U.S. patent application number 16/703016 was filed with the patent office on 2020-04-02 for ligand functionalized polymers.
This patent application is currently assigned to 3M INNOVATIVE PROPERTIES COMPANY. The applicant listed for this patent is 3M INNOVATIVE PROPERTIES COMPANY. Invention is credited to Catherine A. Bothof, Robert T. Fitzimons, JR., George W. Griesgraber, Louis C. Haddad, Yi He, James I. Hembre, Jerald K. Rasmussen, Erin A. Satterwhite, Kannan Seshadri.
Application Number | 20200102345 16/703016 |
Document ID | / |
Family ID | 1000004509795 |
Filed Date | 2020-04-02 |
United States Patent
Application |
20200102345 |
Kind Code |
A1 |
Rasmussen; Jerald K. ; et
al. |
April 2, 2020 |
LIGAND FUNCTIONALIZED POLYMERS
Abstract
Ligand functionalized substrates, methods of making ligand
functionalized substrates, and methods of using functionalized
substrates are disclosed.
Inventors: |
Rasmussen; Jerald K.;
(Woodville, WI) ; Seshadri; Kannan; (Woodbury,
MN) ; Fitzimons, JR.; Robert T.; (Minneapolis,
MN) ; Hembre; James I.; (Plymouth, MN) ;
Bothof; Catherine A.; (Stillwater, MN) ; Satterwhite;
Erin A.; (Chatham, NJ) ; Griesgraber; George W.;
(Eagan, MN) ; He; Yi; (Roseville, MN) ;
Haddad; Louis C.; (Mendota Heights, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
3M INNOVATIVE PROPERTIES COMPANY |
St. Paul |
MN |
US |
|
|
Assignee: |
3M INNOVATIVE PROPERTIES
COMPANY
|
Family ID: |
1000004509795 |
Appl. No.: |
16/703016 |
Filed: |
December 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15987441 |
May 23, 2018 |
10526366 |
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16703016 |
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15591490 |
May 10, 2017 |
10005814 |
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15987441 |
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13848336 |
Mar 21, 2013 |
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15591490 |
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13026331 |
Feb 14, 2011 |
8435776 |
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13848336 |
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61310005 |
Mar 3, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y10T 428/2998 20150115;
Y10T 428/249978 20150401; C08L 79/02 20130101; C08L 77/04 20130101;
C12N 11/08 20130101; G01N 33/545 20130101; C08F 120/60 20130101;
G01N 33/54393 20130101; C08G 73/028 20130101; C08F 126/02 20130101;
Y10T 442/20 20150401; C07K 1/14 20130101; C12N 7/02 20130101; C08G
73/022 20130101; Y10T 428/2935 20150115; C08G 69/10 20130101; C08G
73/0206 20130101; Y10T 428/249991 20150401; C08L 83/08 20130101;
B01D 21/01 20130101 |
International
Class: |
C07K 1/14 20060101
C07K001/14; C08G 69/10 20060101 C08G069/10; C08G 73/02 20060101
C08G073/02; C08L 77/04 20060101 C08L077/04; C08L 79/02 20060101
C08L079/02; C08L 83/08 20060101 C08L083/08; G01N 33/543 20060101
G01N033/543; G01N 33/545 20060101 G01N033/545; C12N 7/02 20060101
C12N007/02; C12N 11/08 20060101 C12N011/08; B01D 21/01 20060101
B01D021/01; C08F 120/60 20060101 C08F120/60; C08F 126/02 20060101
C08F126/02 |
Claims
1.-23. (canceled)
24. A complex comprising: a) a water soluble or water-dispersible
aminopolymer functionalized with pendent or catenary guanidinyl
groups; and b) a neutral or negatively charged target biological
species derived from a cell culture or fermentation process.
25. The complex of claim 24, wherein the aminopolymer
functionalized with pendent or catenary guanidinyl groups is of the
formula: ##STR00003## wherein R.sup.2 is a H, C.sub.1-C.sub.12
alkyl, C.sub.5-C.sub.12 (hetero)aryl, or a residue of the polymer
chain; each R.sup.3 is independently H, C.sub.1-C.sub.12 alkyl, or
C.sub.5-C.sub.12 (hetero)aryl, each R.sup.4 is H, C.sub.1-C.sub.12
alkyl or alkylene, C.sub.5-C.sub.12 (hetero)aryl or
(hetero)arylene, cyano, or --C(.dbd.NH)--N(R.sup.2)-Polymer,
Polymer is the aminopolymer chain; and n is 1 or 2.
26. The complex of claim 24, wherein said biomacromolecules are
selected from proteins, enzymes, nucleic acids, and endotoxins.
27. The complex of claim 24, wherein said biological species is
selected from bacteria, viruses, cells, cell debris, and
spores.
28. The complex of claim 24, wherein the amino polymer is selected
from the group consisting of polyethylenimine, polylysine,
polyaminoamides, polyallylamine, polyvinylamine,
polydimethylamine-epichlorohydrin-ethylenediamine,
polyaminosiloxanes and dendrimers formed from polyamidoamine
(PAMAM) and polypropylenimine.
29. The complex of claim 24, wherein 0.1 to 100 mole percent of the
available amino groups of the aminopolymer are functionalized with
guanidinyl groups.
30. The complex of claim 24, wherein the guanidinyl groups of the
functionalized aminopolymer are pendent from the polymer chain.
31. The complex of claim 24, wherein guanidinyl groups of the
functionalized aminopolymer are in the aminopolymer chain.
32. The complex of claim 27, wherein the cells are selected from
archaea, bacteria, and eucaryota.
33. The complex of claim 24, wherein the amount of
ligand-functionalized polymer relative to the amount of target
biological species is 0.01% to 100% by weight.
34. The complex of claim 24, wherein a portion of the amino groups
of the ligand-functionalized polymer further comprise alkyl or acyl
groups.
35. The complex of claim 24, wherein the functionalized
aminopolymer is crosslinked.
36. The complex of claim 24, wherein the functionalized
aminopolymer is uncrosslinked.
37. A method of separating a target biological species from a fluid
comprising the target biological species comprising: a. contacting
the fluid with a suspension or solution of water-soluble or
water-dispersible aminopolymer functionalized with pendent or
catenary guanidinyl groups represented by the following general
formula: ##STR00004## wherein R.sup.2 is H, C.sub.1-C.sub.12 alkyl,
C.sub.5-C.sub.12 (hetero)aryl, or a residue of the polymer chain;
each R.sup.3 is independently H, C.sub.1-C.sub.12 alkyl, or
C.sub.5-C.sub.12 (hetero)aryl, each R.sup.4 is H, C.sub.1-C.sub.12
alkyl or alkylene, C.sub.5-C.sub.12 (hetero)aryl or
(hetero)arylene, cyano, or --C(.dbd.NH)--N(R.sup.2)-Polymer, n is 1
or 2; and "Polymer-N(R.sup.2)--" is the linkage formed between an
amine group of the polyamino polymer and guanylating agent; b.
forming a complex in the fluid that comprises the aminopolymer
functionalized with guanidinyl groups and the target biological
species; and c. separating the complex from the fluid; wherein the
target biological species is selected from biomacromolecules and
microbiological species and wherein the fluid is a cell culture or
fermentation broth.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/310005, filed Mar. 3, 2010, the
disclosure of which is incorporated by reference herein in its
entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to ligand-functionalized
polymers, and methods for preparing the same. The functionalized
polymers are useful in selectively binding and removing biological
materials, such as viruses, from biological samples.
BACKGROUND
[0003] Detection, quantification, isolation and purification of
target biomaterials, such as viruses and biomacromolecules
(including constituents or products of living cells, for example,
proteins, carbohydrates, lipids, and nucleic acids) have long been
objectives of investigators. Detection and quantification are
important diagnostically, for example, as indicators of various
physiological conditions such as diseases. Isolation and
purification of biomacromolecules are important for therapeutic
uses and in biomedical research. Biomacromotecules such as enzymes
which are a special class of proteins capable of catalyzing
chemical reactions are also useful industrially; enzymes have been
isolated, purified, and then utilized for the production of
sweeteners, antibiotics, and a variety of organic compounds such as
ethanol, acetic acid, lysine, aspartic acid, and biologically
useful products such as antibodies and steroids.
[0004] In their native state in vivo, structures and corresponding
biological activities of these biomacromolecules are maintained
generally within fairly narrow ranges of pH and ionic strength.
Consequently, any separation and purification operation must take
such factors into account in order for the resultant, processed
biomacromolecule to have potency.
