U.S. patent application number 11/236650 was filed with the patent office on 2006-02-02 for composite chromatographic sorbent of mineral oxide beads with hydroxyapatite-filled pores.
This patent application is currently assigned to Pall Corporation. Invention is credited to Egisto Boschetti, Pierre Girot.
Application Number | 20060021941 11/236650 |
Document ID | / |
Family ID | 23301519 |
Filed Date | 2006-02-02 |
United States Patent
Application |
20060021941 |
Kind Code |
A1 |
Boschetti; Egisto ; et
al. |
February 2, 2006 |
Composite chromatographic sorbent of mineral oxide beads with
hydroxyapatite-filled pores
Abstract
A new adsorbent of a porous mineral oxide material with apatite
crystals, preferably hydroxyapatite crystals, in the pores of the
mineral oxide material is disclosed. The adsorbent is useful for
protein and nucleic acid separations
Inventors: |
Boschetti; Egisto; (Croissy
sur Seine, FR) ; Girot; Pierre; (Paris, FR) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
700 THIRTEENTH ST. NW
SUITE 300
WASHINGTON
DC
20005-3960
US
|
Assignee: |
Pall Corporation
East Hills
NY
11548-1209
|
Family ID: |
23301519 |
Appl. No.: |
11/236650 |
Filed: |
September 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10305399 |
Nov 27, 2002 |
6972090 |
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11236650 |
Sep 28, 2005 |
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60333149 |
Nov 27, 2001 |
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Current U.S.
Class: |
210/656 ;
210/198.2; 210/502.1; 436/161 |
Current CPC
Class: |
B01J 20/0292 20130101;
B01J 20/28097 20130101; B01J 20/3268 20130101; B01J 20/0296
20130101; B01J 2220/52 20130101; B01J 20/048 20130101; B01J 20/3071
20130101; B01J 20/327 20130101; B01J 20/3234 20130101; B01J
20/28085 20130101; B01J 20/0211 20130101; B01J 20/282 20130101;
B01J 20/08 20130101; C07K 1/20 20130101; B01J 20/3274 20130101;
B01J 20/3064 20130101; B01J 20/286 20130101; B01J 2220/42 20130101;
B01J 2220/58 20130101; B01J 20/3078 20130101; B01J 20/3272
20130101; B01J 20/3282 20130101; B01J 20/3242 20130101; B01J
20/3236 20130101; B01J 20/3028 20130101; B01J 20/28004 20130101;
B01J 20/103 20130101; B01J 20/28078 20130101; B01J 20/3293
20130101; B01J 20/3204 20130101; B01J 20/06 20130101 |
Class at
Publication: |
210/656 ;
210/198.2; 210/502.1; 436/161 |
International
Class: |
B01D 15/08 20060101
B01D015/08 |
Claims
1-12. (canceled)
13. 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 composite
chromatography sorbent comprising: porous mineral oxide beads that
have a pore volume which exceeds about 10% of the bead volume and
an average pore diameter of at least about 500 .ANG., wherein the
pores of the beads contain apatite crystals which have been formed
within the pores of the beads by solutions that are allowed to
penetrate the pores, and wherein said composite chromatography
sorbent is packed within said tubular member between said first and
second porous members.
14. The chromatography column of claim 13, wherein the column
volume is between about 50 liters and about 5000 liters.
15. The chromatography column of claim 13, further comprising:
means for flowing a liquid sample upward through said composite
chromatography sorbent.
16. The chromatography column of claim 15, further comprising: a
series of stages between said inlet end and said outlet end.
17. A chromatographic separation method comprising: contacting a
solution comprising biomolecules with a composite chromatography
sorbent, wherein said composite chromatography sorbent comprises
porous mineral oxide beads that have a pore volume which exceeds
about 10% of the bead volume and an average pore diameter of at
least about 500 .ANG., wherein the pores of the beads contain
apatite crystals, wherein the solution permeates the pores of the
mineral oxide beads, and wherein some biomolecules in the solution
are bound to the apatite crystals and other, different biomolecules
remain in the solution.
18. A chromatographic separation method comprising: a first flowing
solution comprising biomolecules through 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 composite chromatography sorbent
comprising porous mineral oxide beads that have a pore volume which
exceeds about 10% of the bead volume and an average pore diameter
of at least about 500 .ANG., wherein the pores of the beads contain
apatite crystals, and wherein said composite chromatography is
packed within said tubular member between said first and second
porous members, wherein the solution permeates the pores of the
mineral oxide beads, and wherein some biomolecules in the solution
are bound to the apatite crystals and other, different biomolecules
remain in the solution.
19. The method of claim 18, further comprising: a second flowing
solution through the chromatography column to elute the
biomolecules bound to the apatite crystals.
20. The method of claim 18, wherein the biomolecules are selected
from the group consisting of polypeptides, nucleic acids,
antibodies, and glyco-iso-forms.
21. The method of claim 18, wherein the composite chromatography
sorbent is formed by a method comprising: (i) providing porous
mineral oxide beads that have a pore volume which exceeds about 10%
of the bead volume and an average pore diameter of at least about
500 .ANG.; (ii) contacting the porous mineral oxide beads with a
maximum of one pore volume of a solution of either (A) calcium
chloride or (B) potassium or sodium phosphate so that it permeates
the pores of the beads; (iii) drying the beads from (ii); (iv)
contacting the dried beads with a maximum of one pore volume of a
solution of the other of either (A) calcium chloride or (B)
potassium or sodium phosphate so that it permeates the pores of the
beads, thereby forming calcium phosphate in the pores; (v) washing
the beads from (iv) with water to eliminate excess calcium or
phosphate ions; (vi) contacting the washed beads from (v) with a
solution of sodium hydroxide; (vii) washing the beads from (vi)
with water; and (viii) contacting the washed beads from (vii) with
a solution of disodium phosphate to form hydroxyapatite crystals in
the pores of the beads.
