U.S. patent application number 13/258018 was filed with the patent office on 2012-02-16 for hydrophobic monomers, hydrophobically-derivatized supports, and methods of making and using the same.
Invention is credited to James I. Hembre, Cary A. Kipke, Jerald K. Rasmussen, Peter D. Wickert.
Application Number | 20120039920 13/258018 |
Document ID | / |
Family ID | 42936786 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120039920 |
Kind Code |
A1 |
Rasmussen; Jerald K. ; et
al. |
February 16, 2012 |
HYDROPHOBIC MONOMERS, HYDROPHOBICALLY-DERIVATIZED SUPPORTS, AND
METHODS OF MAKING AND USING THE SAME
Abstract
A composition is disclosed comprising a hydrophobic monomer
having the structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O)-
XR.sup.3 wherein n is an integer of 0 or 1; R.sup.1 is
independently selected from at least one of: a hydrogen atom,
alkyls aryls, and alkylaryls, wherein the alkyls, aryls, and
alkylaryls have a total of 10 carbon atoms or less; R.sup.3 is a
hydrophobic group selected from at least one of: alkyls, aryls,
alkylaryls and ethers, wherein the alkyls, aryls, alkylaryls and
ethers have a total number of carbon atoms ranging from 4 to 30;
R.sup.4 H or CH.sub.3; X is O or NH. In some embodiments the
hydrophobic monomer is derived from an amine or an alcohol
(HXR.sup.3) that has a hydrophilicity index of 25 or less. A
polymerizable composition comprising the hydrophobic monomer is
disclosed, which optionally may comprise a cross-linking monomer
and/or a non-cross-linking monomer. This polymerizable mixture may
be used to from hydrophobically-derivatized supports, which may be
used in applications such as hydrophobic interaction
chromatography.
Inventors: |
Rasmussen; Jerald K.;
(Woodville, WI) ; Kipke; Cary A.; (Woodbury,
MN) ; Hembre; James I.; (Plymouth, MN) ;
Wickert; Peter D.; (St. Paul, MN) |
Family ID: |
42936786 |
Appl. No.: |
13/258018 |
Filed: |
March 19, 2010 |
PCT Filed: |
March 19, 2010 |
PCT NO: |
PCT/US2010/027978 |
371 Date: |
September 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61165132 |
Mar 31, 2009 |
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Current U.S.
Class: |
424/184.1 ;
427/385.5; 435/183; 521/146; 521/149; 526/304; 526/305; 526/306;
530/344; 530/387.1; 530/417; 536/25.4; 564/155; 564/158;
564/159 |
Current CPC
Class: |
B01D 67/0088 20130101;
B01D 71/56 20130101; B01J 20/285 20130101; B01D 2323/30 20130101;
B01J 20/286 20130101; B01D 67/0093 20130101; B01D 15/206 20130101;
A61P 37/04 20180101; C08F 20/54 20130101; C07C 237/22 20130101;
B01D 67/0006 20130101; B01D 69/125 20130101; B01D 15/327 20130101;
B01D 2325/38 20130101 |
Class at
Publication: |
424/184.1 ;
526/304; 526/305; 526/306; 521/146; 521/149; 427/385.5; 564/155;
564/158; 564/159; 530/417; 435/183; 530/344; 530/387.1;
536/25.4 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C08J 9/00 20060101 C08J009/00; B05D 5/00 20060101
B05D005/00; B05D 7/24 20060101 B05D007/24; A61P 37/04 20060101
A61P037/04; C07C 237/22 20060101 C07C237/22; C07K 1/20 20060101
C07K001/20; C12N 9/00 20060101 C12N009/00; C07H 1/06 20060101
C07H001/06; C08F 22/38 20060101 C08F022/38; B05D 3/00 20060101
B05D003/00 |
Claims
1. A composition comprising a hydrophobic monomer having the
structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O)-
XR.sup.3 wherein n is an integer of 0 or 1; R.sup.1 is
independently selected from at least one of: alkyls, aryls, and
alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total
of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected
from at least one of alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X
is NH; wherein the hydrophobic monomer is derived from an amine or
an alcohol (HXR.sup.3) that has a hydrophilicity index of 25 or
less.
2. The composition of claim 1 wherein R.sup.1 is independently
selected from at least one of: methyl, ethyl, phenyl, or
combinations thereof.
3. The composition of claim 1 wherein R.sup.3 is selected from at
least one of: benzyl, phenethyl, phenoxyethyl, phenylpropyl, butyl,
pentyl, hexyl, octyl, dodecyl, octadecyl, phenylbutyl, or
combinations thereof.
4. The composition of claim 1 wherein the hydrophobic monomer has a
structure selected from at least one of:
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.4C.sub.6H.-
sub.5;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NHCH.sub.2C.sub.6H.su-
b.5;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.2C.sub-
.6H.sub.5;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.-
2OC.sub.6H.sub.5;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.3C.sub.6H.-
sub.5;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.3CH.-
sub.3;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.5CH.-
sub.3;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.7CH.-
sub.3;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.11CH-
.sub.3;
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.17C-
H.sub.3; or combinations thereof.
5. A polymerizable mixture comprising the hydrophobic monomer of
claim 1.
6. A polymerizable mixture of claim 5 further comprising a
cross-linking monomer.
7. The polymerizable mixture of claim 6, wherein the cross-linking
monomer is selected from at least one of:
N,N'-alkylenebis(meth)acrylamides, alkylenebis(meth)acrylates,
divinylaromatics, polyallylesters, or combinations thereof.
8. The polymerizable mixture of claim 5, further comprising a
non-cross-linking monomer.
9. The polymerizable mixture of claim 8, wherein the
non-cross-linking monomer is selected from at least one of:
dimethylacrylamide, acrylamide, methacrylamide,
hydroxyethyl(meth)acrylate, or combinations thereof
10.-11. (canceled)
12. The polymerizable mixture of claim 5, further comprising a
porogen.
13. (canceled)
14. An article comprising a reaction product of the polymerizable
mixture according to claim 5.
15. The article of claim 14, wherein the article is a
hydrophobically-derivatized support.
16. The article of claim 14, wherein the hydrophobic monomer is
uniformly distributed throughout the article.
17. A method of using the article of claim 14, wherein the article
is used to purify at least one of: proteins, antibodies, fusion
proteins, vaccines, peptides, enzymes, DNA, RNA, or combinations
thereof.
18. A method of making a hydrophobically-derivatized support
comprising: (a) providing a mixture comprising: (ii) a hydrophobic
monomer having the structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O)-
XR.sup.3 wherein n is an integer of 0 or 1; R.sup.1 is
independently selected from at least one of: a hydrogen atom,
alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and
alkylaryls have a total of 10 carbon atoms or less; R.sup.3 is a
hydrophobic group selected from at least one of: alkyls, aryls,
alkylaryls and ethers, wherein the alkyls, aryls, alkylaryls and
ethers have a total number of carbon atoms ranging from 4 to 30;
R.sup.4 is H or CH.sub.3; and X is O or NH; (ii) a cross-linking
monomer; and (iii) optionally, a non-cross-linking monomer; and (b)
polymerizing the mixture; and (c) confining the polymerized mixture
to generate a chromatography medium or a filtration medium.
19.-20. (canceled)
21. The method of claim 18, wherein the polymerized mixture are
particles.
22. The method of claim 18, wherein the polymerized mixture is
coated, or grafted onto at least one of: a woven web, non-woven
web, microporous fibers, microporous membranes, film, or
combinations thereof.
23. A method of making a hydrophobically-derivatized support
comprising: (a) providing a hydrophobic monomer having the
structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O)-
XR.sup.3 wherein n is an integer of 0 or 1; R.sup.1 is
independently selected from at least one of: a hydrogen atom,
alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and
alkylaryls have a total of 10 carbon atoms or less; R.sup.3 is a
hydrophobic group selected from at least one of: alkyls, aryls,
alkylaryls and ethers, wherein the alkyls, aryls, alkylaryls and
ethers have a total number of carbon atoms ranging from 4 to 30;
R.sup.4 is H or CH.sub.3; X is O or NH; (b) providing a substrate;
(c) contacting the hydrophobic monomer to the substrate; and (d)
polymerizing the hydrophobic monomer.
24. The method of claim 23, wherein the polymerizing results in a
coating on the substrate.
25. The method of claim 23, wherein the polymerizing results in a
grafting from the substrate.
26. (canceled)
Description
TECHNICAL FIELD
[0001] The present disclosure generally relates to hydrophobic
monomers, and their use, for example, in
hydrophobically-derivatized supports. The present disclosure also
generally relates to the method of making and using these
hydrophobically-derivatized supports in applications such as
hydrophobic interaction chromatography.