[0005] The use of certain ionic polymers, especially cationic
polymers, for the flocculation of cell and/or cell debris, as well
as for the precipitation of proteins, is known. Similarly, ionic
polymers have been used to modify filtration media to enhance the
removal of impurities from process streams in depth filtration or
membrane absorber type applications. The effectiveness of these
flocculants is typically reduced as the conductivity of the media
being processed increases, i.e. as the salt content increases.
There is a need in the art for polymeric materials with increased
affinity for biological species under high ionic strength
conditions.
[0006] Chromatographic separation and purification operations can
be performed on biological product mixtures, based on the
interchange of a solute between a moving phase, which can be a gas
or liquid, and a stationary phase. Separation of various solutes of
the solution mixture is accomplished because of varying binding
interactions of each solute with the stationary phase; stronger
binding interactions generally result in longer retention times
when subjected to the dissociation or displacement effects of a
mobile phase compared to solutes which interact less strongly and,
in this fashion, separation and purification can be effected.
[0007] Most current capture or purification chromatography is done
via conventional column techniques. These techniques have severe
bottlenecking issues in downstream purification, as the throughput
using this technology is low. Attempts to alleviate these issues
include increasing the diameter of the chromatography column, but
this in turn creates challenges due to difficulties of packing the
columns effectively and reproducibly. Larger column diameters also
increase the occurrence of problematic channeling. Also, in a
conventional chromatographic column, the absorption operation is
shut down when a breakthrough of the desired product above a
specific level is detected. This causes the dynamic or effective
capacity of the adsorption media to be significantly less than the
overall or static capacity. This reduction in effectiveness has
severe economic consequences, given the high cost of some
chromatographic resins.
[0008] Polymeric resins are widely used for the separation and
purification of various target compounds. For example, polymeric
resins can be used to purify or separate a target compound based on
the presence of an ionic group, based on the size of the target
compound, based on a hydrophobic interaction, based on an affinity
interaction, or based on the formation of a covalent bond. There is
a need in the art for polymeric substrates having enhanced affinity
for viruses and other biological species to allow selective removal
from a biological sample. There is further need in the art for
ligand functionalized membranes that overcome limitations in
diffusion and binding, and that may be operated at high throughput
and at lower pressure drops.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to ligand-functionalized
polymers, and methods of making the same. More specifically, the
ligand-functionalized polymer includes a polyamine polymer, which
has been modified to provide grafted ligand groups having the
requisite affinity for binding neutral or negatively charged
biomaterials, such as cells, cell debris, bacteria, spores,
viruses, nucleic acids, and proteins.
[0010] In some embodiments, the ligand-functionalized polymer may
be used as a flocculant whereby a biological sample, such as a cell
culture fluid, is contacted causing negative and/or neutral species
to bind to the polymer and precipitate from the solution or
suspension. In another embodiment, a base substrate, such as a
microporous membrane, may be coated with the ligand-functionalized
polymer.
[0011] Methods of making a ligand functionalized substrate are
provided. In some embodiments, the method comprises reacting a
polyamine polymer with a guanylating agent, optionally in the
presence of an acid catalyst.
[0012] A functionalized polymer is provided, having grafted pendent
ligand groups, of the formula:
##STR00001##
wherein [0013] R.sup.2 is a H, C.sub.1-C.sub.12 alkyl,
C.sub.5-C.sub.12 (hetero)aryl, or a residue of the polymer chain;
[0014] each R.sup.3 is independently H, C.sub.1-C.sub.12 alkyl, or
C.sub.5-C.sub.12 (hetero)aryl, [0015] each R.sup.4 is H,
C.sub.1-C.sub.12 alkyl or alkylene, C.sub.5-C.sub.12 (hetero)aryl
or (hetero)arylene, cyano, or --C(.dbd.NH)--N(R.sup.2)-Polymer, and
[0016] n is 1 or 2.
[0017] It will be recognized that the "Polymer-N(R.sup.2)-" group
of Formula I is the linkage formed between an amine group of
polyamino polymer and the guanylating agent.
[0018] As used herein, "alkyl" or "alkylene" includes
straight-chained, branched, and cyclic alkyl groups and includes
both unsubstituted and substituted alkyl groups. Unless otherwise
indicated, the alkyl groups typically contain from 1 to 20 carbon
atoms. Examples of "alkyl" as used herein include, but are not
limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl,
t-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl,
cyclohexyl, cycloheptyl, adamantyl, and norbornyl, and the like.
Unless otherwise noted, alkyl groups may be mono- or
polyvalent.
[0019] As used herein, "aryl" or "arylene" is an aromatic group
containing 5-12 ring atoms and can contain optional fused rings,
which may be saturated, unsaturated, or aromatic. Examples of an
aryl groups include phenyl, naphthyl, biphenyl, phenanthryl, and
anthracyl. Heteroaryl is aryl containing 1-3 heteroatoms such as
nitrogen, oxygen, or sulfur and can contain fused rings. Some
examples of heteroaryl groups are pyridyl, furanyl, pyrrolyl,
thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl,
and benzthiazolyl. Unless otherwise noted, aryl and heteroaryl
groups may be mono- or polyvalent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1 to 4 are plots of the Geobacillus stearolhennophilus
flocculation data of Example 35.
DETAILED DESCRIPTION OF THE INVENTION
[0021] In the article and methods of this invention,
ligand-functionalized polymers are provided which have enhanced
affinity, especially in high ionic strength media, for neutral or
negatively charged biological materials such as host cell proteins,
DNA, RNA, viruses, and other microorganisms. The affinity for such
biomaterials allows positively charged materials, such as
antibodies, to be purified, as they are not bound to the ligand
functional groups. The ligand functionalized substrate allows the
materials, lacking the affinity for the ligand groups are passed.
In some embodiments the ligand functionalized polymer is used as a
flocculant to selectively bind target biomaterials, precipitate
them from solution, and the precipitated adduct subsequently
separated.
Polyamine Polymer
[0022] The base polymer comprises a polyamine polymer; i.e. a
polymer having primary or secondary amino groups that may be
pendent or catenary, i.e. in the polymer chain. The aminopolymers
contain primary or secondary amine groups and can be prepared by
chain growth or step growth polymerization procedures with the
corresponding monomers. These monomers can also, if desired, be
copolymerized with other monomers. The polymer can also be a
synthesized or naturally occurring biopolymer. If any of these
polymers, irrespective of source, do not contain primary or
secondary amine groups, these functional groups can be added by the
appropriate graft chemistry.
[0023] Useful aminopolymers are water soluble or water-dispersible.
As used herein, the term "water soluble" refers to a material that
can be dissolved in water. The solubility is typically at least
about 0.1 gram per milliliter of water. As used herein, the term
"water dispersible" refers to a material that is not water soluble
but that can be emulsified or suspended in water.
[0024] Examples of amino polymers suitable for use, which are
prepared by chain growth polymerization include, but are not
limited to: polyvinylamine, poly(N-methylvinylamine),
polyallylamine, polyallylmethylamine, polydiallylamine,
poly(4-aminomethylstyrene), poly(4-aminostyrene),
poly(acrylamide-co-methylaminopropylacrylamide), and
poly(acrylaniide-co-aminoethylmethacrylate).
[0025] Examples of amino polymers suitable for use, which are
prepared by step growth polymerization include, but are not limited
to: polyethylenimine, polypropylenimine, polylysine,
polyaminoamides, polydimethylamine-epichlorohydrin-ethylenediamine,
and any of a number of polyaminosiloxanes, which can be built from
monomers such as aminopropyltriethoxysilane,
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,
N-trimcthoxysilylpropyl-N-methylamine, and
bis(trimcthoxysilylpropyl)amine.
[0026] Useful aminopolymers that have primary or secondary amino
end groups include, but are not limited to, those formed from
polyamidoamine (PAMAM) and polypropylenimine: e.g. DAB-Am and PAMAM
dendrimers (or hyperbranched polymers containing the amine or
quaternary nitrogen functional group). Exemplary dendrimeric
materials formed from PAMAM are commercially available under the
trade designation Starburst.TM. (PAMAM) dendrimer" (e.g.,
Generation 0 with 4 primary amino groups, Generation 1 with 8
primary amino groups, Generation 2 with 16 primary amino groups,
Generation 3 with 32 primary amino groups, and Generation 4 with 64
primary amino groups) from Aldrich Chemical, Milwaukee, Wis.,
Dendrimeric materials formed from polypropylenimine is commercially
available under the trade designation "DAB-AM" from Aldrich
Chemical. For example, DAB-Am-4 is a generation 1 polypropylenimine
tetraamine dendrimer with 4 primary amino groups, DAB-Am-8 is a
generation 2 polypropylenimine octaamine dendrimer with 8 primary
amino groups, DAB-Am-16 is a generation 3 polypropylenimine
hexadecaamine with 16 primary amino groups, DAB-Am-32 is a
generation 4 polypropylenimine dotriacontaamine dendrimer with 32
primary amino groups, and. DAB-Am-64 is a generation 5
polypropylenimine tetrahexacontaamine dendrimer with 64 primary
amino groups.