22. The method according to claim 21, wherein the beads are washed
with a phosphoric acid solution before (ii).
23. A chromatographic separation method comprising: a first flowing
solution comprising biomolecules through 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; (c) a composite chromatography sorbent comprising
porous mineral oxide beads that have a pore volume which exceeds
about 10% of the bead volume and an average pore diameter of at
least about 500 .ANG., wherein the pores of the beads contain
apatite crystals, and wherein said composite chromatography is
packed within said tubular member between said first and second
porous members; and (d) means for flowing a liquid sample upward
through said composite chromatography sorbent, wherein the solution
permeates the pores of the mineral oxide beads, and wherein some
biomolecules in the solution are bound to the apatite crystals and
other, different biomolecules remain in the solution.
24. The method of claim 23, further comprising: a second flowing
solution through the chromatography column to elute the
biomolecules bound to the apatite crystals.
25. The method of claim 23, wherein the biomolecules are selected
from the group consisting of polypeptides, nucleic acids,
antibodies, and glyco-iso-forms.
26. The method of claim 23, wherein the composite chromatography
sorbent is formed by a method comprising: (i) providing porous
mineral oxide beads that have a pore volume which exceeds about 10%
of the bead volume and an average pore diameter of at least about
500 .ANG.; (ii) contacting the porous mineral oxide beads with a
maximum of one pore volume of a solution of either (A) calcium
chloride or (B) potassium or sodium phosphate so that it permeates
the pores of the beads; (iii) drying the beads from (ii); (iv)
contacting the dried beads with a maximum of one pore volume of a
solution of the other of either (A) calcium chloride or (B)
potassium or sodium phosphate so that it permeates the pores of the
beads, thereby forming calcium phosphate in the pores; (v) washing
the beads from (iv) with water to eliminate excess calcium or
phosphate ions; (vi) contacting the washed beads from (v) with a
solution of sodium hydroxide; (vii) washing the beads from (vi)
with water; and (viii) contacting the washed beads from (vii) with
a solution of disodium phosphate to form hydroxyapatite crystals in
the pores of the beads.
27. The method according to claim 26, wherein the beads are washed
with a phosphoric acid solution before (ii).
28-29. (canceled)
30. The chromatography column according to claim 13, wherein the
apatite crystals are hydroxyapatite crystals.
31. The chromatography column according to claim 13, wherein the
mineral oxide is zirconia.
32. The chromatography column according to claim 13, wherein the
apatite crystals are hydroxyapatite crystals and the mineral oxide
is zirconia.
33. The separation method of claim 17, wherein the apatite crystals
are hydroxyapatite crystals.
34. The separation method of claim 17, wherein mineral oxide is
zirconia.
35. The separation method of claim 17, wherein the apatite crystals
are hydroxyapatite crystals and the mineral oxide is zirconia.
36. The method of claim 18, wherein the apatite crystals are
hydroxyapatite crystals.
37. The method of claim 18, wherein mineral oxide is zirconia.
38. The method of claim 18, wherein the apatite crystals are
hydroxyapatite crystals and the mineral oxide is zirconia.
39. The method of claim 23, wherein the apatite crystals are
hydroxyapatite crystals.
40. The method of claim 23, wherein the mineral oxide is
zirconia.
41. The method of claim 23, wherein the apatite crystals are
hydroxyapatite crystals and the mineral oxide is zirconia.
42. The chromatography column of claim 13, wherein the mineral
oxide is selected from the group consisting of alumina, titania,
hafnia, silica, zirconia and mixtures thereof.
43. The chromatography column of claim 13, wherein the mineral
oxide comprises zirconia.
44. The chromatography column of claim 13, wherein the mineral
oxide comprises silica.
45. The chromatography column of claim 13, wherein the beads are
coated with a layer of a hydrophilic polymer.
46. The chromatography column of claim 13, wherein the apatite
crystals comprise: (a) calcium ions; and (b) a metal ion or a
metalloid ion.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a new adsorbent of a porous
mineral oxide material with apatite crystals, preferably
hydroxyapatite crystals, in the pores of the mineral oxide
material. The adsorbent is useful for protein and nucleic acid
separations.
[0002] Apatite is a calcium phosphate material in crystalline form
having the general formula Ca.sub.5(F, Cl, OH, 1/2 CO.sub.3)
(PO.sub.4).sub.3. One of the more common types of apatite is
hydroxyapatite which has the formula
[Ca.sub.2(PO.sub.4).sub.2].sub.3Ca(OH).sub.2. It is useful as a
packing material to be filled in columns for chromatographic
separation of biopolymers, for example, proteins, enzymes, and
nucleic acids. Its ability to adsorb such molecules depends on both
the structure of the crystal itself and on the exposed surface area
of the crystals.
[0003] The technique for the preparation of hydroxyapatite
utilizable for column chromatography was first developed by
Tiselius et al. [Arch. Biochem. Biophys., 65:132-155 (1956)].
