U.S. patent application number 13/496277 was filed with the patent office on 2012-09-20 for hollow particulate body.
This patent application is currently assigned to SPHERITECH LTD. Invention is credited to Donald A. Wellings.
Application Number | 20120237606 13/496277 |
Document ID | / |
Family ID | 41277832 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237606 |
Kind Code |
A1 |
Wellings; Donald A. |
September 20, 2012 |
HOLLOW PARTICULATE BODY
Abstract
A particulate body having a hollow particle and a surface
polymer disposed on the outside of the hollow particle and suitable
for use in solid phase synthesis, especially production of peptides
and oligonucleotides. The particulate body may be used as a
chromatography stationary phase column and the buoyancy of the body
allows the column to be packed efficiently from the bottom reducing
the risk of damage to the stationary phase. The buoyancy of the
particulate body may also allow species for example a catalyst to
be suspended in a liquid phase to allow reactions, for example
hydrolysis of vegetable oil and esterification to produce biodiesel
to be carried out with a reduced risk of catalyst loss from a
reaction zone.
Inventors: |
Wellings; Donald A.;
(Runcorn, GB) |
Assignee: |
SPHERITECH LTD
Runcorn, Cheshire
GB
|
Family ID: |
41277832 |
Appl. No.: |
13/496277 |
Filed: |
September 16, 2010 |
PCT Filed: |
September 16, 2010 |
PCT NO: |
PCT/EP2010/005698 |
371 Date: |
June 4, 2012 |
Current U.S.
Class: |
424/501 ;
428/402; 428/404; 428/407; 435/134; 435/4; 436/501; 502/159;
521/54; 530/334; 536/25.3 |
Current CPC
Class: |
C08J 9/32 20130101; C11C
1/045 20130101; C12P 7/649 20130101; Y02E 50/13 20130101; B01J
20/28011 20130101; C12P 7/6418 20130101; B01J 20/28021 20130101;
C11C 3/003 20130101; C08J 9/224 20130101; Y10T 428/2982 20150115;
Y10T 428/2993 20150115; B01J 20/3268 20130101; C12N 11/08 20130101;
Y02E 50/10 20130101; Y10T 428/2998 20150115; B01J 20/286 20130101;
B01J 13/22 20130101 |
Class at
Publication: |
424/501 ;
436/501; 435/134; 536/25.3; 530/334; 435/4; 502/159; 521/54;
428/402; 428/407; 428/404 |
International
Class: |
B32B 5/16 20060101
B32B005/16; G01N 33/53 20060101 G01N033/53; C12P 7/64 20060101
C12P007/64; C08J 7/04 20060101 C08J007/04; C07K 2/00 20060101
C07K002/00; C07K 1/04 20060101 C07K001/04; C12Q 1/25 20060101
C12Q001/25; B01J 31/06 20060101 B01J031/06; A61K 9/14 20060101
A61K009/14; C07H 21/00 20060101 C07H021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 2009 |
GB |
0916281.9 |
Claims
1. A particulate body comprising a hollow particle comprising a
polymer and, optionally a surface polymer disposed on the outside
of the hollow particle.
2. The particulate body of claim 1 wherein the body is for use in a
liquid phase and is buoyant in the liquid phase.
3. The particulate body of claim 1 wherein the particulate body has
a density of less than 1 g/cm.sup.3.
4. The particulate body of claim 1 wherein the particulate body has
a density in the range 0.01 to 0.05 g/cm.sup.3,
5. The particulate body of claim 1 wherein the polymer is selected
from polystyrene, polyacrylonitrile, polyacrylate,
polymethacrylate, polyvinylchloride and copolymers comprising a
plurality of monmers selected from styrene, acrylonitrile,
acrylate, methacrylate and vinylchloride.
6. The particulate body of claim 1 wherein the polymer comprises
nitrile groups and/or derivatised nitrile groups.
7. The particulate body of claim 1 wherein the hollow particle is
generally spherical or ellipsoidal.
8. The particulate body of claim 1 wherein the body comprises a
spherical, ellipsoidal or other uniform shaped agglomerate
containing multiple hollow particles in a surface polymer
coating.
9. The particulate body of claim 1 wherein the hollow particle has
a particle size of 1 to 500 .mu.m.
10. The particulate body of claim 1 comprising a surface polymer
which is bound covalently to the hollow particle directly or
indirectly.
11. The particulate body of claim 10 comprising one or more surface
polymer selected from a polyacrylamide, a polystyrene, a cellulose,
an agarose, a polyacrylate, a polydimethylacrylamide, a
polymethylmethacrylate, a polymethacrylate, a polyurea, a
polyacryloylmorpholine, a polyvinylalcohol, a silica, a
polybetahydroxy ester and a polyacrylonitrile.
12. The particulate body of claim 1 wherein the said polymer of the
hollow particle or, where present, the surface polymer comprises an
amine functional group for use in peptide synthesis,
oligonucleotide synthesis or solid phase synthesis.
13. The particulate body of claim 1 wherein the body comprises an
inert material coating the hollow particle.
14. The particulate body of claim 1 comprising a surface polymer
bound to or, where the inert material is porous, retained within
the pores of the inert material.
15. The particulate body of claim 13 wherein the inert material is
selected from a polyhipe and a porous silica.
16. The particulate body of claim 1 further comprising a functional
material supported by or reacted with the hollow particle polymer
or the surface polymer.
17. The particulate body of claim 16 wherein the functional
material is selected from a catalyst, an initiator species for
peptide synthesis, an initiator species for oligonucleotide
synthesis, an initiator species for solid phase organic synthesis,
a pharmaceutical active, an agrochemical active, a protein or other
biological macromolecule.
18. (canceled)
19. The particulate body of claim 1 comprising a plurality of
layers of surface polymer a first layer comprising a hydrophilic
surface polymer and a second layer comprising a hydrophobic surface
polymer.
20. The particulate body of claim 19 comprising a pharmaceutical or
an agrochemical active component wherein the plurality of layers of
surface polymer providing controlled release of the active
component.
21. A medical diagnostic for detecting an analyte comprising the
particulate body of claim 1 and comprising a functional material
bound or retained by the body capable of interaction with the
analyte to be detected.
22. The medical diagnostic of claim 21 wherein the functional
material comprises an enzyme supported by the surface polymer.
23. A monolith comprising a plurality of particulate bodies
according to claim 1 arranged in a three dimensional shape.
24. A method for producing a particulate body material comprising
the steps of providing a hollow particle contacting the particle
with a monomer or solution of a monomer, effecting polymerisation
of the monomer so as to form a surface polymer coating on the
hollow particle(s).
25. The method of claim 24 in which the monomer or a solution of
the monomer, is added to the hollow particles and polymerisation is
carried out in the presence of a solvent which is immiscible with
the monomer or monomer solvent.
26.-29. (canceled)
30. A method for the production of a fatty acid comprising
contacting a vegetable oil with a buoyant, hollow particulate body
having an enzyme capable of hydrolyzing the vegetable oil bound to
the body in a reaction zone to produce fatty acid and glycerol,
withdrawing glycerol from the reaction zone leaving the enzyme in
the reaction zone.
