U.S. patent application number 13/210568 was filed with the patent office on 2011-12-22 for chromatographic material for the absorption of proteins at physiological ionic strength.
This patent application is currently assigned to PALL CORPORATION. Invention is credited to Egisto Boschetti, Pierre Girot.
Application Number | 20110313147 13/210568 |
Document ID | / |
Family ID | 34825923 |
Filed Date | 2011-12-22 |
United States Patent
Application |
20110313147 |
Kind Code |
A1 |
Boschetti; Egisto ; et
al. |
December 22, 2011 |
CHROMATOGRAPHIC MATERIAL FOR THE ABSORPTION OF PROTEINS AT
PHYSIOLOGICAL IONIC STRENGTH
Abstract
Ion exchange and hydrophobic interaction chromatographic
materials are constructed by tethering a terminal binding
functionality to a solid support via a hydrophobic linker. The
backbone of the linker typically comprises sulfur-containing
moieties. Suitable terminal binding functionalities are tertiary
amines, quaternary ammonium salts, or hydrophobic groups. These
chromatographic materials possess both hydrophobic and ionic
character under the conditions prescribed for their use. The
separation of proteins from crude mixtures at physiological ionic
strength can be accomplished with a chromatographic material of
this type by applying pH or ionic strength gradients, thereby
effecting protein adsorption and desorption.
Inventors: |
Boschetti; Egisto; (Croissy
sur Seine, FR) ; Girot; Pierre; (Paris, FR) |
Assignee: |
PALL CORPORATION
Port Washington
NY
|
Family ID: |
34825923 |
Appl. No.: |
13/210568 |
Filed: |
August 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10583509 |
Jun 16, 2006 |
8021889 |
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PCT/US05/01304 |
Jan 14, 2005 |
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13210568 |
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60537342 |
Jan 20, 2004 |
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Current U.S.
Class: |
536/56 ; 556/54;
564/201; 564/292; 564/501; 564/508; 568/39; 568/55 |
Current CPC
Class: |
B01J 2219/00702
20130101; G01N 30/02 20130101; G01N 30/02 20130101; B01J 20/3242
20130101; B01J 39/26 20130101; B01D 15/362 20130101; B01J
2219/00626 20130101; G01N 2035/00158 20130101; B01J 20/289
20130101; B01J 2219/00637 20130101; B01J 2219/00612 20130101; B01J
20/3285 20130101; B01J 2219/0074 20130101; C07K 1/18 20130101; B01J
2219/0061 20130101; B01J 2220/54 20130101; G01N 33/54353 20130101;
B01J 2219/00605 20130101; G01N 30/02 20130101; B01J 41/20 20130101;
B01D 15/361 20130101; C07K 1/16 20130101; Y10S 435/803 20130101;
B01D 15/362 20130101; B01D 15/327 20130101; B01J 2219/00527
20130101; B01D 15/327 20130101 |
Class at
Publication: |
536/56 ; 564/501;
564/508; 564/201; 564/292; 568/39; 568/55; 556/54 |
International
Class: |
C08B 15/00 20060101
C08B015/00; C07C 217/06 20060101 C07C217/06; C07F 7/00 20060101
C07F007/00; C07C 323/16 20060101 C07C323/16; C07C 323/12 20060101
C07C323/12; C07C 323/25 20060101 C07C323/25; C07C 323/52 20060101
C07C323/52 |
Claims
1. A chromatographic material comprising: (a) a terminal binding
functionality; (b) a hydrophobic linker comprising at least one
functionality that is different from the terminal binding
functionality; and (c) a solid support, wherein the hydrophobic
linker links the terminal binding functionality to the solid
support; and the chromatographic material is capable of binding
bovine albumin at physiological ionic strength, wherein the
chromatographic material has the following general formula I:
##STR00014## wherein R.sub.1, R.sub.2, R.sub.4 and R.sub.5, at each
occurrence, are independently selected from the group consisting of
H, C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl, C.sub.1-6-alkaryl,
--NR'C(O)R'', --C(O)NR'R'', and hydroxy, wherein R' and R'' are
independently selected from C.sub.1-6-alkyl, and wherein no more
than one of R.sub.1 and R.sub.2 is hydroxy, R.sub.6 is selected
from the group consisting of H, C.sub.1-6-alkyl, aryl,
C.sub.1-6-alkaryl, --C(O)OH, --S(O).sub.2OH, and --P(O)(OH).sub.2;
R.sub.3 and R.sub.3', together with X and Y, respectively, may
independently be absent or present, and if present, then R.sub.3
and R.sub.3' are independently selected from the group consisting
of H, C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl, and C.sub.1-6-alkaryl,
wherein X and Y, independently of each other, represent anions; het
and het' are heteroatom moieties independently selected from the
group consisting of --O--, --S--, --S(O)--, and --S(O).sub.2--; a,
a', a'', and a''' are independently selected from the integers 0
through 6; b and b' are independently 0 or 1; c is 0 or 1, and if c
is 1, then (R.sub.3)X is absent; d and d' are independently 0 or 1;
and the wavy line represents the solid support.
2. (canceled)
3. The chromatographic material according to claim 1, wherein at
least one of a, a', a'', and a''' is 2.
4. The chromatographic material according to claim 3, wherein at
least two of a, a', a'', and a''' are 2.
5. The chromatographic material according to claim 4, further
wherein at least one of a, a', a'', and a''' is 3.
6. The chromatographic material according to claim 1, wherein at
least one of a, a', a'', and a''' is 3.
7. The chromatographic material according to claim 6, wherein at
least two of a, a', a'', and a''' are 3.
8. The chromatographic material according to claim 6, wherein a is
3.
9. The chromatographic material according to claim 8, wherein het
is S and b is 1.
10. The chromatographic material according to claim 9, wherein a'
is selected from the group consisting of 2, 3, 4, 5, and 6.
11. The chromatographic material according to claim 10, wherein b'
is 0.
12. The chromatographic material according to claim 11, wherein c
and d are both 0.
13. The chromatographic material according to claim 12, wherein d'
is 1 and (R.sub.3')Y is absent.
14. The chromatographic material according to claim 12, wherein d'
is 1 and (R.sub.3')Y is present.
15. The chromatographic material according to claim 12, wherein d'
is 0.
16. The chromatographic material according to claim 11, wherein c
is 1 and d is 1.
17. The chromatographic material according to claim 16, wherein d'
is 1 and (R.sub.3')Y is absent.
18. The chromatographic material according to claim 16, wherein d'
is 1 and (R.sub.3')Y is present.
19. The chromatographic material according to claim 16, wherein d'
is 0.
20. The chromatographic material according to claim 10, wherein
R.sub.1 and R.sub.2 are independently selected from H and
C.sub.1-6-alkyl.
21. The chromatographic material according to claim 20, wherein
each of R.sub.1 and R.sub.2 are H.
22. The chromatographic material according to claim 8, wherein d'
is 1.
23. The chromatographic material according to claim 22, wherein
R.sub.3', R.sub.5, and R.sub.6 are independently selected from the
group consisting of H, C.sub.1-6-alkyl, aryl, and
C.sub.1-6-alkaryl.
24. The chromatographic material according to claim 23, wherein
R.sub.3', R.sub.5, and R.sub.6 are independently selected from
C.sub.1-6-alkyl and aryl.
25. The chromatographic material according to claim 24, wherein
R.sub.3', R.sub.5, and R.sub.6 are independently selected from
C.sub.1-6-alkyl.
26. The chromatographic material according to claim 25, wherein
R.sub.3', R.sub.5, and R.sub.6 are independently selected from
methyl and ethyl.
27. The chromatographic material according to claim 24, wherein one
of a'' and a''' is 1 and the other is 1 or 2.
28. The chromatographic material according to claim 25, wherein
(R.sub.3')Y is absent.
29. The chromatographic material according to claim 8, wherein d'
is 0.
30. The chromatographic material according to claim 29, wherein
R.sub.6 is H, C.sub.1-6-alkyl, aryl, or C.sub.1-6-alkaryl.
31. The chromatographic material according to claim 30, wherein
R.sub.6 is selected from C.sub.1-6-alkyl and aryl.
32. The chromatographic material according to claim 31, wherein
R.sub.6 is phenyl.
33. The chromatographic material according to claim 31, wherein one
of a'' and a''' is 1 and the other is 1 or 2.
34. The chromatographic material according to claim 29, wherein
R.sub.6 is --C(O)OH, --S(O).sub.2OH, and --P(O)(OH).sub.2.
35. The chromatographic material according to claim 34, wherein one
of a'' and a''' is 1 and the other is 1 or 2.
36. The chromatographic material according to claim 1, wherein: a
is 3; a' is 2; b is 1; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a and (CR.sub.1R.sub.2).sub.a' is H, except
that one of R.sub.2 in (CR.sub.1R.sub.2).sub.a is optionally
OH.
37. The chromatographic material according to claim 36, wherein one
of R.sub.2 in (CR.sub.1R.sub.2).sub.a is OH.
38. The chromatographic material according to claim 37, wherein
each of a'', a''', and b' is 0.
39. The chromatographic material according to claim 1, wherein: a''
is 3; a''' is 2; b' is 1; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' is H,
except that one of R.sub.2 in (CR.sub.1R.sub.2).sub.a'' is
optionally OH.
40. The chromatographic material according to claim 39, wherein one
of R.sub.2 in (CR.sub.1R.sub.2).sub.a'' is OH.
41. The chromatographic material according to claim 40, wherein
each of a, a', and b'' is 0.
42. The chromatographic material according to claim 1, wherein: a
is 3; a' is 3; b is 1; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a and (CR.sub.1R.sub.2).sub.a' is H.