Hydroxyapatite for column chromatographic use has been prepared by
various methods. Conventionally, hydroxyapatites are synthesized by
(1) wet synthesis in which a water-soluble calcium salt and
phosphate are allowed to react in aqueous solution, (2) dry
synthesis in which calcium phosphate and calcium carbonate are
allowed to react in the presence of water vapor at 900.degree. to
1400.degree. C., or (3) hydrothermal synthesis in which calcium
hydrogen-phosphate is hydrolyzed, for example, at 200.degree. C.
and 15 atmospheres. The hydroxyapatites produced in conventional
processes have been in the form of plates which have to be finely
divided, particularly when used as column packing material for
chromatographic separation. The plates are divided into tiny pieces
varied in shape and size. The irregular pieces of hydroxyapatite
cannot be packed uniformly or densely in the column for
chromatographic separation.
[0004] Hydroxyapatite in the form of plate-like crystals or
agglomerates of microcrystals also is inferior in mechanical
strength and tends to be destroyed during the packing operation and
measurement. Chromatographic characteristics of the hydroxyapatite
vary according to the packing method used, leading to variability
in separations and bed collapse.
[0005] In recent years, a process for producing microspherical
hydroxyapatite was proposed to overcome these shortcomings,
utilizing the so-called spray-drying method which is widely used
for manufacturing granules of a powdery substance (Japanese
Laid-open Patent Appln. Nos. Sho. 62-206445 and 62-230607).
According to the process disclosed in Japanese Laid-open Patent
Appln. No. 62-206445, microcrystals of hydroxyapatite having a
diameter of less than 1 .mu.m as primary particles are physically
coagulated by spray drying to form substantially spherical
particles of 1-10 .mu.m in diameter as second particles.
[0006] When the spherical hydroxyapatite particles obtained
according to these processes are subjected to classification by
screening to collect particles of a definite particle size as a
packing material for liquid chromatography, the spherical particles
tend to be destroyed because of their poor mechanical strength and
are broken to pieces when packed densely in a column under high
pressure. Consequently, the spherical hydroxyapatite particles
formed by spray drying have to be subjected to a heat treatment
carried out at a high temperature for a long period of time in
order to impart mechanical strength sufficient enough to withstand
high pressure on packing. The severe heat treatment, however,
causes the spherical particles tend to be bonded to one another in
a mutually fused state to form partially solid state granules.
[0007] Japanese Laid-open Patent Appin. No. Sho. 62-230607
discloses a process for preparing spherical agglomerates of apatite
in which a gelled hydroxyapatite slurry is sprayed into an
atmosphere kept at 100-200.degree. C. to form spherical
agglomerates of hydroxyapatite having a diameter of 1-10 .mu.m.
Hydroxyapatite trapped in a hydrogel network is relatively soft,
and binding capacity is modest because of the limited amount of
hydroxyapatite crystals present in a given volume of sorbent, about
40%. The presence of a hydrogel that surrounds crystals of
hydroxyapatite prevents the direct contact with very large
molecules such as plasmids.
[0008] Thus, the conventional processes involve a number of
problems not only in the preparation of spherical hydroxyapatite
particles but also in the use of the particles as a packing
material for chromatographic purposes.
[0009] Mineral oxide beads for use in chromatography are known, and
can have more strength than hydroxyapatite sorbents. For
applications in which another substance is introduced into the
bead, a pore size larger than 500 A is required to allow for
unhindered diffusion of large molecules. It is difficult to obtain
a large pore diameter, however, without adversely affecting
porosity and strength. Moreover, mineral oxide surfaces exhibit
various types of interactions with proteins, including
electrostatic, van der Waals, and Lewis acid-base, that can alter
the quality of a separation or even denature a biomolecule.
[0010] There is a need for relatively small porous particles which
provide the separation capabilities of apatite yet retain their
shape, their chemical and mechanical properties in specific
environments useful for biomolecule separation in columns as well
as in suspensions, and which offer a substantial density difference
with liquids used in adsorption and chromatography. Such apatite
materials excellent in mechanical strength and chromatographic
characteristics have not, as yet, been described.
SUMMARY OF THE INVENTION
[0011] In accordance with the present invention, a chromatography
sorbent is provided which combines the separation capabilities of
hydroxyapatite crystals with the strength of a mineral oxide
network. This composite sorbent exhibits superior properties when
used in chromatographic separation, or in substance separation or
development when used as the adsorbent packed or charged into a
column or as a stationary phase agent in column chromatography.
Chromatography using this sorbent as the adsorbent in batch
separations or packed in either a fixed bed or fluidized bed column
achieves high acuteness and precision separation and fractionation
of substances having a minute difference in structure from one
another. This was difficult to achieve with the use of prior-art
adsorbents. Such substances may include biological macromolecule
materials having a molecular weight of 10.sup.4 to 10.sup.9 Dalton,
such as proteins, including immunoglobulins, interferon or enzymes,
or nucleic acids, such as RNA, DNA or plasmids or viruses. The
composite sorbent is indispensable for high purity separation and
refining of a variety of ultimately useful substances obtained by
gene recombination, cell fusion or cell culture en masse.
[0012] The composite chromatographic sorbent comprises mineral
oxide beads with pores filled with apatite, particularly
hydroxyapatite. The mineral oxide beads of the composite sorbent
are characterized by high porosity, low surface area, high mean
pore diameter, and high mechanical stability. Moreover, they show a
density that facilitates packing of fixed-bed columns, increases
the particle sedimentation velocity in batch, and permits the use
of high velocity in fluidized-bed operations. The apatite crystals
are protected by a very strong skeleton of mineral oxide,
preferably zirconia.
[0013] Specifically, the present invention encompasses a composite
chromatography sorbent comprising porous mineral oxide beads that
have a pore volume which exceeds about 10% of the bead volume,
preferably about 30% and about 70%, and more preferably about 30%
and about 60%, and an average pore diameter of at least about 500
.ANG., preferably between about 1000 .ANG. and about 4000 .ANG.,
and more preferably between about 1000 .ANG. and about 3000 .ANG..
