the present invention
with multi-modal functionality
[0276] pH-dependent binding is one of the features of mixed-mode
media. The later allows to use pH is an effective tool for protein
elution without any change in conductivity of mobile phase. Because
of the mixed-mode sorbents operate by a combination of hydrophobic
and electrostatic interaction, mobile phase pH is greatly
responsible for the shift from one type interaction to another.
[0277] Multimodal cation-exchange membrane prepared as described in
Example 1 was used to examine effect of pH on hIgG binding
capacity. 85 mM sodium acetate buffer containing 250 mM sodium
chloride was used as a binding buffer. pH was varied from 4.0 to
6.0. hIgG was dissolved in binding buffer with various pH to
prepare 0.5 mg/mL solution. Binding experiments were performed as
described in Example 1. FIG. 3 illustrates hIgG dynamic binding
capacity at 10% breakthrough as a function of pH.
[0278] As pH is increased, binding capacity decreases, that
consistent with fundamental mechanism.
Example 6
[0279] This example illustrates use of mixed-mode membrane in
bind-elute mode to purify monoclonal antibodies
[0280] Enhanced removal of antibody aggregates from a preparation
or protein A purified monoclonal antibody can be successfully
achieved by using mixed-mode media in bind-elute mode. "Bind-elute"
mode is an operational approach to chromatography in which the
buffer conditions are established so that both a target protein
(e.g., non-aggregated antibody) and undesired contaminants (e.g.,
aggregated antibody) bind to the mixed mode chromatography support.
Fractionation of intact non-aggregated protein is achieved
subsequently by changing the conditions such that the target of
interest is eluted from the support while contaminants remain
bound. These contaminants may optimally be removed by an
appropriate cleaning buffer.
[0281] Multimodal cation-exchange membrane prepared as described in
Example 1 was used to examine membrane performance in bind-elute
mode to purify monoclonal antibodies. 85 mM sodium acetate buffer
containing 250 mM sodium chloride, pH 4.5 was used as a binding
buffer A. Protein A purified monoclonal antibody (mAbs) was
dissolved in binding buffer A to prepare 0.5 mg/mL solution.
Membrane was equilibrated at a flow rate of 1 mL/min with buffer A
as described in Example 1. mAbs solution was applied to the
membrane to achieve 80% of 10% breakthrough dynamic binding
capacity, washed with equilibration buffer A, and then eluted in
two steps. First step included the use of 100 mM MES buffer
containing 135 mM NaCl, pH 5.7 as elution buffer B while 25 mM
TRIS/HCl buffer containing 250 mM NaCl, pH 8.2 was used in a second
step as elution buffer C (FIG. 4). Eluents were analyzed on SEC
column TSKgel G3000SW.sub.x1 (Tosoh Bioscience). 100 mM sodium
phosphate buffer containing 100 mM sodium sulphate, pH 6.7 was used
as a mobile phase. 800 .mu.L sample was applied into the column at
0.80 mL/min.
[0282] FIG. 4 presents performance of mixed-mode membrane in
bind-elute mode. Two-step elution allows separating non-aggregated
mAbs from aggregates (FIG. 5-7).
[0283] As can be seen from FIG. 6 first fraction of mAbs eluted
with buffer B contain significantly lower amount of aggregates
compare to the feed solution. This mixed-mode membrane selectively
reduced amount of aggregates from 1.51% to 0.11% or by 93%. Second
fraction of mAbs eluted with buffer C showed a significant amount
of aggregates (FIG. 7).
Example 7
[0284] This example illustrates multicycles use of mixed-mode
membrane in bind-elute mode.
[0285] Multimodal cation-exchange membrane prepared as described in
Example 1 was used to examine membrane performance in multicycles.
Bind-elute experiments were run as described in Example 1. Membrane
was equilibrated with 100 mM sodium citrate buffer, pH 4.5,
conductivity -21.9 mS/cm. The same buffer was used as a binding
buffer A. Protein A purified monoclonal antibody (mAbs) was
dissolved in binding buffer to prepare 0.5 mg/mL solution. mAbs
solution was applied to the membrane to achieve 20% breakthrough
dynamic binding capacity, washed with equilibration buffer A, then
eluted in two steps as described in Example 6. First step included
the use of 100 mM MES buffer containing 135 mM NaCl, pH 5.7 as
elution buffer B while 25 mM TRIS/HCl buffer containing 250 mM NaCl
was used in a second step as elution buffer C. mAbs feed solution
and eluents were analyzed on SEC column TSKgel G3000SW.sub.x1
(Tosoh Bioscience). 100 mM sodium phosphate buffer containing 100
mM sodium sulphate, pH 6.7 was used as a mobile phase. 800 .mu.L
sample was applied into the column at 0.80 mL/min.
[0286] The effluent was monitored for UV absorbance at 280 nm to
characterize the binding capacity of the membrane. The membrane was
then cleaned with 0.1 M NaCl/0.1 M NaCl and equilibrated with
buffer A for the second run. The run was repeated as described
above. The membrane was then cleaned with 0.1 M NaCl/0.1M NaCl and
equilibrated with buffer A for the third run as described above.