BACKGROUND
[0002] Hydrophobic interaction chromatography (HIC) is a
chromatography technique based on the separation of molecules based
on their hydrophobicity. Generally, sample molecules in a high salt
buffer are loaded on the HIC column. The salt in the buffer
interacts with water molecules to reduce the solvation of the
sample molecules in solution, thereby exposing hydrophobic regions
in the sample molecules, which are consequently adsorbed by the
stationary phase of the HIC column. The more hydrophobic the
molecule, the less salt needed to promote binding. Usually, a
decreasing salt gradient is used to elute samples from the column
in order of increasing hydrophobicity. Sample elution may also be
achieved by the addition of mild organic modifiers (e.g., solvents)
or detergents to the elution buffer, by changing the pH, or by the
addition of chaotropic agents.
[0003] The HIC stationary phase typically comprises agarose,
silica, or organic polymer resins, which may be modified by
hydrophobic ligands. One such HIC stationary phase is prepared by
reacting a hydrophobic ligand comprising a nucleophile, to a
particle (e.g., a bead) comprising an azlactone moiety. For
example, U.S. Pat. No. 5,993,935 (Rasmussen et al.) describes the
covalent bonding of azlactone moieties on the surface of particles
with nucleophilic ligands by direct interaction (i.e., without the
need for an intermediate activation step).
[0004] U.S. Pat. No. 5,561,097 (Gleason, et al.) describes a method
of controlling the density of low molecular weight ligands, which
are covalently bonded to azlactone moieties on the surface of
supports (e.g., particles). The density is controlled by conducting
the covalent bonding reaction in the presence of a quencher. The
azlactone-functionalized support is typically prepared by
polymerization of an azlactone monomer or precursor to a support
with subsequent cyclization to the azlactone. The
azlactone-functionalized support is then reacted with a ligand
(such as benzyl amine) to produce a derivatized support. Although
extremely low levels of side reactions such as hydrolysis take
place during the course of the derivatization reaction, some
hydrolysis of the azlactone may indeed take place, generating
carboxylic acid groups. When the end product is an ion exchange
resin, this minor amount of side reaction is not a concern.
However, in some applications, such as HIC stationary phases, the
presence of any ionic functionality, even trace amounts, can lead
to changes in performance, for example, in dynamic binding capacity
and/or resolution.
[0005] HIC stationary phases are also susceptible to hydrolysis
when exposed to basic conditions if they are derived from
hydrophobic (meth)acrylate esters and/or (meth)acrylamide monomers.
For example, one molar sodium hydroxide is often used to clean
chromatography columns between uses, however these basic conditions
can hydrolize the (meth)acrylate ester and/or (meth)acrylamide
polymer. This hydrolysis leads to the formation of carboxylic acid
functionality on the support, and thus to a degradation in
chromatographic performance.
SUMMARY
[0006] In one aspect, the present disclosure provides a composition
comprising a hydrophobic monomer having the structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O-
)XR.sup.3
wherein n is an integer of 0 or 1; R.sup.1 is independently
selected from at least one of: a hydrogen atom, alkyls, aryls, and
alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total
of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected
from at least one of alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X
is O or NH; wherein the hydrophobic monomer is derived from an
amine or an alcohol (HXR.sup.3) that has a hydrophilicity index of
25 or less.
[0007] In another aspect, the present disclosure provides a
polymerizable mixture comprising a hydrophobic monomer having the
structure:
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O-
)XR.sup.3
wherein n is an integer of 0 or 1; R.sup.1 is independently
selected from at least one of: a hydrogen atom, alkyls, aryls, and
alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total
of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected
from at least one of: alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X
is O or NH.
[0008] In one embodiment, the polymerizable mixture further
comprises a cross-linking monomer and/or a non-cross-linking
monomer.
[0009] In yet another aspect, an article is provided comprising the
reaction product of a hydrophobic monomer and optionally a
cross-linking monomer and/or a non-cross-linking monomer.
[0010] In one embodiment, the article is a
hydrophobically-derivatized support.
[0011] In another aspect, a method of using the hydrophobic
interaction chromatography particle is provided to purify at least
one of proteins, antibodies, fusion proteins, vaccines, peptides,
enzymes, DNA, RNA, or combinations thereof.
[0012] In yet another aspect, a method of making a
hydrophobically-derivatized support is provided comprising: [0013]
(a) providing a mixture comprising: [0014] (ii) a hydrophobic
monomer having the structure:
[0014]
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).s-
ub.nC(O)XR.sup.3
wherein n is an integer of 0 or 1; R.sup.1 is independently
selected from at least one of: a hydrogen atom, alkyls, aryls, and
alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total
of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected
from at least one of: alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X
is O or NH; [0015] (ii) a cross-linking monomer; and [0016] (iii)
optionally, a non-cross-linking monomer; and [0017] (b)
polymerizing the mixture.
[0018] In another aspect, a method of making a
hydrophobically-derivatized support is provided comprising: [0019]
(a) providing a hydrophobic monomer having the structure:
[0019]
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).s-
ub.nC(O)XR.sup.3
wherein n is an integer of 0 or 1; R.sup.1 is independently
selected from at least one of: a hydrogen atom, alkyls, aryls, and
alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total
of 10 carbon atoms or less; R.sup.3 is a hydrophobic group selected
from at least one of: alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30; R.sup.4 is H or CH.sub.3; and X
is O or NH; [0020] (b) providing a substrate; and [0021] (c)
contacting the hydrophobic monomer to the substrate; and [0022] (d)
polymerizing the hydrophobic monomer.
[0023] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0024] The words "preferred" and "preferably" refer to embodiments
of the disclosure that may afford certain benefits, under certain
circumstances. However, other embodiments may also be preferred,
under the same or other circumstances. Furthermore, the recitation
of one or more preferred embodiments does not imply that other
embodiments are not useful, and is not intended to exclude other
embodiments from the scope of the disclosure.
[0025] The terms "a", "an", and "the" are used interchangeably with
"at least one" to mean one or more of the elements being
described.
[0026] The term "and/or" means one or all of the listed elements or
a combination of any two or more of the listed elements.
[0027] The term "alkyl" refers to a monovalent group that is a
radical of an alkane, which is a saturated hydrocarbon. The alkyl
can be linear, branched, cyclic, or combinations thereof and
typically has 1 to 30 carbon atoms. In some embodiments, the alkyl
group contains at least 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 25
carbon atoms; at most 30, 28, 26, 25, 20, 15, 10, 8, 6, 5, 4, or 3
carbon atoms. Examples of alkyl groups include, but are not limited
to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
tert-butyl, n-pentyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl, and
ethylhexyl.
[0028] The term "alkylene" refers to a divalent group that is a
radical of an alkane. The alkylene can be straight-chained,
branched, cyclic, or combinations thereof. The alkylene often has 1
to 30 carbon atoms. In some embodiments, the alkylene group
contains at least 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 25 carbon
atoms; at most 30, 28, 26, 25, 20, 15, 10, 8, 6, 5, 4, or 3 carbon
atoms. The radical centers of the alkylene can be on the same
carbon atom (i.e., an alkylidene) or on different carbon atoms.
[0029] The term "aryl" refers to a monovalent group that is
aromatic and carbocyclic or heterocyclic. The aryl can have one to
five rings that are connected to or fused to the aromatic ring. The
other ring structures can be aromatic, non-aromatic, or
combinations thereof and typically has 1 to 30 carbon atoms. In
some embodiments, the aryl group contains at least 1, 2, 3, 4, 5,
6, 8, 10, 15, 20, or 25 carbon atoms; at most 30, 28, 26, 25, 20,
15, 10, 8, 6, 5, 4, or 3 carbon atoms. Examples of aryl groups
include, but are not limited to, phenyl, biphenyl, terphenyl,
anthryl, naphthyl, acenaphthyl, anthraquinonyl, phenanthryl,
anthracenyl, pyrenyl, perylenyl, and fluorenyl.
[0030] The term "alkylaryl" refers to a monovalent group that is a
combination of an alkyl and an aryl group. The alkylaryl can be an
aralkyl, that is, an alkyl substituted with an aryl, or alkaryl,
that is, an aryl substituted with an alkyl. The alkylaryl can have
one to five rings that are connected to or fused to the aromatic
ring and can comprise linear, branched, or cyclic segments, or
combinations thereof. The alkylaryl group typically has 1 to 30
carbon atoms. In some embodiments, the alkylaryl group contains at
least 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 25 carbon atoms; at most
30, 28, 26, 25, 20, 15, 10, 8, 6, 5, 4, or 3 carbon atoms.
[0031] The term "(meth)acrylamide" refers to compounds containing
either an acrylamide or a methacrylamide structure or combinations
thereof. Similarly, the term "(meth)acrylate" refers to compounds
containing either an acrylate or a methacrylate structure or
combinations thereof.