The pores of the beads contain apatite, and preferably
hydroxyapatite, crystals. Preferably, the mineral oxide is selected
from the group consisting of alumina, titania, hafnia, silica,
zirconia and mixtures thereof, and most preferably it is zirconia.
In a preferred embodiment, the mineral is silica, the pore volume
is between about 40% and about 70% of the bead volume, and the
average pore diameter is between about 2000 .ANG. and about 5000
.ANG..
[0014] In one embodiment, the beads of the composite chromatography
sorbent are coated with a layer of hydrophilic polymer, preferably
a hydrophilic polymer selected from the group consisting of
polyoxyethylene, polyoxypropylene, cross-linked polysaccharides and
vinyl polymers.
[0015] In a preferred embodiment, the apatite crystals comprise
calcium ions and a metal ion or a metalloid ion. Preferably, the
metal ion or metalloid ion is strontium, barium or fluoride.
[0016] The present invention also encompasses chromatography
apparatus and methods. A chromatography column comprises a tubular
member having an inlet end and an outlet end; first and second
porous members disposed within the tubular member; and a composite
chromatography sorbent according to the invention packed within the
tubular member between the first and second porous members.
Preferably, the column volume is between about 50 liters and about
5000 liters. The column additionally may comprise means for flowing
a liquid sample upward through the composite chromatography
sorbent.
[0017] The column may be used in a chromatographic separation
method comprising flowing a solution comprising biomolecules
through the chromatography column so that the solution permeates
the pores of the mineral oxide beads, wherein some biomolecules in
the solution are bound to the apatite crystals and other, different
biomolecules remain in the solution. This may be followed by
flowing another solution through the chromatography column to elute
the biomolecules bound to the apatite crystals. In one embodiment
the biomolecules are polypeptides, and in another embodiment the
biomolecules are nucleic acids. Substances other than proteins and
nucleic acids are included within the scope of the term
biomolecule, such as glycopeptides.
[0018] The composite sorbent may also be used in batch
chromatography apparatus and methods. In a batch method, a solution
comprising biomolecules is brought into contact with the composite
chromatography sorbent, so that the solution permeates the pores of
the mineral oxide beads, wherein some biomolecules in the solution
are bound to the apatite crystals and other, different biomolecules
remain in the solution.
[0019] The present invention also provides a method of making a
composite chromatography sorbent according to the invention,
comprising: (1) providing porous mineral oxide beads that have a
pore volume which exceeds about 10% of the bead volume and an
average pore diameter of at least about 500 .ANG.; (2) contacting
the porous mineral oxide beads with a maximum of one pore volume of
a solution of either (i) calcium chloride or (ii) potassium or
sodium phosphate so that it permeates the pores of the beads; (3)
drying the beads; (4) contacting the dried beads with a maximum of
one pore volume of a solution of the other of either (i) calcium
chloride or (i) potassium or sodium phosphate so that it permeates
the pores of the beads, thereby forming calcium phosphate in the
pores; (5) washing the beads with water to eliminate excess calcium
or phosphate ions; (6) contacting the washed beads with a solution
of sodium hydroxide; (7) washing the beads with water; and (8)
contacting the washed beads with a solution of disodium phosphate
to form hydroxyapatite crystals in the pores of the beads. In a
preferred embodiment, the beads are washed with a phosphoric acid
solution before contacting the porous mineral oxide beads with a
maximum of one pore volume of a solution of either (i) calcium
chloride or (ii) potassium or sodium phosphate.
[0020] Both the foregoing general description and the following
detailed description are exemplary and explanatory and are intended
to provide further explanation of the invention as claimed. Other
objects, advantages, and novel features will be readily apparent to
those skilled in the art from the following detailed description of
the invention.
[0021] Detailed Description of Preferred Embodiments
[0022] The composite chromatography sorbent according to the
invention uses mineral oxide beads to impart mechanical stability
to an apatite sorbent. The mineral oxide beads have a pore volume
of at least about 10% of the bead volume and an average pore
diameter of at least about 500 .ANG..
[0023] In a preferred embodiment, mineral oxide beads with a higher
pore volume, preferably at least about 30%, more preferably at
least about 40%, and most preferably at least about 50%, are used.
The preparation of mineral oxide beads having high pore volumes is
described in WO 99/51335, the contents of which are incorporated
herein in their entirety. The beads can be small discrete beaded
particles as well as irregular shaped particles, showing high pore
volume and high mechanical and chemical stability. Because of their
stability and high porosity, they are particularly useful in packed
bed, fluidized bed or stirred batch adsorption or chromatographic
separation for large macromolecules.
[0024] The mineral oxide beads are prepared by combining a
tetravalent metal oxide with a trivalent metal salt or oxide as a
pore inducing agent. The combination results in the formation of
unstable suspensions which, after agglomeration to form spherical
or irregular particles, show both macroporosity and large pore
sizes. The porosity and pore size is greater than that which can be
obtained in the absence of the trivalent metal salt or oxide.
[0025] The mineral oxide preferably is an oxide of titania,
zirconia, silica or hafnium, preferably silica or zirconia, and
most preferably zirconia. The mineral oxide can also be a mixture
of two or more tetravalent metal oxides. Preferably the mineral
oxide powders are in the form of a powder, and most preferably a
powder with a particle size of about 0.1 to about 10 .mu.m.