[0027] Examples of aminopolymers suitable for use, which are
biopolymers include chitosan, and starch, where the latter is
grafted with reagents such as methylaminoethylehloride,
[0028] Other categories of aminopolymers suitable for use include
polyacrylamide homo-or copolymers with amino monomers including
aminoalkyl(meth)acrylate, (meth)acrylatnidoalkylamine, and
diallylamine.
[0029] Preferred aminopolymers include polyaminoamides,
polyethyleneimine, polyvinylamine, polyallylamine, and
polydiallylamine.
[0030] Suitable commercially available aminopolymers include, but
are not limited to, polyamidoamines such as ANQUAMINE.TM.360, 401,
419, 456, and 701 (Air Products and Chemicals, Allentown, Pa.);
LUPASOL .TM. polyethylenimine polymers such as FG, PR 8515,
Waterfree, P, PS (BASF Corporation, Resselaer, N.Y.);
polyethylenimine polymers such as CORCAT.TM. P-600 (ElT Company,
Lake Wylie, S.C.); polyoxyalkyleneamines such as JEFFAMINE,.TM.
D-230, D-400, D-2000, HK-511 (XTJ-511), XTJ-510 (D-4000), XTJ-500
(ED-600), XTJ-502 (ED-2003), T-403, XTJ-509 (T-3000), and T-5000
(Huntsman Corporation, Houston, Tex.); and polyamide resins such as
the VERSAMID series of resins that are formed by reacting a
dimerized unsaturated fatty acid with alkylene diamines (Cognis
Corporation, Cincinnati, Ohio).
[0031] The ligand functional polymer may be prepared by
condensation of the polyamine polymer with a guanylating agent.
Known guanylating agents include: cyanamide; O-alkylisourea salts
such as O-methylisourea sulfate, O-methylisourea hydrogen sulfate,
O-methylisourea acetate, O-ethylisourea hydrogen sulfate, and
O-ethylisourea hydrochloride; chloroformamidine hydrochloride;
1-amidino-1,2,4-triazole hydrochloride;
3,5-dimethylpyrazole-1-carboxamidine nitrate;
pyrazole-1-carboxamidine hydrochloride;
N-amidinopyrazole-1-carboxamidine hydrochloride; and carbodiimides,
such as dicyclohexylcarbodiimide,
N-ethyl-N'-(3-climethylaminopropyl)carbodiimide, and
diisopropylcarbodiimide. The polyamine polymer may also be acylated
with guanidino-functional carboxylic acids such as guanidinoacetic
acid and 4-guanidinobutyric acid in the presence of activating
agents such as EDC (N-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride), or EEDQ
(2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline). Additionally, the
ligand functional polymer may be prepared by alkylation with
chloroacetone guanyl hydrazone, as described in U.S. Pat. No.
5,712,027.
[0032] Reagents for the preparation of biguanide-functional
polymers include sodium dicyanamide, dicyanodiamide and substituted
cyanoguanidines such as
N.sup.3-p-chlorophenyl-N.sup.1-cyanoguanidine,
N.sup.3-phenyl-N.sup.1-cyanoguanidine,
N.sup.3-alpha-naphthyl-N.sup.1-cyanoguanidine,
N.sup.3-methyl-N.sup.1-cyanoguanidine,
N.sup.3,N.sup.3-dimethyl-N.sup.1-cyanoguanidine,
N.sup.3-(2-hydroxyethyl)-N.sup.1-cyanoguanidine, and
N.sup.3-butyl-N.sup.1-cyanoguanidine. Alkylene- and
arylenebiscyanoguanidines may be utilized to prepare biguanide
functional polymers by chain extension reactions. The preparation
of cyanoguanidines and biscyanoguanidines is described in detail in
Rose, F. L. and Swain, G. J. Chem Soc., 1956, pp. 4422-4425. Other
useful guanylating reagents are described by Alan R. Katritzky et
al., Comprehensive Organic Functional Group Transformation, Vol. 6,
p. 640. Generally, such guanylation reagents are used in amounts
sufficient to functionalize 0.5 to 100 mole percent, preferably 2.5
to 50 mole percent, of the available amino groups of the
aminopolymer.
[0033] The resulting polymer will have pendent or catenary
guanidinyl groups of the formula:
##STR00002##
wherein [0034] R.sup.2 is a H, C.sub.1-C.sub.12 alkyl,
C.sub.5-C.sub.12 (hetero)aryl, or a residue of the polymer chain;
[0035] each R.sup.3 is independently H, alkyl, or C.sub.5-C.sub.12
(hetero)aryl, [0036] each R.sup.4 is H, C.sub.1-C.sub.12 alkyl or
alkylene, (hetero)aryl or (hetero)arylene, cyano, or
--C(.dbd.NH)--N(R.sup.2)-Polymer, and [0037] n is 1 or 2.
[0038] In some embodiments, it may be advantageous to functionalize
the amine containing polymer with other ligands in addition to the
guanidinyl ligand. For example, it may be useful to include a
hydrophobic ligand, an ionic ligand, or a hydrogen bonding ligand.
This can be particularly advantageous for the capture of certain
biological species, especially under conditions of high ionic
strength.
[0039] The additional ligands are readily incorporated into the
ligand functional polymers by alkylation or acylation procedures
well known in the art, such as by using halide, sulfonate, or
sulfate displacement reactions, r by using epoxide ring opening
reactions. Useful alkylating agents for these reactions include,
for example, dimethylsulfate, butyl bromide, butyl chloride, benzyl
bromide, dodecyl bromide, 2-chloroethanol, bromoacetic acid.
2-chloroethyltrimethylammonium chloride, styrene oxide, glycidyl
hexadecyl ether, glycidyltrimethylammonium chloride, and glycidyl
phenyl ether. Useful acylating agents include, for example, acid
chlorides and anhydrides such as benzoyl chloride, acetic
anhydride, succinic anhydride, and decanoyl chloride, and
isocyanates such as trimethylsilylisocyanate, phenyl isocyanate,
butyl isocyanate, and butyl isothiocyanate. In such embodiments 0.1
to 20 mole percent, preferably 2 to 10 mole percent, of the
available amino groups of the aminopolymer may be alkylated and/or
acylated.
[0040] The disclosure further provides a functionalized substrate
comprising a base substrate and an ungrafted coating of the ligand
functionalized polymer thereon. Preferably the base substrate is a
porous base substrate having interstitial and outer surfaces.
[0041] The base substrate may be formed from any suitable metallic,
thermoplastic, or thermoset material. The material may be an
organic or inorganic polymeric 3 material. Suitable organic
polymeric materials include, but are not limited to,
poly(meth)acrylates, poly(meth)acrylamides, polyolefins,
poly(isoprenes), poly(butadienes), fluorinated polymers,
chlorinated polymers. polyamides, polyimides, polyethers,
poly(ether sulfones), poly(sulfones), poly(vinyl acetates),
copolymers of vinyl acetate, such as poly(ethylene)co-poly(vinyl
alcohol), poly(phosphazenes), poly(vinyl esters), poly(vinyl
ethers), poly(vinyl alcohols), and poly(carbonates). Suitable
inorganic polymeric materials include, but are not limited to,
quartz, silica, glass, diatomaceous earth, and ceramic
materials.
[0042] Suitable polyolefins include, but are not limited to,
poly(ethylene), poly(propylene), poly(1-butene), copolymers of
ethylene and propylene, alpha olefin copolymers (such as copolymers
of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and
1-decene), poly(ethylene-co-1-butene) and
poly(ethylene-co-1-butene-co-1-hexene).
[0043] Suitable fluorinated polymers include, but are not limited
to, poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of
vinylidene fluoride (such as poly(vinylidene
fluoride-co-hexafluoropropylene), and copolymers of
chlorotrifluoroethylene (such as
poly(ethylene-co-chlorotrifluoroethylene).
[0044] Suitable polyamides include, but are not limited to,
poly(iminoadipoyliminohexamethylene),
poly(iminoadipoyliminodecamethylene), and polycaprolactam. Suitable
polyimides include, but are-not limited to,
poly(pyromellitimide).
[0045] Suitable poly(ether sulfones) include, but are not limited
to, poly(diphenylether sulfone) and
poly(diphenylsulfone-co-diphenylene oxide sulfone).