[0032] The terms "polymer" and "polymeric material" refer to both
materials prepared from one monomer such as a homopolymer or to
materials prepared from two or more monomers such as a copolymer,
terpolymer, etc. Likewise, the term "polymerize" refers to the
process of making a polymeric material that can be a homopolymer,
copolymer, terpolymer, or the like. The terms "copolymer" and
"copolymeric material" refer to a polymeric material prepared from
at least two monomers and includes terpolymers, quadpolymers,
etc.
[0033] The terms "room temperature" and "ambient temperature" are
used interchangeably to mean temperatures in the range of
20.degree. C. to 25.degree. C.
[0034] The above summary of the present disclosure is not intended
to describe each disclosed embodiment or every implementation of
the present disclosure. The description that follows more
particularly exemplifies illustrative embodiments. In several
places throughout the application, guidance is provided through
lists of examples, which can be used in various combinations. In
each instance, the recited list serves only as a representative
group and should not be interpreted as an exclusive list.
BRIEF DESCRIPTION OF DRAWINGS
[0035] FIG. 1 depicts the hydrophobic interaction chromatograms
(absorbance versus elution volume) for Example 17 (Ex. 17) and
Example 18 (Ex. 18).
[0036] FIG. 2 depicts the dynamic binding capacity chromatograms
(absorbance versus elution volume) for Examples 19 (Ex. 19) and
Examples 20 (Ex. 20) and Comparative Example B (Comp. Ex. B) and
Comparative Example C (Comp. Ex. C).
[0037] FIG. 3 depicts hydrophobic interaction chromatograms
(absorbance versus elution volume) for Example 21 (Ex. 21) and
Example 22 (Ex. 22).
[0038] FIG. 4 depicts a selected peak in a hydrophobic interaction
chromatogram (absorbance versus elution volume) for Example 24 (Ex.
24) and Comparative Example D (Comp. Ex. D).
DETAILED DESCRIPTION
[0039] There is a need to synthesize hydrophobic free-radically
polymerizable (meth)acrylamide monomers that are easily synthesized
and isolated. Additionally, there is a need to manufacture a
polymer support that comprises hydrophobic character with reduced
ionic character. There is also a need to manufacture hydrophobic
supports, which, for example, can be used as an HIC stationary
phase, that have both high dynamic binding capacity and good
protein resolution characteristics.
[0040] This disclosure provides hydrophobic free-radically
polymerizable (meth)acrylamide monomers. In some embodiments, these
hydrophobic free radically polymerizable (meth)acrylamide monomers
can be polymerized with other monomers to produce hydrophobic
supports. In some embodiments, these hydrophobic supports may be
used to separate biological and non-biological samples.
[0041] The hydrophobic monomers according to this disclosure have
the structure according to formula (I):
CH.sub.2.dbd.CR.sup.4C(O)NHC(R.sup.1R.sup.1)(C(R.sup.1R.sup.1)).sub.nC(O-
)XR.sup.3 (I)
wherein n is an integer of 0 or 1; R.sup.1 is independently
selected from at least one of a hydrogen atom, alkyls, aryls, and
alkylaryls; R.sup.3 is a hydrophobic group selected from at least
one of: alkyls, aryls, alkylaryls and ethers; R.sup.4 is H or
CH.sub.3; and X is O or NH.
[0042] In one embodiment, the hydrophobic monomer is derived from
an amine or alcohol (HXR.sup.3) that has a hydrophilicity index of
25 or less.
[0043] In one embodiment, R.sup.1 is independently selected from at
least one of: hydrogen atoms, alkyls, aryls, and alkylaryls,
wherein the alkyls, aryls, and alkylaryls have a total of 10 carbon
atoms or less, 9 carbon atoms or less, 8 carbon atoms or less, 7
carbon atoms or less, 6 carbon atoms or less, 5 carbon atoms or
less, 4 carbon atoms or less, or even 3 carbon atoms or less.
Examples of R.sup.1 include: a hydrogen atom, a methyl group, an
ethyl group, and a phenyl group.
[0044] In one embodiment, R.sup.3 is a hydrophobic group selected
from at least one of: alkyls, aryls, alkylaryls and ethers, wherein
the alkyls, aryls, alkylaryls and ethers have a total number of
carbon atoms ranging from 4 to 30. In some embodiments, the alkyls,
aryls, alkylaryls and ethers contain at least 4, 5, 6, 8, 10, 12,
15, or 20 carbon atoms; at most 30, 28, 26, 24, 20, 15, 12, 10, 8,
or 6 carbon atoms. Examples of R.sup.3 include: a benzyl group, a
phenethyl group, a phenoxyethyl group, a phenylpropyl group, a
butyl group, a pentyl group, a hexyl group, an octyl group, a
dodecyl group, an octadecyl group, and a phenylbutyl group.
[0045] The hydrophobic monomers of this disclosure are synthesized
at room temperature by a nucleophilic reaction between an alkenyl
azlactone with a primary amine or alcohol ligand. The alkenyl
azlactone includes 5-member and 6-member azlactones with an alkenyl
substituent, such as those disclosed in formulas (II) and (III)
below, wherein R.sup.1 and R.sup.4 are the same as those defined
above.
##STR00001##
[0046] Exemplary alkenyl azlactones include:
4,4-dimethyl-2-vinyl-4H-oxazol-5-one (vinyldimethylazlactone),
2-isopropenyl-4H-oxazol-5-one,
2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,
4,4-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,
4,5-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one, and
combinations thereof.
[0047] During the synthesis of the hydrophobic monomer, the primary
amine or alcohol reacts with the carbonyl of the alkenyl azlactone,
opening the azlactone ring and forming an adduct. The reaction
solvent can be organic (such as alcohols, ethers, hydrocarbons,
esters, halogenated solvents, or combinations thereof), aqueous, or
mixed, but should be capable of dissolving or at least partially
dissolving the alkenyl azlactone and the primary amine or alcohol
ligand. Although the azlactone moiety is quite stable towards
hydrolysis, it is known that ring opening by water can occur as a
minor side-reaction. This hydrolysis can lead to the formation of
carboxyl groups, which may impart ionic character. Therefore, in
one embodiment, the covalent bonding of the alkenyl azlactone with
the primary amine or alcohol ligand is conducted in an organic
solvent to ensure little to no hydrolysis of the alkenyl
azlactone.
[0048] The azlactone moiety reacts rapidly with the primary amine
or alcohol of the ligand forming a direct covalent bond with no
displacement of a by-product molecule. Thus, purification of the
resulting hydrophobic monomer is minimized. Typically, the
hydrophobic monomer precipitates from the reaction solvent in very
pure form (for example, greater than 90% purity, or even greater
than 99% purity) and can be isolated by simple filtration and
drying. Optionally, the hydrophobic monomer can be recrystallized
to further enhance its purity, although this is generally not
necessary.
[0049] For purposes of this disclosure, the selection of the
primary amine or alcohol ligand utilized in the synthesis of the
hydrophobic monomer will determine the hydrophobicity of the
resulting hydrophobic monomer. The primary amine or alcohol ligand
comprises a hydrophobic group selected from at least one of:
alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls,
alkylaryls and ethers have a total number of carbon atoms ranging
from 4 to 30. In some embodiments, the alkyls, aryls, alkylaryls
and ethers contain at least 4, 5, 6, 8, 10, 12, 15, or 20 carbon
atoms; at most 30, 28, 26, 24, 20, 15, 12, 10, 8, or 6 carbon
atoms.
[0050] In one embodiment, the hydrophobic monomer has a calculated
hydrophilicity index (HI) of 25 or less, 20 or less, 15 or less, or
even 10 or less. The HI is an empirical concept that is described
in detail in U.S. Pat. No. 4,451,619 (Heilmann, et al.), herein
incorporated by reference. In general, this concept allows one to
determine the effect that an added primary amine or alcohol ligand
will have on the hydrophilicity or hydrophobicity of the final
product, (i.e., a hydrophobic monomer, a polymerizable mixture, or
a hydrophobically-derivatized support). For purposes of this
disclosure, HI is calculated based on the primary amine or alcohol
ligand of R.sup.3 (i.e., HXR.sup.3). The HI of the hydrophobic
monomer according to this disclosure is defined as:
HI=total molecular weight of the hydrophilic groups in
HXR.sup.3.times.100 molecular weight of HXR.sup.3
[0051] The hydrophilic groups are generally those that are
functionally capable of forming hydrogen bonds with water. Examples
of hydrophilic groups include: --N--, --NH--, --NH.sub.2, --OH,
--O--, C.dbd.O, --CO.sub.2H, --CO.sub.2.sup.-M.sup.+ (where M.sup.+
is an alkali or alkaline earth metal ion), --SO.sub.3H,
--SO.sub.3.sup.-M.sup.+, --CONH2, --SH, --NR.sub.3.sup.+X.sup.-
(where R.dbd.C.sub.1-4 alkyl and X.sup.- is typically a halide),
--NHCONH--, and the like.