[0026] The trivalent metal can be used in the form of an oxide, a
salt, or mixtures of oxide and salt. A particularly preferred salt
is nitrate. The metal can be any metal which exhibits a +3 valence,
such as Group IIIB metals, rare earth metals, and the like.
Preferred trivalent metals are aluminum, gallium, indium, scandium,
yttrium, lanthanum, cerium, neodymium, erbium, ytterbium and
actinium. Also included are compositions in which the trivalent
metal oxide or salt is a mixture of two or more such oxides or
salts. Such mixtures include salt/oxide, salt/salt, and oxide/oxide
mixtures of the same or different trivalent metals.
[0027] Only a limited pore volume reduction is observed when firing
the compositions at very high temperatures. In contrast, mineral
oxide beads obtained without the use of the trivalent metal salt or
oxide have lower pore volumes when fired at very high temperatures,
due to a severe reduction of pore volume resulting from the firing
process. The trivalent metal salt or oxide additionally stabilizes
a crystalline form of the mineral oxide and prevents grain growth
and cracking of the final material.
[0028] Optionally, an agent that induces particle agglomeration to
make a beaded final material, such as an agglomeration promoting
material or a binder, may be included. These may be salts of
trivalent or tetravalent metals, and can contain the same
tetravalent or trivalent metals just described. In a preferred
embodiment, the binder comprises a mixture of nitrates, including a
tetravalent metal nitrate and trivalent metal nitrate. For example,
when zirconium oxide is used as a mineral oxide bead constituent
and cerium oxide is used as the trivalent pore inducing agent, it
is convenient to use a mixture of zirconium nitrate and cerium
nitrate as a binder. Other suitable binders include materials which
form mineral hydrogels that can encapsulate mineral oxide elemental
particles, for example, silica gels. A mineral hydrogel may also be
used in combination with one or more additional binders.
[0029] Composite mineral oxides with enhanced pore volume are made
by preparing a liquid suspension of a tetravalent mineral oxide.
The liquid portion of the suspension can be water or any other
appropriate solvent. The mineral oxide should be in the form of a
powder, with a particle size of between about 0.1 and about 10
.mu.m, with the particular particle size chosen depending on the
desired pore size of the porous particles. This suspension is mixed
with one or more pore inducing trivalent agents. The suspension
optionally also contains one or more binders.
[0030] In a typical composition which includes one or more metal
oxide or salt binders, the binders are first mixed in a liquid such
as water, then the mineral oxide and the pore inducing agent are
added while stirring, producing a suspension. The stirring should
be gentle to avoid introducing air bubbles into the mixture.
[0031] The amount of pore inducing agent which is used in the
initial suspension is roughly proportional to the amount of mineral
oxide used. In the final product, the oxide of the tetravalent
metal will constitute about 50 to about 99% of the final particles,
with the remaining about 1 to about 50% made up of pore inducers
and optional binders. In the initial suspension, however, the
mineral oxide particles, the major constituent of the porous beads,
are at a concentration of about 10 to about 95% by weight, based on
the total weight of components used. More preferably, the mineral
oxide should be about 20 to about 60% by weight. The pore inducing
agent concentration is between about 5 and about 50% by weight. The
optimal concentration varies, depending on the nature of the
specific compounds used. The concentration of the agglomeration
promoting material or binder is between about 0 and about 20% by
weight, and also depends on the nature of the binders. Optionally,
organic compounds may also be added to the initial suspension in
order to alter the viscosity of the solution.
[0032] The suspension containing all of the desired components is
then used to form beads. A variety of techniques well known in the
art, such as spray drying, emulsion-polycondensation and sol-gel
processes can be used to effect the agglomeration on the
compositions. After the elemental particles are agglomerated into a
beaded shape, they are heated at high temperatures to stabilize the
architecture of the porous mineral bead by partial fusion of the
elemental particles. The heating rate, the calcination temperature
and the soak time used depend on the nature of the mineral oxides
and mineral pore inducers. A controlled sintering is desirable in
order to obtain stronger particles without elimination of the
porosity. Typically, temperatures between about 800 and about
1400.degree. C., for a duration of about 1 to about 10 hours, and
with a heating rate ranging between about 1 and about 100.degree.
C./hour, are used. A sequential calcination treatment also can be
used, to first remove volatile components such as water, organic
materials, nitrates and the like, and then to sinter the elemental
particles.
[0033] The fired beads then are cooled to room temperature, and
subsequently washed with, for example, acidic, alkaline, neutral or
diluted hydro-organic solutions. The particles optionally can be
subjected to a sieving step to adjust the particle size
distribution, as desired. Typical pore volumes of at least about
30%, about 40% or about 50% can be obtained according to the
invention. The upper limit of pore volume is about 70%.
[0034] The beads with larger pore volumes and/or average pore
diameters are particularly suited for the introduction of apatite
crystals, preferably hydroxyapatite, to prepare a composite
chromatography sorbent. Pore volume varies based on the bead
material. For example, when the mineral oxide is selected from the
group consisting of zirconia, titania and hafnia, the pore volume
is between about 30% and about 60% of the bead volume. When the
mineral is silica and the pore volume is between about 40% and
about 70% of the bead volume.
[0035] Pore size also varies depending on the bead material, and
can be selected based on the material to be separated by the
composite sorbent. Larger average pore diameter is selected for
applications in separating larger biomolecules, such as plasmids,
in which an average pore diameter greater than 2000 .ANG. may be
required. The average pore diameter generally is between about 1000
.ANG. and about 4000 .ANG.. When the mineral is selected from the
group consisting of zirconia, titania and hafnia, the average pore
diameter is between about 1000 .ANG. and about 3000 .ANG., whereas
when the mineral is silica the average pore diameter is between
about 2000 .ANG. and about 5000 .ANG..