31. The method of claim 30 further comprising the step of
introducing an alcohol to the reaction zone, contacting the alcohol
with the fatty acid in the presence of an esterification catalyst
to produce biodiesel.
32. A process for the synthesis of a reacted product comprising
charging a reaction zone with a liquid of density D.sub.l, the
particulate body of claim 1 having a density D.sub.t wherein
D.sub.t is less than D.sub.l and wherein the surface of the
particulate body comprises reactive sites feeding one or more
reactants to the reaction zone under conditions for the one or more
reactants to react with the reactive sites on the body to provide a
reacted product.
33. The process of claim 32 comprising at least one further step of
feeding a reactant to the reaction zone to react with the reacted
product to produce a further reacted product and optionally
repeating this step with the same or a different reactant to
produce a macromolecule.
34. The process of claim 33 for producing a macromolecule selected
from an oligonucleotide, a peptide and where the macromolecule is a
peptide the reactants are amino acids or species able to provide an
amino acid moiety in the reacted product and where the
macromolecule is a nucleic acid sequence, the reactants are
nucleosides or species able to provide a nucleic acid moiety in the
reacted product.
35. The process any one of claims 32 to 311 claim 32 wherein the
surface polymer is selected from polydimethylacrylamide,
polyethyelene glycol and polystyrene.
36. A process for the simultaneous preparation of two products in a
single reaction zone comprising charging the reaction zone with a
liquid of density D.sub.l, a particulate body of claim 1 having a
density D.sub.t and providing a body for a top synthesis reaction,
and a solid body having a density D.sub.b and providing a body for
a bottom synthesis reaction, wherein D.sub.t is less than D.sub.l
and D.sub.l is less than D.sub.b, feeding reactants for the top
synthesis reaction and for the bottom synthesis reaction to the
reaction zone under conditions for the top synthesis reaction and
bottom synthesis reaction to occur to produce a top synthesis
product and a bottom synthesis product and optionally separating
the particulate body from the solid body whereby the top synthesis
product and bottom synthesis product are separated.
Description
[0001] This invention relates to a hollow particulate body, a
method of preparing the body and the use of the body in solid phase
processes and in particular to a hollow, buoyant, particulate
polymer body. The body is useful in a wide range of physical and
chemical processes especially where interaction with a substrate is
required for example solid phase synthesis, solid phase extraction,
solid phase reagents, immobilization of species, cell culture,
catalysis, chromatography and in medical diagnostics.
[0002] Solid support materials useful in solid phase synthetic
processes are known. A wide range of physical and chemical
processes employ solid support materials including by way of
example synthesis of organic molecules, in particular peptides and
oligonucleotides, immobilization of species, support of catalysts,
ion exchange, extraction of species from a material, diagnostics
and chromatography.
[0003] Typically, multi-stage synthesis of an organic molecule
involves numerous isolation steps to separate intermediates,
produced at each stage, before progressing to the subsequent stage.
These processes are often time-consuming, expensive and may be
inefficient as regards yield. The intermediates often require
purification to remove excess reagents and reaction by-products and
procedures such as precipitation, filtration, bi-phase solvent
extraction solid phase extraction, crystallization and
chromatography may be employed.
[0004] Solid phase synthesis offers some advantages over solution
phase synthesis. For example, isolation procedures used in solution
phase synthesis may to some extent be avoided by reversibly
attaching the target molecule to a solid support. Excess reagents
and some of the side-products may be removed by filtration and
washing of the solid support. The target molecule may be recovered
in essentially quantitative yield in some processes which is
typically particularly difficult in solution phase synthesis. In
addition, the time required to perform operations on a solid
support is typically much less than that required carrying out the
equivalent stage in a solution phase synthesis.
[0005] Immobilization of species in a range of processes is also
known. For example, polymer supports are commonly used for the
immobilization of catalysts for use in traditional organic
chemistry including chemo and bio catalysis. Immobilized enzymes
may be employed to perform organic chemical reactions or for chiral
resolution, for example the use of immobilized Penicillin amidase
for the resolution of secondary alcohols (E. Baldaro et al. Tet.
Asym. 4, 1031, (1993) and immobilized Penicillin G amidase is also
used for the hydrolysis of Benzylpenicillin in the manufacture of
Amoxicillin (Carleysmith, S. W. and Lilly, M.D. Biotechnol.
Bioeng., 21, 1057-73, 1979).
[0006] Solid supports are also used to immobilize biological
macromolecules for medical and diagnostic applications. This
includes immobilization of proteins, monoclonal and polyclonal
antibodies. Cell culture is commonly carried out on solid supports
with specific surface characteristics and morphology. Immobilized
enzymes in the beads can be employed as sensors to generate a
signal. An example is the detection of glucose by the glucose
oxidase/peroxidase coupled enzyme system, in which the presence of
glucose generates hydrogen peroxide which in turn is the substrate
for peroxidase for the oxidation of a wide variety of substrates to
provide a coloured, fluorescent or luminescent signal.
[0007] A variety of fluors whose fluorescence is sensitive to
specific cations or anions may be utilized to indicate
concentrations of specific ions including hydrogen ions for pH
measurement.
[0008] Polymeric particles are often used in chromatography where
the solid supports are termed stationary phases. In certain modes
of chromatography the cost of stationary phases may be restrictive.
In other modes the physical nature of the stationary phase can
reduce the effectiveness of the technology. For instance, the soft
polymers often used for affinity, ion-exchange and gel permeation
chromatography cannot be used at high flow rates because of the
deformable nature of the particles. The rigid macroporous polymers
used for many other modes of chromatography can often be
mechanically friable and subsequently suffer from a short
lifetime.
[0009] The application of solid supports or stationary phases in
chromatographic separations is very extensive for example complex
high-technology separations used in the pharmaceutical and
biotechnology industry and larger scale processes used in the
mining industry. Some of the pharmaceutical industry's most
valuable drugs are purified by preparative chromatography and
improved chromatographic separation would be technically beneficial
and economically advantageous. In the mining and precious metal
recovery industry a large portion of the world's palladium, a
critical component in a wide range of industrial applications and
processes including catalytic converters and manufacture of high
value products, may be refined using immobilized crown ethers
(Traczyk, F. P.; Bruening, R. L.; Izatt, N. E. "The Application of
Molecular Recognition Technology (MRT) for Removal and Recovery of
Metal Ions from Aqueous Solutions"; In Fortschritte in der
Hydrometallurgie; 1998, Vortrage beim 34. Metallurgischen Seminar
des Fachausschusses fuer Metallurgische Ausund Weiterbildung der
GDMB; 18-20 Nov. 1998; Goslar).
[0010] The use of polymeric particles in solid phase extraction and
in the preparation of solid phase reagents is also known in the
chemical, pharmaceutical and biotechnology industry.
[0011] Known solid phase supports generally comprise polymer
particles of a particular size and physical nature to suit the
application. For ease of use these polymer particles are often
spherical and have a defined particle size distribution. The
spherical nature of the particles improves the flow and filtration
characteristics of the polymer. Although the uses of solid supports
have operational advantages there are disadvantages to the solid
phase approach. For example, commercially available supports
commonly used for solid phase synthesis of peptides and
oligonucleotides may be expensive, for example due to the complex
manufacturing processes. Microporous polymers and macroporous
polymers are generally used. Microporous polymers have a relatively
low level of cross-linker which allows the polymer particles to
solvate and consequently swell in suitable solvents. Macroporous
polymers have a high level of cross-linker in the polymer matrix
and contain large pores. These polymer particles are generally
rigid and have good flow characteristics and are suitable for use
in packed columns.