[0052] Primary amine or alcohol ligands that tend to impart a
hydrophilic character to final product typically have an HI of
greater than 30, while the primary amine or alcohol ligands that
impart a hydrophobic character typically have an HI of less than
20. Primary amine or alcohol ligands with an HI between 20 and 30
are typically classified as "neutral" or "borderline".
[0053] Table 1 lists the HI of a number of primary amine and
alcohol ligands that have been found to be useful for the purposes
of this disclosure. Interestingly, some ligands having a
"borderline" HI (for example, butylamine and phenoxyethylamine) can
be used in applications such as protein purification.
TABLE-US-00001 TABLE 1 Hydrophilicity Index for Primary Amine
Ligands Total Hydrophilic Molecular Component Ligands weight MW HI
Benzylamine 107 16 15 Phenethylamine 121 16 13 Phenoxyethylamine
137 32 23 Phenylpropylamine 135 16 12 Phenylbutylamine 149 16 11
Butylamine 73 16 22 Hexylamine 101 16 16 Octylamine 129 16 12
Octadecylamine 270 16 6 Phenylbutanol 150 17 11
[0054] In some applications, hydrophobic monomers derived from
primary amine ligands are preferred over those derived from
alcohols due to the presence of two amide functional groups, which
result in a lower susceptibility towards hydrolysis.
[0055] Exemplary hydrophobic monomers include: [0056]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.4C.sub.6H.-
sub.5; [0057]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)O(CH.sub.2).sub.4C.sub.6H.s-
ub.5; [0058]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NHCH.sub.2C.sub.6H.sub.5;
[0059]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.2C.-
sub.6H.sub.5; [0060]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.2OC.sub.6H-
.sub.5; [0061]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.3C.sub.6H.-
sub.5; [0062]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.3CH.sub.3;
[0063]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.5CH-
.sub.3; [0064]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.7CH.sub.3;
[0065]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.11C-
H.sub.3; [0066]
CH.sub.2.dbd.CHC(O)NHC(CH.sub.3)(CH.sub.3)C(O)NH(CH.sub.2).sub.17CH.sub.3-
; or combinations thereof.
[0067] The hydrophobic monomers of this disclosure may have a
tendency in solution to self-associate or, when in the presence of
other monomers or polymers, associate with the other monomers or
polymers. This association can be the result of two separate
interactions. First, the hydrophobic groups of the hydrophobic
monomers may associate with one another, or with the other monomers
or polymers, especially in aqueous media. Secondly,
hydrogen-bonding interactions can occur between the amide
functionalities of the hydrophobic monomers or between the
hydrophobic monomers and the other monomers or polymers. The
hydrogen-bonding interactions are particularly prevalent with the
hydrophobic monomers derived from amine ligands, wherein two amide
groups are present. The tendency of the hydrophobic monomers to
associate may be advantageous, for example, enabling one to control
a polymer microstructure (such as, for example, the distribution of
hydrophobic groups within a polymerized support) and/or the
properties of the final product (such as, for example,
hydrophobicity and/or viscosity) by manipulating the polymerization
conditions.
[0068] In addition to the hydrophobic portion, which can
participate in hydrophobic interactions, the hydrophobic monomer
also comprises an unsaturated site (e.g., a double bond), which is
derived from the alkenyl substituent of the alkenyl azlactone. In
one aspect of this disclosure, this site of unsaturation enables
the hydrophobic monomer to participate in free radical
polymerization schemes. Thus, these hydrophobic monomers may be
added to a polymerizable mixture, which then may be used to make a
hydrophobically-derivatized support.
[0069] In one aspect, the polymerizable mixture comprises the
hydrophobic monomer.
[0070] In one embodiment, the polymerizable mixture further
comprises a cross-linking monomer. The cross-linking monomer
comprises a plurality of polymerizable groups, which during
polymerization, extend the chain length of the polymer backbone and
during curing, physically join (or cross-link) the polymer
backbones. Cross-linking aids in the mechanical stability of the
resulting article.
[0071] The cross-linking monomers include, for example,
N,N'-alkylenebis(meth)acrylamides, alkylenebis(meth)acrylates,
divinylaromatics, polyallylesters or combinations thereof.
Exemplary cross-linking monomers include: ethylenically unsaturated
esters such as ethylene diacrylate, ethylene dimethacrylate,
trimethylolpropane triacrylate and trimethacrylate; and .alpha.-
and .beta.-unsaturated amides, such as methylene bis(acrylamide),
methylene bis(methacrylamide), N,N'-diacryloylpiperazine,
N,N'-diacryloyl-1,2-diaminoethane, and
N,N'-dimethacryloyl-1,2-diaminoethane; or combinations thereof. In
some applications, such as HIC-type applications, the
N,N'-alkylenebis(meth)acrylamides are preferred due to their
hydrophilicity and increased hydrolytic stability.
[0072] In one embodiment, the polymerizable mixture further
comprises a non-cross-linking monomer. The non-cross-linking
monomer is used to propagate the polymer backbone (i.e., extend the
chain length), but does not generally participate in physically
joining polymer backbones or may be used to solubilize the
hydrophobic monomer. In one embodiment, the non-cross-linking
monomers are uniformly distributed throughout the
hydrophobically-derivatized support and assist in uniformly
distributing the hydrophobic monomers in the
hydrophobically-derivatized support.
[0073] The non-cross-linking monomers include: (meth)acrylate,
(meth)acrylamide monomers, or combinations thereof. Exemplary
non-cross-linking monomers include: dimethylacrylamide, acrylamide,
methacrylamide, hydroxyethyl(meth)acrylate, or combinations
thereof. The use of these non-cross-linking monomers can provide
significant enhancements to the properties of the
hydrophobically-derivatized support. For example, although not
wanting to be bound by theory, the concentration and type of
non-cross-linking monomers are thought to influence the porosity of
the hydrophobically-derivatized support and/or the distribution of
the hydrophobic monomer.
[0074] In one embodiment, the hydrophobic monomers of formula (I)
and non-crosslinking monomers may be used in the preparation of
hydrophobically-associating polymers, which are useful as aqueous
fluid rheology or flow modifiers. In one embodiment, these
hydrophobically-associating polymers may be used, for example, as
flocculation aids for waste water treatment and dewatering sludge,
and for rheology control for secondary and tertiary oil recovery.
In another embodiment, these hydrophobically-associating polymers
may also be used as separation media for capillary electrophoresis
in DNA or RNA sequencing and separations.
[0075] The amount of hydrophobic monomer, cross-linking monomer,
and/or non-cross-linking monomer may be important in the properties
of the polymerized mixture and the resulting
hydrophobically-derivatized support. Generally, the amount of
hydrophobic monomer added controls the hydrophobicity of the
resulting hydrophobically-derivatized support. The hydrophobic
monomer can be added at 0.1 to 30% by weight relative to the total
monomer amount. In some embodiments, the hydrophobic monomer is at
least 0.1, 0.2, 0.5, 1, 1.5, 3, 5, 10, 15, 20, or 25% by weight; at
most 30, 25, 20, 15, 10, 5, 3, 1.5, 1, 0.5% by weight relative to
the total monomer amount. Generally, the amount of cross-linking
monomer added controls the rigidity and swelling ability of the
particle. The cross-linking monomer can be added at 0-99.9% by
weight relative to the total monomer amount. In some embodiments,
the cross-linking monomer is at least 0, 0.1, 0.5, 1, 1.5, 3, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 98% by weight; at most
99.9, 99.5, 99, 98, 95, 90, 80, 75, 70, 60, 50, 40, 30, 20, 10, 5,
3, 1, or 0.5% by weight relative to the total monomer amount.
Generally, the amount of non-cross-linking monomer aids in the
determination of final copolymer properties, including
hydrophilicity, solubility, porosity, crosslink density, rigidity,
etc., depending upon the final application. The non-cross-linking
monomer may be added at 0-99.9% by weight relative to total monomer
amount. In some embodiments, the non-cross-linking monomer is at
least 0, 0.1, 0.5, 1, 1.5, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80,
90, 95, or 98% by weight; at most 99.9, 99.5, 99, 98, 95, 90, 80,
75, 70, 60, 50, 40, 30, 20, 10, 5, 3, 1, or 0.5% by weight relative
to the total monomer amount.
[0076] In one embodiment, the amount of the cross-linking monomer
is greater than the amount of the non-cross-linking monomer. In one
embodiment, the amount of the non-cross-linking monomer is greater
than the amount of the hydrophobic monomer. In yet another
embodiment, the amount of the cross-linking monomer is greater than
the amount of the non-cross-linking monomer, which is greater than
the amount of the hydrophobic monomer. For example, in an HIC
application, the amount of cross-linking monomer is 60% or more by
weight relative to the total monomer amount to enable rigidity of
the hydrophobically-derivatized support to withstand the pressure
tolerances, and the amount of hydrophobic monomer is 10% or less by
weight relative to the total monomer amount to ensure release of
the analyte from the HIC stationary phase.