[0036] In order to fill the pores with hydroxyapatite, mineral
oxide beads optionally, but preferably, first are washed with a
solution of phosphoric acid to eliminate impurities and then
incubated with mono-potassium phosphate. The beads are then washed
and dried. If desired, the pore volume can be determined by known
methods. The beads are next contacted with a solution of either (i)
calcium chloride or (ii) potassium or sodium phosphate, and the
solution is allowed to penetrate the pores. The beads are dried and
then contacted with a solution of either (i) calcium chloride or
(ii) potassium or sodium phosphate. If the beads were contacted
with calcium chloride in the first step, then they are contacted
with potassium or sodium phosphate in the second step, and vice
versa. The solution is allowed to penetrate the pores, and after
allowing sufficient time for the calcium phosphate crystalline
structure to form within the pores, the beads are washed to
eliminate excess calcium or phosphate ions. The beads then are
contacted with a solution of sodium hydroxide, and are again
washed. Finally, the beads are contacted with a solution of
disodium phosphate to form hydroxyapatite crystals in the pores of
the beads.
[0037] When the mineral beads are contacted with the calcium
chloride and potassium or sodium phosphate solutions, it is
preferable to use a maximum of one pore volume of solution, and
preferably exactly one pore volume. The use of one pore volume
exactly generates the maximum amount of hydroxyapatite crystals in
the pores of the mineral oxide beads, without having crystals grow
outside the pores of the beads.
[0038] In an alternative embodiment, the hydroxyapatite made with
phosphate ions can be doped with small amounts of other metal ions.
The doped metal ions can used to vary the adsorption properties of
the composite sorbent.
[0039] Apatites other than hydroxyapatite can be grown in the
pores. In this case, calcium could be replaced by strontium, barium
or other elements. The resulting apatites would have different
adsorption properties than hydroxyapatite. Crystalline apatites
other than hydroxyapatite, such as apatite derivatives with F, Cl
or CO.sub.3, are known can be grown in the pores of the mineral
oxide beads. For example, the preparation of fluorapatite is
described in Matsumoto et al., Caries Res., 34(1):26-32 (2000);
Okazaki et al., Biomaterials, 20(15):1421-6 (1999); Okazaki et al.,
Biomaterials, 19(10):919-23 (1998); Okazaki et al., Biomaterials,
19(7-9):611-6 (April;-May, 1998).
[0040] The apatite crystals, and more preferably the hydroxyapatite
crystals, comprise calcium ions and a metal ion or a metalloid ion.
In embodiments using a metal or metalloid ion, preferred metal ions
or metalloid ions is strontium, barium or fluoride.
[0041] Prior to the formation of apatite crystals in the pores of
the mineral oxide beads, the beads may first be coated with a layer
of hydrophilic polymer. Preferably, the hydrophilic polymer is
selected from the group consisting of polyoxyethylene,
polyoxypropylene, cross-linked polysaccharides and vinyl polymers.
The coating reduces non-specific binding for biomolecules.
[0042] Different chromatography techniques can be used to separate
biomolecules using the composite sorbent according to the
invention. These techniques comprise contacting a solution
containing the biological macromolecules with the composite sorbent
leading to the selective adsorption or molecules in the solution by
the sorbent. In the event of the desired macromolecule(s) being
fixed to the resin, the elution of the latter allows it or them to
be separated and collected in a purified and concentrated form. If
the desired macromolecule remains in the treated solution (the
other macromolecules being fixed to the sorbent) then the desired
separation is obtained directly.
[0043] When using batch chromatography, the composite sorbent is
added directly to the solution of biomolecules, and the
sorbent-biomolecule mixture is gently agitated for a time
sufficient to allow the biomolecules to bind to the sorbent. The
biomolecule-bound-sorbent may then be removed by centrifugation or
filtration, and the biomolecules subsequently eluted in a separate
step.
[0044] Alternatively, column chromatography may be used. In fixed
bed column chromatography, the composite sorbent is packed into a
column, and the solution which contains the biomolecules to be
separated is applied to the sorbent by pouring it through the
sorbent at a rate that allows the biomolecules to bind to the
sorbent. 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 sorbent to some degree.
[0045] In fluidized bed column chromatography, a rising filtration
flow and large rather than dense particles are used in order to
maintain an equilibrium against the rising forces. An essentially
vertical column composed of between 2 and 5 stages placed on top of
the other is used, and the solution successively passes through
stage and is drawn off by an overflow on the upper part of the
upper stage. Each stage, with the exception of the uppermost one,
is separated by two perforated 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.
[0046] 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 larger diameter resins that are neutral to
gravity or buoyant. These larger diameter sorbents have less
surface area per unit volume than smaller sorbents used in fixed
bed columns, and correspondingly have less surface binding
capacity. The most significant problem of the fluidized bed is
mixing. Since the column does not contain any static mixing means,
the bed is conventionally mixed by means of air jets or by
recycling the liquid to be separated through the column at a high
flow rate. The high flow rate and limited mixing inhibit the
uniform phase change required during elution of the product from
the resin.
[0047] 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 resin
deterioration. In fact, the fluidized bed allows free passage of
impurities in the solution with no risk of clogging; no cleaning is
necessary so the life-span of the resins is greatly increased.
However, the chromatographic sorbents for biological macromolecules
typically are not suitable for fluidized bed chromatography, being
too small in granulometry, or having a density too close to that of
water. This makes it impossible to fluidize without drawing
particles into the flux. Another problem with fluidized bed
chromatography of biological macromolecules relates to the large
space between molecules, would result in a decrease in efficiency
in a fluidized bed environment.