[0012] Polymeric particles may typically be made by a dispersion or
emulsion polymerization process in which a solution of monomers is
dispersed in an immiscible solvent (continuous phase) prior to
initiation of the polymerization. The polymer particles formed are
typically then filtered, washed and classified to isolate the
required particle size distribution.
[0013] These processes are disadvantageous in some respects
including monomer loss to the continuous phase, generation of a
range of particle sizes and the undesirable generation of fine
particles during the polymerization, laborious particle size
classification, for example sieving and air classification.
[0014] In addition to undesirable costs of manufacture and wastage
during preparation certain disadvantages may arise with the
physical properties of the known polymeric particles, particularly
poor physical stability. Microporous polymeric particles are
generally soft and generally not suitable for use in chromatography
applications at a high flow rate in a packed column bed. In
addition, the soft particles may be compressed undesirably and
cause fouling, for example during filtration often leading to
compressive intrusion into the sinter or mesh being used at the
bottom of the column. Rigid macroporous and macroreticular
particles are more suited to high flow rates in packed column beds.
However, due to the rigid nature the particles may be fragile and
fragment under physical stress.
[0015] These problems are exacerbated by conventional packing
procedures which may involve inserting the polymer particles into
the column from the bottom upwards such that the polymer particles
are subjected to undesirably large stresses and which may cause
physical weakening or damage to the particles and render the column
less efficient for chromatographic processes.
[0016] We have now found that these and other problems associated
with known polymer particles may be ameliorated by providing a
particulate body comprising a preformed support polymer with a
hollow centre optionally having a polymer disposed on its surface.
The hollow centre provides a particulate body of a low density
suitably providing buoyancy in a liquid phase.
[0017] WO2008/012064 describes a solid support comprising a
polymer-impregnated bead wherein the bead has a hole in it and the
polymer is disposed in the hole. The bead is solid throughout and
acts as a framework to support the polymer in the hole. In
producing the solid support, the polymer is said to be formed on
the bead and in the hole and the polymer on the outside of the bead
is removed for example by abrasion. WO2008/012064 however does not
disclose or suggest that the bead is hollow or in the form of a
shell.
[0018] In a first aspect, the invention provides a buoyant
particulate body comprising a hollow particle comprising a polymer
and, optionally a surface polymer disposed on the outside of the
hollow particle.
[0019] Suitably the particulate body is buoyant. The particulate
body is suitable for use in a liquid phase wherein particulate body
has a density less than that of the liquid phase. Preferably, the
particulate body has a density of less than 1 g/cm.sup.3, more
preferably less than 0.8 g/cm.sup.3 and especially less than 0.7
g/cm.sup.3 so enabling buoyancy in diethyl ether. In a further
preferment, the particulate body has a density of less than 0.5
g/cm.sup.3 and more especially of 0.3 g/cm.sup.3 or less. Suitably,
the particulate body has a density of at least 0.005 g/cm.sup.3,
and suitably at least 0.01 g/cm.sup.3. Preferably the particulate
body has a density in the range 0.01 to 0.05 g/cm.sup.3, more
preferably 0.01 to 0.03 g/cm.sup.3 for example 0.015 g/cm.sup.3.
Desirably, the particulate body may be used in batch-wise
operations allowing for rapid processing
[0020] The term "particulate body" as employed herein includes a
particulate support and may be employed as a support, for example
in solid-phase synthesis.
[0021] The term "polymer as employed herein includes organic
polymers, for example polystyrene and polyacrylonitrile. Preferably
the polymer of the hollow particulate body comprises a functional
group which may be derivatised, for example a nitrile group.
[0022] In the context of this invention, the term "hollow" means
that the particle has a polymer wall which entirely encloses a
space such that the particle is a shell with a gas filled or an
empty centre.
[0023] Advantageously, the hollow particles are rigid and
mechanically robust and may be utilized at high flow rates in
packed column beds.
[0024] The hollow particle may suitably comprise an inert material
comprising a polymer and preferably consists essentially of a
polymer.
[0025] In the preferred embodiment the hollow particle comprises a
polymer selected from polystyrene, polyacrylonitrile,
polymethacrylate, polyvinylchloride and mixtures of polymers.
Copolymers may be employed and preferably the copolymer comprises a
plurality of monmers selected from styrene, acrylonitrile,
acrylate, methacrylate and vinylchloride, for example
polyacrylonitrile comprising polymethacrylate and/or
polyvinylchloride. Blends of different homopolymers or copolymers
may also be employed as desired.
[0026] Suitable hollow polymer particles may be obtained
commercially for example from Akzo Nobel, Expancel, Box 300, S-850
13, Sundsvall, Sweden and are sold under the trademark
Expancel.RTM..
[0027] Suitably, the particulate body of the current invention is
spherical, near to spherical or ellipsoidal. Advantageously, the
body is spherical. A spherical body is beneficial in many
applications and facilitates for example, packing in columns and
improved flow characteristics over a bed during filtration.
However, irregular, oval and other shapes of particle can be
used.
[0028] There are many grades of hollow polymer particles available
which vary in outer diameter and inner diameter, that is the
diameter of the inner wall of the shell so allowing the thickness
of the polymer particle wall of the shell according to the desired
use. Suitably, the particulate body has a particle size of 1 to 500
.mu.m, preferably 5 to 150 .mu.m, and the particle size will be
selected according to the desired application. In one preferred
embodiment, the particle size is from 1 to 50 .mu.m, preferably 5
to 20 .mu.m for example 10 .mu.m. A particle size in this range is
suitable for use in high pressure liquid chromatography. In another
preferred embodiment, the particle size is suitably 50 to 500
.mu.m, preferably 50 to 200 .mu.m, for example 100 .mu.m. A
particulate body having a particle size in this range is suitable
for use in solid phase synthesis, especially for peptide synthesis,
DNA synthesis and RNA synthesis.
[0029] This density of each grade of hollow particle is readily
controlled during manufacture and varied by the ratio of inner
diameter to the outer diameter of the hollow particle. The higher
this ratio the less dense and more buoyant the final product will
be.
[0030] Suitably, the polymer in the hollow particle presents a
surface to which a species may bond covalently or modified to bind
covalently or otherwise or to which a coating may be applied and
preferably bind to the polymer of the hollow particle. The particle
surface is may be modified to provide a covalent bond for use
directly in certain applications, for example in synthesis of
peptides or nucleic acid sequences. In one embodiment a surface
polymer may be used to coat or bind to the outside of the particle.
The polymer may be bound covalently to the particle directly or
indirectly. Where the particle is made of a material having active
sites, the polymer may be bound directly. Where the particle is
made of a more inert polymer, it may be desirable to treat the
particle to provide active sites to which the surface polymer may
bind.