[0077] In another embodiment, the polymerizable mixture may
comprise a porogen. A porogen may be added to the polymerizable
mixture to control the pore structure of the
hydrophobically-derviatized support, especially when the
hydrophobically-derviatized support is a particle or a coating.
[0078] Pore formation or porosity in polymeric materials is
described in detail by Sherrington, Chem. Commun., 2275-2286
(1998). With some materials, especially gel-type materials,
porosity is formed during the polymerization or curing process as a
result of the entanglement and/or crosslinking of the polymer
chains. Typically this porosity is very low or nonexistent unless
the polymer network is highly swollen by a solvent. Alternately,
porogens can be added to the composition to create permanent pores.
Added porogens typically influence the timing of phase separation
of the forming polymer network from the rest of the monomer phase
mixture. Examples of porogens include: water, alcohols (such as,
for example, methanol, ethanol, and isopropanol), ethylene glycol,
propylene glycol, polyols having at least three hydroxy groups
(such as, for example, glycerol, inositol, glucose, sucrose,
maltose, dextran, pentaerithritol, trimethylolethane,
timethylolpropane, dip entaerithritol, and tripentaerithritol), and
polymeric porogens (such as, for example, polyethylene glycol,
polypropylene glycol, polyacrylic acid, polysaccharide, and the
like), dispersed organic aggregate (such as, for example,
ethoxylated hydrocarbons), or combinations thereof.
[0079] Other factors may also be important in controlling the pore
structure of the hydrophobically-derivatized support including, for
example, the interaction between the co-monomer composition and the
selection of the porogen(s), the mass ratio between the
non-cross-linking monomer and the cross-linking monomer, the
chemical structure of the non-cross-linking monomer, or
combinations thereof.
[0080] As mentioned above, the polymerizable mixture may be used to
form a hydrophobically-derivatized support. This
hydrophobically-derivatized support may be obtained by at least one
of: graft polymerizing the polymerizable mixture onto a substrate,
coating the polymerizable mixture onto a substrate and polymerizing
the polymerizable mixture on the surfaces of the substrate, or
polymerizing and cross-linking the polymerizable mixture to form
particles, which may be used as supports themselves or may added to
other porous substrates.
[0081] The degree of hydrophobicity of the
hydrophobically-derivatized support is controlled by the nature of
the hydrophobic ligand, the amount of hydrophobic ligand present on
the support surface, and/or the distribution of the hydrophobic
groups on the support surface (which in the case of
hydrophobically-derivatized particles, is controlled primarily by
pore structure and swell volume of the particle).
[0082] In one embodiment, the surface of a pre-existing support is
exposed to high energy radiation to generate free radical reaction
sites on the surface as disclosed in U.S. Pat. No. 5,344,701
(Gagnon et al.). Exposure of the pre-existing support with the
polymerizable mixture can take place simultaneously with or
subsequent to the irradiation of the pre-existing support.
Depending on the type of radiation and other process conditions,
the polymerizable mixture can either be grafted to the surface of
the pre-existing support or can be formed as a coating on the
pre-existing support or can become particles enmeshed within void
spaces of the support. In the former instance, the hydrophobic
monomer is covalently bound to the pre-existing support. The
pre-existing support may be treated with plasma, corona, beta,
gamma, electron-beam, x-ray, ultraviolet, and other electromagnetic
radiation as is known in the art. The radiation may occur in the
presence of other compounds, such as, for example, oxygen, or
photoinitiators. The pre-existing supports, depending on the final
use, may be porous or non-porous, continuous or non-continuous, and
flexible or inflexible. Examples of pre-existing supports may
include: woven webs, nonwoven webs, fibrous webs, microporous
membranes, fibers, hollow fibers, tubes, microporous films,
nonporous films, or combinations thereof. The pre-existing supports
may be made from a variety of materials including ceramics, glass,
metallic, polymeric materials, or combinations thereof. Examples of
suitable polymeric materials include: polyalkylenes such as
polyethylene and polypropylene; halogenated polymers such as
polyvinyl chloride and polyvinylidene fluoride; polyamides such as
nylons; polystyrenes; poly(ethylene vinyl acetate); polyacrylates
such as polymethyl methacrylate; polycarbonate; cellulosics such as
cellulose acetate butyrate; polyesters such as poly(ethylene
terphthalate); poly imidines; polyurethanes; or combinations
thereof.
[0083] In another embodiment, the polymerizable mixture is coated
onto the surface of a pre-existing support. Typically, the alkenyl
moiety of the hydrophobic monomer is not covalently bound to the
surface of the pre-existing support, therefore, the polymerizable
mixture also comprises a cross-linking monomer and optionally a
non-cross-linking monomer to cross-link the polymerizable mixture
onto the pre-existing support. The pre-existing supports are
similar to those described above. The polymerization and resultant
cross-linking may be initiated by chemical and/or physical means
including, for example, redox chemistry, thermal initiation, UV
irradiation or by ionizing radiation (such as, for example, e-beam
and gamma radiation), or by other means as is well known in the
art.
[0084] In one embodiment, the hydrophobic monomer is polymerized to
produce hydrophobically-derivatized particles. The hydrophobic
monomer, a cross-linking monomer, and optionally a
non-cross-linking monomer are mixed together and polymerized as an
inverse suspension. As is apparent to one skilled in the art, the
initiation system, suspending medium, stirring rate and the
suspending agent are all essentially independent and important
variables in the polymerization process. In one embodiment, the
monomers are dissolved in a water/alcohol solution, this solution
is suspended as droplets in an organic, immiscible medium, and
sodium persulfate and tetramethylethylenediamine are used to
initiate the polymerization. Substitution of the various components
by comparable materials can certainly be made, and such
substitutions would not be outside the spirit and scope of the
present disclosure.
[0085] The hydrophobically-derivatized particles of this disclosure
can have a spherical shape, a regular shape, or an irregular shape.
Size of the azlactone-derived functionalized particles can vary
widely within the scope of the disclosure. Generally the size of
the azlactone-derived functionalized particles ranges from 0.1
micrometer (.mu.m) to 5 millimeters (mm) in average diameter.
[0086] In one embodiment, the hydrophobically-derivatized particles
are confined. For example, the hydrophobically-derivatized
particles can be placed in a vessel (such as a tube), enclosing at
least one end of the vessel with a frit to create a chromatographic
column. Suitable columns are known in the art and can be
constructed of such materials as glass, polymeric material,
stainless steel, titanium and alloys thereof, or nickel and alloys
thereof. Methods of filling the column to effectively pack
particles in the column are known in the art. The chromatographic
column, when packed with the hydrophobically-derivatized particles,
can be used in HIC applications.
[0087] Although the average particle size in chromatography can be
as large as 2000 micrometers, the average particle size is
typically no greater than 500 micrometers. If the average particle
size is larger than about 500 micrometers, the efficiency of the
chromatographic process may be low, especially for the purification
or separation of large biomacromolecules such as proteins that
often have low diffusion rates into the pores of chromatographic
particles.
[0088] In another embodiment, the hydrophobically-derivatized
particles are dispersed within a continuous, porous matrix. The
continuous, porous matrix is typically at least one of a woven or
non-woven fibrous web, porous fiber, porous membrane, porous film,
hollow fiber, film, or tube. Suitable continuous, porous matrixes
are further described in U.S. Pat. No. 5,993,935 (Rasmussen et
al.).
[0089] In yet another embodiment, the hydrophobically-derivatized
particles are disposed on a surface of a filtration medium. The
filter element can be positioned within a housing to provide a
filter cartridge. Suitable filtration medium and systems that
include a filter cartridge are further described, for example, in
U.S. Pat. No. 5,468,847 (Heilmann et al.). Such a filter cartridge
can be used, for example, to purify or separate biomolecules.
Typically, less rigid particles or smaller porous particles can be
utilized within a filter cartridge compared to within a
chromatographic column due to the lower pressure drops inherent in
the filter cartridge system.
[0090] In one aspect of the present disclosure, the
hydrophobically-derivatized supports have a reduced amount of ionic
groups at the surface of the hydrophobically-derivatized support.
Although, not wanting to be bound by theory, it is believed that
the hydrophobically-derivatized supports are able to be prepared
with even less ionic functionality than the methods currently known
in the art. Less ionic functionality on the support surface is
thought to be a result of the hydrophobic monomers being prepared
and purified prior to any contact with water (i.e., conducting the
synthesis of the hydrophobic monomer in organic solvent with no
hydrolysis), and conducting the polymerization reactions with
monomers that are resistant to hydrolysis.
[0091] Azlactones are known to be susceptible to attack by water,
which can lead to the formation of carboxyl groups, which may
change the selectivity of the surface, imparting both ion-exchange
and hydrophilic character to the support's surface. In the present
disclosure, the azlactone ring may be opened and covalently bonded
to the hydrophobic ligands under non-aqueous conditions, which may
limit the generation of ionic groups at the surface of the support.