The run was repeated as described above. FIG. 8 presents
performance of a multimodal membrane in multiple cycles.
[0287] SEC column analysis showed 1.5% aggregates in mAbs feed
solution.
[0288] As can be seen from FIG. 8, Natrix multimodal membrane
showed identical performance in three runs in terms of dynamic
binding capacity and selective aggregates removal. Aggregates
concentration in mAbs post protein A was reduced from 1.51% to
0.11% or by 93%.
Example 8
[0289] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality
[0290] A multimodal strong anion-exchange media has a great
potential to be used in post-protein A purification of monoclonal
antibodies (mAbs) at process scale. The goal is to remove key
contaminants such is DNA, host cell proteins (HCP), leached protein
A, aggregates and viruses in a single step.
[0291] A 12.6 wt % solution was prepared by dissolving
(ar-vinylbenzyl)trimethylammonium chloride as a monomer and
trimethylolpropane triacrylate (TRIM-A) as a cross-linker in a
solvent mixture containing 52.0 wt % 1,2-propanediol and 48.0 wt %
di(propylene glycol) methyl ether acetate. Cross-linking agent
TRIM-A was added to achieve cross-linking density of 13% (mol/mol).
The photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0292] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and dried at room temperature.
[0293] Membrane thus obtained was characterized in terms of water,
solute flux (100 mM sodium phosphate buffer, containing 150 mM
sodium chloride, pH 7.0) and protein A binding capacity as
described Example 1. Protein A was dissolved in 100 mM sodium
phosphate buffer containing 150 mM sodium chloride, pH 7.0
[0294] The composite material produced by this method showed water
flux of 1,850.00 kg/m.sup.2 h at 100 kPa, solute flux of 2,270.00
kg/m.sup.2 h and protein A binding capacity of 85.8 mg/mL at 10%
breakthrough.
Example 9
[0295] This example illustrates use of mixed-mode membrane in
flow-through mode to purify monoclonal antibodies
[0296] Removal antibody aggregates from a preparation or protein A
purified monoclonal antibody can be successfully achieved by using
mixed-mode media in a flow-through mode. The later refers to an
operational approach to chromatography in which the buffer
conditions are established so that intact non-aggregated protein to
be purified flows through the mixed mode chromatography support
upon application, while aggregates and other large molecules
(including viruses) are selectively retained, thus achieving their
removal.
[0297] Multimodal anion-exchange membrane prepared as described in
Example 8 was used to examine membrane performance in flow-through
mode to purify monoclonal antibodies. Membrane was equilibrated at
a flow rate of 1 mL/min with 100 mM sodium phosphate buffer
containing 250 mM sodium chloride (buffer A) as described in
Example 1. Protein A purified monoclonal antibody (mAbs) was
dissolved in buffer A and 300 mg mAbs/mL was applied to the
membrane at flow rate of 1 mg/mL. mAbs feed and flowthrough
fraction were analyzed on SEC column TSKgel G3000SW.sub.x1 (Tosoh
Bioscience). 100 mM sodium phosphate buffer containing 100 mM
sodium sulphate, pH 6.7 was used as a mobile phase. 800 .mu.L
sample was applied into the column at 0.80 mL/min. The aggregate
level was reduced from 1.51% to 0.32%.
Example 10
[0298] This example illustrates a method of preparing a
anion-exchange material of the present invention with multi-modal
functionality A 25 wt % solution was prepared by dissolving
2-(diethylamino)ethyl methacrylate,
(ar-vinylbenzyl)trimethylammonium chloride, ethylene glycol phenyl
ether methacrylate and 2-aminoethyl methacrylate in a molar ratio
of 1:0.36:0.52:0.1, respectively, in a solvent mixture containing
72.5 wt % 1,3-Butanediol and 27.5 wt % N,N'-dimethylacetamide.
Ethylene glycol dimethacrylate was used as cross-linking agent to
achieve cross-linking density of 12.2% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0299] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and dried at room temperature.
[0300] Membrane thus obtained was characterized in terms of water,
solute flux (100 mM sodium phosphate buffer, containing 250 mM
sodium chloride, pH 7.0) as described in Example 1 and aggregates
removal from monoclonal antibodies in flow-through mode as
described in Example 9. Protein A purified monoclonal antibody
(mAbs) was dissolved in buffer A and 400 mg/mL was applied to the
membrane at flow rate of 1 mg/mL. mAbs feed and flowthrough
fraction were analyzed on SEC column TSKgel G3000SW.sub.x1 (Tosoh
Bioscience). 100 mM sodium phosphate buffer containing 100 mM
sodium sulphate, pH 6.7 was used as a mobile phase. 800 .mu.L
sample was applied into the column at 0.80 mL/min.
[0301] The composite material produced by this method showed water
flux of 3,750.00 kg/m.sup.2 h and solute flux of 4,050.00
kg/m.sup.2 h. The aggregate level was reduced from 1.51% to
0.47%.