[0046] Suitable copolymers of vinyl acetate include, but are not
limited to, poly(ethylene-co-vinyl acetate) and such copolymers in
which at least some of the acetate groups have been hydrolyzed to
afford various poly(vinyl alcohols).
[0047] The base substrate may be in any form such as particles,
fibers, films or sheets. Suitable particles include, but are not
limited to, magnetic particles, organic particles, inorganic
particles, and porous and nonporous particles. Preferably the base
substrate is porous. Suitable porous base substrates include, but
are not limited to, porous particles, porous membranes, porous
nonwoven webs, and porous fibers.
[0048] In some embodiments, the porous base substrate is formed
from propylene homo- or copolymers, most preferably propylene
homopolymers. Polypropylene polymers are often a material of choice
for porous articles, such as nonwovens and microporous films, due
to properties such as non-toxicity, inertness, low cost, and the
ease with which it can be extruded, molded, and formed into
articles.
[0049] In many embodiments, the porous base substrate has an
average pore size that is typically greater than about 0.2
micrometers in order to minimize size exclusion separations,
minimize diffusion constraints and maximize surface area and
separation based on binding of a target molecule. Generally, the
pore size is in the range of 0.1 to 10 micrometers, preferably 0.5
to 3 micrometers and most preferably 0.8 to 2 micrometers when used
for binding of viruses. The efficiency of binding other target
molecules may confer different optimal ranges.
[0050] Suitable porous base substrates include, but are not limited
to, porous and microporous membranes, nonwoven webs, and fibers. In
some embodiments, the porous base substrate is a microporous
membrane such as a thermally-induced phase separation (TIPS)
membrane. TIPS membranes are often prepared by forming a homogenous
solution of a thermoplastic material and a second material above
the melting point of the thermoplastic material. Upon cooling, the
thermoplastic material crystallizes and phase separates from the
second material. The crystallized thermoplastic material is often
stretched. The second material is optionally removed either before
or after stretching. Microporous membrane are further disclosed in
U.S. Pat. No. 4,539,256 (Shipman), U.S. Pat. No. 4,726,989
(Mrozinski), U.S. Pat. No. 4,867,881 (Kinzer), U.S. Pat. No.
5,120,594 (Mrozinski), U.S. Pat. No. 5,260,360 (Mrozinski et al.),
and U.S. Pat. No. 5,962,544 (Waller), all of which are assigned to
3M Company (St. Paul, Minn.). Further, the microporous film can be
prepared from ethylene-vinyl alcohol copolymers as described in
U.S. Pat. No. 5,962,544 (Waller).
[0051] Some exemplary TIPS membranes comprise poly(vinylidene
fluoride) (PVDF), polyolefins such as polyethylene homo- or
copolymers or polypropylene homo- or copolymers, vinyl-containing
polymers or copolymers such as ethylene-vinyl alcohol copolymers
and butadiene-containing polymers or copolymers, and
acrylate-containing polymers or copolymers. For some applications,
a TIPS membrane comprising PVDF is particularly desirable. TIPS
membranes comprising PVDF are further described in U.S. Pat. No.
7,338,692 (Smith et al.).
[0052] In another exemplary embodiment the porous bases substrate
comprises a nylon microporous film or sheet, such as those
described in US. Pat. No. 6,056,529 (Meyering et al.), U.S. Pat.
No. 6,267,916 (Meyering et al.), U.S. Pat. No. 6,413,070 (Meyering
et al.), U.S. Pat. No. 6,776,940 (Meyering et al.), U.S. Pat. No.
3,876,738 (Marinacchio et al.), U.S. Pat. Nos. 3,928,517, 4,707,265
(Knight et al.), and U.S. Pat. No. 5,458,782 (Hou et al.).
[0053] In other embodiments, the porous base substrate is a
nonwoven web which may include nonwoven webs manufactured by any of
the commonly known processes for producing nonwoven webs. As used
herein, the term "nonwoven web" refers to a fabric that has a
structure of individual fibers or filaments which are randomly
and/or unidirectionally interlaid in a mat-like fashion.
[0054] For example, the fibrous nonwoven web can be made by wet
laid, carded, air laid, spunlaced, spunbonding or melt-blowing
techniques or combinations thereof. Spunbonded fibers are typically
small diameter fibers that are formed by extruding molten
thermoplastic polymer as filaments from a plurality of fine,
usually circular capillaries of a spinneret with the diameter of
the extruded fibers being rapidly reduced. Meltblown fibers are
typically formed by extruding the molten thermoplastic material
through a plurality of fine, usually circular, die capillaries as
molten threads or filaments into a high velocity, usually heated
gas (e.g. air) stream which attenuates the filaments of molten
thermoplastic material to reduce their diameter. Thereafter, the
meltblown fibers are carried by the high velocity gas stream and
are deposited on a collecting surface to from a web of randomly
disbursed meltblown fibers. Any of the non-woven webs may be made
from a single type of fiber or two or morefibers that differ in the
type of thermoplastic polymer and/or thickness.
[0055] Further details on the manufacturing method of non-woven
webs of this invention may be found in Wente, Superfine
Thermoplastic Fibers, 48 INDUS. ENG. CHEM. 1342 (1956), or in Wente
et al., Manufacture Of Superfine Organic Fibers, (Naval Research
Laboratories. Report No. 4364, 1954).
[0056] In one embodiment the base substrate may have a coating of
the ligand functional (co)polymer on a surface thereon. Useful
coating techniques include applying a solution or dispersion of the
(co)polymer, optionally including a crosslinker, onto the base
substrate. Polymer application is generally followed by evaporating
the solvent to form the polymer coating. Coating methods include
the techniques commonly known as dip, spray, knife, bar, slot,
slide, die, roll, or gravure coating. Coating quality generally
depends on mixture uniformity, the quality of the deposited liquid
layer, and the process used to dry or cure the liquid layer.
[0057] In some embodiments, the polyamine polymer is first coated
on the base substrate and subsequently reacted with a guanylating
agent, such as pyrazole carboxamidine hydrochloride.
[0058] In other embodiments, the ligand functional (co)polymer
itself is coated on the base substrate. Useful crosslinkers in
these instances include amine reactive compounds such as bis- and
polyaldehydes such as glutaraldehyde, bis- and polyepoxides such as
butanedioldiglycidylether and ethyleneglycoldiglycidylether,
polycarboxylic acids and to their derivatives (e.g., acid
chlorides), polyisocyanates, formaldehyde-based crosslinkers such
as hydroxymethyl and alkoxymethyl functional crosslinkers, such as
those derived from urea or melamine, and amine-reactive silanes,
such as 3-glycidoxypropyltrimethoxysilane,
3-glycidoxypropyltriethoxysilane, 5,6-epoxyhexyltriethoxysilane,
(p-chloromethyl)phenyltrimethoxysilane,
chloromethyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and
3-thiocyanatopropyltriethoxysilane,
[0059] In other embodiments, the ligand functional copolymer is
coated on the base substrate by polyelectrolyte layer-by-layer
coating techniques, such as those described in EP 472,990.
[0060] In some embodiments, the base substrate has amine-reactive
functional groups, such as halide, epoxy, ester, isocyanate groups,
on the surface thereof. These surface functional groups may react
with extant amine functional groups on the ligand functional
aminopolymer. In another embodiment, the surface of the base
substrate may be provided with amine-reactive functional groups,
that can react with the amine groups of the ligand functionalized
polymer.
[0061] The amine-reactive functional groups may be provided by any
of the techniques known to one in the art. In one embodiment the
base substrate may have a coating of a (co)polymer comprising
amine-reactive functional groups on a surface thereon. Especially
useful (co)polymers in this regard are azlactone functional
(co)polymers such as those described in U.S. Pat. No. 7,101,621.
Useful coating techniques include applying a solution or dispersion
of the (co)polymer, optionally including a crosslinker, onto the
base substrate. Polymer application is generally followed by
evaporating the solvent to form the polymer coating. Coating
methods include the techniques commonly known as dip, spray, knife,
bar, slot, slide, die, roll, or gravure coating. Coating quality
generally depends on mixture uniformity, the quality of the
deposited liquid layer, and the process used to dry or cure the
liquid layer.
[0062] In some embodiments the polymer comprising amine-reactive
groups may be grafted to the surface of a substrate by ionizing
radiation-initiated graft polymerization of a monomer having a
free-radically polymerizable group and a second functional group
reactive with the ligand functional polymer, as described in
Assignee's copending application (64729US002). Such monomers may
include azlactone-functional monomers, to isocyanatoethyl
(meth)acrylate or glycidyl (meth)acrylate. Alternatively, a
carbonyl functional monomer may be grafted to the surface of a
substrate by ionizing radiation-initiated graft polymerization,
followed by functionalization by reaction with the ligand
functional polymer of Formula I, as described in Assignee s
copending U.S. Application. No. 61/305740.