43. The chromatographic material according to claim 42, wherein
each of a'', a''', and b' is 0.
44. The chromatographic material according to claim 1, wherein: a''
is 3; a''' is 3; b' is 1; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' is H.
45. The chromatographic material according to claim 44, wherein
each of a, a', and b is 0.
46. The chromatographic material according to claim 1, wherein a is
3; a' is 5; b is 1; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a and (CR.sub.1R.sub.2).sub.a' is H.
47. The chromatographic material according to claim 46, wherein: b'
is 0; one of a'' and a''' is 2 or 3, the other being 0; and each
R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a'' and
(CR.sub.1R.sub.2).sub.a''' is H, except that one of R.sub.2 in
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' is
optionally OH.
48. The chromatographic material according to claim 47, wherein a''
or a''' is 3 and one of R.sub.2 in (CR.sub.1R.sub.2).sub.a'' and
(CR.sub.1R.sub.2).sub.a''' is OH.
49. The chromatographic material according to claim 1, wherein a or
a' is 3, the other being 0; a'' or a''' is 3; each of b, b', and c
is 0; and d is 1.
50. The chromatographic material according to claim 49, wherein d'
is 1.
51. The chromatographic material according to claim 1, wherein:
each of R.sub.1 and R.sub.2 are H; R.sub.3, R.sub.3', R.sub.4,
R.sub.5, and R.sub.6 are independently selected from the group
consisting of H, C.sub.1-6-alkyl, aryl, and C.sub.1-6-alkaryl; het
is S; a is 3; a' is selected from the group consisting of 2, 3, 4,
5, and 6; one of a'' and a''' is 1 and the other is 1 or 2; and b
is 1 and b' is 0.
52. The chromatographic material according to claim 1, selected
from the group consisting of: ##STR00015##
53. The chromatographic material according to claim 1, wherein the
wherein the solid support is an organic material.
54. The chromatographic material according to claim 53, wherein the
organic material is one selected from the group consisting of
cellulose, agarose, dextran, polyacrylates, polystyrene,
polyacrylamide, polymethacrylamide, copolymers of styrene and
divinylbenzene, and mixtures thereof.
55. The chromatographic material according to claim 1, wherein the
solid support is an inorganic material.
56. The chromatographic material according to claim 55, wherein the
inorganic material is one selected from the group consisting of
silica, zirconia, alumina, titania, ceramics, and mixtures
thereof.
57. The chromatographic material according to claim 1, wherein the
solid support is in the form of a bead or particle.
58. The chromatographic material according to claim 1, wherein the
solid support is a planar solid support.
59-98. (canceled)
99. The chromatographic material of claim 1, wherein R.sub.1,
R.sub.2, R.sub.4, and R.sub.5, at each occurrence, are
independently selected from the group consisting of H,
C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-C.sub.1-6-alkoxy, --NR'C(O)R'', --C(O)NR'R'', and
hydroxy, wherein R' and R'' are independently selected from
C.sub.1-6-alkyl, and wherein no more than one of R.sub.1 and
R.sub.2 is hydroxy; and, R.sub.6 is selected from the group
consisting of H, C.sub.1-6-alkyl, --C(O)OH, --S(O).sub.2OH, and
--P(O)(OH).sub.2.
100. The chromatographic material of claim 1, wherein R.sub.6 is
selected from the group consisting of C.sub.1-6-alkyl, --C(O)OH,
--S(O).sub.2OH, and --P(O)(OH).sub.2.
101. The chromatographic material of claim 1, wherein R.sub.1,
R.sub.2, R.sub.4, and R.sub.5, at each occurrence, are
independently selected from the group consisting of H,
C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl, C.sub.1-6-alkaryl, and
hydroxy, wherein no more than one of R.sub.1 and R.sub.2 is
hydroxy; R.sub.6 is selected from the group consisting of H,
C.sub.1-6-alkyl, aryl, and C.sub.1-6-alkaryl; R.sub.3 and R.sub.3',
together with X and Y, are both absent; and d and d' are both
0.
102. The chromatographic material of claim 1, wherein a is 3; a''
is 0 or 3; d is 1; a', a''', b, b', c, and d' are all 0; (R.sub.3)X
is absent; each R.sub.1, R.sub.2, and R.sub.4, is H; and R.sub.6 is
selected from the group consisting of C.sub.1-6-alkyl and aryl.
103. The chromatographic material of claim 1, wherein a is 3; d is
1; a', a'', a''', b, b', c, and d' are all 0; (R.sub.3)X is absent;
each R.sub.1, R.sub.2, and R.sub.4, is H; and R.sub.6 is selected
from the group consisting of C.sub.1-6-alkyl.
104. The chromatographic material of claim 1, wherein a is 3; a''
is 3; d is 1; a', a''', b, b', c, and d' are all 0; (R.sub.3)X is
absent; R.sub.1 and R.sub.2 at each occurrence, are independently
selected from the group consisting of H and hydroxy; wherein no
more than one of R.sub.1 and R.sub.2 is hydroxy; R.sub.4 is H; and
R.sub.6 is selected from the group consisting of aryl.
105. The chromatographic material of claim 104, wherein a is 3; a''
is 3; d is 1; a', a''', b, b', c, and d' are all 0; (R.sub.3)X is
absent; each R.sub.1, R.sub.2, and R.sub.4, is H; and R.sub.6 is
selected from the group consisting of aryl.
106. The chromatographic material of claim 104, wherein R.sub.6 is
phenyl.
107. The chromatographic material of claim 105, wherein R.sub.6 is
phenyl.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates generally to the field of ion
exchange and hydrophobic interaction chromatographic materials in
the context of separation science and analytical biochemistry.
[0002] The increasing need for bulk quantities of biologically
relevant molecules (i.e., biomolecules) such as proteins has
spawned a variety of techniques for isolating such biomolecules
from physiological isolates. Traditional techniques in this regard
include precipitation methods, electrophoretic separations, and
membrane filtration. One of the more promising separation
methodologies advanced, however, is liquid chromatography.
[0003] Chromatographic separations of complex biomolecules
typically require one or more modifications of the sample that
contains the biomolecules. The interactions between biomolecules
and a chromatographic sorbent include electrostatic attraction and
repulsion, ion exchange, hydrophobic associations, charge transfer,
and van der Waals attraction. These forces often compete with each
other to impose a delicate balance between conditions that are
suitable for a biomolecule to adsorb onto a chromatographic sorbent
and those conditions under which the biomolecule may desorb. Crude
physiological isolates, such as whole blood, exhibit non-ideal pH
and ionic strength, for example, for desired proteins to adsorb to
typical sorbents. As a consequence of these interactions, it is
necessary to adjust the pH and/or ionic strength of physiological
samples to achieve biomolecule adsorption. It may be additionally
necessary to add chemical additives or to dilute or concentrate the
samples prior to chromatographic separations.
[0004] A useful subset of chromatographic resins that are subject
to these limitations are ion exchange resins, which primarily
attract biomolecules such as proteins via opposing charges on the
resins and proteins. In this regard, typical ion exchange resins
adsorb proteins at pH 4 to 10 and at low to very low ionic
strength. The pH of a sample containing a protein dictates the net
charge of the protein, which, of course, must be opposite to the
charge of the chromatographic resin. Thus, crude biological samples
must be adjusted to higher or lower pH as a prerequisite to confer
net charges for protein separations.
[0005] Achieving the correct pH of a sample is not always
sufficient to promote the adsorption of a protein to an ion
exchange resin. The ionic strength of a sample exerts a powerful
influence on adsorption since the counterions that are present in
the form of salts can compete with the protein charges for the
charged resin. If the ionic strength of a sample is too high, then
proteins will not adsorb to the resin. The physiological ionic
strength of most biological extracts are 15-20 mS/cm, whereas most
chromatographic separation conditions require the ionic strength to
be between about 1 mS/cm and 10 mS/cm. Johansson et al., for
example, describe a number of multi-modal ligands that feature
primary or secondary amines as anion-exchange groups, or certain
carboxylic acids as cation exchange groups, for the adsorption of
biomolecules. However, these ligands require high ionic strength
for biomolecule adsorption. See B.-L. Johansson et al. "Preparation
and Characterization of Prototypes for Multi-Modal Separation Media
Aimed for Capture of Negatively Charged Biomolecules at High Salt
Conditions," 814 J. Chromatography A 71-81 (1998) and B.-L.
Johansson et al. "Preparation and Characterization of Prototypes
for Multi-Modal Separation Aimed for Capture of Positively Charged
Biomolecules at High Salt Conditions," 1016 J. Chromatography A
35-49 (2003).
[0006] Another useful subset of chromatographic resins relies upon
hydrophobic interactions between the resins and sample molecules.
In hydrophobic interaction chromatography (HIC), the interactions
generally require high salt buffer concentrations to reduce the
solvation of the molecules in solution, thereby revealing
hydrophobic regions in the sample molecules that are consequently
adsorbed by the hydrophobic resin. See U.S. Pat. No. 5,641,870. As
with ion exchange chromatography, it is often difficult to achieve
the correct balance of ionic strength and pH to afford useful
separations of sample molecules as discussed, for example, by S. C.
Burton et al. "Hydrophobic Charge Induction Chromatography: Salt
Independent Protein Adsorption and Facile Elution With Aqueous
Buffer," 814 J. Chromatography A 71-81 (1998). Many conventional
HIC resins thus operate at neutral pH, but require high salt
conditions, which pose practical difficulties as discussed
below.
[0007] For these reasons, as noted above, the separation of
proteins and other biomolecules from physiological isolates
requires processing of the isolates to enforce the correct pH and
ionic strength requirements of ion exchange and HIC resins.