Example 11
[0302] This example illustrates a method of preparing a
cation-exchange material of the present invention with multi-modal
functionality
[0303] A 19.3 wt % solution was prepared by dissolving
2-carboxyethyl acrylate, 2-acrylamido-2-methyl-1-propanesulfonic
acid, 2-(methylthio)ethyl methacrylate, 2-aminoethyl methacrylate
hydrochloride and ethylene glycol phenyl ether methacrylate in a
molar ratio of 1:0.34:0.05:0.05:0.22, respectively, in a solvent
mixture containing 26.7 wt % N,N'-dimethylacetamide, 61.5 wt %
di(propylene glycol)dimethyl ether, 7.1 wt % 1,2-propanediol and
4.7 wt % water. N,N'-methylenebisacrylamide was used as
cross-linking agents to achieve cross-linking density of 5.4%
(mol/mol). The photo-initiator Irgacure 2959 was added in the
amount of 1 wt % with respect to the mass of the monomers.
[0304] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and then placed in a solution containing 0.1 N sodium
hydroxide and 0.1 N sodium chloride to transfer the membrane into
Na.sup.+-form. Thereafter membrane was washed with RO water and
dried at room temperature.
[0305] Membrane thus obtained was characterized in terms of solute
flux (85 mM sodium acetate buffer/250 mM NaCl, pH 4.5), hIgG
binding capacity and hIgG recovery as described in Example 1.
[0306] The composite material produced by this method showed solute
flux of 2,744.00 kg/m.sup.2 h and hIgG binding capacity of 181
mg/mL at 10% breakthrough. The recovery of hIgG exceeded 95%.
Example 12
[0307] This example illustrates selectivity of a cation-exchange
material of the present invention with multi-modal
functionality
[0308] Selectivity is the most important factor in a separation.
Mixed-mode media combines both ion-exchange and hydrophobic
characteristics so that its selectivity can be manipulated in order
for the retention magnitude of each retention mode to be adjusted
by changing mobile phase ionic strength, pH and the organic solvent
content, either individually or concurrently.
[0309] Mixed-mode membrane prepared in Example 11 was used in
selectivity study. Membrane was tested using a single layer
inserted into a stainless steel disk holder attached to typical
HPLC equipment. Chromatographic study on selective separation of
lysozyme and cytochrome C was carried out using 20 mM sodium
phosphate buffer, pH 6.5 as the mobile phase (Buffer A). Linear
gradient elution was performed from buffer A to buffer B, using 20
mM sodium phosphate buffer containing 1.0 M sodium chloride as
elution buffer B. Waters 600E HPLC system was used for carrying out
the membrane chromatographic study. A 100 .mu.L loop was used for
injecting a 50 .mu.L sample of protein mixture (5 mg/mL lysozyme
and 3 mg/mL Cytochrome C). The UV absorbance (at 280 nm) of the
effluent stream from the membrane holder and the system pressure
were continuously recorded. The flow rate was 2 mL/min. All
chromatographic studies were performed at 25.degree. C. FIG. 10
shows selective separation of proteins on mixed-mode membrane of
the present invention.
Example 13
[0310] This example illustrates caustic stability of a
cation-exchange material of the present invention with multi-modal
functionality
[0311] Sodium hydroxide is widely accepted for cleaning, sanitizing
and storing chromatography media and systems. The benefits of its
use include efficacy, low cost, ease of detection, removal and
disposal
[0312] Sodium hydroxide has been shown to be effective in removing
proteins and nucleic acids. It is also effective for inactivating
most viruses, bacteria, yeasts, and endotoxins
[0313] In order to maintain selectivity and binding capacity,
chromatography media and systems have to be cleaned and are
typically cleaned under alkaline conditions, e.g., with sodium
hydroxide. For example, a standard process which is used for
cleaning and restoring the media is a cleaning-in-place (CIP)
alkaline protocol, which typically involves treatment of the
membranes with 0.5 M NaOH. Thus, membranes developed for
chromatography applications MUST be able to withstand conventional
alkaline cleaning for a prolonged period of time.
[0314] A multimodal cation-exchange membrane prepared as described
in Example 11 was used to examine an effect of membrane exposure
into 0.5 M sodium hydroxide on its performance. Membrane was placed
into 0.5 M sodium hydroxide containing 0.1 M NaCl for period of
time ranging from 30 min to 24 hrs. Thereafter, the membrane was
washed with RO water and equilibrated with 85 mM sodium acetate
buffer containing 250 mM sodium chloride, pH 4.5. Then, solute flux
and hIgG binding capacity were measured as described in Example 1.
FIG. 11 illustrates effect of membrane exposure into 0.5 M sodium
hydroxide containing 0.1 M sodium chloride onto its solute flux and
hIgG binding capacity.
[0315] As can be seen from FIG. 11, membrane showed great caustic
stability. No significant changes in membrane performance after
exposure into 0.5 M NaOH/0.1 M NaCl for up to 24 hrs were
observed.
Example 14
[0316] This example illustrates use of mixed-mode membrane in
bind-elute mode to purify monoclonal antibodies
[0317] Multimodal cation-exchange membrane prepared as described in
Example 11 was used to illustrate the use of mixed-mode membrane in
bind-elute mode to purify monoclonal antibodies.