[0063] The method of grafting (or coating) a ligand functionalized
polymer to the surface of the substrate alters the original nature
of the base substrate, as the substrate bears a grafted or
ungrafted coating of the ligand functional polymer. The present
invention enables the formation of ligand functionalized polymer
substrates having many of the advantages of a base substrate (e.g.,
mechanical and thermal stability, porosity), but with enhanced
affinity for biological species such as viruses, resulting from the
monomers and steps used to form a given functionalized
substrate.
[0064] The porous substrates having a coating of
ligand-functionalized polymer are particularly suited as filter
media, for the selective binding and removal of target biological
species including proteins, cells, cell debris, microbes, nucleic
acids, and/or viruses from biological samples. The present
disclosure further provides a method for the removal of target
biological species from a biological sample by contacting the
sample with the ligand polymer functionalized substrate as
described herein. As used herein "target biological species" may
include, a contaminant or a species of interest.
[0065] The ligand functionalized (co)polymer (either the polymer
per se, or a substrate having a coating thereof) is useful for the
purification of biological or other fluid samples comprising
biologically derived species (biological species). Biological
species include, but are not limited to, cells, cell debris,
proteins, nucleic acids, endotoxins, and viruses. Cells and cell
debris include those derived from archaea, bacteria, and
eucaryotes. Bacteria include, but are not limited to,
Gram-negatives such as. Pseudomonas species, Escherichia coli,
Helicobacter pylori, and Serratia marcesens; Gram-positives such as
Staphylococcus species, Enterococcus species, Clostridium species,
Bacillus species, and Lactobacillus species; bacteria that do not
stain traditionally by Gram's method such as Mycobacterium species,
and non-vegetative forms of bacteria such as spores. Eucaryotes
include, but are not limited to, animal cells, algae, hybridoma
cells, stem cells, cancer cells, plant cells, fungal hyphae, fungal
spores, yeast cells, parasites, parasitic oocysts, insect cells,
and helminthes. Proteins, include, but are not limited to, natural
proteins, recombinant proteins, enzymes, and host cell proteins.
Viruses include, but are not limited to, enveloped species such as
Herpesviruses, Poxviruses, Adenoviruses, Papovaviruses,
Coronaviruses, retroviruses such as HIV, and Plasmaviridae; and
non-enveloped species such as Caliciviridae, Corticoviridae,
Myoviridae, and Picornaviridae,
[0066] In some embodiments, the biological species being removed
from the fluid is the object of the purification. For example, a
recombinant protein or enzyme may be prepared in cell culture or by
fermentation, the (co)polymer can be added to flocculate the
protein or enzyme, and the precipitate can be separated as the
first step in the purification process for the protein or enzyme.
In another example, the (co)polymer or a substrate with a coating
thereof, may be used to capture microorganisms from a fluid as the
first step in a process of concentrating, enumerating, and/or
identifying the microorganisms.
[0067] In other embodiments, the biological species being removed
from the fluid is a contaminant that must be removed prior to
additional processing steps for the fluid. The polymer can be used
as a flocculant to facilitate the removal of cells and cell debris
from a cell culture or fermentation broth prior to, subsequent to,
or in place of a centrifuge or depth filtration operation. For
example, the (co)polymer can be used to flocculate cells in a cell
culture broth prior to centrifugation, and thereby improve the
efficiency with which the centrifugation process separates the cell
mass from the liquid centrate. Alternatively, it can be added to
the liquid centrate after a centrifugation step to flocculate
suspended cell debris and dissolved host cell proteins and nucleic
acids, thereby increasing the efficiency of a subsequent depth
filtration step. It can be used to flocculate or precipitate
suspended bacteria, viruses, or other microorganisms. It can be
used to precipitate either desired or contaminating proteins or
nucleic acids from solution. Significantly, the ligand functional
(co)polymers, or substrates having a coating thereof, are useful
under conditions of high salt concentration or high ionic strength,
i.e., they are "salt tolerant". The term "salt" is meant to include
all low molecular weight ionic species which contribute to the
conductivity of the solution. The importance of utility of the
ligand functional (co)polymers in the presence of salt is that many
process solutions used in biopharmaceutical or enzyme manufacture
have conductivities in the range of 15-30 inSkin (approximately
150-300 mM salt) or more. Salt tolerance can be measured in
comparison to that f the conventional quaternary amine or Q ligand
(e.g. trimethylammonium ligand), whose primarily electrostatic
interactions with many biological species rapidly deteriorates at
conductivities three- to six-fold less than the target range. For
example, membranes derivatized with the conventional Q ligand
exhibit a drop in .PHI.X174 viral clearance from a six
log-reduction value (LRV) to a one (1) LRV in going from 0 to 50 mM
NaCl (ca. 5-6 mS/cm conductivity). Viruses such as .PHI.X174 which
have pIs close to 7 (are neutral or near-neutral) are extremely
difficult to remove from process streams. Similar problems are
observed when attempting to remove other biological species from
process fluids. For example, when attempting to remove positively
charged proteins such as host cell proteins through the use of
filtration devices functionalized with conventional Q ligands, the
process fluid may have to be diluted two-fold or more in order to
reduce the conductivity to an acceptable range. This is expensive
and dramatically increases the overall processing time.
[0068] When used as a flocculant, the amount of ligand functional
polymer that is added relative to the amount of sample can vary
over a wide range. Generally, the amount added will produce a final
concentration of (co)polymer in the mixture of from about 0.01
micrograms/mL to about 5000 micrograms/mL. The optimal amount of
polymer added will depend upon the concentration of the species one
desires to flocculate. Typically, the amount of polymer relative to
the amount of species being flocculated will be in the range of
0.01% to 100% by weight, preferably 0.05%-30% by weight, more
preferably about 0.1%-10% by weight. The optimal amount is readily
assessed by challenging the sample with a series of polymer
concentrations as is well known in the art. While the above
concentration ranges are typical, one skilled in the art will
realize that other ranges may work in some instances. Flocculation
efficiency also depends upon the physical and chemical
characteristics of the species being flocculated. For example, we
have found that optimal flocculation of the near neutral virus
.PHI.X174 from aqueous suspension occurs at a polymer to virus
weight ratio of about 800-1000%.
[0069] The biological sample is contacted with the ligand
functionalized polymer (either the polymer per se, or a substrate
having a coating thereof) for a time sufficient to interact and
form a complex with the target biological species disposed
(dissolved or suspended) in the solution when the solution
comprises from 0 to about 50 mM salt, preferably when the solution
comprises from 0 to about 150 mM salt, more preferably when the
solution comprises from 0 to about 300 mM salt or higher, such that
the concentration of the target biological species remaining
disposed in the solution is less than 50% of its original
concentration. It is more preferred that the solution is contacted
with the ligand functionalized polymer for a time sufficient to
interact and form a complex with the target biological species
disposed in the solution when the solution comprises from 0 to
about 50 mM salt, preferably when the solution comprises from 0 to
about 150 mM salt, more preferably when the solution comprises from
0 to about 300 mM salt or higher, such that the concentration of
the target biological species remaining disposed in the solution is
less than 10% of its original concentration. It is still more
preferred that the solution is contacted with the ligand
functionalized polymer for a time sufficient to interact and form a
complex with the target biological species disposed in the solution
when the solution comprises from 0 to about 50 mM salt, preferably
when the solution comprises from 0 to about 150 mM salt, more
preferably when the solution comprises from 0 to about 300 mM salt
or higher, such that the concentration of the target biological
species remaining disposed in the solution is less than 1% of its
original concentration.
[0070] In many embodiments the ligand functionalized polymer, being
positively charged in aqueous media, will bind near neutral or
negatively charged species to the ligand functional group of
Formula H while other species (e.g., positively charged proteins
such as monoclonal antibodies) will be excluded or repelled from
the ligand functionalized substrate. In addition, as previously
described, the substrate may be directly or indirectly grafted with
one or more ionic monomers. In particular, the ligand
functionalized polymer may comprise grafted ionic groups that are
positively charged at the selected pH of the biological sample
solution to enhance electrostatic charge repulsion of proteins,
such as monoclonal antibodies, many of which are charged positive
at neutral pH, and ligand functional groups of Formula II to
provide salt tolerance.
[0071] In some embodiments the ligand functionalized polymer and
coated substrate containing the bound biological species are
disposable. In such embodiments, the binding of the biological
species to the ligand functionalized polymer is preferably
essentially irreversible because there is no need to recover the
bound biological species. Nonetheless, if desired, one can reverse
the binding of biological species by increasing the ionic strength
or changing the pH of an eluting solution.