Conventional solutions to overcome the ionic strength problem in
particular include diluting a sample with very low ionic strength
buffers (or even water) and dialyzing the sample against a buffer
with the desired ionic strength. Both of these operations, however,
are time consuming and are often incompatible with large scale
biomolecule separations.
[0008] More recent attempts to overcome the above-mentioned
difficulties focus upon protein separation based on dual-mode
ligands, which seek to incorporate, for example, ionic and
hydrophobic features into the same capture ligand, and thereby
lessen the need to employ high salt conditions for protein capture.
See L. Guerrier et al. "New Method for the Selective Capture of
Antibodies Under Physiological Conditions," 9 Bioseparation 211-221
(2000) for a discussion of resins that combine mild hydrophobic
association and charge induction for protein capture. Other resins,
by contrast to the present invention, rely upon thiophilic ligands
that incorporate heterocyclic moieties, such as mercaptonicotinic
acid as disclosed by G. H. Scholz et al. "Salt-Independent Binding
of Antibodies from Human Serum to Thiophilic Heterocyclic Ligands,"
709 J. Chromatography B: Biomedical Sciences and Applications
189-196 (1998).
[0009] In principle, powerful analytical methods can be realized
with the ion exchange and HIC adsorbents discussed above. For
example, the rapid identification of disease markers by
analyte/adsorbent interactions would supplant the tedious and time
consuming work required in conventional clinical diagnostics in
order to prepare reagents that specifically bind to such markers.
Additionally, the direct and rapid identification of differentially
expressed proteins would be a significant benefit to the field,
thereby circumventing, for example, the long process of polypeptide
isolation and subsequent immunization to produce desired
immunoglobulins. The above-mentioned shortcomings of conventional
adsorbents limit the sensitivity and resolution of such analytical
tools, however.
[0010] Accordingly, a continued need exists in the art for improved
ion exchange chromatographic and HIC materials that exhibit high
binding capacity and specificity, that can be regenerated
extensively without suffering physiochemical degradation, and that
can function under physiological pH and/or ionic strength. A need
also exists for improved biochemical analytic tools that are useful
for the rapid identification of biologically important
molecules.
SUMMARY OF THE INVENTION
[0011] To address these and other needs, the present invention
provides a chromatographic material comprising (a) a terminal
binding functionality; (b) a hydrophobic linker comprising at least
one functionality that is different from the terminal binding
functionality; and (c) a solid support, wherein the hydrophobic
linker links the terminal binding functionality to the solid
support. Within these provisions, the chromatographic material is
capable of binding bovine albumin at physiological ionic
strength.
[0012] The chromatographic material of the invention preferably
conforms to general formula (I):
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.4, and R.sub.5, at each occurrence,
are independently selected from the group consisting of H,
C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl, C.sub.1-6-alkaryl,
--NR'C(O)R'', --C(O)NR'R'', and hydroxy. R' and R'' are
independently selected from C.sub.1-6-alkyl, and no more than one
of R.sub.1 and R.sub.2 is hydroxy. R.sub.6 is selected from the
group consisting of H, C.sub.1-6-alkyl, aryl, C.sub.1-6-alkaryl,
--C(O)OH, --S(O).sub.2OH, and --P(O)(OH).sub.2. R.sub.3 and
R.sub.3', together with X and Y, respectively, may independently be
absent or present, and if present, then R.sub.3 and R.sub.3' are
independently selected from the group consisting of H,
C.sub.1-6-alkoxy, C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl, and
C.sub.1-6-alkaryl. X and Y, independently of each other, represent
anions. Het and het' are heteroatom moieties independently selected
from the group consisting of --O--, --S--, --S(O)--, and
--S(O).sub.2--. Subscripts a, a', a'', and a''' are independently
selected from the integers 0 through 6; b and b' are independently
0 or 1; c is 0 or 1, and if c is 1, then (R.sub.3)X is absent; and
d and d' are independently 0 or 1. In formula (I), the wavy line
represents the solid support.
[0013] In certain embodiments, at least one or two of a, a', a'',
and a''' is 2, preferably where at least one of a, a', a'', and
a''' is also 3. In other embodiments, at least one or two of a, a',
a'', and a''' is 3.
[0014] Preferred chromatographic materials are those wherein a is
3, het is S, and b is 1. Within these constraints, a' is preferably
2, 3, 4, 5, or 6; b' is 0; c and d are both 0 or 1; and d' is 1. In
other materials, d' can be 0 or 1. Preferably, R.sub.1 and R.sub.2
are independently selected from H and C.sub.1-6-alkyl, more
preferably each of R.sub.1 and R.sub.2 is H.
[0015] In one group of preferred terminal groups, R.sub.3',
R.sub.5, and R.sub.6 are independently selected from the group
consisting of H, C.sub.1-6-alkyl, aryl, and C.sub.1-6-alkaryl,
preferably from C.sub.1-6-alkyl and aryl, and most preferably from
C.sub.1-6-alkyl. Exemplary groups in this context are methyl and
ethyl. In this embodiment it is preferred that one of a'' and a'''
1 while the other is 1 or 2, and that (R.sub.3')Y is absent.
[0016] Another preferred subset of chromatographic materials are
those wherein d' is 0 and R.sub.6 is preferably H, C.sub.1-6-alkyl,
aryl, or C.sub.1-6-alkaryl, more preferably, C.sub.1-6-alkyl and
aryl. R.sub.6 is most preferably phenyl. Alternatively, R.sub.6 is
--C(O)OH, --S(O).sub.2OH, or --P(O)(OH).sub.2. In combinations, one
of a'' and a''' preferably is 1 and the other is 1 or 2.
[0017] In another embodiment, a is 3, a' is 2, a is 3; a' is 2; b
is 1; and each R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a and
(CR.sub.1R.sub.2).sub.a' is H, except that one of R.sub.2 in
(CR.sub.1R.sub.2).sub.a is optionally OH. Preferably, one of
R.sub.2 in (CR.sub.1R.sub.2).sub.a is OH. More preferably, each of
a'', a''', and b' is 0.
[0018] In another embodiment, a'' is 3; a''' is 2; b' is 1; and
each R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a'' and
(CR.sub.1R.sub.2).sub.a''' is H, except that one of R.sub.2 in
(CR.sub.1R.sub.2).sub.a'' is optionally OH. Preferably, one of
R.sub.2 in (CR.sub.1R.sub.2).sub.a'' is OH. More preferably, each
of a, a', and b'' is 0.
[0019] In still another embodiment, a is 3; a' is 3; b is 1; and
each R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a and
(CR.sub.1R.sub.2).sub.a' is H. Preferably, each of a'', a''', and
b' is 0.
[0020] In yet another embodiment, a'' is 3; a''' is 3; b' is 1; and
each R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a' and
(CR.sub.1R.sub.2).sub.a''' is H. Preferably, each of a, a', and b
is 0.
[0021] In another embodiment, a is 3; a' is 5; b is 1; and each
R.sub.1 and R.sub.2 in (CR.sub.1R.sub.2).sub.a and
(CR.sub.1R.sub.2).sub.a' is H.
[0022] In an additional embodiment, b' is 0; one of a'' and a''' is
2 or 3, the other being 0; and each R.sub.1 and R.sub.2 in
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' is H,
except that one of R.sub.2 in (CR.sub.1R.sub.2).sub.a'' and
(CR.sub.1R.sub.2).sub.a''' is optionally OH. Preferably, a'' or
a''' is 3 and one of R.sub.2 in (CR.sub.1R.sub.2).sub.a'' and
(CR.sub.1R.sub.2).sub.a''' is OH.
[0023] In another embodiment, a or a' is 3, the other being 0; a''
or a''' is 3; each of b, b', and c is 0; and d is 1. Preferably, d'
is also 1.
[0024] In still another embodiment, each of R.sub.1 and R.sub.2 are
H; R.sub.3, R.sub.3', R.sub.4, R.sub.5, and R.sub.6 are
independently selected from the group consisting of H,
C.sub.1-6-alkyl, aryl, and C.sub.1-6-alkaryl; het is S; a is 3; a'
is selected from the group consisting of 2, 3, 4, 5, and 6; one of
a'' and a''' is 1 and the other is 1 or 2; and b is 1 and b' is
0.
[0025] The most preferred chromatographic materials are represented
by the following formulae:
##STR00002##
[0026] In certain embodiments, the solid support is an organic
material. Preferably, the organic material is one selected from the
group consisting of cellulose, agarose, dextran, polyacrylates,
polystyrene, polyacrylamide, polymethacrylamide, copolymers of
styrene and divinylbenzene, and mixtures thereof.
[0027] In other embodiments, the solid support an inorganic
material. Preferably, the inorganic material is one selected from
the group consisting of silica, zirconia, alumina, titania,
ceramics, and mixtures thereof.
[0028] The solid support can be in the form of a bead or particle.
Alternatively, the solid support is planar. In the latter case, the
chromatographic material can be in the form of a biochip. Preferred
solid supports in this context include a metal, metal oxide,
silicon, glass, a polymer, and a composite material. Biochips are
particularly preferred wherein a multitude of terminal binding
functionalities and the hydrophobic linkers to which the terminal
binding functionalities are linked are segregated into a plurality
of addressable locations on the solid support. In this scenario, at
least two different addressable locations comprise the same
terminal binding functionality and hydrophobic linker. The biochip
can be a mass spectrometer probe.
[0029] The invention also provides a chromatography column,
comprising (a) a tubular member having an inlet end and an outlet
end; (b) first and second porous members disposed within said
tubular member; and (c) a chromatographic material according to
this invention packed within the tubular member between the first
and second porous members. In some embodiments, the chromatography
column volume is between about 1 microliter and about 5000 liters.