[0318] The membrane chromatographic experiments were carried out
using an AKTA.TM. Purifier liquid chromatographic system (GE Life
Sciences). The UV absorbance, pH and conductivity of the effluent
stream from the membrane holder and the system pressure were
continuously monitored. The feed solution was prepared by spiking
of 1 mg/mL Protein A purified monoclonal antibodies (mAbs) with 5.2
.mu.g/mL host cell protein (HCP) and 100 .mu.g/mL herring sperm DNA
dissolved in 85 mM sodium acetate buffer containing 250 mM sodium
chloride, pH 4.5 (Buffer A). The feed was loaded using a sample
pump. Before injecting the feed solution into the membrane holder,
the buffer A was passed through the membrane till stable readings
were obtained. A target mAbs were eluted in two steps. In a first
step 30 mM sodium phosphate buffer containing 15 mM sodium
chloride, pH 6.5 was used as elution buffer (Buffer B) and in the
second step 25 mM TRIS/HCl buffer containing 1.0 M sodium chloride
was used as elution buffer (buffer C). Eluent from the first
elution step (buffer B) was collected in 8 fractions (A1-A8). HCP
levels and DNA concentrations were measured in combined fractions
(A1-A5) and (A6-A8) and in the feed fraction.
[0319] HCP levels were determined with a broadly reactive Chinese
Hamster Ovary (CHO) HCP ELISA kit (Cygnus Technologies F550).
Calibration was performed with the HCP used in the chromatographic
experiments. Calibrators and samples were diluted with Sample
Diluent (Cygnus 1028). Measurements were performed on a Thermo
Scientific Multiskan Ascent.RTM. Plate Reader.
[0320] DNA concentrations were measured with a DNA Assay (Life
Technologies Quant-iT.TM. PicoGreen.RTM. dsDNA Assay Kit P11496).
.lamda. DNA standard and samples were diluted with 85 mM sodium
acetate buffer containing 250 mM sodium chloride, pH 4.5.
Measurements were performed on a Thermo Scientific Fluoroskan
Ascent.RTM. Plate Reader.
[0321] Samples from feed solution and flowthrough fraction were
also analyzed using absorption spectrophotometry. Absorption
measurements were taken at wavelengths of 260 and 280 nanometers
(nm). A.sub.260/A.sub.280 absorption ratios were computed from the
measurements. An A.sub.260/A.sub.280 of a greater than equal to 1.8
was interpreted to indicate the sample analyzed therein was
relatively free of protein.
[0322] mAbs feed and elution fractions A6-A8 were also analyzed on
SEC column TSKgel G3000SW.sub.x1 (Tosoh Bioscience) (FIG. 13). 100
mM sodium phosphate buffer containing 100 mM sodium sulphate, pH
6.7 was used as a mobile phase. 800 .mu.L sample was applied into
the column at 0.75 mL/min.
[0323] A summary of HCP/DNA clearance data is presented in FIG.
14.
[0324] As can be seen from FIG. 13 and FIG. 14 mixed-mode membrane
described in Example 11 showed the superior performance in terms of
HCP/DNA clearance as well as aggregates clearance. The membrane can
be used in a capture step in antibody processes.
Example 15
[0325] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality
[0326] A 13.7 wt % solution was prepared by dissolving
(ar-vinylbenzyl)trimethylammonium chloride, 2-aminoethyl
methacrylate hydrochloride and ethylene glycol phenyl ether
methacrylate in a molar ratio of 1:0.46:0.085, respectively, in a
solvent mixture containing 52.7 wt % 1,2-propanediol, 41.9 wt %
tri(propylene glycol) propyl ether and 5.4 wt % water.
Trimethylolpropane trimethacrylate was used as cross-linking agents
to achieve cross-linking density of 9.4% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0327] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and dried at room temperature.
[0328] Membrane thus obtained was characterized in terms of water,
solute flux (100 mM sodium phosphate buffer, containing 150 mM
sodium chloride, pH 7.0), protein A and herring sperm DNA binding
capacities as described Example 1. Protein A was dissolved in 100
mM sodium phosphate buffer containing 150 mM sodium chloride, pH
7.0 and herring sperm DNA in 20 mM sodium phosphate buffer
containing 150 mM sodium chloride, pH 6.5.
[0329] The composite material produced by this method showed water
flux of 3,735.00 kg/m.sup.2 hr at 100 kPa, solute flux of 4,330.00
kg/m.sup.2 hr and protein A binding capacity of 125.5 mg/mL and
herring sperm DNA binding capacity of 20 mg/mL at 10%
breakthrough.
Example 16
[0330] This example illustrates use of mixed-mode membrane in
flow-through mode to purify hIgG.
[0331] Multimodal anion-exchange membrane prepared as described in
Example 15 was used to examine membrane performance in flow-through
mode to purify hIgG from aggregates. Membrane was equilibrated at a
flow rate of 1 mL/min with 20 mM sodium phosphate buffer containing
150 mM sodium chloride (buffer A) as described in Example 1. hIgG
was dissolved in buffer A and 300 mg hIgG/mL was applied to the
membrane at flow rate of 1 mg/mL. Aggregates were eluted with 100
mM sodium acetate buffer, pH 4.2 (FIG. 12). hIgG feed and
flowthrough fraction were analyzed on SEC column TSKgel
G3000SW.sub.x1 (Tosoh Bioscience). 100 mM sodium phosphate buffer
containing 100 mM sodium sulphate, pH 6.7 was used as a mobile
phase. 800 .mu.L sample was applied into the column at 0.80 mL/min.