[0072] The substrate, having a grafted or ungrafted coating of the
ligand functionalized polymer may be any previously described, but
is preferably a microporous membrane. The membrane pore size
desired is from 0.1 to 10 .mu.m, preferably 0.5 to 3 micrometers
and most preferably 0.8 to 2 micrometers. A membrane with a high
surface area for the internal pore structure is desired, which
typically corresponds to fine pore sizes. However, if the pore size
is too small, then the membrane tends to plug with fine
particulates present in the sample solution.
[0073] If desired, efficiency of binding and capture may be
improved by using a plurality of stacked, ligand functionalized
polymer coated porous membranes as a filter element. Thus the
present disclosure provides a filter element comprising one or more
layers of the porous, ligand functionalized polymer coated
substrate. The individual layers may be the same or different, and
may have layers of different porosity, and degree of grafting by
the aforementioned grafting monomers. The filter element may
further comprise an upstream prefilter layer and downstream support
layer. The individual filter elements may be planar or pleated as
desired. Examples of suitable prefilter and support layer materials
include any suitable porous membranes of polypropylene, polyester,
polyamide, resin-bonded or binder-free fibers (e.g., glass fibers),
and other synthetics (woven and non-woven fleece structures):
sintered materials such as polyolefins, metals, and ceramics; yams;
special filter papers (e.g., mixtures of fibers, cellulose,
polyolefins, and binders); polymer membranes; and others.
[0074] In another embodiment, there is provided a filter cartridge
including the above-described filter element. In yet another
embodiment, there is provided a filter assembly comprising the
filter elements and a filter housing. In a further embodiment, this
invention relates to a method of capture or removal f a target
biological species comprising the steps of:
[0075] a) providing the filter element comprising one of more
layers of the ligand functionalized base substrate of this
disclosure, and
[0076] b) allowing a moving biological solution containing a target
biological species to impinge upon the upstream surface of the
filter element for a time sufficient to effect binding of a target
biological species.
[0077] The present invention is described above and further
illustrated below by way of examples, which are not to be construed
in any way as imposing limitations upon 1.5 the scope of the
invention. On the contrary, it is to be clearly understood that
resort may be had to various other embodiments, modifications, and
equivalents thereof which, after reading the description herein,
may suggest themselves to those skilled in the art without
departing from the spirit of the present invention and/or the scope
of the appended claims.
EXAMPLES
Example 1
Alkylation of Polyethylenimine (PEI)
[0078] Dodecyl bromide (2.32 grams) was added to PEI (40 grams of a
10% by weight solution of PEI (MW=10,000. from Polysciences, Inc.,
Warrington, Pa.) in ethanol in an 8 ounce glass bottle and sealed.
The mixture was heated in a water bath at 50.degree. C. for 20
hours, after which time .sup.1H-NMR indicated complete conversion
to the alkylated product
Example 2
Guanylation of Alkylated Polyetheyleneamine
[0079] A portion of the solution of alkylated product (20 grams) of
Example 1 was mixed with pyrazole carboxamidine hydrochloride (0.17
gram, from Sigma-Aldrich, Milwaukee, Wis.). The mixture was allowed
to react overnight at ambient temperature, after which time
.sup.1H-NMR indicated conversion of 2.5% of the amine groups to
guanidine groups had occurred.
Examples 3-21
[0080] Similar procedures were used to produce alkylated and
guanylated PEI's as listed in Table 1.
TABLE-US-00001 TABLE 1 Modified Polyethylenimines PEI % % Example
MW Alkylating agent Alkylated Guanylated 1 10,000 dodecyl bromide
10 0 2 10,000 dodecyl bromide 10 2.5 3 10,000 dodecyl bromide 5 0 4
10,000 dodecyl bromide 5 2.5 5 10,000 none 0 2.5 6 10,000 none 0
12.5 7 10,000 none 0 25 8 10,000 benzyl bromide 10 0 9 10,000
dimethylsulfate 10 0 10 10,000 butyl bromide 10 0 11 70,000 none 0
10 12 70,000 none 0 25 13 70,000 none 0 50 14 70,000 butyl bromide
10 0 15 70,000 dodecyl bromide 10 0 16 70,000 benzyl bromide 10 0
17 70,000 dimethylsulfate 10 0 18 70,000 butyl bromide 10 50 19
70,000 dodecyl bromide 10 50 20 70,000 benzyl bromide 10 50 21
70,000 dimethylsulfate 10 50
Example 22-32
Modified Poly(Allylamine)s
[0081] Using procedures similar to those described in Example 1,
poly(allylamine) (MW 60,000. Polysciences) was reacted with a
variety of alkylating agents and pyrazole carboxamidine
hydrochloride to provide a series of alkylated, guanylated, or
alkylated and guanylated polymers (Table 2).
TABLE-US-00002 TABLE 2 Modified Poly(allylamines) % % Example
Alkylating agent Alkylated Guanylated 22 dodecyl bromide 10 0 23
butyl bromide 10 0 24 benzyl bromide 10 0 25 dimethylsulfate 10 0
26 none 0 10 27 none 0 25 28 none 0 50 29 dodecyl bromide 10 50 30
butyl bromide 10 50 31 benzyl bromide 10 50 32 dimethylsulfate 10
50
Comparative Example 1
Poly(Methacrylamidopropyltrimethylammonium Chloride) (pMAPTAC)
[0082] MAPTAC (160 grams of a 50% by weight solution in water, from
Aldrich, Milwaukee, Wis.), ethanol (40 grams) and sodium persulfate
(0.4 gram) were charged to a 16 ounce glass bottle. The mixture was
purged with a slow stream of nitrogen gas for 10 minutes, sealed,
and then tumbled in a water bath equilibrated to 55.degree. C. for
24 hours to convert the monomer to polymer. This polymer solution
was diluted with deionized water (80 grams) and ethanol (40 grams)
and mixed well. A sample for evaluation as a flocculant was
prepared by dilution of a portion of this polymer to 1% solids by
weight with deionized water. pH 7.
Example 33
[0083] A solution of bovine serum albumin (BSA, Sigma-Aldrich) was
prepared in 10 mM MOPS, pH 7.5, and determined to have a
concentration of BSA of 4.02 mg/mL. A series of BSA solutions were
prepared containing various concentrations of sodium chloride
according to Table 3.
TABLE-US-00003 TABLE 3 BSA Solutions BSA 5M MOPS [NaCl] solution
NaCl buffer (mM, final) (mL) (.mu.L) (.mu.L) 0 10 0 500 50 10 100
400 100 10 200 300 150 10 300 200 200 10 400 100 250 10 500 0
[0084] Solutions of the polymers from Examples 5, 6, and 7 were
diluted with deionized water to 1% solids by weight, pH 7. A 1%
solids solution:of PEI (10,000 MW) in DI water, pH 7, was also
prepared as a control.
[0085] A 5 mL polypropylene centrifuge tube was charged with 2.0 mL
of BSA solution, followed by 125 .mu.L of diluted polymer solution.
The centrifuge tube was sealed and tumbled end over end for 30
minutes, then centrifuged at 2000 rcf for 10 minutes. A BSA
standard solution was prepared by mixing 2 mL of original BSA
solution with 125 .mu.L of MOPS buffer. A serial 1:1 dilution was
performed to provide a total of 7 BSA standards. These seven
standards were pipetted (200 .mu.L) in triplicate into wells of a
96-well microtitration plate, along with triplicate samples of the
supernates from each polymeric flocculant being evaluated. Three
wells containing DI water as a blank were also included. The plate
was analyzed using a SpectraMAX 250 Microplate Spectrophotometer
System (Molecular Devices Corp, Sunnyvale, Calif.) using a
wavelength of 293 nm. Comparison of the absorptions of the
flocculant solutions to those of the standards provided a measure
of the flocculation efficiency. Results are recorded as the
percentage of starting BSA remaining in solution; thus, the lower
the number, the better the flocculant. Results are presented in the
following Table 4:
TABLE-US-00004 TABLE 4 % BSA Remaining 0 mM 50 mM 100 mM 150 mM 200
mM 250 mM Polymer NaCl NaCl NaCl NaCl NaCl NaCl PEI (10,000) 0.0
40.6 87.2 96.1 101.6 99.7 Example 5 0.9 6.2 29.3 53.9 83.4 96.8
Example 6 0.0 5.6 28.0 51.2 79.0 93.8 Example 7 0.3 18.2 40.3 60.8
78.1 88.3 Comparative 0.0 42.1 73.3 101.9 103.1 99.3 Example 1
This example illustrates that incorporation of as little as 2.5%
guanidine groups into PEI dramatically improves its ability to
precipitate proteins in the presence of sodium chloride.