Preferably, the column volume is between about 1 liter and about
100 liters. The column may comprise one or more fluid control
devices for flowing a liquid sample upward or downward through the
chromatographic material.
[0030] The invention also provides for a method for the separation
of at least one substance from a sample. The method entails (a)
contacting a chromatographic material according to this invention
with a liquid sample that comprises at least one substance, whereby
the substance adsorbs to the chromatographic material; (b)
adjusting the pH, ionic strength, or both such that the substance
desorbs from the resin. Preferably, the method further comprises
washing the chromatographic material obtained in (a) with an
equilibrium buffer.
[0031] In the most preferred embodiments, the substance to be
separated is a biological substance. The biological substance is
preferably selected from of proteins, viruses, nucleic acids,
carbohydrates, oligosaccharides, polysaccharides, lipids, and
lipopolysaccharides. More preferably, the biological substance is a
protein, such as an immunoglobulin, hormone, clotting factor,
cytokine, peptide, polypeptide, or enzyme. The most preferred
substance is an immunoglobulin.
[0032] In one aspect of the method, the liquid sample is at
physiological ionic strength. The liquid sample may also be at
physiological pH. Preferred ionic strengths in this regard are
between about 0.1 M and about 0.2 M. Additionally, the
concentration of the biological substance can be the physiological
concentration.
[0033] In some embodiments, the method further comprises adjusting
the ionic strength of the liquid sample to physiological ionic
strength prior to step (a). In other embodiments, the method
entails only increasing the ionic strength.
[0034] The method may be accomplished via several modes. These
include fixed bed, fluidized bed, or batch chromatography.
[0035] The invention also contemplates a method of detecting an
analyte, comprising (a) contacting an addressable location of the
present chromatographic material with a sample comprising the
analyte. This fixes the analyte to the chromatographic material.
The analyte is detected by virtue of its binding to the terminal
binding functionality and hydrophobic linker, preferably in a mass
spectrometer. In the latter scenario, the addressable location is
positioned proximately to a laser beam in the mass spectrometer,
preferably where the detecting comprises irradiating the
chromatographic material at the addressable location with a laser
pulse for a time and power sufficient to desorb and ionize the
analyte. The mass spectrometer can be a gas phase ion
spectrometer.
[0036] In some embodiments of the method, the sample is a blood
sample. Preferably, the blood sample is a serum sample.
[0037] The invention also contemplates a process for making the
chromatographic material of this invention. The method generally
comprises activating the solid support by contacting the solid
support with one functionality of a bifunctional reagent that
comprises part or all of the hydrophobic linker to bind the reagent
to the solid support. The activated solid support is then reacted
with a reagent that comprises the terminal binding functionality to
form a bond between the hydrophobic linker and the terminal binding
functionality. The bifunctional reagent may comprise at least two
functional groups including but not limited to chloro, bromo, iodo,
epoxide, carboxyl, ester, aldehyde, ketone, amido, alkenyl, cyano,
and imino.
[0038] In one embodiment, the contacting step provides discreet
spots of activated solid support. In this context, the
chromatographic material preferably is in the form of a
biochip.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] The present invention provides a chromatographic material
that is an effective adsorbent for use in separating and isolating
a variety of substances, including biologically relevant molecules.
The chromatographic material of this invention may be used, for
example, in preparative techniques, such as column chromatography,
and in analytical devices, such as biochips.
[0040] One advantage of the present chromatographic material
described herein is its high selectivity and specificity for
biological substances such as proteins, together with the avoidance
of costly and often detrimental cleaning processes required for
prior art substrates. A second advantage is that the
chromatographic material of the invention is ideally suited for use
with biological samples at physiological ionic strength pH and/or
physiological pH, thereby obviating the need for ionic strength
and/or pH adjustments and the addition of lyotropic salts for
desorption as prescribed by conventional materials. A third
advantage is the high biological molecule binding capacity of the
present substrates, which, in view the low cost of reagents
employed to prepare them, presents significant economic gains over
the use of specialized prior art chromatographic materials.
Consequently, it is possible to manipulate smaller volumes of a
sample, to reduce processing time, or to process a large amount of
a sample per unit column volume.
I. Chromatographic Material
[0041] The chromatographic material of this invention comprises a
solid support and a terminal binding functionality that is attached
to the solid support via a hydrophobic linker. The terminal binding
functionality serves to attract in part a substance in a sample.
The hydrophobic linker comprises at least one functionality that is
different from the terminal binding functionality, and typically
contains a sulfur and/or a nitrogen atom. Overall, therefore, the
linker presents a hydrophobic, and possibly thiophilic, region
between the solid support and terminal binding functionality. The
functionalities can give rise to a range of intermolecular forces;
thus, the chromatographic material provides complementary modes of
selectively attracting substances.
[0042] A. Functionalities
[0043] The terminal binding functionality may be selected according
to the properties of the substance that is desired to be separated
and according to the complementary characteristics of the
hydrophobic linker. For example, where the terminal binding
functionality is an anion or cation exchange group, it is desirable
to maintain ancillary modes of sample interaction by tailoring the
linker to be at least hydrophobic. In this embodiment, the
hydrophobic linker can contain an anion or cation exchange group,
but in accordance with the provisions of the invention, the linker
preferably contains at least one functionality that is different
from the anion or cation exchange group, respectively. A preferred
functionality in this regard is a sulfur-containing group, such as
--S-- or --S(O).sub.2--.
[0044] In other embodiments, the terminal binding functionality
itself is a hydrophobic group. Thus, although the linker generally
is composed of hydrophobic regions, the linker must contain at
least one functionality that is not the same as the hydrophobic
terminal binding functionality. Functionalities in this regard
include, of course, anion and cation exchange groups, possibly
together with the sulfur-containing moieties mentioned above.
[0045] The "terminal binding functionality," as contemplated
herein, resides by definition at the end of the hydrophobic linker,
and therefore is believed to exert the greatest contribution to the
adsorbent properties of the inventive chromatographic material. In
some embodiments, the terminal binding functionality is an anion
exchange group, such as amines and quaternary ammonium salts. In
other embodiments, the terminal binding functionality is a cation
exchange group. In yet other embodiments, hydrophobic groups, as
defined below, are most suitable for the terminal binding
functionality.
[0046] The requirements for the hydrophobic linker include the
presence of at least one functionality that is different from the
terminal binding functionality. It is believed that this
functionality, by virtue of it being embedded within the
hydrophobic linker, confers secondary adsorbent properties to the
inventive chromatographic material.
[0047] The term "hydrophobic," as used herein, generally refers to
a non-polar chemical moiety that is understood in the art to repel
polar entities such as water, or equally, to attract other
hydrophobic entities such as hydrophobic regions in proteins.
Exemplary hydrophobic groups contemplated for this invention
include but are not limited to alkyl, aryl, and alkaryl groups.
Typical hydrophobic groups are simple hydrocarbon chains such as
ethyl and propyl, or when embedded into another group, ethylene or
propylene spacers, respectively. While no general standard exists
for evaluating hydrophobicity, it is understood within the context
of this invention that at least a 2-carbon chain is sufficient to
create a hydrophobic region. The overall hydrophobic character of a
group, however, is not negated by the presence of a limited number
of polar groups, but it is recognized in the art that the presence
of ionic or polar groups generally require longer or large
hydrophobic groups to maintain the overall hydrophobic character.
See U.S. Pat. No. 3,917,527. Thus, for example, the hydrophobic
linker of this invention may be substituted with an amine or
quaternary ammonium group, an ether or thioether, an amide, or a
hydroxyl group.
[0048] Preferred embodiments of the invention conform to the
general formula (I):
##STR00003##
as described generally above. In this formula, R.sub.1, R.sub.2,
R.sub.4, and R.sub.5 are independently selected from H,
C.sub.1-6-alkoxy, C.sub.1-6-alkyl-C.sub.1-6-alkoxy, aryl,
C.sub.1-6-alkaryl, --NR'C(O)R'', --C(O)NR'R'', and hydroxy.
Preferably, R.sub.1, R.sub.2, R.sub.4, and R.sub.5 are
independently selected from H and C.sub.1-6-alkyl. The most
preferred embodiments are those in which R.sub.1 and R.sub.2 are H,
while R.sub.4 and R.sub.5 are C.sub.1-6-alkyl.
[0049] Depending upon the desired terminal binding functionality,
R.sub.6 is selected from the group consisting of H,
C.sub.1-6-alkyl, aryl, C.sub.1-6-alkaryl, --C(O)OH, --S(O).sub.2OH,
and --P(O)(OH).sub.2. The terminal binding functionality as a whole
is thus represented generally by --(NR.sub.5)(R.sub.3')Y--R.sub.6
in formula (I). In one preferred embodiment, for example, d' is 1,
thus giving the terminal binding functionality as an amine (when
(R.sub.3')Y is absent) or a quaternary ammonium salt (when
(R.sub.3')Y is present). In these embodiments, R.sub.6 is
preferably C.sub.1-6-alkyl.
[0050] In other embodiments, d' is 0, thus providing for a terminal
binding functionality that is represented predominantly by R.sub.6.
In these cases, R.sub.6 is preferably chosen from H,
C.sub.1-6-alkyl, aryl, and C.sub.1-6-alkaryl groups when a
hydrophobic terminal binding functionality is desired. Where the
terminal binding functionality is a cation exchange group, R.sub.6
is accordingly chosen from --C(O)OH, --S(O).sub.2OH, and
--P(O)(OH).sub.2.
[0051] The moieties (R.sub.3)X and (R.sub.3')Y, when they are
present in formula (I), form quaternary ammonium salts with the
respective nitrogen atoms to which each moiety is bound. As
required by formula (I), X and Y represent anions. No particular
requirements restrict the identity of these anions, so long as they
are compatible with the prescribed use of the chromatographic
material. Exemplary anions in this regard include fluoride,
chloride, bromide, iodide, acetate, nitrate, hydroxide, sulfate,
carbonate, borate, and formate.