The aggregate level was reduced from 15.1% to 8.3%.
Example 17
[0332] This example illustrates a method of preparing a strong
cation-exchange material of the present invention with multi-modal
functionality.
[0333] A 13.2 wt % solution was prepared by dissolving
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide and N-phenylacrylamide in a molar ratio of
1:0.18:0.1, respectively, in a solvent mixture containing 17.8 wt %
N,N'-dimethylacetamide, 54.1 wt % di(propylene glycol)dimethyl
ether, 17.2 wt % 1,2-propanediol and 10.9 wt % water.
N,N'-Hexamethylenebis(methacrylamide) was used as a cross-linking
agent to achieve cross-linking density of 12% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0334] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO and dried at room temperature.
[0335] Membrane thus obtained was characterized in terms of solute
flux (85 mM sodium acetate buffer/250 mM NaCl, pH 4.5), hIgG
binding capacity (hIgG was dissolved in 85 mM sodium acetate
buffer/NaCl, pH 4.5 and conductivity of 15 mS/cm) and hIgG recovery
as described in Example 1.
[0336] The composite material produced by this method showed solute
flux of 1,564.00 kg/m.sup.2 hr and hIgG binding capacity of 91.5
mg/mL at 10% breakthrough. The recovery of hIgG exceeded 95%.
Example 18
[0337] This example illustrates selectivity of a cation-exchange
material of the present invention with multi-modal
functionality.
[0338] Mixed-mode membrane prepared in Example 17 was used in
selectivity study. Membrane was tested using a single layer
inserted into a stainless steel disk holder attached to typical
HPLC equipment. Chromatographic study on selective separation of
myoglobin, ribonuclease A and lysozyme was carried out using 20 mM
sodium phosphate buffer, pH 6.5 as the mobile phase (Buffer A).
Linear gradient elution was performed from buffer A to buffer B,
using 20 mM sodium phosphate buffer containing 1.0 M sodium
chloride as elution buffer B. Waters 600E HPLC system was used for
carrying out the membrane chromatographic study. A 100-.mu.L loop
was used for injecting a 50-.mu.L sample of protein mixture (5.4
mg/mL ribonuclease A, 3.3 mg/mL myoglobin and 7 mg/mL lysozyme in a
volume ratio of 1:0.25:0.15). The UV absorbance (at 280 nm) of the
effluent stream from the membrane holder and the system pressure
were continuously recorded. The flow rate was 3 mL/min. All
chromatographic studies were performed at 25.degree. C. FIG. 15
shows selective separation of proteins on mixed-mode membrane of
the present invention.
Example 19
[0339] This example illustrates effect of nature of co-monomer used
on performance of a strong cation-exchange material of the present
invention with multi-modal functionality.
[0340] A series of multimodal strong cation-exchange membranes were
prepared as described in Example 17 using various co-monomers.
Thus, a 13.2 wt % solution was prepared by dissolving
2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt,
N-isopropyl acrylamide (NIPAM) (or
N-[tris(hydroxymethyl)methyl]acrylamide (THMAAm) or
N-(3-methoxypropyl) acrylamide (MPAAm) or N,N'-dimethylacrylamide
(DMAAm)) and N-phenylacrylamide in a molar ratio of 1:0.18:0.1,
respectively, in a solvent mixture containing 17.8 wt %
N,N'-dimethylacetamide, 54.1 wt % di(propylene glycol)dimethyl
ether, 17.2 wt % 1,2-propanediol and 10.9 wt % water.
N,N'-Hexamethylenebis(methacrylamide) was used as cross-linking
agent to achieve cross-linking density of 12% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0341] A composite materials were prepared from the solution and
the support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The samples were irradiated for 10 min
at 350 nm. The resulting composite materials were thoroughly washed
with RO and dried at room temperature.
[0342] Membranes thus obtained were characterized in terms of flux
(85 mM sodium acetate buffer/250 mM NaCl, pH 4.5 and 25 mM
TRIS/HCl, 250 mM NaCl, pH 8.21), hIgG binding capacity and hIgG
recovery as described in Example 1.
[0343] FIGS. 16 and 17 illustrate effect of nature co-monomer used
on membrane performance.
[0344] The recovery of hIgG exceeded 95% for all membranes
examined.
[0345] Using N-isopropyl acrylamide as a co-monomer in the strong
cation-exchange mixed-mode formulation resulted in the membrane
with both good permeability characteristics and binding
capacity.
Example 20
[0346] This example illustrates effect of nature of cross-linker
used on performance of a strong cation-exchange material of the
present invention with multi-modal functionality.