By having fewer competing side reactions, a more pure hydrophobic
support can be generated (i.e., less ion-exchange character
exhibited by the hydrophobically-derivatized support). Thus, the
hydrophobically-derivatized supports of the present disclosure can
be more sensitive in hydrophobic interactions and not be influenced
by other functional group interactions (e.g. ionic). This provides
a more specific separation that is based only on hydrophobic
interactions.
[0092] Further, the hydrophobically-derivatized supports of the
present disclosure display hydrophobicities that are comparable to
prior art materials, but comprise much lower hydrophobic ligand
densities. For the purposes of this discussion, "ligand density"
means micromoles of ligand per milliliter of packed support
material. While not wanting to be bound by theory, it is believed
that the hydrophobically-derivatized supports of the present
disclosure display a more random and even distribution of the
hydrophobic ligands on the surface of the support, thus leading to
a more efficient utilization of the hydrophobic ligand for
interaction with the analyte (e.g., protein) of interest.
[0093] In one embodiment, neither the polymerizable mixture nor the
hydrophobically-derivatized support comprises a quencher. Because
the hydrophobic monomer comprises the hydrophobic ligand covalently
bonded to the azlactone, a quencher, such as described in U.S. Pat.
No. 5,561,097 (Gleason, et al.) is not needed when forming the
hydrophobically-derivatized supports.
[0094] Due to the hydrophobic nature of the
hydrophobically-derivatized supports, the
hydrophobically-derivatized supports may be used for the
purification of biological materials, for example, proteins,
antibodies, fusion proteins, vaccines, peptides, enzymes, DNA, RNA,
or combinations thereof, as well as non-biological molecules with
hydrophobic characteristics, in applications such as HIC.
[0095] The hydrophobically-derivatized supports of the present
disclosure may have advantages over prior art HIC supports. In the
present disclosure, an alkenyl azlactone is reacted with a
hydrophobic ligand comprising a nucleophile, for example an amine,
to form the hydrophobic monomer (or adduct). By using the
hydrophobic ligand as part of the hydrophobic monomer in the
polymerization step, instead of adding the hydrophobic ligand to a
support already comprising an attached azlactone group (such as
disclosed in U.S. Pat. Nos. 5,993,935 and 5,561,097), the
hydrophobically-derivatized supports of the present disclosure may
be achieved with improved uniformity in the distribution of the
hydrophobic ligand over the surface of the support and with fewer
ionic sites present at the surface of the support. These
characteristics may be critical in some HIC applications. The
hydrophobically-derivatized supports of the present disclosure may
be more resistant to hydrolysis than HIC supports prepared by
polymerizing hydrophobic (meth)acrylate esters and/or (meth)
acrylamide monomers due to the steric hindrance provided by the
--C(R.sup.1R.sup.1) group interposed between the two carbonyl
groups.
EXAMPLES
[0096] The following examples are merely for illustrative purposes
and are not meant to limit in any way the scope of the appended
claims. All parts, percentages, ratios, and the like in the
examples are by weight, unless noted otherwise. All raw materials
are commercially available or known to those skilled in the art
unless otherwise stated or apparent. The structures of all novel
hydrophobic monomers were confirmed by H.sup.1 nuclear magnetic
resonance and infrared spectroscopy.
Materials
TABLE-US-00002 [0097] Table of Materials Name Description Vinyl
dimethylaz- Purchased from SNPE, Inc., Princeton, N.J. lactone
N-acryloylmethyl- 3M, prepared by the procedure described in U.S.
alanine Pat. No. 4,304,705 (Heilmann, et al.) PEG 2,000 A
polyethylene glycol having a molecular weight of 1800-2200 g/mole
commercially available from Merck Schuchadt OHG, Hohenbrunn,
Germany. PEG 6000 A polyethylene glycol having a molecular weight
of 5000-7000 g/mole commercially available from Merck Schuchadt
OHG, Hohenbrunn, Germany. PEG 10,000 A polyethylene glycol having a
molecular weight of 9000-11250 g/mole commercially available from
Merck Schuchadt OHG, Hohenbrunn, Germany.
Preparation of Hyrdophobic Monomers
Example 1
[0098] The following procedure was used to prepare a
4-phenylbutylamine/VDM adduct. Methyl-tert-butyl ether (MTBE, 100
ml (milliliter)) was added to a 1,000 ml, 3-necked flask, equipped
with a condenser, overhead mixing paddle at 400 rotations per
minute (rpm), and nitrogen inlet, in an ice bath.
Vinyldimethylazlactone (VDM, 10.44 g (gram)) was added to the
flask. 4-Phenylbutylamine (10 g) was added to an addition funnel.
The bottle in which the 4-phenylbutylamine was stored before
addition to the addition funnel was rinsed with portions of MTBE
(10 ml total). The MTBE used to rinse the bottle was added to the
addition funnel. The 4-phenylbutylamine was added drop-wise over
10-minutes to the flask that contained the VDM. After addition of
the 4-phenylbutylamine to the VDM was completed, the addition
funnel was rinsed with 10 ml of MTBE. A white precipitate (product)
formed almost immediately. The reaction was then allowed to proceed
with mixing, under nitrogen, at 0.degree. C. for 60 minutes.
[0099] The flask and at least 300 ml of MTBE were transferred to a
freezer set at about -20.degree. C. Crystallization of the white
precipitate (product) was allowed to occur for 1 to 2 hours. The
solid product was filtered on a fitted funnel with three 100 ml
washes of the chilled MTBE. The product was dried overnight in a
vacuum oven at 60.degree. C. and about 25 inches Hg vacuum. The %
yield was about 90%. The purity was checked by silica thin layer
chromatography (TLC) with MTBE as the mobile phase. Both of the
reactants were much more soluble in MTBE than the product. The melt
point of the product, or adduct, was 89-91.degree. C.
Example 2
[0100] The following procedure was used to prepare a
benzylamine/VDM adduct. A similar procedure as that described for
Example 1 above was followed except 49.3 g of benzylamine (instead
of 4-phenylbutylamine), 69.9 g of VDM, and 393 ml of diethyl ether
(instead of MTBE) were used. The % yield was about 84%.
Example 3
[0101] The following procedure was used to prepare a
phenethylamine/VDM adduct. A similar procedure as that described
for Example 1 above was followed except 43.8 g of phenethylamine
(instead of 4-phenylbutylamine), 36.3 g of VDM, and 300 ml of MTBE
were used. The % yield was about 94%.
Example 4
[0102] The following procedure was used to prepare a
phenoxyethylamine/VDM adduct. A similar procedure as that described
for Example 1 above was followed except 10 g of phenoxyethylamine
(instead of 4-phenylbutylamine), 10.6 g of VDM, and 150 ml of MTBE
were used. The % yield was about 92%.
Example 5
[0103] The following procedure was used to prepare a
3-phenylpropylamine/VDM adduct. A similar procedure as that
described for Example 1 above was followed except 24.7 g of
3-phenylpropylamine (instead of 4-phenylbutylamine), 24.4 g of VDM,
and 150 ml of MTBE were used. The % yield was about 77%.
Example 6
[0104] The following procedure was used to prepare a butylamine/VDM
adduct. A similar procedure as that described for Example 1 above
was followed except 43.9 g of butylamine (instead of
4-phenylbutylamine), 83.4 g of VDM, and 500 ml of diethylether
(instead of MTBE) were used. The % yield was greater than 85%.
Example 7
[0105] The following procedure was used to prepare an
octylamine/VDM adduct. A similar procedure as that described for
Example 1 above was followed except 77.6 g of octylamine (instead
of 4-phenylbutylamine), 83.4 g of VDM, and 650 ml of diethylether
(instead of MTBE) were used. The % yield was greater than 85%.
Example 8
[0106] The following procedure was used to prepare a
dodecylamine/VDM adduct. A similar procedure as that described for
Example 1 above was followed except 26.9 g of dodecylamine (instead
of 4-phenylbutylamine), 13.9 g of VDM, and 250 ml of diethylether
(instead of MTBE) were used. The % yield was greater than 85%.
Example 9
[0107] The following procedure was used to prepare an
octadecylamine/VDM adduct. A similar procedure as that described
for Example 1 above was followed except 26.95 g of octadecylamine
(instead of 4-phenylbutylamine), 13.9 g of VDM, and 250 ml of
diethylether (instead of MTBE) were used. The product was isolated
by evaporating the diethylether solvent, and the % yield was
greater than 95%.
Example 10
[0108] The following procedure was used to prepare a hexylamine/VDM
adduct. A similar procedure as that described for Example 1 above
was followed except 60.7 g of hexylamine (instead of
4-phenylbutylamine), 83.4 g of VDM, and 675 of ml diethylether
(instead of MTBE) were used. The % yield was greater than 85%.