[0031] The particle may be derivatised to provide active sites for
reaction with a surface polymer. In a preferred embodiment, the
polymer comprises nitrile groups and these may be derivatised or
converted to other functional groups. For example nitriles may be
converted to carboxylic acid by hydrolysis with acid or alkali, to
methyl esters using methanolic hydrochloric acid, to an amine by
reduction using for instance lithium aluminium hydride in
tetrahydrofuran. Nitriles may also be reacted directly with amines
to form amidines, for example ethylene diamines and bis-amino PEGS
may be employed to incorporate a primary amine surface. Nitriles
may also be derivatised to produce an aldehyde or alcohol
functional group or other functional groups as desired.
[0032] The surface polymer may be any suitable material according
to the desired application. In a preferred embodiment, the surface
polymer is selected from a range of polymer types including but not
limited to a polyacrylamide, a polystyrene, a cellulose, an
agarose, a polyacrylate, a polydimethylacrylamide, a
polymethylmethacrylate, a polymethacrylate, a polyurea, a
polyacryloylmorpholine, a polyvinylalcohol, a silica, a
polybetahydroxy ester and a polyacrylonitrile.
[0033] The hollow particle polymer or the surface polymer may be
reacted further to provide particular functionality for a given
application. Suitably, the hollow particle polymer or surface
polymer is reacted with a compound having at least two functional
groups, one for reacting with the hollow particle polymer or
surface polymer and the other to provide free functionality for use
in the desired application. In a preferred embodiment, the polymer,
for example polydimethylacrylamide and polyacryloylmorpholine
copolymers with N-acryloyl sarcosine methyl ester, is reacted with
a diamine compound, for example ethylene diamine. Amine
functionalised bodies for example are suitable for use in peptide
synthesis, oligonucleotide synthesis, for example DNA and RNA and
solid phase organic chemistry. Amino functional polymers may be
employed for peptide synthesis and oligonucleotide (DNA/RNA)
synthesis wherein a linkage agent is attached and the peptide or
oligonucleotide assembled stepwise using techniques familiar to
those skilled in the art.
[0034] An amine functionalised body may be further functionalised,
for example by conversion to a carboxylic acid using succinic acid
as desired. By way of example, an amine functionalised body may be
treated with N-hydroxysuccinimide and
1-Ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride in
preparation for immobilising a protein, for example protein A.
[0035] In a further embodiment, the body comprises the polymer
particle and a surface polymer and also an inert material coating
the particle. An especially preferred inert material is Polyhipe.
Polyhipe is a high internal phase emulsion polymer and is porous
and highly absorbent. This material is particularly preferred for
applications in which a material is to be absorbed by the
particulate body.
[0036] A particulate body according to the invention may also
comprise a functional material supported by the surface polymer.
Examples of suitable functional materials include a catalyst, an
initiator species for peptide synthesis, a pharmaceutical active,
an agrochemical active, a macromolecule, an enzyme, a nucleic acid
sequence and a protein.
[0037] In one embodiment, multiple layers of surface polymer may be
applied to the hollow particle to provide different properties for
each layer. In a preferred embodiment, the invention provides a
buoyant particulate body comprising a hollow particle comprising a
polymer and a plurality of layers of surface polymer disposed on
the hollow particle. Each layer may encase all or part of the
underlying hollow particulate body or the body with a surface
polymer. In an especially preferred embodiment, the particulate
body comprises a hydrophilic surface polymer, for example
polyacrylamide, and a hydrophobic polymer, for example polystyrene.
Desirably the hydrophilic polymer encases in part or preferably the
whole of the hollow particle and the hydrophobic layer encases, in
part or preferably the whole of the hydrophilic layer.
Advantageously, multiple surface coatings allow the properties of
the particulate body to be tailored.
[0038] In another embodiment, the invention provides a buoyant
particulate body comprising a hollow particle comprising a polymer
and a plurality of layers of surface polymer disposed on the hollow
particle and the particulate body further comprises an active
component, for example a pharmaceutical and an agrochemical, the
plurality of layers of surface polymer providing controlled release
of the active component.
[0039] The active component may be any known pharmaceutical or
agrochemical active component, suitable for controlled release from
a particle.
[0040] The invention is particularly useful in supporting precious
metal catalysts, for example palladium catalysts. A particular
advantageous example is palladium acetate supported on
polyurea.
[0041] The particulate body of the invention may be produced by an
efficient and relatively simple process. The invention provides in
a further aspect a method for producing a particulate body material
comprising the steps of providing a particle comprising a polymer
having a hollow centre contacting the particle with a monomer or
solution of a monomer, effecting polymerisation of the monomer so
as to form a surface polymer coating on the surface of the hollow
particle.
[0042] Suitably the polymerisation is initiated by processes known
to those skilled in the art. For example, particles mixed with a
monomer or a solution of the monomer are added to a solvent which
is immiscible with the monomer solvent and heated to effect
polymerisation. Where the monomer solution is aqueous, the solvent
is for example kerosene.
[0043] If preferred the surface polymer may be covalently linked to
the hollow particle either during the polymerization or subsequent
to the polymerization. Alternatively, one or more of the
constituent monomers which are precursors to the surface polymer
can be covalently linked to the particle surface prior to
initiation of the polymerization.
[0044] The particulate body of the invention may be used in any
chemical or physical process in which a solid support is used.
[0045] The particulate body may be employed in applications
involving electro-conducting and light emitting polymers. The
particulate body containing light emitting polymers may be arranged
on display panels.
[0046] There are a number of practical problems associated with the
use of traditional solid polymer particles which in part relate to
the relatively high density of the solid particles. The lower
density of the hollow particulate body of the present invention
affords advantage in a wide range of applications.
[0047] The particulate body of the invention may be employed in a
novel process for the production of biofuels. It is known to
produce biodiesel by produced by a chemical process. However, this
requires the use of methanol and caustic soda, that is a feedstock
derived from fossil sources, energy, and requires significant
capital investment and management of waste products from the
process. Biodiesel may also be produced by enzyme hydrolysis which
is believed to be more economic than the chemical process and
environmentally more acceptable but the known enzyme process has
the drawback that enzyme may be lost from the process. During the
process, fatty acid and glycerol is produced and glycerol is
withdrawn from the bottom of the reactor as a heavy fraction in the
reaction process. Unfortunately, significant quantities of the
enzyme may also be located towards the bottom of the reactor and be
inadvertently withdrawn with the glycerol.
[0048] In a further aspect, the invention provides for the use of a
buoyant, hollow particulate body to retain a component or catalyst
in the reaction zone, the body having the reaction component or
catalyst bound to the body, wherein the reaction produces a heavy
component of greater density than the particulate body and
component or catalyst and the heavy component is withdrawn from the
reaction zone leaving the particulate body and component or
catalyst in the reaction zone.
[0049] In a preferred embodiment the particulate component is used
to retain a catalyst, for example an enzyme in the reaction
zone.
[0050] The hollow buoyant particulate body having the enzyme bound
to its surface reduces the loss of enzyme from the process by
enabling the enzyme to reside away from the zone in which glycerol
is withdrawn due to the buoyancy of the particulate body. This
principle may be applied to any reaction in which a component of
the reaction or a catalyst is to be retained in the reactor and
wherein the component or catalyst may be carried by the buoyant
particulate body according to the invention.