[0347] A series of multimodal strong cation-exchange membranes were
prepared using various cross-linkers. Thus, A 14.5 wt % solution
was prepared by dissolving 2-acrylamido-2-methyl-1-propanesulfonic
acid sodium salt, N-isopropyl acrylamide and ethylene glycol phenyl
ether methacrylate in a molar ratio of 1:0.1:0.15, respectively, in
a solvent mixture containing 23.7 wt % N,N'-dimethylacetamide, 55.5
wt % tri(propylene glycol) methyl ether, 8.7 wt % 1,2-propanediol
and 12.1 wt % water. N,N'-Methylenebisacrylamide (BIS) (or
N,N'-hexamethylenebis(methacrylamide (Hexa-BIS) or
2,4,6-triallyloxy-1,3,5-triazine (T-XL-1) or
1,3,5-triallyl-1,3,5-triazine-2,4,6-(1H,3H,5H)-trione (T-XL-2) or
1,3,5-triacryloylhexahydro-1,3,5-triazine (T-XL-3) or glyoxal
bis(diallylacetal) (GBDA or GBDE)) were used as cross-linking agent
to achieve cross-linking density of 10% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0348] A composite materials were prepared from the solution and
the support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The samples were irradiated for 10 min
at 350 nm. The resulting composite materials were thoroughly washed
with RO and dried at room temperature.
[0349] Membranes thus obtained were characterized in terms of flux
(85 mM sodium acetate buffer/250 mM NaCl, pH 4.5 and 25 mM
TRIS/HCl, 250 mM NaCl, pH 8.2), hIgG binding capacity and hIgG
recovery as described in Example 1.
[0350] FIGS. 18 and 19 illustrate effect of nature cross-linker
used on membrane performance.
[0351] The recovery of hIgG exceeded 95% for all membranes
examined.
[0352] As can be seen from the data presented above, in FIG. 18 and
FIG. 19, nature of cross-linking agent used plays a significant
role in controlling the size of the membrane pores, the pore volume
fraction and the interconnections. By employing trifunctional
cross-linker 1,3,5-triacryloylhexahydro-1,3,5-triazine, membrane
with good permeability characteristics was obtained.
Example 21
[0353] This example illustrates a gel morphology of the
anion-exchange material of the present invention with multi-modal
functionality prepared according to Example 8.
[0354] Gel morphology of mixed-mode membrane prepared according to
Example 8 was examined with an environmental scanning electron
microscope (ESEM, Philips Electroscan, model E-2020, Electroscan
Corp., USA). A small sample of the membrane (3 mm.times.3 mm) was
soaked in DI water, surface water was removed with wet filter paper
and the wet membrane was placed in the sample chamber. An
accelerating voltage of 20 kV was used in conjunction with a large
spot size and magnifications less than 1500.times., to limit both
heating effects and sample damage. A working distance of 9-13 mm
was employed to minimize the scattering of the beam. In the
specimen chamber, the pressure was maintained between 1 and 4 Torr
and the temperature was maintained at 3.+-.0.5.degree. C. using a
Peltier-cooled sample stage. The sample chamber was periodically
flushed with water vapor to maintain a satisfactory partial
pressure of water, ensuring constant hydration of the membrane and
preventing any drying of the sample that potentially can lead to
some changes in membrane gel morphology. ESEM micrograph showed a
developed macroporous structure of the anion-exchange mixed-mode
membrane (FIG. 20).
Example 22
[0355] This example illustrates a gel morphology of the
cation-exchange material of the present invention with multi-modal
functionality prepared according to Example 11.
[0356] Gel morphology of mixed-mode membrane prepared according to
Example 11 was examined using an environmental scanning electron
microscope as described in Example 21 (FIG. 21).
[0357] As can be seen from FIG. 21, a cation-exchange mixed-mode
membrane prepared according to Example 11 has a macroporous
structure.
Example 23
[0358] This example illustrates a gel morphology of the
cation-exchange material of the present invention with multi-modal
functionality prepared according to Example 17.
[0359] Gel morphology of mixed-mode membrane prepared according to
Example 17 was examined using an environmental scanning electron
microscope as described in Example 21 (FIG. 22).
[0360] As can be seen from FIG. 22, ESEM micrograph demonstrates a
macroporous structure of a cation-exchange mixed-mode prepared
according to Example 17.
Example 24
[0361] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0362] A 15.5 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water) and N-(3-N,N-dimethylaminopropyl) methacrylamide in a
molar ratio of 1:1.17, respectively, in a solvent mixture
containing 44.04 wt % N,N'-dimethylacetamide, 43.71 wt %
di(propylene glycol) methyl ether acetate and 12.25 wt % water.
N,N'-methylenebisacrylamide was used as cross-linking agent to
achieve cross-linking density of 6.71% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0363] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO water at least three times.
[0364] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0365] The composite material produced by this method showed solute
flux of 3,970.00 kg/m.sup.2 h and BSA binding capacity of 87.4
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0366] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc., PMI). Membrane coupon (25 mm diameter) was cut from the
sample. Excess water was removed with wetted filter paper and the
average wet thickness was recorded using the micrometer.
Thereafter, membrane coupon was placed on a support screen and
installed on sample stage over airport in the CFP machine. Autotest
was run according to a standard CFP procedure. The test showed mean
flow pore diameter of 0.71 .mu.m and bubble point pore diameter of
1.81 .mu.m.