[0048] Based on these factors, batch and fixed bed chromatography
have been the methods of choice in prior art separation techniques
for biological macromolecules. The present composite sorbent, on
the other hand, can be used in a batch, fixed bed, or fluidized bed
chromatography.
[0049] The composite sorbent according to the invention is used to
separate biomolecules contained in a "source liquid," which is a
liquid containing at least one and possibly two or more biological
substances or products of value which are sought to be purified
from other substances also present. In the practice of the
invention, source liquids may for example be aqueous solutions,
organic solvent systems, or aqueous/organic solvent mixtures or
solutions. The source liquids are often complex mixtures or
solutions containing many biological molecules such as proteins,
antibodies, hormones, and viruses as well as small molecules such
as salts, sugars, lipids, etc. While a typical source liquid of
biological origin may begin as an aqueous solution or suspension,
it may also contain organic solvents used in earlier separation
steps such as solvent precipitations, extractions, and the like.
Examples of source liquids that may contain valuable biological
substances amenable to the purification method of the invention
include, but are not limited to, a culture supernatant from a
bioreactor, a homogenized cell suspension, plasma, plasma
fractions, milk, colostrum and cheese whey.
[0050] The source liquid contains at least one "biomolecule" to be
purified from the source liquid. Biomolecules are biological
products and include, for example, nucleic acids, immunoglobulins,
clotting factors, vaccines, antigens, antibodies, selected proteins
or glycoproteins, peptides, enzymes, etc. The biomolecule may be
present in the source liquid as a suspension or in solution. For
convenience, the term "biomolecule" is used herein in the singular,
but it should be understood that it may refer to more than one
substance that is to be purified, either together as co-products or
separately (e.g., sequentially) as discrete recovered
components.
[0051] An "elution liquid" or "elution buffer" is used to
dissociate the biomolecules, such as glyco-iso-forms, away from the
composite sorbent. The elution liquid acts to dissociate the
biomolecules without denaturing them irreversibly. Typical elution
liquids are well known in the chromatography art and may have
higher concentrations of salts, free affinity ligands or analogs,
or other substances that promote dissociation of the target
substance from the chromatography sorbent. "Elution conditions"
refers to process conditions imposed on the biomolecule-bound
chromatography sorbent that dissociate the undenatured biomolecules
from the chromatography sorbent, such as the contacting of the
biomolecule-bound chromatography sorbent with an elution liquid or
elution buffer to produce such dissociation.
[0052] A "cleaning liquid" or "cleaning buffer" is used to wash the
chromatography sorbent after the completion of the separation
process. The cleaning liquid may contain a detergent, a
virus-inactivating agent, or relatively high concentrations of
salts, and may have a higher or lower pH than the liquids used
during the purification process. Its purpose is to fully
decontaminate the chromatography sorbent to render it ready for
reuse. Typical cleaning liquids are well-known in the
chromatography art.
[0053] Between uses, the composite sorbent is stored in a "storage
liquid" or "storage buffer." Storage liquids, in addition to
buffering ions, may also contain microbicides or other
preservatives. Such storage liquids are well known in the
chromatography art.
[0054] The composite sorbent can be used in batch separations, or
it can be packed into a chromatography column, either a fixed bed
or fluidized bed. The column comprises a tubular member having an
inlet end and an outlet end, and first and second porous members,
such as a glass frit, disposed within the tubular member. The
composite chromatography sorbent is packed within the tubular
member between the first and second porous members. In a fluidized
bed column, there typically are multiple stages. In a preferred
embodiment, the column volume is between about 50 liters and about
5000 liters. For fluidized bed chromatography, the column
additionally comprises a means for flowing a liquid sample upward
through the composite chromatography sorbent.
[0055] Batch chromatographic separations comprised mixing the
composite sorbent with the source liquid in a suitable container,
and gently stirring. Chromatographic separations using column
chromatography comprise the steps of flowing a first solution
comprising biomolecules through the column such that the
biomolecules in the solution permeate the pores of the mineral
oxide beads and are bound to the apatite crystals therein; and then
flowing a second solution through the column to elute the
biomolecules bound to the apatite crystals. In a fixed bed column
the source liquid flows downwardly by gravity, while in a fluidized
bed column the source liquid is propelled upwardly through the
column. The biomolecules to be separated usually are polypeptides
or nucleic acids. The composite sorbent is particularly useful for
difficult protein separations, including antibody separation. It
also is excellent for plasmids for the elimination of RNA and of
open circles.
[0056] The following examples relate to specific embodiments within
the scope of the present invention, but are not limiting.
EXAMPLE 1
Preparation of Zirconia Particles by Sol-Gel
[0057] A silica sol is prepared by mixing sequentially and
progressively 150 ml of sodium silicate 35% with 200 ml of water
and 100 ml of water and 100 ml of glacial acetic acid. Dry solid
irregular zirconia powder (350 mg of 0.3 to 3 .mu.m size) is
dispersed in this suspension. Cerium oxide (10 g) and cerium
nitrate (10 g) are then added under vigorous stirring. Under the
above conditions the gelation process occurs at ambient temperature
within 15 to 60 minutes.
[0058] After complete gelation, which takes a few hours, the gel is
divided into small pieces by press-filtering it through a 200 .mu.
sieve. The particles are suspended in clear water and recovered by
filtration, washed and then dried at 80.degree. C. under an air
stream.
[0059] The silica gel that entraps the solid zirconia and ceria
composite microparticles is progressively dehydrated. At this
point, the particles are soft and show only very modest porosity.