[0051] In a preferred embodiment, the invention provides a method
for the production of biodiesel comprising contacting vegetable oil
with a buoyant, hollow particulate body having an enzyme for
example a lipase such as Cal B, bound to the body in a reaction
zone to produce fatty acid and glycerol, withdrawing glycerol from
the reaction zone leaving the enzyme in the reaction zone. The
hollow particulate body having the bound enzyme is buoyant and
retained in the reaction zone as the glycerol, being a heavy
component, is withdrawn from the bottom of the reaction zone. The
esterification to form biodiesel can also be envisaged to take
place in the same reaction vessel using the same immobilized
enzyme.
[0052] The particulate body is particularly useful for solid phase
synthesis of an organic species, particularly macromolecules.
[0053] In a further aspect, the invention provides a process for
the synthesis of a reacted product comprising charging a reaction
zone with a liquid of density D.sub.l, a particulate body according
to the invention having a density D.sub.t wherein D.sub.t is less
than D.sub.l and wherein the surface of the particulate body
comprises reactive sites feeding one or more reactants to the
reaction zone under conditions for the one or more reactants to
react with the reactive sites on the body to provide a reacted
product.
[0054] Suitably, the reacted product itself possesses a reactive
site for reaction with a further reactant. The process suitably
comprises at least one further step of feeding a reactant to the
reaction zone to react with the reacted product to produce a
further reacted product. This step may be repeated with different
reactants selected according to the intended macromolecule product
whereby the reacted product is a macromolecule.
[0055] Suitably, the macromolecule is an oligonucleotide or
oligosaccharide but is especially beneficial in the synthesis of a
peptide or a nucleic acid sequence. Where the macromolecule is a
peptide the reactants are amino acids or species able to provide an
amino acid moiety in the reacted product. Where the macromolecule
is a nucleic acid sequence, for example DNA or RNA, the reactants
are nucleosides or species able to provide a nucleic,acid moiety in
the reacted product.
[0056] The particulate body of the invention is especially useful
in peptide and nucleotide synthesis. A particulate body according
to the invention is suitably washed with N,N-dimethylformamide
(DMF). A linkage agent, for example Fmoc-Am-Rink-OH and
2-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) may be dissolved in a solvent, for example
DMF. 4-Methylmorpholine (NMM) may be added and the mixture
pre-activated before adding to the washed particulate body.
[0057] The coupling reaction is suitably complete by Ninhydrin
assay preferably within 30 minutes. The support is suitably washed
with DMF and amino acids then coupled to it by reacting a compound
of formula Fmoc-X--OH where X is the desired amino acid with the
linkage agent on the particulate support.
[0058] Fmoc-X--OH is then coupled to the particulate body and
linkage agent and treated with a solvent, for example
piperidine/DMF. Each amino acid in the desired peptide sequence is
then added in sequence by the same procedure until the peptide
chain is complete.
[0059] The peptide is then suitably cleaved using a known method.
For example, the particulate body is suitably washed for example
with dichloromethane and trifluoroacetic acid (TFA) containing
water, for example 5% v/v is suitably added. The solution suitably
turns red indicating that the cleavage is progressing. After 10
minutes further TFA is suitably added and the mixture left to
cleave for 1hour. The particulate body is suitably washed with TFA.
The combined TFA cleavage solutions and washes are desirably
reduced to an oil for example on a rotary evaporator. The oil is
triturated with diethyl ether to form a white solid. The ether is
suitably moved by decantation and the peptide air dried
overnight.
[0060] The polymer of the hollow particulate body or the surface
polymer, where present, is desirably selected from
polyacrylonitrile, polydimethylacrylamide, polyethyelene glycol and
polystyrene which are particularly advantageous in synthesis of
peptides.
[0061] In solid phase synthesis, the process for using known solid
polymeric particles in applications such as peptide synthesis
typically involves suspending the particles in the appropriate
solvent above a porous filter plate and stirring the particles
gently so as not to mechanically damage the particles. Known
particles are dense and settle on to the filter in the solvents
commonly used. The manufacturing process for the particles often
generates fines that cause blockages in the filter plate leading
slow filtration or the need to replace or clean the filter. In
addition, the stirring of solid particles may cause fracture
leading to generation of fines that exaggerate the problems of
filter blockage.
[0062] In the pharmaceutical and associated industries strict
quality regulations under current good manufacturing process (cGMP)
require that the filter plate is replaced following each batch of
product in order to avoid contamination of subsequent batches with
material dislodged from the filter plate.
[0063] The buoyant particulate body according to the invention
simplifies solid phase synthesis through the use of simpler
equipment than conventionally employed.
[0064] Agitation of the particulate body is simplified and the use
of a filter plate is negated. Using the present buoyant particulate
body, solid phase synthesis can be performed in a standard solution
phase reactor. This is an advantage in the laboratory where solid
phase synthesis can be performed in its simplest form in a
separating funnel. At process scale the need for bespoke solid
phase reactors with specialist filtration and agitation systems can
be avoided. Use of the particulate body of the invention
advantageously enables cGMP operations to be simplified with easier
cleaning and cleaning verification coupled with reduced down-time
by avoiding the conventional filter plate replacement step.
[0065] In a preferred embodiment, the particulate body of the
invention may be employed in the preparation of two products in the
same reactor. For example, assembling a peptide or nucleic acid
sequence in a reactor containing buoyant particles and traditional
dense particles would allow separation of the particles, by
decantation for instance, at any stage. This would allow
continuation of the assembly of one or both of the peptide
sequences independently. This might be particularly useful for
preparation of analogues or in solid phase combinatorial
chemistry.
[0066] The invention provides a process for the simultaneous
preparation of two products in a single reaction zone comprising
charging the reaction zone with a liquid of density D.sub.l, a
particulate body according to the invention having a density
D.sub.t and providing a body for a top synthesis reaction, and a
solid body having a density D.sub.b and providing a body for a
bottom synthesis reaction, wherein D.sub.t is less than D.sub.l and
D.sub.l is less than D.sub.b, feeding reactants for the top
synthesis reaction and for the bottom synthesis reaction to the
reaction zone under conditions for the top synthesis reaction and
bottom synthesis reaction to occur to produce a top synthesis
product and a bottom synthesis product and optionally separating
the particulate body from the solid body whereby the top synthesis
product and bottom synthesis product are separated.
[0067] Suitably the top synthesis reaction and bottom synthesis
reaction are independently selected from a peptide synthesis
reaction and a nucleotide synthesis reaction.
[0068] The invention further provides for the use of a particulate
body according to the invention as a solid phase in a
chromatography process.
[0069] Conventionally, chromatography columns are generally packed
by preparing a slurry of the particles, or stationary phase in a
suitable solvent and transferring this into the column with the
lower column filter plate present. Uneven settling of the bed in
chromatography columns can cause uneven and even cracked stationary
phase beds resulting in poor and irreproducible separations.
Columns often have to be emptied and repacked several times to
achieve the required performance. This can be laborious and leads
to down time which a particular disadvantage in process scale
operations.