Example 34
[0086] Guanylated PEIs from Examples 11-13 were assayed for BSA
precipitation by the procedure described in Example 33, except that
250 .mu.L of 1% solids polymer solution was used instead of 125
.mu.L. Results are shown in Table 4, compared to a control
unmodified 70,000 MW PEI.
TABLE-US-00005 TABLE 5 % BSA Remaining 50 mM 100 mM 150 mM 200 mM
250 mM Polymer NaCl NaCl NaCl NaCl NaCl PEI (70,000) 54.4 28.8 35.4
60.0 87.9 Example 11 11.1 11.5 14.4 27.6 41.1 Example 12 17.7 7.4
9.0 13.1 95.6 Example 13 17.6 16.7 20.2 27.8 39.1
Example 35
[0087] A Geobacillus stearothermophilus cell culture broth was
provided by 3M consisting of approximately 1.4% by weight cell
debris and spores. Test samples of broth were prepared containing
0, 100, 200, and 300 mM NaCl by a procedure similar to that
described in Example 33. Solutions of polymers were prepared at
0.5% solids in DI water from 70,000 MW PEI and from the modified
polymers of Examples 13, 16, and 20. A dilution series of each of
these polymers was prepared (1:4, 1:4, 1:2, 1:2, 1:2) to provide a
total of 6 polymer concentrations. Then 2 mL of broth sample was
mixed with 0.5 mL of polymer solution, and the mixture was tumbled
for 30 minutes, then centrifuged at 200 rcf for 5 minutes.
Standards were prepared by mixing 2 mL of broth with 0.5 mL of DI
water, carrying the mixture through the same mixing/centrifugation
procedure, then preparing a 2-fold serial dilution (6 samples) from
the supernate. Supernates from the test solutions and from the
standards were pipetted into a 96-welt microtitration plate and
assayed by absorbance measurement at 650 nm. Comparison of the
absorptions of the flocculant solutions to those of the standards
provided a measure of the flocculation efficiencies. Results are
presented in the FIGS. 1-4 which show the removal of turbidity at
different salt concentrations and different polymer concentrations
for an unmodified PEI polymer (FIG. 1), a guanylated PEI polymer
(FIG. 2), an alkylated PEI polymer (FIG. 3), and a polymer that has
been alkylated and guanylated (FIG. 4).
[0088] Similar results were observed for Examples 14, 15, 17, 18,
19, and 21; that is, alkylation of the PEI resulted in broadening
the concentration range in which the flocculant was effective in
higher salt concentrations, while alkylation plus guanylation were
synergistic in this regard.
[0089] By making minor modifications to the assay procedure,
similar flocculation studies on a Bacillus atrophaeus cell culture
broth (2.2% by weight vegetative cells, cell debris, and spores),
and a Clostriduni sporagenes purified spore suspension (0.013%
solids) were conducted and similar results were observed.
Example 36
Virus Flocculation
[0090] Aqueous suspensions of .PHI.X174 bacteriophage (ca. 10.sup.9
pfu/mL) were prepared in 10 mM TRIS((hydroxymethyl)aminomethane) pH
8.0 containing 0, 50 mM, and 150 mM NaCl. Aqueous solutions of
flocculator polymers were prepared in DI water. pH 7, at 0.001%
polymer by weight 16 .mu.L of polymer solution were added to a 2 mL
sample of bacteriophage suspension in a centrifuge tube. The tube
was sealed, vortexed, and rotated end-over-end for 2 hours. The
tubes were then centrifuged at 3000 rcf for 10 minutes, and the
resultant suspensions were filtered through a 0.45 micron sterile
syringe filter (GHP Acrodisc, Pall Life Sciences). A 10-fold
dilution series was prepared.
[0091] One mL of each dilution was mixed with 1 mL E. coli culture
(grown to an optical density of 0.3-0.6 when measured at 550 nm).
After waiting 5 minutes, a sterile pipet was used to mix 4.5 mL TSA
Top agar with the dilution/E. coli mixture and plated on TSB
plates. After the top agar had solidified, the plates were inverted
and placed in a 37.degree. C. incubator overnight. Plates were then
removed from the incubator and .PHI.X174 plaques were counted and
recorded. A dilution series of the original virus suspension was
also evaluated in a similar manner. Comparison of the results
allowed estimation of the LRV (log reduction in viral load) as a
result of the flocculant treatment. Results for several polymers
are listed in Table 6:
TABLE-US-00006 TABLE 6 Virus LRV PhiX174 LRV 0 mM 50 mM 150 mM
Polymer NaCl NaCl NaCl Comparative 6.3 0.1 0.5 Example 1 Example 27
5.8 3.5 2.9 Example 11 8.5 3.4 2.0 Example 12 8.5 3.8 1.9 Example
13 >8 6.3 2.9
Example 37
[0092] Five coating baths were prepared: [0093] Coating Bath #1:
0.5% wtiwt poly(2-acrylamido-2-methyl-1-propanesulfonic acid,
sodium salt) in deionized water; [0094] Coating Bath #2: 1% wt/wt
polyethylenimine (10,000 M.sub.n) in deionized water; [0095]
Coating Bath #3-5: 1% wt/wt polymer of Examples 5, 6, or 7,
respectively, in deionized water. [0096] Aminosi lane coated glass
microscope slides (obtained from Newcomer Supply, Middleton, Wis.)
were dip coated, in sequence, with coating solution #1, rinsed with
deionized water, and dried at room temperature. Slides were then
dip coated in coating solution #2 (control), #3, #4, or #5, rinsed
with deionized water, and dried at room temperature. The coated
slides could then be used to capture bacteria or spores from
aqueous media for enumeration or for identification. A control
aminosilane slide coated with only coating solution #1 was found to
bind almost no bacteria or spores.
[0097] Slides coated with PEI and the modified PEIs of Examples 5-7
were evaluated for their ability to capture Clostridium sporogenes
spores by the following procedure: [0098] Prepare a suspension of
pure C. sporogenes spores at approximately 1.times.10.sup.8 CFU/mL
in 40% ethylalcohol, 60% water. Centrifuge between 0.5 and 1.0 mL
of the spore suspension (volume will depend on number of materials
tested), discard the supernatant and re-suspend 1:1 in 70% ethyl
alcohol in water. Next place three replicates of coated microscope
slides into a square Petri dish and apply 10 .mu.l of the spore
suspension 0.5 cm from the bottom in the center of the slide. Leave
for 1 minute then rinse each slide under a steady stream of
ultrapure water from a Milli-Q system at a flow rate of
approximately 1.2 liters per minute for 10 seconds. Now dry the
slide using a gentle flow of nitrogen gas. Using a light
microscope, place the spore-exposed portion of the slide under the
10.times. objective and randomly capture images of three separate
areas within the exposed region. Count the number of spores in each
image; this can be done by hand or alternately using image
processing software such as AxioVision (Zeiss). Calculate the
average per slide based on the counts from the three images on the
same slide. Finally, calculate the average and standard deviation
per material from the three replicates. [0099] The results are
displayed in Table 7:
TABLE-US-00007 [0099] TABLE 7 Coating: PEI Example 5 Example 6
Example 7 # of Spores 664 1392 1347 1050 Captured:
Example 38
p-Chlorophenylbiguanide Derivative of PEI
[0100] A solution of 2.00 grams of PEI (MW=10,000, from
Polysciences, Inc., Warrington, Pa.) in 10 mL of ethoxyethanol was
treated with 2.93 mL of 1.0 N aqueous hydrochloric acid solution.
N.sup.3-p-chlorophenyl-N.sup.1-cyanoguanidine (570 mg, 2.94 mmol)
was added and the reaction mixture was heated to 140.degree. C.
overnight. Thin layer chromatography indicated that all of the
N.sup.3-p-chlorophenyl-N.sup.1cyanoguanidine had been consumed. The
reaction mixture was concentrated under reduced pressure to give an
orange syrup. .sup.1H-NMR indicated conversion to the
p-chlorophenylbiguanide product. The resulting syrup was dissolved
in water to give a 20% by wt solution based on the PEI initial
mass.
[0101] Evaluation of this polymer by the BSA precipitation test
described in Example 33, using 250 .mu.L flocculant solution,
showed good BSA removal at all salt concentrations, up to 250 mM,
tested.
