Patent Application
20120237606
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.
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.
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.
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.
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).
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.
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.
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.
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, Vorträge beim 34. Metallurgischen Seminar des Fachausschusses fuer Metallurgische Ausund Weiterbildung der GDMB; 18-20 Nov. 1998; Goslar).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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/cm3, more preferably less than 0.8 g/cm3 and especially less than 0.7 g/cm3 so enabling buoyancy in diethyl ether. In a further preferment, the particulate body has a density of less than 0.5 g/cm3 and more especially of 0.3 g/cm3 or less. Suitably, the particulate body has a density of at least 0.005 g/cm3, and suitably at least 0.01 g/cm3. Preferably the particulate body has a density in the range 0.01 to 0.05 g/cm3, more preferably 0.01 to 0.03 g/cm3 for example 0.015 g/cm3. Desirably, the particulate body may be used in batch-wise operations allowing for rapid processing
The term “particulate body” as employed herein includes a particulate support and may be employed as a support, for example in solid-phase synthesis.
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.
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.
Advantageously, the hollow particles are rigid and mechanically robust and may be utilized at high flow rates in packed column beds.
The hollow particle may suitably comprise an inert material comprising a polymer and preferably consists essentially of a polymer.
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.
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®.
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.
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 μm, preferably 5 to 150 μm, and the particle size will be selected according to the desired application. In one preferred embodiment, the particle size is from 1 to 50 μm, preferably 5 to 20 μm for example 10 μm. 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 μm, preferably 50 to 200 μm, for example 100 μm. 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The active component may be any known pharmaceutical or agrochemical active component, suitable for controlled release from a particle.
The invention is particularly useful in supporting precious metal catalysts, for example palladium catalysts. A particular advantageous example is palladium acetate supported on polyurea.
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.
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.
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.
The particulate body of the invention may be used in any chemical or physical process in which a solid support is used.
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.
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.
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.
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.
In a preferred embodiment the particulate component is used to retain a catalyst, for example an enzyme in the reaction zone.
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.
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.
The particulate body is particularly useful for solid phase synthesis of an organic species, particularly macromolecules.
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 Dl, a particulate body according to the invention having a density Dt wherein Dt is less than Dl 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The buoyant particulate body according to the invention simplifies solid phase synthesis through the use of simpler equipment than conventionally employed.
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.
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.
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 Dl, a particulate body according to the invention having a density Dt and providing a body for a top synthesis reaction, and a solid body having a density Db and providing a body for a bottom synthesis reaction, wherein Dt is less than Dl and Dl is less than Db, 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.
Suitably the top synthesis reaction and bottom synthesis reaction are independently selected from a peptide synthesis reaction and a nucleotide synthesis reaction.
The invention further provides for the use of a particulate body according to the invention as a solid phase in a chromatography process.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
The invention is illustrated by reference to the accompanying drawings in which:
The invention is illustrated by the following non-limiting examples.
Expancel 920 DEX 80 d30 (80 μm 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° C. for 3 h. The hollow particles were washed with water (3×100 cm3), concentrated hydrochloric acid (3×100 cm3) and methanol (3×100 cm3) before air drying.
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.
Expancel 920 DEX 80 d30 (80 μm 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° C. for 20 h. The hollow particles were washed with water (3×100 cm3), concentrated hydrochloric acid (3×100 cm3) and methanol (3×100 cm3) before air drying.
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.
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° C. for 16 h. Excess lithium aluminium hydride was destroyed by slow addition of water (100 cm3) then the polymer was washed with water (5×200 cm3), THF (2×100 cm3), methanol (5×200 cm3) and diethyl ether (1×100 cm3) before air drying. Yield 5.8 g.
The polymer was positive to ninhydrin assay used to determine primary amine content.
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 (3×100 cm3) and treated with piperidine in DCM (100 cm3, 20% v/v) for 30 minutes. The polymer was then washed with DCM (3×100 cm3), MeOH (3×100 cm3), water (3×100 cm3), MeOH (3×100 cm3) and diethyl ether (1×100 cm3) before air drying. Yield 6.3 g (−0.6 mmol/g).
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 (3×30 cm3), MeOH (3×30 cm3), water (3×30 cm3), MeOH (3×30 cm3) and diethyl ether (1×30 cm3) before air drying.
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.
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.
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.
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.
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° C. for 4 h. The polymer was washed thoroughly with water and MeOH before air drying.
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.
The polymer was positive to ninhydrin assay used to determine primary amine content.
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° C. for 6h. The polymer was washed thoroughly with water and MeOH before air drying.
The polymer was positive to ninhydrin assay used to determine primary amine content.
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.
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° 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).
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.
The polymer was positive to ninhydrin assay used to determine primary amine content.
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.
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.
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.
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.
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.
Coupling of Amino Acids Fmoc-X—OH where X is the Desired Amino Acid
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.
Peptide Cleavage
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.
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0916281.9 | Sep 2009 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/005698 | 9/16/2010 | WO | 00 | 6/4/2012 |