[0070] The buoyant particulate body of the invention allows the
process for preparation of the slurry and transfer of the slurry to
the column to be simplified. The buoyancy of the particulate body
allows the stationary phase to be formed by floatation of the body
which affords a simpler process and a more evenly packed column.
Advantageously, the stationary phase may be easily mobilized by
re-floating the particles within the column avoiding the need to
empty and re-slurry if desired. In addition, the process for
emptying columns is simplified since the particles can be floated
within the column and removed by decantation. This is of particular
advantage for process scale operations.
[0071] In a further preferred embodiment, a two or more particulate
bodies each having a different density may be employed to pack a
column to provide a mixed bed column with defined bands. For
example, affinity separation of IgG on a Protein A based stationary
phase could be combined with an ion exchange separation to remove
leached Protein A in the same column. Following such a process the
stationary phases may be separated by taking advantage of differing
density of the two or more particulate bodies. In a further
embodiment, the invention may be employed to perform chiral
separations for example where the different particulate bodies
carry R and S chiral selectors in the same column.
[0072] The particles may also be loaded or packed into a column and
the interstitial spaces filled with a monolith polymer to form a
monolith. The invention further provides a particulate body
monolith comprising a plurality of particulate body particles
according to the invention packed to provide a monolith, preferably
in a column arrangement.
[0073] As desired, the interstitial spaces between the particulate
body in a monolith are suitably filled with a different polymer to
that of the particulate body and surface polymer.
[0074] In another embodiment the interstitial spaces between the
particles in a monolith may be filled with a different component
such as a cell culture nutrient for example. In this example the
cells may be cultured on the surface polymer coating the
particle.
[0075] In a preferred embodiment the invention provides a
particulate, body comprising a hollow particle comprising
polyacrylonitrile and, optionally a surface polymer disposed on the
outside of the hollow particle.
[0076] Polyacrylonitrile is advantageous as it is generally
chemically stable but is soluble in dimethylformamide (DMF) and a
hollow particulate body having a polyacrylonitrile wall may be
penetrated by a dilute DMF solution but remain intact. This enables
a material to be carried through the wall of the hollow particulate
body to the interior and therein subjected to a process within the
confines of the internal dimensions of the hollow particle. This
enables particles of a very narrow particle size distribution to be
produced.
[0077] The invention provides for use of a hollow particle
comprising polyacrylonitrile for the production of particles having
a narrow particle size distribution. In a further aspect the
invention provides a method of producing polymer particles
comprising contacting a solution comprising DMF and water, and a
monomer with a hollow particulate body comprising a
polyacrylonitrile shell so that the solution passes through the
shell to the interior of the body, subjecting the solution to
conditions to initiate polymerisation of the monomer to form
polymer particles within the hollow particulate body and contacting
the particulate body with DMF so as to dissolve the particulate
shell so providing the polymer particle.
[0078] Suitably the DMF solution comprises DMF at a level of 20 to
80% w/w and preferably 35 to 65%, optimally 40 to 60% for example
50%, depending on the polymer to be penetrated. The particular
dilution of DMF should be selected to allow penetration of the
solution through the polymer wall, which may be in the form of a
membrane. If the solution is too weak, penetration will be less
likely to occur and if too strong, the polymer membrane may
rupture. Should it be desired to rupture the polymer, a stronger
solution of DMF than that required to penetrate the polymer will be
employed.
[0079] Advantageously, particles having a narrow particle size
distribution and desirable porosity may be obtained.
Conventionally, particles having a narrow particle size
distribution have been obtained by sieving which is however capital
intensive and may cause physical degradation of the particles. It
is known to grow polymer particles however this approach has the
drawback that the particles may lack porosity.
[0080] The particulate body of the invention is also useful for
solid phase extraction to remove species from a liquor which is
contacted with the body, whether in batch form or as a flow over
the body, for example ion extraction and ion exchange. Solid phase
extraction is typically performed in columns or in systems with
filter plates for separation of the solid phase from the mixture
under extraction. The problems observed for solid phase synthesis
and chromatography referred to herein may similarly be observed
with solid phase extraction. The buoyant particulate body of the
invention provides similar advantages as afforded in chromatography
and solid phase synthesis.
[0081] The buoyant particulate body of the invention may be
employed to separate species using solvent immiscibility. In a
binary system comprising an aqueous phase and an organic phase, for
example dichloromethane, a hydrophilic buoyant particulate body
according to the invention floated in the aqueous phase and a
hydrophobic particulate body floated in the dichloromethane phase
may provide discrete zones in which processes, for example
separation, extraction or synthesis may be carried out. This
arrangement suitably effectively provides a four phase extraction
system.
[0082] The body of the invention may be used to immobilize species
including antibodies, oligonucleotides, enzymes or fluors and may
be positioned in an array, with each body assaying a different
component of a solution. A particulate body having ligands
covalently attached to their surface, or via a surface polymer
bound to the surface may be employed as `wells`. Specific binding
of a target ligand such as antigen or complimentary DNA or RNA
sequence may then be detected using established methods.
[0083] The particulate body of the invention also may be employed
to immobilize a biocatalyst. Biocatalysts are often used in columns
or in systems with filter plates for separation of the solid phase
from the mixture under extraction. The problems observed for solid
phase synthesis and chromatography referred to herein may similarly
be observed with solid phase extraction. The buoyant particulate
body of the invention provides similar advantages as afforded in
chromatography and solid phase synthesis.
[0084] Conventionally immobilized biocatalysts, for example
immobilized enzymes, on solid bodies may disadvantageously settle
on the base of the reactor leading to reduced contact of the
biocatalyst with the substrate. The particulate body of the present
invention may be readily agitated to ensure the surface the maximum
usable area of the biocatalyst remains available to the
substrate.
[0085] The present invention also envisages systems with two or
more different immobilized biocatalysts or cofactors in the same
column or reactor. Advantageously, an immiscible solvent system
similar to that described for solid phase extraction may also be
employed to provide different reaction zones for biocatalysts
immobilized on different particulate bodies. The buoyant
particulate body of the invention may also have applications in
systems where countercurrent or vortex separation systems are
used.
[0086] The particulate body of the invention is especially useful
in immobilising species including solid phase reagents, metal and
other catalysts, bio-catalysts, enzymes, proteins, antibodies
including polyclonal and monoclonal antibodies, whole cells and
polymers. The invention is particularly advantageous in supporting
enzymes, for example the lipase Cal A works well, particularly in
combination with polydimethylacrylamide and other similar
hydrophilic polymers. The present invention is also especially
useful in the immobilisation of affinity ligands such as Protein
A.
[0087] In a further application, the particulate body of the
invention may also be used in chemocatalysis, for example by
immobilizing transition metal catalysts and ligands.
[0088] In yet a further application, the present invention may be
used in cell culture. Mass culture of animal cell lines is
fundamental to the manufacture of viral vaccines and many products
of biotechnology. Biological products produced by recombinant DNA
technology in animal cell cultures include enzymes, synthetic
hormones, immunobiologicals (monoclonal antibodies, interleukins,
lymphokines) and anticancer agents. Many simpler proteins can be
produced using rDNA in bacterial cultures; more complex proteins
that are glycosylated (carbohydrate-modified) currently must be
made in animal cells. An important example of such a complex
protein is the hormone erythropoietin. The cost of growing
mammalian cell cultures is high, so companies are constantly
looking to improve techniques.