Example 11
[0109] The following procedure was used to prepare a
4-phenyl-1-butanol/VDM adduct. Heptane (50 ml) was added to a 250
ml round bottom flask, equipped with a condenser and magnetic stir
bar, in an ice bath. 4-Phenylbutanol (5.00 g) was added to the
flask, followed by 5 drops of diazabicycloundecene (DBU) as
catalyst. VDM (5.00 g) was added to an addition funnel along with
heptane (25 ml). The contents of the dropping funnel were added
dropwise over 5 minutes to the flask. After the addition was
completed, the mixture was stirred for 45 minutes, then the ice
bath was removed. A colorless oil (product) had formed. The
reaction was then allowed to proceed with mixing for an additional
2 hours. The heptane supernate was poured off, additional heptane
(50 ml) was added, the mixture was stirred another 15 minutes, then
left to stand with no stirring for 10 minutes. The heptane
supernate again was poured off, and the residual oil was stripped
on a rotary evaporator with heating at 35.degree. C. to give 9.28 g
of colorless oil (96.5% yield), which crystallized at room
temperature.
Preparation of Particles
Example 12
[0110] Heptane (174 ml) and 1.4 ml of polymer stabilizer solution
((0.1 g of a polymer comprising a ratio of 92.5 isooctylacrylate to
7.5 VDM, which has been ring opened with ammonia) per ml of
toluene) were added to a 1 L Mortonized round bottom flask equipped
with an overhead stirrer, thermocouple, reflux condenser, and
nitrogen gas inlet. The overhead stirrer was adjusted to a stir
rate of approximately 300 rpm and the reaction flask was heated to
35.degree. C. under a slow nitrogen gas purge. Methylene
bis-acrylamide (MBA, 11.31 g), 2.09 g of acrylamide (AAm), and 0.60
g of 4-phenylbutylamine/VDM adduct (PhBVDM, prepared according to
Example 1 above) were added to a 250 ml Erlenmeyer flask equipped
with a stir bar. Isopropyl alcohol (62.5 ml) and 42 ml of water
were added to dissolve the solids. 10 g of a 50% aqueous solution
of PEG 2,000 was then added. Upon dissolution of all solids, sodium
persulfate was added to the stirred solution (0.56 g in 3 ml
water). The aqueous phase was added to the organic phase and mixed
until the reaction mixture reached 35.degree. C.
Tetramethylethylenediamine (0.55 ml) was added to initiate the
polymerization. The polymerization reaction was stirred for 2 hours
while particles formed.
[0111] The particles were course filtered and washed twice with
acetone (250 ml each), twice with methanol (250 ml each), and then
twice with acetone (250 ml each). The resulting particles were
transferred to a 500 ml Erlenmeyer flask. Acetone (300 ml) was
added to suspend the particles. The suspended particles were
sonicated for approximately 15 minutes, then filtered. The
resulting particles were classified to a mean particle size of
approximately 60 .mu.m (micrometers) using a series of stacked
sieves.
Example 13
[0112] A similar procedure as that described in Example 12 was
followed except PEG 6,000 was added as a porogen additive instead
of PEG 2,000.
Example 14
[0113] Heptane (348 ml) and 2.8 ml of polymer stabilizer solution
were added to a 1 L Mortonized round bottom flask equipped with an
overhead stirrer, thermocouple, reflux condenser, and nitrogen gas
inlet. The overhead stirrer was adjusted to a stir rate of
approximately 300 rpm and the reaction flask was heated to
35.degree. C. under a slow nitrogen gas purge. MBA (21.36 g), 5.50
g of dimethylacrylamide (DMA), and 1.14 g of PhBVDM were added to a
250 ml Erlenmeyer flask equipped with a stir bar. 125 ml of
isopropyl alcohol and 84 ml of water were used to dissolve the
solids. 20 g of a 50% aqueous solution of PEG 6,000 was then added.
Upon dissolution of all solids, sodium persulfate was added to the
stirred solution (1.10 g in 6 ml water). The aqueous phase was
added to the organic phase and mixed until the reaction reached
35.degree. C. Tetramethylethylenediamine (1.10 ml) was added to
initiate the reaction. The polymerization reaction was stirred for
2 hours until particles were formed.
[0114] The particles were course filtered and washed twice with
acetone (250 ml each), twice with methanol (250 ml each), and twice
with acetone (250 ml each). The resulting particles were
transferred to a 500 ml Erlenmeyer flask. Acetone (300 ml) was
added to suspend the particles. The suspended particles were
sonicated for approximately 15 minutes, then filtered. The
resulting particles were classified to a mean particle size of
approximately 60 .mu.tm using a series of stacked sieves.
Example 15
[0115] A similar procedure as that described in Example 14 was
followed except PEG 10,000 was added as a porogen additive instead
of PEG 6,000.
Example 16
[0116] Heptane (348 ml), toluene (188 ml) and 1.4 ml of polymer
stabilizer solution were added to a 1L Mortonized round bottom
flask equipped with an overhead stirrer, thermocouple, reflux
condenser, and nitrogen gas inlet. The overhead stirrer was
adjusted to a stir rate of approximately 360 rpm and the reaction
flask was heated to 35.degree. C. under a slow nitrogen gas purge.
MBA (12.89 g) and 1.11 g of benzylamine/VDM adduct (prepared
according to Example 2 above) were added to a 250 ml Erlenmeyer
flask equipped with a stir bar. Isopropyl alcohol (65 ml) and 47 ml
of water were added to dissolve the solids. Ethylene glycol (25 ml)
was then added. Upon dissolution of all solids, sodium persulfate
(0.55 g in 3 ml water) was added to the stirred solution. The
aqueous phase was added to the organic phase and mixed until the
reaction reached 35.degree. C. Tetramethylethylenediamine (0.55 ml)
was added to initiate the reaction. The polymerization reaction was
stirred for 2 hours until beads formed.
[0117] The particles were course filtered and washed twice with
acetone (250 ml each), twice with methanol (250 ml each), and then
twice with acetone (250 ml each). The resulting particles were
transferred to a 500 ml Erlenmeyer flask. Acetone (300 ml) was
added to suspend the particles. The suspended particles were
sonicated for approximately 15 minutes, and then filtered. The
resulting particles were classified to a mean particle size of
approximately 65 .mu.m using a series of stacked sieves.
Comparative Example A
[0118] Heptane (348 ml), toluene (188 ml) and 1.4 ml of polymer
stabilizer solution were added to a 1L Mortonized round bottom
flask equipped with an overhead stirrer, thermocouple, reflux
condenser, and nitrogen gas inlet. The overhead stirrer was
adjusted to a stir rate of approximately 360 rpm and the reaction
flask was heated to 35.degree. C. under a slow nitrogen gas purge.
MBA (13.3 g) and N-acryloylmethylalanine (AMA, 0.7 g) were added to
a 250 ml Erlenmeyer flask equipped with a stir bar. Isopropyl
alcohol (65 ml) and 47 ml of water were added to dissolve the
solids. Ethylene glycol (25 ml) was then added. Upon dissolution of
all solids, sodium persulfate (0.55 g in 3 ml water) was added to
the stirred solution. The aqueous phase was added to the organic
phase and mixed until the reaction reached 35.degree. C.
Tetramethylethylenediamine (0.55 ml) was added to initiate the
reaction. The polymerization reaction was stirred for 2 hrs until
particles formed.
[0119] The particles were course filtered and washed twice with
acetone (250 ml each), twice with methanol (250 ml each), and then
twice with acetone (250 ml each). The resulting particles were
transferred to a 500 ml Erlenmeyer flask. Acetone (300 ml) was
added to suspend the particles. The suspended particles were
sonicated for approximately 15 minutes, and then filtered. The
resulting particles were classified to a mean particle size of
approximately 65 .mu.m using a series of stacked sieves.
[0120] Following preparation, the resulting particles were washed
thoroughly with acetone and suspended in 500 ml dry
dimethylsulfoxide. To this slurry was added acetic anhydride (25
ml) and triethylamine (2 ml). The particles were agitated by
rocking for an hour, filtered, and then washed extensively with
acetone and MTBE. The resultant azlactone-functional reactive beads
were suspended in an aqueous 1 M solution of benzylamine and
allowed to react for 1 hour. The beads were then filtered and
washed extensively with distilled water.
Experimental Methods
[0121] Preparation of Chromatography Column: Chromatography columns
were prepared by slurry packing the exemplary particles into a 3.0
mm.times.150 mm glass tube supplied by Omifit, Cambridge, CB1 3HD
England. Porous Teflon fits (25 .mu.m average pore size, Small
Parts, Inc., Miami Lakes, Fla.) were placed at both ends of the
tube to form a chromatography column.
[0122] Preparation of Chromatography System: The chromatography
column was assembled in an FPLC (fast protein liquid chromatograph,
obtained under the trade designation "AKTA FPLC", GE Healthcare,
Uppsala, Sweden equipped with a UV detector and a conductivity
detector.