Examples 39-44
Carbodiimide Modifications of PEI
[0102] A solution of 1.05 grams of PEI (MW=10,000, from
Polysciences, Inc., Warrington, Pa.) in 10 mL of tert-butyl alcohol
was placed in a vial and treated with enough
dicyclohexycarbodiimide (314 mg, 1.52 mmol) to react with 6.3% of
the amine groups. The vial was sealed and the mixture was heated at
100.degree. C. overnight. The reaction mixture was concentrated
under reduced pressure to give a colorless syrup. .sup.1H-NMR
indicated conversion to the dicyclohexyl guanide product (Example
39). Likewise, PEI samples functionalized with 12.5% and 25%
dicyclohexyl guanides were also prepared (Examples 40 and 41,
respectively). The resulting syrups with 6.3 and 12.5% dicyclohexyl
guanides were dissolved in dilute hydrochloric acid to give a 10%
by wt solution based on the initial PEI mass. The product with 25%
dicyclohexyl guanides was dissolved in 1:1 ethanol/dilute
hydrochloric acid to give a 5% by wt solution based on the initial
PEI mass.
[0103] A solution of 0.99 grams of PEI (MW=10,000, from
Polysciences, Inc., Warrington, Pa.) in 10 mL of tert-butyl alcohol
was placed in a vial and treated with enough
N[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (280
mg, 1.46 mmol) to react with 6.3% of the amine groups. The vial was
sealed and the mixture was heated at 100.degree. C. overnight. The
reaction mixture was concentrated under reduced pressure to give a
colorless syrup. .sup.1H-NMR indicated conversion to the desired
guanide product (Example 42). Likewise, PEI samples functionalized
with 12.5% and 25% N-[3-(dimethylamino)propyl9 -3-ethylcarbodiimide
were also prepared (Examples 43 and 44, respectively). The
resulting syrups were dissolved water to give a 10% by wt solution
based on the initial PEI mass.
[0104] Polymers of Examples 39 and 41 were diluted to 1% solids and
evaluated for BSA precipitation using 250 .mu.L flocculant
solution. Results are displayed in. Table 8, along with those for
unmodified PEI:
TABLE-US-00008 TABLE 8 % BSA Remaining 50 mM 100 mM 150 mM 200 mM
250 mM Polymer NaCl NaCl NaCl NaCl NaCl PEI (10,000) 4.0 12.5 40.0
83.7 92.8 Example 39 3.8 10.5 27.3 63.6 80.9 Example 41 4.6 8.0
17.6 39.9 47.0
Example 45
[0105] The polymer of Example 11 was diluted to 0.5% solids with
isopropanol. Four portions of this solution (50 grams each) were
formulated with enough butanediol diglycidyl ether (BUDGE) to react
with 2.5%, 5%, 10%, and 20%, respectively, of the amine groups of
the polymer. Samples (ca. 10 cm.times.10 cm) of a nylon 66 membrane
(single reinforced layer nylon three zone membrane, nominal pore
size 1.8 .mu.m, from 3M Purification Inc, Meridan, Conn.), were dip
coated with the polymer solution, excess coating solution was
removed using a #14 wire-wound coating rod, then allowed to dry for
15 minutes. In some instances, a second coating layer was applied.
The coated membranes were then placed in 500 mL polyethylene
bottles filled with deionized water and allowed to mix overnight to
extract any non-crosslinked coating. Disks (24 mm diameter) were
punched out of the membranes and placed in 5 mL centrifuge tubes.
Bovine serum albumin solution (BSA, Sigma Aldrich) was prepared to
a concentration of 1.1 mg/ml in 25 mM TRIS buffer. pH 8.0
(tris(hydroxymethyl)aminomethane, Sigma). 4.5 ml of the BSA
solution was pipetted into each centrifuge tube, the tubes were
capped, and the tubes were tumbled overnight. The supernatant
solutions were analyzed by a UV-VIS spectrometer at 279 nm with
background correction applied at 325 nm. Static binding capacities
for the samples are listed in Table along with that for an uncoated
membrane.
TABLE-US-00009 TABLE 9 % Crosslinker # of Coating Static BSA
Capacity (BUDGE) Layers (mg/mL) 2.5 1 15 2.5 2 20 5 1 17 5 2 26 10
1 19 10 2 35 20 1 27 20 2 46 0 0 1
Examples 46-48
Cyanoguanidine-Derivatized PEI
[0106] A solution of 2.01 grams of PEI (MW=10.000, from
Polysciences, Inc., Warrington, Pa.) in 11.7 mL, of 0.1 N aqueous
hydrochloric acid was placed in a pressure flask and treated with
enough sodium dicyanamide (104 mg, 1.17 mmol) to react with 2.5% of
the amine groups. The flask was sealed and the mixture was heated
at 120.degree. C. for 5 hours. .sup.1H-NMR indicated conversion to
the cyanoguanidine product (Example 46).
[0107] Likewise, a 1.96 g solution of PEI dissolved in 9 mL of
water was treated with 2.9 mL of 1.0 N hydrochloric acid and sodium
dicyanamide (255 mg, 2.86 mmol) to give a product where 6.3% of the
amines were converted to cyanoguanides and a 1.99 g solution of PEI
dissolved in 6 mL of water was treated with 5.8 mL of 1.0 N
hydrochloric acid and sodium dicyanamide (520 mg, 5.84 mmol) to
give a product where 12.5% of the amines were converted to
cyanoguanides (Examples 47 and 48 respectively).
Examples 49-51
Urea Modification of PEI
[0108] A solution of 3.00 grams of PEI (MW=10,000, from
Polysciences, Inc., Warrington, Pa.) in 15 mL of CH.sub.2Cl.sub.2
was treated with enough trimethylsilyl isocyante (235
.quadrature.L, 1.74 mmol) to react with 2.5% of the amine groups.
After stirring for 1 h, the reaction mixture was treated with a few
drops of methanol and concentrated under reduced pressure.
.sup.1H-NMR indicated conversion to the urea product (Example 49).
The resulting syrup was dissolved in water to give a 20% by wt
solution based on the PEI initial mass. Likewise, PEI
functionalized with 6.3% and 12.5% ureas were also prepared
(Examples 50 and 51, respectively).
Examples 52-54
[0109] The urea modified PEI materials from Examples 49-51, were
each reacted with enough pyrazole-1-carboxamidine hydrochloride, by
procedures similar to that used in Example 1, to convert 5% of the
amine groups to guanidines. .sup.1H-NMR indicated conversion to the
expected derivatized products (Examples 52-54, respectively).
Example 55
PEI Biguanide
[0110] A solution of 92 mg of PEI (MW=10,000, from Polysciences,
Inc., Warrington. Pa.) in 0.9 mL of water was placed in a vial and
treated with N-amidinopyrazole-carboxamidine hydrochloride (100 mg,
0.53 mmol) to react with 25% of the amine groups. The vial was
sealed and the mixture was heated at 100.degree. C. overnight.
.sup.1H-NMR indicated conversion to the desired guanide
product.
Example 56
Poly(Polyethylenimine Biguanide)
[0111] A solution of 1.00 grams of PEI (MW=600, from Polysciences,
Inc., Warrington, Pa.) in 10 mL of water was placed in a pressure
flask and treated with sodium dicyanamide (139 mg, 1.56 mmol) and
200 .quadrature.L of acetic acid. The flask was scaled and the
mixture was heated at 140.degree. C. overnight. .sup.1H-NMR
indicated conversion to the polybiguanidine product.
[0112] In a similar manner, PEI polymers of different molecular
weights can be utilized, and differing ratios of PEI to sodium
dicyanamide can be utilized, to prepare a variety of poly(PEI
biguanide)s.
Examples 57-59
Modification with Guanidinoacetic Acid
[0113] Guanidinoacetic acid (6.0 grams) was dissolved in 1 N
aqueous hydrochloric acid (51.4 mL).
2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (14 grams) was
dissolved in an ethanol (25 grams)/methanol (11 grams) mixture. The
two solutions were then mixed, and allowed to react for 10 minutes.
A portion of this mixture (15.3 grains) was added to a PEI solution
(16.67 grams of a 30% solids 70,000 MW PEI solution in water). This
mixture was allowed to react for 6 hours to acylate 6.3% of the
amine groups of the PEI (Example 57). By similar procedures,
modified polymers having 12.5% and 20% of the amine groups acylated
were prepared (Examples 58 and 59, respectively).
[0114] A portion of the polymer solution of Example 59 was diluted
to 1% by weight in deionized water, pH 7, and evaluated in the BSA
precipitation test, providing excellent flocculation at all salt
concentrations.
Example 60
Using standard microbiological procedures, cultures of the
following were prepared:
[0115] a) Escherichia coli (cells and cell debris)
[0116] b) Chinese hamster ovary (CHO) cells
[0117] c) Baker s yeast
[0118] When flocculation experiments were conducted similarly to
those described in Example 35 on these mixtures, the ligand
functional polymers of the invention consistently displayed good
flocculating ability in the presence of sodium chloride
concentrations in excess of 50 mM.
* * * * *