[0089] Cells can be grown in suspension or as adherent cultures.
However, adherent cells require a surface, which may be coated with
extracellular matrix components to increase adhesion properties and
provide other signals needed for growth and differentiation.
Generally cells derived from solid tissues are adherent.
Organotypic culture involves growing cells in a three-dimensional
environment as opposed to two-dimensional culture dishes. This 3D
culture system is biochemically and physiologically more similar to
in vivo tissue, but is technically challenging to maintain because
of many factors (e.g. diffusion).
[0090] In a further aspect, the invention provides for the use of a
buoyant particulate body according to the invention to culture
cells on the surface of the body. Suitably, stem cells may be
cultured on the particulate body of the invention to reduce
uncontrolled differentiation and to control desired
differentiation. The handling characteristics of the particulate
body and high utilization of surface area through buoyancy of the
body is advantageous in this application.
[0091] The invention is particular useful in medical diagnostic
tests such as immunoassay. Accordingly the invention further
provides a medical diagnostic for detecting the presence of a
compound the diagnostic comprising a particulate body according to
the invention and a functional material such as an enzyme, for
example horseradish peroxidase, supported by the surface polymer in
the particulate body for selectively reacting with or binding to
the compound to be detected.
[0092] Many medical diagnostics rely upon solid supports to
immobilize various diagnostic ligands. The particulate body of the
present invention may be used in a medical diagnostic procedure
where physical separation of the solid phase through a liquid
phase.
[0093] In a further application, the particulate body may be used
as an absorbent. In this application, it is especially advantageous
if the body contains an inert, absorbent material bound to the
particulate body and to which the surface polymer is bound.
Polyhipe is a particularly preferred inert material. The
particulate body may be used to absorb household spillages, for
example tea, coffee and wine, or may be used in larger-scale
applications for example, to absorb oil from spillages. The
absorbent body may be used to absorb the spillage and then
physically removed or, in the case of oil spillage in a body of
water, effectively trap the oil and retain the oil in a buoyant
mass for collection and disposal.
[0094] The particulate body of the invention may be used as a
carrier to carry a compound which is to be released over a period
of time, for example a pharmaceutical or agrochemical compound or
composition. This use provides a means of tailoring a dosage regime
of the compound according to the loading of the compound in the
body. In the case of a pharmaceutical, this may be advantageous in
assisting the correct dosage of an active, for example with
continuous slow release rather than requiring a patient to take
periodic large doses, for example in chemotherapy.
[0095] The invention is illustrated by reference to the
accompanying drawings in which:
[0096] FIGS. 1 and 2 each shows illustrative embodiments of the
particulate body of the invention in cross section.
[0097] FIG. 1 shows a hollow particulate body having a uniform
shape comprising a hollow particle (1) which has a surface polymer
(2) uniformly coated on the particle (1).
[0098] FIG. 2 shows a hollow particulate body in which the hollow
particle (1) is coated with surface polymer (2) and which has an
irregular shape.
[0099] FIG. 3 shows a hollow particulate body comprising a
plurality of hollow particles (1) which are coated with surface
polymer (2) such that the particulate body has a uniform shape.
[0100] FIG. 4 shows a hollow particulate body comprising a
plurality of hollow particles (1) which are coated with surface
polymer (2) such that the particulate body has a non-uniform or
irregular shape.
[0101] The invention is illustrated by the following non-limiting
examples.
EXAMPLES
Example 1
Conversion of Surface Nitrile to Carboxylic Acid
[0102] Expancel 920 DEX 80 d30 (80 .mu.m polyacrylonitrile
balloons)(100 cm3) were treated with potassium hydroxide solution
(20% w/v in 200 cm3 of 4:1 v/v water: methanol) at 80.degree. C.
for 3 h. The hollow particles were washed with water (3.times.100
cm3), concentrated hydrochloric acid (3.times.100 cm3) and methanol
(3.times.100 cm3) before air drying.
[0103] The carboxylic acid content was determined by titration of
the polymer (250 mg) with aqueous sodium hydroxide (5.85 mmol/dm3)
using phenolphthalein indicator. The result was also confirmed by
addition of excess sodium hydroxide solution (5.85 mmol/dm3)
followed by back titration with aqueous hydrochloric acid (6
mmol/dm3). The carboxylic acid loading was 0.30 mmol/g.
Example 2
Conversion of Surface Nitrile to Carboxylic Acid
[0104] Expancel 920 DEX 80 d30 (80 .mu.m polyacrylonitrile
balloons)(100 cm3) were treated with potassium hydroxide solution
(20% w/v in 200 cm3 of 4:1 v/v water: methanol) at 80.degree. C.
for 20 h. The hollow particles were washed with water (3.times.100
cm3), concentrated hydrochloric acid (3.times.100 cm3) and methanol
(3.times.100 cm3) before air drying.
[0105] The carboxylic acid content was determined by titration of
the polymer (250 mg) with aqueous sodium hydroxide (5.85 mmol/dm3)
using phenolphthalein indicator. The result was also confirmed by
addition of excess sodium hydroxide solution (5.85 mmol/dm3)
followed by back titration with aqueous hydrochloric acid (6
mmol/dm3). The carboxylic acid loading was 1.0 mmol/g.
Example 3
Conversion of Surface Nitrile to Primary Amine
[0106] Expancel 920 DEX 80 d30 (200 cm3) was dispersed in dry
tetrahydrofuran (THF)(400 cm3 ) and lithium aluminium hydride in
THF (30 cm3 of 2 mmol/dm3) was added slowly over 20 minutes. The
reaction was left at 50.degree. C. for 16 h. Excess lithium
aluminium hydride was destroyed by slow addition of water (100 cm3)
then the polymer was washed with water (5.times.200 cm3), THF
(2.times.100 cm3), methanol (5.times.200 cm3) and diethyl ether
(1.times.100 cm3) before air drying. Yield 5.8 g.
[0107] The polymer was positive to ninhydrin assay used to
determine primary amine content.
Example 4
Increasing Amine Loading
[0108] The product from Example 3 was reacted with
Fmoc-Lys(Fmoc)-OH (1.8 g, 3 mmol) in the presence of
diisopropylcarbodiimide (0.76 g, 6 mmol) and N-methylmorpholine
(0.61 g, 6 mmol) in dichloromethane (DCM)(100 cm3) as solvent for 1
h. The polymer was washed with DCM (3.times.100 cm3) and treated
with piperidine in DCM (100 cm3, 20% v/v) for 30 minutes. The
polymer was then washed with DCM (3.times.100 cm3), MeOH
(3.times.100 cm3), water (3.times.100 cm3), MeOH (3.times.100 cm3)
and diethyl ether (1.times.100 cm3) before air drying. Yield 6.3 g
(-0.6 mmol/g).
Example 5
Reaction with Alpha-Bromoisobutyryl Bromide (BIB)
[0109] The polymer from Example 4 (1.9 g) was dispersed in DCM (30
cm3) and BIB (2 cm3, 8.8 mmol) added followed by pyridine (2 cm3).