[0052] The balance of formula (I), therefore, generally represents
the hydrophobic linker. Consistent with the definition of a
hydrophobic group as defined hereinabove, the linker is hydrophobic
overall, which property is achieved preferably by incorporating
alkylene chains into the linker, corresponding to the selection of
a, a', a'', and a'. Preferably, at least one of a, a', a'', and
a''' is 2 or 3, more preferably at least two of a, a', a'', and
a''' are 2 or 3, and most preferably a is 3 while a' is 2, 3, 4, 5,
or 6.
[0053] In preferred embodiments, the linker is thiophilic in
addition to being hydrophobic. Accordingly, one or both of het and
het' in formula (I) are chosen from increasingly thiophilic groups
--S--, --S(O)--, and --S(O).sub.2--, S being most preferred. In the
most preferred chromatographic material, het is S while het' is
absent.
[0054] The inventors have discovered that certain subsets of
chromatographic materials are particularly efficacious. This is so
because the materials present significant patches or regions of
hydrophobicity in the hydrophobic linker, which property is
generally achieved by coupling alkylene fragments together. Thus,
at least two of (CR.sub.1R.sub.2).sub.a, (CR.sub.1R.sub.2).sub.a',
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' represent
two unsubstituted ethylene groups (i.e., --CH.sub.2--CH.sub.2--).
Alternatively, the hydrophobic linker can comprise at least two
unsubstituted propylene groups. That is, at least two of
(CR.sub.1R.sub.2).sub.a, (CR.sub.1R.sub.2).sub.a',
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' represent
two propylene groups (i.e., --CH.sub.2--CH.sub.2--CH.sub.2--). In
another embodiment, the hydrophobic linker can comprise at least
one unsubstituted ethylene group and at least one mono-substituted
propylene group. For example, at least one of
(CR.sub.1R.sub.2).sub.a, (CR.sub.1R.sub.2).sub.a',
(CR.sub.1R.sub.2).sub.a'' and (CR.sub.1R.sub.2).sub.a''' is
--CH.sub.2--CH.sub.2-- and at least one is --C.sub.3H.sub.5(OH)--.
In another embodiment, the hydrophobic linker can comprise at least
two mono-substituted propylene groups. For example, at least two of
(CR.sub.1R.sub.2).sub.a, (CR.sub.1R.sub.2).sub.a',
(CR.sub.1R.sub.2).sub.a'', and (CR.sub.1R.sub.2).sub.a''' are
--C.sub.3H.sub.5(OH). In these embodiments the alkylene groups can
be separated by a heteroatom or a group comprising a heteroatom,
such as --O--, --S--, --NH-- or --C(O)N(H)--. All combinations of
these are contemplated.
[0055] More specifically, one embodiment incorporates an
unsubstituted propylene group and an unsubstituted ethylene group
that are separated by het or het' in general formula (I), in which,
for example, a (or a'') is 3, a' (or a''' is 2), and b (or b') is
1. In this embodiment, it is possible, however, to substitute the
propylene group with one hydroxyl group and maintain the overall
hydrophobicity of the linker.
[0056] In another preferred embodiment, the hydrophobic linker
comprises two unsubstituted propylene groups that are separated by
het or het'. Thus referring to general formula (I), a and a' are
both 3 while b is 1, or a'' and a''' are both 3 while b' is 1.
[0057] In yet another preferred embodiment, the hydrophobic linker
comprises an unsubstituted propylene group and at least an
unsubstituted pentylene group that are separated by het, thus
corresponding to a being 3, a' being 5, and b being 1 in general
formula (I). In this embodiment, the propylene group can be
substituted once with a hydroxyl group.
[0058] In still another preferred embodiment, the hydrophobic
linker comprises two unsubstituted propylene groups that are
separated by one amino moiety. Referring therefore to general
formula (I), a or a' is 3, the other being 0; a'' or a''' is 3; het
and het' are absent; and c is 0 while d is 1.
[0059] In general formula (I), the wavy line represents the solid
support to which the hydrophobic linker is attached. It is
understood for the purpose of clarity, however, that general
formula (I) depicts only one (1) linker-terminal binding
functionality as being tethered to the solid support. The inventive
chromatographic materials actually exhibit linker-terminal binding
functionality densities of about 50 to about 150 .mu.mol/mL
chromatographic material, preferably about 80 to about 150
.mu.mol/mL, and more preferably 100 to about 150 .mu.mol/mL.
[0060] "Alkyl", as used herein, refers to a straight or branched
hydrocarbon having 1 to 12 carbon atoms, preferably 1 to 6 carbon
atoms. Exemplary alkyl groups are methyl, ethyl, propyl, butyl,
pentyl, and hexyl. An alkyl fragment that is part of a chain is
necessarily divalent and is referred to as an "alkylene" group.
[0061] "Aryl," as used herein, refers to a cyclic, fused or
non-fused, fully aromatic hydrocarbon that has 6 to 12 carbon
atoms. Exemplary aryl groups include but are not limited to phenyl,
naphthyl, and biphenyl.
[0062] "Alkaryl" thus refers to an alkyl group that is substituted
by an aryl group, as each are defined above. Exemplary alkaryl
groups include but are not limited to benzyl and phenethyl.
[0063] "Alkoxy," as used herein, refers to a group of the formula
--O-alkyl, wherein alkyl is defined above. Exemplary alkoxy groups
include but are not limited to methoxy and ethoxy.
[0064] "Alkylalkoxy," as used herein, is an alkyl moiety that is
substituted by an alkoxy moiety, i.e., corresponding to the general
formula -alkylene-O-alkyl.
[0065] As mentioned above, a key advantage of the present invention
is that the chromatographic material binds a variety of biological
substances at physiological ionic strength. Accordingly, it is
understood that the invention preferably does not include
chromatographic sorbents that do not bind bovine albumin at
physiological ionic strength.
[0066] Without limiting themselves to any particular theory, the
inventors believe that the chromatographic material of this
invention operates via combined interactions between the
chromatographic material and a substance. In the context of
separating biological substances, it is believed that mild
hydrophobic and/or thiophilic interactions between the
chromatographic material and a biological substance reinforce the
stronger electrostatic attractions arising from anion or cation
exchange terminal groups. It is also believed that mild ionic and
thiophilic interactions between the chromatographic material and a
biological substance reinforce the strength of interactions arising
from hydrophobic terminal groups. The primary advantage that flows
from these combined interactions is that salts at physiological
ionic strength do not disrupt the selective adsorbent properties of
the chromatographic material.
[0067] B. Solid Support
[0068] This invention contemplates a solid support to which the
hydrophobic linker and terminal binding functionality are attached.
Two different formats are contemplated in particular. In one
format, the solid support is of the form typically used for
chromatography media, that is, a bead or particle. These beads or
particles are derivatized with the combination hydrophobic linker
and terminal binding functionality. The beads or particles form a
chromatography medium that one can use to pack a column, for
example. In another format, the solid support takes the form of a
chip, that is, a solid support having a generally planar surface to
which the hydrophobic linker and terminal binding functionality can
be attached, covalently or otherwise. Chips that are adapted to
engage a probe interface of a detection device such as a mass
spectrometer are also called "probes."
[0069] 1. Beads and Particles
[0070] In accordance with the teachings of this invention, the
chromatographic material first comprises a solid support, which may
comprise an organic material. Exemplary organic materials are
polysaccharides, such as cellulose, starch, agar, agarose, and
dextran. Hydrophilic synthetic polymers are contemplated, including
substituted or unsubstituted polyacrylamides, polymethacrylamides,
polyacrylates, polymethacrylates, polyvinyl hydrophilic polymers
such as polyvinyl alcohol, polystyrene, polysulfone, and copolymers
or styrene and divinylbenzene, and mixtures thereof. Alternatively,
inorganic materials may be used as the solid support material. Such
inorganic materials include but are not limited to porous mineral
materials, such as silica; hydrogel-containing silica, zirconia,
titania, alumina; and other ceramic materials. It is also possible
to use mixtures of these materials, or composite materials formed
by copolymerization of or by an interpenetrated network of two
materials, such as those disclosed in U.S. Pat. No. 5,268,097, No.
5,234,991, and No. 5,075,371.
[0071] The solid support may be in the form of beads or irregular
particles of about 0.1 mm to about 1000 mm in diameter.
Alternatively, the solid support can be fashioned into fibers,
membranes, or sponge-like materials permeated with holes in the
micron to multi-millimeter sizes.
[0072] 2. Biochip
[0073] A preferred embodiment has the chromatographic material,
thus described, in a "biochip" or microarray format, where the
material presents a generally planar surface to which is attached a
capture reagent: in the present context, a combination of a
hydrophobic linker and a terminal binding functionality. Thus, a
biochip presents a defined region or site--more typically, a
collection of defined regions or sites--on which analytes may be
captured selectively. Upon capture, analytes can be detected and,
optionally, characterized by a variety of techniques, described in
more detail below.
[0074] Thus, the solid support can comprise a metal, such as gold,
aluminum, iron, titanium, chromium, platinum, copper and their
respective alloys. Such metals can be derivatized on their surfaces
with silicon dioxide, for instance, to provide reactive groups for
linking. One method of derivatizing a metal surface is to sputter a
metal oxide, such as silicon oxide, onto the metal surface.
Alternatively, the solid support can comprise silicon, glass or an
organic polymer, such as a plastic. In certain embodiments, the
solid support can be transparent.