[0123] Protein Analysis: The chromatography column in the
chromatography system was equilibrated with a mobile phase of 50 mM
(millimolar) sodium phosphate, pH 7 with 1.0 M sodium citrate at a
flow rate of 0.088 mL/min. 200 .mu.L (microliter) of a solution
containing 0.30 mg (milligram)/m1 myoglobin (from Sigma-Aldrich
Chemical Company; Milwaukee, Wis.), 0.24 mg/ml .beta.-lactoglobulin
(from USB Corporation, Cleveland, Ohio), 0.11 mg/ml lysozyme (from
Sigma-Aldrich Chemical Company), and 0.14 mg/ml bovine serum
albumin (BSA) (from Sigma-Aldrich Chemical Company) in 50 mM sodium
phosphate, pH 7 with 1.0 M sodium citrate was injected onto the
chromatography column. A gradient elution (40 column volumes) from
the initial buffer condition (high salt) to 50 mM sodium phosphate,
pH 7 (low salt) was applied. Using UV detection, the eluent was
monitored at a 280 nm (nanometer) wavelength.
[0124] Dynamic Binding Capacity Analysis: The chromatography column
in the chromatography system was equilibrated with a mobile phase
of 0.6M sodium citrate, pH 6.0. A solution of 2.3 mg/mL of human
IgG (hIgG from Equitech, Kerrville, Tex.) in 0.6M sodium citrate at
a pH 6.0 was pumped through the chromatography column at a flow
rate of 170 cm/hr. Using UV detection, the eluent was monitored at
a 280 nm wavelength. The 280 nm absorbance was correlated with IgG
concentration. The dynamic binding capacity (DBC) was determined by
monitoring the IgG breakthrough (10% of maximum protein
concentration eluting from the column).
Analysis Using Particles
Example 17
[0125] The particles prepared in Example 12 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Protein Analysis method as
described above. Shown in FIG. 1 is the chromatogram.
Example 18
[0126] The particles prepared in Example 13 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Protein Analysis method as
described above. Shown in FIG. 1 is the chromatogram.
[0127] Shown in FIG. 1 is an overlay of the chromatogram from
Example 17 (using the particles prepared in Example 12) and Example
18 (using the particles prepared in Example 13). The particles
prepared in Examples 12 and 13 were prepared using the same monomer
composition and reaction conditions, however, the PEG additive had
an effect on the overall hydrophobicity of the particles. As shown
in FIG. 1, the particle prepared in Example 12 with PEG 2,000 was
more hydrophobic (elution of proteins required a lower salt buffer)
than the particle prepared in Example 13 with PEG 6,000. The
resolution of BSA was comparable in both Examples 17 and 18,
however, the largest eluting peak (a co-elution of B-lactoglobulin
and lysozyme) was sharper in Example 17 (the particle prepared with
PEG 2,000).
Example 19
[0128] The particles prepared in Example 12 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Dynamic Binding Capacity
method as described above. Shown in FIG. 2 is the breakthrough
curve.
Example 20
[0129] The particles prepared in Example 13 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Dynamic Binding Capacity
method as described above. Shown in FIG. 2 is the breakthrough
curve.
Comparative Example B
[0130] An aromatic HIC media of highly cross-linked 90 .mu.m
agarose beads derivatized with phenyl groups via an ether linkage
sold under the trade designation "PHENYL SEPHAROSE 6 FAST FLOW (LOW
SUB)" commercially available from GE Healthcare, Chalfont St.
Giles, United Kingdom were packed into a tube to form a
chromatography column using the method as described above and an
analysis was preformed using the Dynamic Binding Capacity method as
described above. Shown in FIG. 2 is the breakthrough curve.
Comparative Example C
[0131] An aromatic HIC media of highly cross-linked 90 .mu.m
agarose beads derivatized with phenyl groups via an ether linkage
sold under the trade designation "PHENYL SEPHAROSE 6 FAST FLOW
(HIGH SUB)" commercially available from GE Healthcare, Chalfont St.
Giles, United Kingdom were packed into a tube to form a
chromatography column using the method as described above and an
analysis was preformed using the Dynamic Binding Capacity method as
described above. Shown in FIG. 2 is the breakthrough curve.
[0132] FIG. 2 is an overlay of the breakthrough curves for Examples
19 and 20 and Comparative Examples B and C. From the breakthrough
curves shown in FIG. 2 the breakthrough was calculated to be as
follows: Example 19=58 mg/mL, Example 20=49 mg/mL, Comparative
Example B=38 mg/mL, and Comparative Example C=54 mg/mL. The
calculated breakthrough indicates that the particles of Example 19
had the most adsorbance of IgG, followed by Comparative Example C,
Example 20, and then Comparative Example B. Also shown in FIG. 2 is
the profile of the breakthrough for each of the examples, which
indicates how the IgG is being adsorbed by the particles/beads.
Examples 19 and 20 provided a sharper breakthrough curve (steepness
of the exponential growth) than Comparative Example C. Comparative
Example B not only had the lowest DBC at 10% breakthrough, but also
did not show a flat baseline, indicating that there was
inconsistent binding of IgG to the beads. The amount of phenyl per
ml of the particles/beads shown in FIG. 2 are as follows: Example
19=14 .mu.mol (micromole) phenyl/ml particle (calculated based on
the amount of phenyl monomer used to make the particle and the
swell volume of the particle), Example 20=13 .mu.mol phenyl/ml
particle (calculated based on the amount of phenyl monomer used to
make the particle and the swell volume of the particle),
Comparative Example B=25 .mu.mol phenyl/ml particle (taken from
product literature), and Comparative Example C=50 .mu.mol phenyl/ml
particle (taken from product literature). Based on the data shown
in FIG. 2, the particles according to the present disclosure have
comparable or better DBC than Comparative Example C, while having
substantially less phenyl groups per ml of particle.
Example 21
[0133] The particles prepared in Example 14 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Protein Analysis method as
described above. Shown in FIG. 3 is the chromatogram.
Example 22
[0134] The particles prepared in Example 15 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Protein Analysis method as
described above. Shown in FIG. 3 is the chromatogram.
[0135] Shown in FIG. 3 is an overlay of the chromatogram from
Example 21 (using the particles prepared in Example 14) and Example
22 (using the particles prepared in Example 15). The particles
prepared in Examples 14 and 15 were prepared using the same monomer
composition and reaction conditions, however, the PEG additive had
an effect on the overall hydrophobicity of the particles. As shown
in FIG. 3, the particle prepared in Example 14 with PEG 6,000 was
more hydrophobic than the particle prepared in Example 15 with PEG
10,000. However, the particle prepared in Example 15 with PEG
10,000 was able to resolve BSA from B-lactoglobulin and lysozme,
whereas the particle prepared in Example 14 with PEG 6,000 showed
no resolution of the BSA peak.
Example 23
[0136] The particles prepared in Example 15 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Dymanic Binding Capacity
method as described above. The particle had a calculated DBC of 34
mg/ml. This value is less than the DBC values for the particles
prepared with AAm as a comonomer (as shown in Examples 19 and 20
above).
Example 24
[0137] The particles prepared in Example 16 were packed into a tube
to form a chromatography column using the method as described above
and an analysis was preformed using the Protein Analysis method as
described above. Shown in FIG. 4 is blow-up of the chromatogram
comprising the IgG peak.
Comparative Example D
[0138] The particles prepared in Comparative Example A were packed
into a tube to form a chromatography column using the method as
described above and an analysis was preformed using the Protein
Analysis method as described above. Shown in FIG. 4 is blow-up of
the chromatogram comprising the IgG peak.
[0139] Shown in FIG. 4 is an overlay of the IgG peak from Example
24 and Comparative Example D. The particles used in Example 24 and
Comparative Example D have the same particle composition; azlactone
linkages covalently bonding acrylamide particles with the
benzylamine hydrophobic group. Thus, one would expect them to have
the same retention time. As shown in FIG. 4, the IgG peak in
Example 24 is retained less than in Comparative Example D. Although
not wanting to be bound by theory, the increased retention of IgG
in Comparative Example D is speculated to be due to the presence of
a small amount of negative charge generated by hydrolysis of the
azlactone during the reaction with benzylamine. Further, as shown
in FIG. 4, the IgG peak in Comparative Example D is broader than
that in Example 24, which is also speculated to be indicative of a
mixed interaction (e.g. hydrophobic interactions and ion exchange
interactions) of the IgG with the stationary phase.
[0140] Various modifications and alterations to this disclosure
will become apparent to those skilled in the art without departing
from the scope and spirit of this disclosure. It should be
understood that this disclosure is not intended to be unduly
limited by the illustrative embodiments and examples set forth
herein and that such examples and embodiments are presented by way
of example only with the scope of the disclosure intended to be
limited only by the claims set forth herein as follows.
* * * * *