Example 25
[0367] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0368] A 13.6 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water), N-(3-N,N-dimethylaminopropyl) methacrylamide and
N-tert-butyl acrylamide a molar ratio of 1:0.53:0.1, respectively,
in a solvent mixture containing 60.32 wt % N,N'-dimethylacetamide,
29.01 wt % di(propylene glycol) methyl ether acetate and 10.67 wt %
water. N,N'-methylenebisacrylamide was used as cross-linking agent
to achieve cross-linking density of 9.91% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0369] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0370] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0371] The composite material produced by this method showed solute
flux of 1,826.00 kg/m.sup.2 h and BSA binding capacity of 151.5
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0372] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.59 .mu.m and bubble point pore diameter of 1.74
.mu.m.
Example 26
[0373] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0374] A 11.8 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water) and N-tert-butyl acrylamide in a molar ratio of 1:0.09,
respectively, in a solvent mixture containing 60.64 wt %
N,N'-dimethylacetamide, 28.87 wt % di(propylene glycol) methyl
ether acetate and 10.49 wt % water. N,N'-methylenebisacrylamide was
used as cross-linking agent to achieve cross-linking density of
7.25% (mol/mol). The photo-initiator Irgacure 2959 was added in the
amount of 1 wt % with respect to the mass of the monomers.
[0375] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0376] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0377] The composite material produced by this method showed solute
flux of 1,235.00 kg/m.sup.2 h and BSA binding capacity of 196.8
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0378] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.31 .mu.m and bubble point pore diameter of 1.50
.mu.m.
Example 27
[0379] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0380] A 12.2 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water), N-tert-butyl acrylamide and diacetone acrylamide in a
molar ratio of 1:0.1:0.1, respectively, in a solvent mixture
containing 61.11 wt % N,N'-dimethylacetamide, 30.15 wt %
di(propylene glycol) methyl ether acetate and 8.75 wt % water.
N,N'-methylenebisacrylamide was used as cross-linking agent to
achieve cross-linking density of 7.25% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0381] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0382] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0383] The composite material produced by this method showed solute
flux of 1,075.00 kg/m.sup.2 h and BSA binding capacity of 184.2
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0384] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials,
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.21 .mu.m and bubble point pore diameter of 0.95
.mu.m.
Example 28
[0385] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0386] A 12.8 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water), N-phenyl acrylamide and diacetone acrylamide in a molar
ratio of 1:0.08:0.16, respectively, in a solvent mixture containing
61.89 wt % N,N'-dimethylacetamide, 29.97 wt % di(propylene glycol)
methyl ether acetate and 8.14 wt % water.
N,N'-methylenebisacrylamide was used as cross-linking agent to
achieve cross-linking density of 10.43% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0387] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0388] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0389] The composite material produced by this method showed solute
flux of 1,135.00 kg/m.sup.2 h and BSA binding capacity of 187.9
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0390] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.29 .mu.m and bubble point pore diameter of 1.15
.mu.m.
Example 29
[0391] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0392] A 13.2 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water), N-(3-N,N-dimethylaminopropyl) methacrylamide and
N-phenyl acrylamide in a molar ratio of 1:0.51:0.09, respectively,
in a solvent mixture containing 62.18 wt % N,N'-dimethylacetamide,
29.55 wt % di (propylene glycol) methyl ether acetate and 11.33 wt
% water. N,N'-methylenebisacrylamide was used as cross-linking
agent to achieve cross-linking density of 10.43% (mol/mol). The
photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers.
[0393] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0394] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0395] The composite material produced by this method showed solute
flux of 2,810.00 kg/m.sup.2 h and BSA binding capacity of 122.4
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0396] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.65 .mu.m and bubble point pore diameter of 1.69
.mu.m.
Example 30
[0397] This example illustrates a method of preparing an
anion-exchange material of the present invention with multi-modal
functionality.
[0398] A 12.2 wt % solution was prepared by dissolving
(3-acrylamido propyl) trimethylammonium chloride (75 wt % solution
in water) and acrylic acid in a molar ratio of 1:1.19,
respectively, in a solvent mixture containing 60.27 wt %
N,N'-dimethylacetamide, 29.73 wt % di(propylene glycol) methyl
ether acetate and 10.00 wt % water. N,N'-methylenebis-acrylamide
was used as cross-linking agent to achieve cross-linking density of
7.25% (mol/mol). The photo-initiator Irgacure 2959 was added in the
amount of 1 wt % with respect to the mass of the monomers.
[0399] A composite material was prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The sample was irradiated for 10 min
at 350 nm. The resulting composite material was thoroughly washed
with RO at least three times.
[0400] Membrane thus obtained was characterized in terms of solute
flux (25 mM Tris/HCl, pH 8.2) and BSA binding capacity using
general procedure as described in Example 1.
[0401] The composite material produced by this method showed solute
flux of 1,850.00 kg/m.sup.2 h and BSA binding capacity of 150.5
mg/mL at 10% breakthrough using 25 mM Tris/HCl pH 8.2 as a binding
buffer.
[0402] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24. The test showed mean flow pore
diameter of 0.57 .mu.m and bubble point pore diameter of 1.44
.mu.m.
Example 31
[0403] This example illustrates effect of hydrophobicity on
membrane performance of cation-exchange material of the present
invention with multi-modal functionality.