[0075] Notably, the arrangement of sites on the surface of a
biochip of the invention preferably permits interrogation of
multiple sites at the same time, to achieve higher throughput and
speed. The use of a biochip is therefore essentially equivalent to
concurrently conducting multiple chromatographic experiments, each
with a different chromatographic column, but the present biochip
has the advantage of requiring only a single system.
[0076] Thus, it is preferable that an inventive biochip comprise a
plurality of addressable locations, and to each such location is
tethered a unique combination of hydrophobic linker and terminal
binding functionality. The biochip can incorporate a single
addressable location or as many as 8, 10, 16, 100, 1000, 10,000 or
more addressable locations, which need only be as large as an
impinging energy source, such as a laser. In this regard,
"addressable" connotes a position on the chromatographic material
that can be located, e.g., by an energy source, using an
appropriate addressing scheme or algorithm. Thus, each addressable
location or subsets of locations can bind a biological substance
preferentially, and the binding can be located by virtue of the
fact that capture occurs at a defined location on the biochip.
[0077] The addressable locations can be arranged in any pattern but
preferably appear in regular patterns, such as lines or orthogonal
arrays, or even as curves, such as circles. Circular arrangements
of the addressable locations are particularly useful on disk-shaped
biochips. Thus arranged, the addressable locations can provide
known gradients of binding capacity on the chromatographic
material.
[0078] In a particularly preferred embodiment, the present
chromatographic material in the form of a biochip is a probe for
use in a detection instrument, such as a mass spectrometer,
therewith providing a powerful analytic tool for the capture and
identification of known and unknown biological analytes.
Illustrative probes are described in U.S. Pat. No. 6,225,047, which
is incorporated herein by reference. For example, a mass
spectrometer probe ("MS probe") refers to a device that, when
positionally engaged in an interrogatable relationship to an
ionization source, e.g., a laser desorption/ionization source, and
in concurrent communication at atmospheric or subatmospheric
pressure with the detector of the preferred Laser
Desorption/Ionization Time-Of-Flight spectrometer, can be used to
introduce ions derived from an analyte into the spectrometer.
Preferred laser sources include nitrogen lasers, Nd-Yag lasers and
other pulsed laser sources. Thus, a MS probe typically is
reversibly engageable (e.g., removably insertable) with a probe
interface that positions the MS probe in an interrogatable
relationship with the ionization source and in communication with
the detector.
[0079] In another embodiment, the biochip comprising an attached
hydrophobic linker and terminal binding functionality is adapted
for SEND (Surface Enhanced Neat Desorption). This is accomplished
by attaching to the solid support molecules that absorb laser
energy and promote desorption and ionization of an analyte into the
gas phase. The energy absorbing molecules are attached to the
surface in such a manner that they do not envelop the analyte in a
matrix crystal and they are not desorbed upon contact with ionizing
energy. SEND is further described in U.S. Pat. No. 6,124,137
(Hutchens and Yip) and WO 03/064594 (Kitagawa).
II. Process for Making The Chromatographic Material
[0080] The terminal binding functionality described above is
chemically immobilized on the solid support by forming covalent
bonds between the solid support and the hydrophobic linker, and
between the hydrophobic linker and terminal binding functionality.
In typical scenarios, the solid support is first treated with a
bifunctional reagent which serves to introduce onto the solid
support reactive groups that form part or all of the hydrophobic
linker. For some solid supports, such as cellulose, composites
containing a hydrogel, or other materials presenting hydroxyl
groups, it is often advantageous to deprotonate the hydroxyl groups
with a hydroxide source, for example, prior to reaction with a
bifunctional reagent. The bifunctional reagent is capable of
reacting both with the solid support and with reagents that contain
the terminal binding functionality. Illustrative bifunctional
reagents, which contain the same or different functional groups,
include but are not limited to epichlorhydrin, epibromhydrin,
dibromo- and dichloropropanol, dibromobutane, ethylene glycol
diglycidylether, butanediol diglycidylether, divinyl sulfone,
allylglycidylether, and allyl bromide. Allyl heterofunctional
compounds, such as allyl bromide, are preferred bifunctional
reagents.
[0081] Once functionalized, the solid support is then washed
extensively with one or more solvents to remove unreacted
bifunctional reagent, reaction byproducts, or both. A typical
solvent used in this regard is water.
[0082] The terminal binding functionalities then are introduced by
way of reagents that contain such functionalities. Such reagents
react with the functional groups that are presented by the
functionalized solid support as described above.
[0083] The particular pairing of a bifunctional reagent with a
terminal binding functionality reagent is guided by well-known
chemistries. For example, solid supports that are functionalized
with epoxides may undergo reactions with mercapto, hydroxy, or
amino-containing reagents to furnish a substrate with
ethylene-containing linking groups. Other solid supports that are
modified with allyl bromide, for example, present alkene groups
that can be reacted directly with mercapto-containing reagents,
thereby providing hydrophobic linkers that contain sulfur atoms.
Alternatively, the alkene groups can be further brominated to
furnish suitably reactive bromo derivatives.
[0084] Preferred embodiments include those in which the hydrophobic
linker comprises sulfur atoms to increase the thiophilicity of the
resultant chromatographic material. Sulfur atoms can be introduced
in several ways, depending upon the source of the sulfur atom. The
first, as described above, is the direct reaction of the solid
support with a sulfur-containing bifunctional reagent such as
divinylsulfone (DVS). In this instance, the reagent comprising the
terminal binding functionality need only to react with the vinyl
group presented by the DVS-activated solid support.
[0085] An alternative way to introduce sulfur atoms into the
hydrophobic linker is by way of the reagents that comprise the
terminal binding functionalities. In preferred embodiments,
suitable reagents in this regard already possess sulfur atoms in
their structures, e.g., trimethylaminoethylmercaptan,
trimethylaminopropylmercaptan, and diethylaminoethylmercaptan.
Other reagents that do not contain sulfur but contain a primary
amine, such as diethylaminopropylamine, dimethylaminopropylamine,
and their corresponding quaternary ammonium salt derivatives, can
be reacted first with N-acetyl-homocysteine thiolactone. In this
scenario, the products are thiol-containing reagents that contain
part of the hydrophobic linkers and comprise the terminal binding
functionalities. The thiol portions of these reagents thus react
according to known methods with suitably activated solid supports,
such as those presenting alkene and bromo groups as described
above.
[0086] In a second alternative route, the solid support, activated
as described above, is treated with an intermediate bifunctional
reagent containing the desired sulfur-containing moiety. The
product of this reaction is then treated with the reagent
comprising the terminal binding functionality. An illustrative
example in this regard is the reaction between an allyl-activated
solid support, as described above, with mercaptohexanoic acid. The
resultant pendant carboxyl groups can be reacted with any
convenient terminal binding functionality reagent that bears, for
example, a primary amine. In embodiments that employ this
methodology, it may be necessary to use coupling reagents such as
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) or
commonly-known carbodiimides such as dicyclohexylcarbodiimide
(DCC).
[0087] The concentration of immobilized hydrophobic linker and
terminal binding functionality can vary between a fraction of a
micromole to several hundred micromoles per milliliter of solid
support, depending upon the concentration of bifunctional reagent
used to make the solid support. Low concentrations of the
immobilized group typically result in low separation capacity of
the chromatographic material, whereas high concentrations generally
lead to increased capacity.
III. Methods of Using the Chromatographic Material
[0088] A benefit of the present invention is the ability of the
inventive chromatographic material to bind analytes at
physiological ionic strength, in contrast to conventional
chromatographic materials that require the use of extreme ionic
strengths. Thus in preferred embodiments, the chromatographic
material of the present invention can be used to separate and
isolate a variety of substances, including biologically relevant
molecules such as proteins, viruses, nucleic acids, carbohydrates,
and lipids. In many instances, the substances can be separated
without any modification to raw feedstock under the conditions
prescribed for the use of the chromatographic material as described
below. Other substances that are suitable for separation include
oligo- and polysaccharides, pigments, lipopolysaccharides,
polypeptides, and synthetic soluble polymers. The biological
substances typically derive from, or are contained in, sources
including but not limited to liquid samples such as saliva, blood,
urine, lymphatic fluid, prostatic fluid, seminal fluid, milk, milk
whey, organ extracts, plant extracts, cell extract, cell culture
media, fermentation broths, serum, ascites fluid, and transgenic
plant and animal extracts.
[0089] In this context, a particularly preferred class of
biological substances is immunoglobulins. The "immunoglobulins"
category embraces whole immunoglobulins, including monoclonal and
polyclonal antibodies, as well as Fab, F(ab').sub.2, F.sub.c and
F.sub.v fragments thereof.
[0090] A. Method of Separating Substances
[0091] The liquid sample containing one or more biological
substances is contacted with the chromatographic material of this
invention for a period of time sufficient to allow at least one
biological substance to bind to the chromatographic material.
Typically, the contact period is between about 30 seconds to about
12 hours.
[0092] As mentioned above, an advantage of the present invention is
that the pH, ionic strength, or both of the liquid sample need not
be adjusted prior to contacting the sample with the chromatographic
material. Additionally, it is not necessary to concentrate, dilute,
or mix the sample with additives such as salts. Thus, it is
possible to directly load a liquid sample onto the chromatographic
material of this invention. The pH of liquid samples can be
adjusted, however, to experimentally determined values that are
optimized for adsorption. Typical capture pH values for a range of
proteins is from about 4 to about 10. Preferably, a pH in the range
of about 4 to about 7 promotes protein adsorption to those
chromatographic materials that feature a cation exchange moiety,
while a pH in the range of about 6 to about 10 will accomplish the
same where anion exchange moieties are employed.
[0093] Many biological substances will readily adsorb to the
chromatographic material at physiological ionic strength.
Physiological ionic strength, as used herein, generally ranges from
about 15 to about 20 mS/cm. Typical salt concentrations that
correspond to this range fall within about 0.1 to about 0.2 M,
preferably 0.14 to about 0.17 M.
[0094] The temperature at which the liquid sample is contacted with
the chromatographic material varies between samples and a given
chromatographic material. Preferably, the temperature is ambient,
but can be changed.
[0095] After the sample is contacted with the chromatographic
material, the chromatographic material is preferably washed with an
equilibration buffer. As defined herein, an equilibration buffer is
a buffer that is preferably of the pH at which the liquid sample
was contacted with the chromatographic material. Furthermore, the
equilibration buffer washes from the chromatographic material any
substance that does not adsorb to the substrate. Suitable
equilibration buffers include acetate buffer and phosphate buffered
saline. The washing may be accomplished by bathing, soaking, or
dipping the chromatographic material with bound biological
substance into the equilibration buffer. Alternatively, the
equilibration buffer may be rinsed, sprayed, or washed over the
chromatographic material.
[0096] The desired biological substance typically is one that
adsorbs to the chromatographic material. However, the invention
contemplates scenarios in which the biological substance of
interest is removed in the equilibration buffer washing. In this
case, the substance may be isolated from the buffer by routine
methods.
[0097] Biological substances that are adsorbed to the
chromatographic material are then desorbed in one embodiment by
adjusting the pH to a value where the substance can desorb. The pH
at which desorption occurs will depend upon the substance and upon
a given chromatographic material. For example, for chromatographic
materials that comprise an anion exchange moiety, desorption
generally occurs over a pH gradient starting at about pH 8 and
decreasing to about pH 3. For chromatographic materials that
comprise a cation exchange moiety, the pH gradient applied starts
at about pH 4 and is increased to about pH 11. For chromatographic
materials that feature primarily hydrophobic groups, the pH
gradient for desorption starts at about pH 7 is decreased to about
pH 3. In the last scenario, preferably an ionic strength gradient
is also applied as described below. The pH can be adjusted by any
routinely available reagent, such as aqueous solutions of Tris-HCl
or carbonate buffers.
[0098] In some instances, as mentioned above, adjustment of the
eluant ionic strength can increase effectiveness of the
chromatographic material. Thus for chromatographic materials that
comprise primarily hydrophobic groups, the ionic strength can be
decreased concomitantly with pH. This is especially so for
materials that additionally comprise --NH-- moieties, which can
give rise to mild ionic charges that become more effective as the
ionic strength is decreased. The use of salt gradients is
well-known in the art. Typically, salt concentrations for the
present chromatographic material need not exceed about 0.5 M.
[0099] The desorbed biological substance is then collected. Typical
purities of biological substances, such as antibodies, that are
purified by the method of this invention range from about 70% to
about 99%, preferably 85% to about 99%, and most preferably about
90% to about 99%.
[0100] The separation method described above can be adapted for use
in a variety of techniques, including preparative methods employing
fixed bed, fluidized bed, and batch chromatographies.
Alternatively, the method can be practiced in the context of high
throughput separation techniques that utilize small devices such as
spin columns or multiwell plate formats where device volumes can be
as small as a few microliters.
[0101] The techniques mentioned above comprise contacting a
solution containing the biological substances with the
chromatographic material, thereby leading to the selective
adsorption of at least one biologicial substance in the solution by
the chromatographic material. In the event of the desired
biological substance(s) being fixed to the chromatographic
material, the elution of the latter allows it or them to be
separated and collected in a purified and concentrated form. If the
desired biological substance remains in the treated solution (the
other biological substances being fixed to the chromatographic
material) then the desired separation is obtained directly by
collecting the eluant.
[0102] When using batch adsorption/separation, the chromatographic
material is added directly to the solution of biological
substances, and the chromatographic material/biological substance
mixture is gently agitated for a time sufficient to allow the
biological substances to bind to the chromatographic material. The
chromatographic material, with adsorbed biological substances, may
then be removed by centrifugation or filtration, and the biological
substances subsequently eluted from the chromatographic material in
a separate step.
[0103] Alternatively, column chromatography may be used. In fixed
bed column chromatography, the chromatographic material is packed
into a column, and the solution which contains the biological
substances to be separated is applied to the chromatographic
material by pouring it through the chromatographic material at a
rate that allows the biological substances to bind to the
chromatographic material.
[0104] Advantages of fixed bed chromatography include minimal
column volume and water consumption. The disadvantage of the column
chromatography method is that the flow rate of liquids through the
column is slow, and, therefore, time-consuming. This flow rate can
be reduced even further if the material being applied to the column
includes particulates, since such particulate material can "clog"
the chromatographic material to some degree.
[0105] In fluidized bed column chromatography, a rising filtration
flow and large/dense particles are used in order to maintain an
equilibrium against the rising forces. An essentially vertical
column composed of between 1 and 5 stages placed on top of the
other is used, and the solution successively passes through each
stage and is drawn off by an overflow on the upper part of the
upper stage. Preferably, the column has three stages. Each stage,
with the exception of the uppermost one, is separated by two
distribution systems, one distributing the solution at the base of
the stage in question, the other distributing the solution towards
the stage located immediately above.
[0106] The advantages of a fluidized bed are higher flow rates at
lower pressures as compared to fixed bed chromatography. Although
the higher flow rates offer certain advantages to the
chromatographic separation, the method has several shortcomings.
The method requires either large particle diameter and/or high
density chromatographic materials that expand only under high
upward liquid velocity. Large diameter resins have less surface
area per unit volume than small chromatographic materials used, and
correspondingly have less surface binding capacity. This is why
small bead chromatographic materials are preferred, in which case
the bead chromatographic materials must be highly dense.
[0107] On the other hand, fluidized bed chromatography avoids many
of the serious disadvantages of fixed beds, which include clogging,
need for cleaning, compression and cleaning-induced chromatographic
material deterioration. In fact, the fluidized bed allows free
passage of solid impurities in the solution with no risk of
clogging; less stringent cleaning is necessary so the life-span of
the chromatographic materials is greatly increased. However, the
chromatographic material for biological substances typically are
not suitable for fluidized bed chromatography, having a density too
close to that of water or being too small in granulometry. This
makes it impossible to fluidize without drawing particles into the
flux. Another problem with fluidized bed chromatography of
biological substances relates to the large space between beads,
which would result in a decrease in efficiency.
[0108] In view of these factors, batch and fixed bed chromatography
have been the methods of choice in prior art separation techniques
for biological substances. The present chromatographic material, on
the other hand, can be used in batch, fixed bed, or fluidized bed
chromatography.
[0109] B. Chromatography Column
[0110] Thus, in a preferred embodiment, the present invention
provides a chromatography column, which is a tubular member packed
with the chromatographic material described herein. The tubular
member can be made of any suitable material, such as glass,
plastic, or metal. The packed chromatographic material is abutted
on each end by porous members that keep the substrate fixed within
the tubular member.
[0111] In some embodiments, gravity flow of an eluant through a
column is sufficient. In other embodiments, the column may comprise
one or more fluid moving devices to achieve an upward flow of
eluant through the column. Such devices include pumps, injectors,
and any other device typically employed in association with
chromatography equipment.
[0112] The chromatography column of this invention can be of any
volume. For example, separations on a laboratory scale may warrant
a column volume as small as about 1 milliliter or even about 1
microliter. Large scale purification and isolation of biological
substances can be performed on columns as large as 5000 liters.
More typical volumes are between 1 liter and 100 liters. The column
is tubular in general shape, but is not otherwise particularly
limited in length or diameter. Depending upon the context in which
the column is employed, the column diameter can vary between about
0.5 mm to about 1000 mm. Additionally, the column length can vary
between about 50 mm to about 1000 mm. Thus, the invention
contemplates columns of a variety of dimensions and corresponding
volumes.
[0113] The column of this invention can be used in tandem with
columns comprising other chromatographic materials, which would be
effective in eliminating different impurities from a sample. Thus,
the advantages of the present column can be viewed as being
complementary to the characteristics of other or conventional
columns. In this context, such a tandem arrangement of columns
would conserve eluants and equilibration buffer, thereby
eliminating the need for additional sample manipulation and
preparation.
[0114] C. Method of Detecting an Analyte
[0115] This invention provides a convenient method of detecting an
analyte. An addressable location of the biochip as described above
is contacted with a sample that contains at least one analyte. The
analyte can be a biological substance, such as those described
herein, which adsorbs to (i.e., is captured at) the addressable
location. The present method thus accommodates the detection of a
plurality of analytes contained in a single sample, each analyte
being bound to a unique location on the biochip.
[0116] The biochip is then preferably washed with an eluant as
described above to remove unbound materials. In this context, the
introduction of eluant to small diameter spots of the
chromatographic material is best accomplished by a microfluidics
process.
[0117] Detection of analytes that remain bound to the biochip can
be accomplished by a variety of methods. These include microscopy
and other optical techniques, mass spectrometry, and electrical
techniques. Light-based detection parameters include, for example,
absorbance, reflectance, transmittance, birefringence, refractive
index, and diffraction measurement techniques.
[0118] Fluorescence detection of labeled analytes is particularly
popular. Methods involving fluorescence include direct and indirect
fluorescent measurement. Specific methods include, for example,
fluorescent tagging in immunological methods such as ELISA or
sandwich assay.
[0119] Other useful techniques include, for example, surface
plasmon resonance, ellipsometry, resonant mirror techniques,
grating coupled waveguide techniques, multipolar resonance
spectroscopy, impedimetric detection, chemiluminescence detection,
and electrical conductivity/reduction-oxidation methods. Methods of
desorbing and/or ionizing analytes from biochips for direct
analysis are well known in the art, and are generally described,
for example, in U.S. Pat. No. 6,225,047.