The reaction was left for 2 h then the polymer was washed with DCM
(3.times.30 cm3), MeOH (3.times.30 cm3), water (3.times.30 cm3),
MeOH (3.times.30 cm3) and diethyl ether (1.times.30 cm3) before air
drying.
Example 6
Polymer Coating of BIB Functionalised Polymer
[0110] The BIB functionalised polymer prepared in Example 5 (0.6 g)
was dispersed in an aqueous monomer solution (100 cm3) containing
dimethylacrylamide (8.43 g, 85 mmol), ethylene bis-acrylamide (1.6
g, 9.8 mmol) and acryloyl sarcosine methyl ester (1.57 g, 10 mmol).
CuBr (186 mg, 1.3 mmol) and
N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) (0.675 g, 3.9
mmol) were dissolved in MeOH (5 cm3) and added to the above
dispersion to initiate the polymerisation.
[0111] After 1.5 h the mixture had thickened. Water (100 cm3) was
added to redisperse the polymer and the reaction left for a further
16 h. The polymer was washed thoroughly with water, DMF, MeOH and
then diethyl ether before air drying. Yield 5.1 g.
Example 7
Conversion of Coated Polymer to Primary Amine
[0112] The polymer coated hollow spheres prepared in Example 6 (2.5
g) were treated with ethylene diamine (50 cm3) overnight. The
polymer was washed thoroughly with water and MeOH before air
drying.
Example 8
Conversion of Coated Polymer to Primary Amine
[0113] The polymer coated hollow spheres prepared in Example 6 (2.5
g) were treated with 1,2-Bis(2-aminoethoxy)ethane (50 cm3)
overnight. The polymer was washed thoroughly with water and MeOH
before air drying.
Example 9
Conversion of Surface Nitrile to Methyl Ester
[0114] Expancel 920 DEX 80 d30 (100 cm3) was dispersed in
concentrated hydrochloric acid in MeOH (300 cm3, 1:1 v/v) and
stirred at 80.degree. C. for 4 h. The polymer was washed thoroughly
with water and MeOH before air drying.
Example 10
Conversion of Surface Methyl Ester to Primary Amine
[0115] The polymer prepared in Example 9 (50 cm3) was dispersed in
ethylene diamine to displace the methyl ester and stirred at room
temperature for 16 h. The polymer was washed thoroughly with water
and MeOH before air drying.
[0116] The polymer was positive to ninhydrin assay used to
determine primary amine content.
Example 11
Conversion of Surface Methyl Ester to Primary Amine
[0117] The polymer prepared in Example 9 (50 cm3) was dispersed in
Jeffamine 800 (bis-amino PEG) to displace the methyl ester and
stirred at 90.degree. C. for 6h. The polymer was washed thoroughly
with water and MeOH before air drying.
[0118] The polymer was positive to ninhydrin assay used to
determine primary amine content.
Example 12
Hollow Polymer Spheres as a Mould
[0119] Expancel 920 DEX 80 d30 (80 cm3) was dispersed in an aqueous
DMF (30 cm3, 1:1 v/v) containing dimethylacrylamide (1.455 g, 14.7
mmol), methylene bis-acrylamide (0.231 g, 1.5 mmol) and acryloyl
sarcosine methyl ester (0.314 g, 2 mmol) and left for 1 h.
[0120] Excess monomer solution was drained off (-10 cm3) and the
polymer particles dispersed in toluene (100 cm3). Ammonium
persulfate solution (0.25 cm3, 10% w/v) was added followed by
tetramethylene ethylene diamine (TEMEDA)(0.25 cm3) and the mixture
stirred at 80.degree. C. for 2 h then left overnight at room
temperature. The polymer was washed thoroughly with DMF, DCM and
diethyl ether before air drying to produce spherical particles of
polydimethylacrylamide based polymer (yield, 0.6 g).
Example 13
Conversion of Polydimethylacrylamide Based Polymer to Primary
Amine
[0121] The polymer coated hollow spheres prepared in Example 12
(0.5 g) were treated with ethylene diamine (20 cm3) overnight. The
polymer was washed thoroughly with water and MeOH before air
drying.
[0122] The polymer was positive to ninhydrin assay used to
determine primary amine content.
Example 14
Coating of Hollow Polymer Spheres with Silica
[0123] Methyl ester functional hollow polymer spheres (50 cm3)
prepared as described in Example 9 were treated with
3-aminopropyltrimethoxysilane (10 cm3) in MeOH (50 cm3) overnight
at room temperature. The particles were washed thoroughly with
water in MeOH (1:1 v/v) then treated with water in MeOH (1:1 v/v)
containing ammonium hydroxide (0.1% v/v) for 3h to initiate
hydrolysis of the methoxysilane to form silica. The polymer was
washed thoroughly with acetone and left to cure in acetone for 1
week before filtering and air drying.
Example 15
Use of a Particulate Body in Peptide Synthesis
[0124] The polymer of Example 6 was used to prepare the peptide
oxytocin using a known, conventional peptide synthesis method by
coupling a linkage agent to the particulate body and then coupling
in sequence the amino acids of oxytocin and cleaving the
synthesised peptide to produce oxytocin.
[0125] The peptide was produced to a high level of purity
comparable or greater than that obtained using a conventional
peptide synthesis support and at lower cost as the particulate body
of the invention is less costly to produce than conventional
peptide synthesis supports.
[0126] A peptide synthesis method suitable for use in producing
oxytocin and other peptides comprises providing a particulate body,
for example having an amine functional polydimethylacrylamide
polymer is suitably washed with N,N-dimethylformamide (DMF) and
subjecting the particulate body to a known peptide synthesis
method, for example as set out below.
[0127] A linkage agent, for example Fmoc-Am-Rink-OH and
2-(1H-benzotriazol-1-yl)-N, N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) may be dissolved in DMF.
4-Methylmorpholine (NMM) may be added and the mixture pre-activated
for 2-3 minutes before adding to the washed particulate body. The
coupling reaction is suitably complete by Ninhydrin assay
preferably within 30 min. The support is suitably washed in with
DMF.
[0128] Coupling of Amino Acids Fmoc-X--OH where X is the Desired
Amino Acid
[0129] Fmoc-X--OH is then coupled to the particulate body and
linkage agent and treated with piperidine/DMF using the procedure
set out for coupling the linkage agent. Each amino acid in the
desired peptide sequence is then added in sequence by the same
procedure until the peptide chain is complete.
[0130] Peptide Cleavage
[0131] The particulate body is suitably washed for example with
dichloromethane and trifluoroacetic acid (TFA) containing water,
for example 5% v/v is suitably added. The solution suitably turns
red indicating that the cleavage is progressing. After 10 minutes
further TFA is suitably added and the mixture left to cleave for 1
hour.
[0132] The particulate body is suitably washed with TFA in a
separating funnel. The combined TFA cleavage solutions and washes
are desirably reduced to an oil for example on a rotary evaporator.
The oil is triturated with diethyl ether to form a white solid. The
ether is suitably moved by decantation and the peptide air dried
overnight.
[0133] The peptide was shown to contain one major component by
reversed phase HPLC and had the expected molecular weight as
determined by MALDI-TOF mass spectrometry.
* * * * *