Then, the particles are fired at 1300.degree. C. for 2 hours. Under
these conditions, the silica gel is totally dehydrated and shrinks
to such an extent that it forms a continuous layer around the solid
sub-particles. The void between subparticles constitutes the
macroporosity.
[0060] After this treatment, the final pore volume represents more
than 50% of the whole porous particle volume. The density of the
dry irregular particles is about 2.1 g/cm.sup.3. After cooling, the
beads do not show any cracks due to volume variation of mineral
crystalline forms.
EXAMPLE 2
Preparation of Zirconia Particles by Suspension Polymerization
[0061] A silica sol is prepared by mixing sequentially and
progressively 150 ml of sodium silicate 35% with 200 ml of water
and 100 ml of water and 100 ml of glacial acetic acid. Dry solid
irregular zirconia powder (350 mg of 0.3 to 3 .mu.m size) is
dispersed in this suspension. Cerium oxide (10 g) and cerium
nitrate (10 g) are then added under vigorous stirring.
[0062] The resulting homogeneous suspension is slowly poured in an
agitated paraffin oil bath containing 2% sorbitan sesquioleate and
dispersed as small droplets. The suspension is heated at 80.degree.
C. while stirring. Under these conditions, the gelation process
occurs at ambient temperature within 15 to 30 minutes.
[0063] The beads of a diameter ranging from 10 to 500 .mu.m
comprise a silica hydrogel trapped within its network solid
microparticles of pre-formed zirconia and ceria. They are recovered
by filtration, washed, and dried at 80.degree. C. under an air
stream. The gel is progressively dehydrated and acts as a binder
for solid zirconia and ceria composite microparticles. The beads
are then fired at 1300.degree. C. for 2 hours, to singer bead
sub-particles with minimal pore volume reduction. After this
treatment, the final pore volume represents more than 50% of the
total bead volume. The density of the dry beads is about 2.1
g/cm.sup.3. After cooling, the beads do not show any cracks due to
volume variation of mineral crystalline forms.
EXAMPLE 3
Preparation of Zirconia Beads by Spray Drying
[0064] A solution is prepared by mixing 231 g of zirconium nitrate
and 143.6 g of yttrium nitrate in 1000 ml of distilled water.
Yttrium oxide (144 g) and zirconia powder (752 g of 0.3 to 3 .mu.m
size) are added under gentle stirring to prevent the introduction
of air bubbles.
[0065] The suspension is then injected into a vertical drying
chamber through an atomization device, such as a revolving disk, a
spray nozzle, or an ultrasonic nebulizer, together with a hot gas
stream, preferably air or nitrogen. The hot gas stream causes rapid
evaporation of water from the microdroplets. The gas is typically
injected at 300-350.degree. C. and exits the dryer at a temperature
slightly above 100.degree. C. Microparticles of original mineral
oxides are consolidated into individual aggregates of spherical
shape. Dry microbeads are then fired at a temperature close to the
melting temperature of the zirconium oxide to irreversibly
consolidate the network. After cooling, the beads do not show any
cracks due to volume variation of mineral crystalline forms. This
operation results in the formation of stable beads with a large
pore volume that exceeds 50% of the bead volume.
EXAMPLE 4
Preparation of Hydroxyapatite Filled Zirconia Beads
[0066] The beads from Example 3 are washed with a 1 M solution of
phosphoric acid to eliminate impurities and then incubated with two
volumes of 1 M monopotassium phosphate overnight at room
temperature under occasional shaking. The treated beads are then
washed with water until neutral pH and dried under vacuum at
60-80.degree. C. to eliminate all residual water. The pore volume
of the dry beads is determined according to well-known methods.
[0067] A solution of calcium chloride is prepared by solubilizing
74 g of CaCl.sub.2.2H.sub.2O in 500 ml of distilled water (final
volume). The dry beads of treated zirconia are mixed with one pore
volume of the solution of calcium chloride. After 30-60 minutes
mixing, to ensure a good penetration of the solution into the pores
of the mineral material, the beads are dried again, as above.
[0068] A solution of disodium phosphate is prepared by solubilizing
180 g of Na.sub.2HPO.sub.4.12H.sub.2O in 500 ml water (final
volume). The dried beads are mixed with one pore volume of the
solution, and the mixture again is thoroughly shaken for 30-60
minutes to ensure good penetration. The temperature is kept at
25-40.degree. C.;, and the material is left overnight.
[0069] The material then is mixed with a large volume of water and
washed several times with water until elimination of the excess
calcium ions (no precipitation of Ca(OH).sub.2 with NaOH should
occur). The washed material is added to several volumes (at least
10) of sodium hydroxide at a concentration of 0.5M. The suspension
is then brought to 95-100.degree. C. for one hour and left
overnight, during which time the temperature decreases to room
temperature.
[0070] The treated material is again extensively washed with water
and mixed with a solution of 3 g/l of disodium phosphate. The pH is
adjusted to 6.8 and the suspension heated to 95-100.degree. C. for
about 20 minutes. The resulting material is finally washed with
water and stored in a phosphate buffer at neutral pH containing 1 M
sodium chloride and 20% ethanol.
[0071] The present invention provides a novel composite adsorbent,
methods of use and manufacture. While specific examples have been
provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will be become
apparent to those skilled in the art upon review of the
specification. The scope of the invention should, therefore, be
determined not with reference to the above description, but instead
should be determined with reference to the appended claims along
with the their full scope of equivalents.
[0072] All publication and patent documents cited in this
application are incorporated by reference in their entirety for all
purposed to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their "invention."
* * * * *