[0120] A particularly preferred method of analysis is
Surface-Enhanced Laser Desorption/Ionization ("SELDI"), which is
described in, for example, U.S. Pat. No. 5,719,060 and No.
6,255,047. In SELDI, an addressable location on the biochip is
presented to an energy source such as a laser, which desorbs and
ionizes the analyte bound at the addressable location. The ionized
analyte is then detected directly in a time-of-flight ("TOF") mass
spectrometer, for example, thereby yielding the mass-to-charge
ratio of the desorbed analyte. By repeatedly shifting and
positioning the biochip within the probe interface to align with
the laser, each addressable location on the biochip can be
similarly analyzed.
[0121] Additionally, an ion mobility spectrometer can be used to
analyze samples. The principle of ion mobility spectrometry is
based on different mobility of ions. Specifically, ions of a sample
produced by ionization move at different rates, due to their
difference in, e.g., mass, charge, or shape, through a tube under
the influence of an electric field. The ions, which are typically
in the form of a current, are registered at the detector which can
then be used to identify the sample. One advantage of ion mobility
spectrometry is that it can operate at atmospheric pressure.
[0122] Furthermore, a total ion current measuring device can be
used to analyze samples. This device can be used when the probe has
a surface chemistry that allows only a single class of analytes to
be bound. When a single class of analytes is bound on the probe,
the total current generated from the ionized analyte reflects the
nature of the analyte. The total ion current from the analyte can
then be compared to stored total ion current of known compounds.
Therefore, the identity of the analyte bound on the probe can be
determined.
[0123] An advantage of the biochips and analytical method of this
invention is that binding and detecting analytes are effective in
picomolar or even attomolar amounts of analyte. In accordance with
the teachings of this invention, it is thus possible to discover
certain subclasses of biological substances referred to as
biomarkers. In the present context, a biomarker is an organic
biological substance, particularly a polypeptide or protein, which
is differentially present in a sample taken from a diseased subject
as compared to a sample taken from a healthy subject. A biomarker
is differentially present in samples taken from diseased subjects
if it is present at an elevated level or a decreased level relative
to the level present in a sample taken from a healthy subject. The
chromatographic material of the present invention, particularly in
the form of a biochip, allows the rapid discovery and
identification of biomarkers.
[0124] This method is useful for protein profiling, in which
proteins in a sample are captured using one or more different
chromatographic materials of this invention and then the captured
analytes are detected. In turn, protein profiling is useful for
difference mapping, in which the protein profiles of different
samples are compared to detect differences in protein expression
between the samples.
[0125] The following examples are given to illustrate the present
invention. It should be understood, however, that the invention is
not to be limited to the specific conditions or details described
in these examples. All references to publicly available documents,
including patents, are incorporated herein by reference as if set
forth fully in their entireties.
Example 1
Preparation of a Dimethylaminopropyl Thiol Derivative of
Cellulose
[0126] 100 ml of cellulose beads were washed extensively with 1M
sodium hydroxide solution and then with water until neutral pH was
obtained. To the drained beads were added 18 ml of 0.5 M sodium
hydroxide and 10 ml of allylbromide. The resultant mixture was then
stirred vigorously overnight at room temperature.
[0127] The beads were washed with water to remove by-products,
yielding allyl-cellulose for a substrate for the attachment of
various functionalities.
[0128] The beads of allyl-cellulose were condensed with 25 g of
dimethylaminopropylthiol to give the material as shown below:
##STR00004##
[0129] The product resin exhibited a dimethylamino moiety density
of 150 mmol/mL of resin. Binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 39 mg/mL.
Example 2
Preparation of a Dimethylaminoethyl Thiol Derivative of
Cellulose
[0130] 100 mL of allyl cellulose beads, obtained according to the
protocol described above in example 1, were condensed with 25 g of
dimethylaminoethylthiol to give the following material:
##STR00005##
[0131] The product exhibited a dimethylamino moiety density of 87
.mu.mol/mL of resin. The binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 21 mg/mL. The terminal
dimethylamino moiety was quaternized by the addition of
diethylaminoethyl chloride at pH 12 for 6 hours to give a
derivative, which exhibited a binding capacity of the resin reached
27 mg of albumin per mL.
Example 3
Coupling of Dimethylaminopropylamine to an Allylated Solid
Support
[0132] 100 mL of allyl cellulose beads obtained according to the
protocol described above in example 1 were condensed with 25 g of
dimethylaminopropylamine to give the following material:
##STR00006##
[0133] The product exhibited a dimethylamino moiety density of 126
.mu.mol/mL of resin. The binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 38 mg/mL.
Example 4
Preparation of a Dimethylaminoethylamine Derivative of
Cellulose
[0134] 100 mL of allyl cellulose beads obtained according to the
protocol described abovein example 1 were first condensed with 25 g
of thiohexanoic acid and. The resultant carboxyl derivative was
then condensed with dimethylaminoethylamine, using EEDQ as a
condensation agent, to give the following product:
##STR00007##
[0135] The product exhibited a dimethylamino moiety density of 95
.mu.mol/mL of resin. The binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 35 mg/mL.
Example 5
Preparation of a Thiocholine Derivative of Cellulose
[0136] 100 mL of allyl cellulose beads obtained according to the
protocol described above in example 1 were condensed with 20 g of
thiocholine to give the following product:
##STR00008##
[0137] The product exhibited a trimethylammonium moiety density of
86 .mu.mol/mL of resin. The binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 22 mg/mL.
Example 6
Preparation of a Dimethylaminopropylamine Derivative of
Cellulose
[0138] 100 mL of allyl cellulose beads obtained according to the
protocol described above in example 1 were first condensed with 25
g of thiohexanoic acid. The resultant carboxyl derivative was
reacted with dimethylaminopropylamine, using EEDQ as a condensation
agent to give the following product:
##STR00009##
[0139] The product exhibited a dimethylamino moiety density of 70
.mu.mol/mL of resin. The binding capacity for bovine albumin at
physiological ionic strength and pH 8.6 was 13 mg/mL.
Example 7
Preparation of a Phenyl Ethyl Derivative of Cellulose
[0140] 100 ml of cellulose beads were washed extensively with 1M
sodium hydroxide solution and then with water until neutral pH was
obtained. To the drained beads were added 18 ml of 0.5 M sodium
hydroxide and 10 ml of allylbromide. The resultant mixture was then
stirred vigorously overnight at room temperature.
[0141] The beads were washed with water to remove by-products,
yielding allyl-cellulose for a substrate for the attachment of
various functionalities.
[0142] The allyl-cellulose beads were brominated by the addition of
potassium bromide and N-bromosuccinimide under acidic conditions
and then mixed with 5 g of phenylethyl mercaptan to give the
following product:
##STR00010##
[0143] The product exhibited a phenylethyl moiety density of about
50 .mu.mol/mL of settled beads. The binding capacity for bovine
albumin at physiological ionic strength and pH 7 was 27 mg/mL. By
contrast, a commercially available phenyl derivative for HIC
chromatography (Phenyl Sepharose) gave a binding capacity of 2
mg/mL.
Example 8
Preparation of an Phenyl-Propyl Hydrophobic Derivative of
Cellulose
[0144] 100 mL of allyl-cellulose beads as prepared according to
example 7 were brominated by addition of potassium bromide and
N-bromosuccinimide in acidic conditions and then mixed with 6 mL of
phenyl-propyl-amine to give the following product:
##STR00011##
[0145] The product exhibited a phenylpropyl moiety density of about
50 .mu.mol/mL of settled beads. The binding capacity for bovine
albumin at physiological ionic strength and pH 7 was of 45
mg/mL.
Example 9
Preparation of an Aliphatic Hydrophobic Derivative of Cellulose
[0146] 100 ml of allyl-cellulose beads prepared according to
example 7 were brominated by the addition of potassium bromide and
N-bromosuccinimide under acidic conditions and then mixed with 6 mL
of hexylamine-amine to give the following product:
##STR00012##
[0147] The product exhibited a hexylamino moiety density of about
55 .mu.mol/mL of settled beads. The binding capacity for bovine
albumin at physiological f ionic strength and pH 7 was 47 mg/mL. By
contrast, a similar product obtained using glucamine (a hydrophilic
ligand) instead of hexylamine gave a binding capacity of 4
mg/mL.
Example 10
Preparation of an Octanol Hydrophobic Derivative of Cellulose
[0148] 100 ml of allyl-cellulose beads prepared according to
example 7 were brominated by the addition of potassium bromide and
N-bromosuccinimide under acidic conditions and then mixed with 6 mL
of 1,8-thiooctanol to give the following product:
##STR00013##
[0149] The product exhibited an octanol moiety density more than 50
.mu.mol/mL of settled beads. The binding capacity for bovine
albumin at physiological ionic strength and pH 7 was higher than 40
mg/mL.
Example 11
Preparation of an Phenyl-Propyl Hydrophobic Derivative of Zirconia
Composite Beads
[0150] 50 ml of zirconia porous beads that were filled with an
agarose gel (6% by volume) were allylated using a similar protocol
as the one described in example 7 using 2 mL allylbromide and 2 mL
1 M sodium hydroxide. The resultant allylated zirconia composite
beads were washed extensively with water to eliminate by-products
and until neutral pH was obtained.
[0151] The allyl-derivative was brominated by the addition of
potassium bromide and N-bromosuccinimide under acidic conditions
and then mixed with 6 mL of phenyl-propyl-amine.
[0152] The product exhibited a phenylpropyl moiety density of about
60 .mu.mol/mL of settled beads. The binding capacity for bovine
albumin at physiological ionic strength and pH 7 was 30 mg/mL.
* * * * *