[0404] A 10.5 wt % solutions were prepared by dissolving
2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS), N-phenyl
acrylamide (PhAAm) or N-tert-butyl acrylamide (BuAAm) (PhAAm and
BuAAm monomers are referred as Hm in FIG. 23 below) and
N-hydroxyethyl acrylamide (HEAAm) in a solvent mixture containing
N,N'-dimethylacetamide (DMAc), di(propylene glycol) methyl (DPM),
di(propylene glycol) dimethyl ether (DMM), propylene carbonate
(PrCarb), 1,2-propanediol (Prdiol) and water.
N,N'-methylenebisacrylamide (BIS) was used as cross-linking agent.
The photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers. The formulations
characteristics are presented in FIG. 23 below.
[0405] A composite materials were prepared from the solution and
the support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The samples were irradiated for 10 min
at 350 nm. The resulting composite materials were placed into
solution containing 0.25 M NaOH and 0.5 M NaCl for 10 min, and then
membranes were thoroughly washed with RO at least three times and
dried at room temperature.
[0406] Membranes thus obtained were characterized in terms of
solute flux (85 mM NaAc/NaCl, conductivity 15 mS/cm, pH 4.4), hIgG
binding capacity (BC) and protein recovery using general procedure
as described in Example 1. A solution of 85 mM NaAc/NaCl,
conductivity 15 mS/cm and pH 4.4 was used as a binding buffer and
25 mM Tris/HCl buffer containing 250 mM NaCl, pH 8.2 was used as
elution buffer.
[0407] Swelling in water was measured as percentage increase in
thickness after dry membrane was soaked in water for 15 min.
[0408] Pore size analysis of membrane was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24.
[0409] The performance characteristics of cation-exchange
mixed-mode membranes with various hydrophobic components are
presented in FIG. 24 below.
[0410] As can be seen from FIG. 24 phenyl-containing membrane
showed lower buffer flux and higher dynamic binding capacity. The
latter is consistent with more hydrophilic nature of the
phenyl-based membrane compare with butyl-based membrane.
Example 32
[0411] This example illustrates an effect of co-monomers nature on
performance of cation-exchange material of the present invention
with multi-modal functionality.
[0412] A 10.5 wt % solutions were prepared by dissolving
2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS), N-tert-butyl
acrylamide (BuAAm) and N-isopropyl acrylamide (NIPAM) or
N,N'-dimethyl acrylamide (DMAAm) or diacetone acrylamide (DAAm) or
N-hydroxyethyl acrylamide (HEAAM) (co-monomers are referred as CM
in FIG. 25 below) in a solvent mixture containing
N,N'-dimethylacetamide (DMAc), di(propylene glycol) methyl (DPM),
di(propylene glycol) dimethyl ether (DMM), propylene carbonate
(PrCarb), 1,2-propanediol (Prdiol) and water.
N,N'-methylenebisacrylamide (BIS) was used as cross-linking agent.
The photo-initiator Irgacure 2959 was added in the amount of 1 wt %
with respect to the mass of the monomers. The formulations
characteristics are presented in FIG. 25 below.
[0413] Composite materials were prepared from the solution and the
support TR0671 B50 (Hollingsworth & Vose) using the
photoinitiated polymerization according to the general procedure
described above (Example 1). The samples were irradiated for 4 min
and 10 min at 350 nm. The resulting composite materials were placed
into solution containing 0.25M NaOH and 0.5M NaCl for 10 min, and
then membranes were thoroughly washed with RO at least three times
and dried at room temperature.
[0414] Membranes irradiated for 10 min were characterized in terms
of solute flux (85 mM NaAc/NaCl, conductivity 15 mS/cm, pH 4.4),
hIgG binding capacity (BC) and protein recovery using general
procedure as described in Example 1. A solution of 85 mM NaAc,
conductivity of 15 mS/cm and pH 4.4 was used as a binding buffer
and 25 mM Tris/HCl buffer containing 250 mM NaCl, pH 8.2 was used
as elution buffer. Swelling in water was measured as percentage
increase in thickness after dry membranes were soaked in water for
15 min.
[0415] Both membranes irradiated for 4 min and 10 min were
characterized in terms of mean flow pore size and bubble point pore
size. Pore size analysis of membranes was performed using a fully
automated Capillary Flow Porometer--CFP-1500-AE (Porous Materials
Inc.) as described in Example 24.
[0416] The performance characteristics of cation-exchange
mixed-mode membranes with various hydrophobic components are
presented in FIG. 26 below.
[0417] All selected co-monomers used in this Example are
hydrophilic, completely/or partially water soluble and very
compatible with AMPS-monomer in terms of polymerization rate. As it
can be seen from FIG. 26, altering the type of comonomer results in
membranes with varying buffer fluxes, hIgG dynamic binding
capacity, pore size and swelling. NIPAM, DAAm and HEAAm-based
membranes prepared at 4 min and 10 min polymerization time had
similar mean flow pore size and bubble point pore size. The latter
indicates high polymerization rate that occurred in these
cases.
INCORPORATION BY REFERENCE
[0418] All of the U.S. patents and U.S. patent application
publications cited herein are hereby incorporated by reference.
EQUIVALENTS
[0419] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *