Solid phase process

Abstract

The invention provides a solid phase process comprising passing a mixture comprising solid phase particles and one or more liquid components through a confined, contact zone having an inlet and an outlet and between the inlet and outlet, a permeable barrier which is permeable to liquids and wherein the solid and liquid component(s) interact, for example chemically react, and a portion of the liquid component(s) or a liquid product of the interaction between the solid and the liquid component(s) passes through the permeable barrier. Apparatus for carrying out the process are also provided. The invention is useful for solid-phase synthesis, particularly for peptides, peptidomimetics and oligonucleotides.

Description

[0001] SOLID PHASE PROCESS

[0002] This invention relates to a solid phase process, particularly to any process that uses a solid phase for example in the synthesis of a chemical or biological product, or uses a solid phase material, for example an immobilized species such as enzymes, immobilized antibodies, immobilized catalysts and the like. The invention provides a filtration process and also a process useful in solid phase extraction methods. The invention particularly relates to a solid-phase process for the synthesis of peptides, peptidomimetics and oligonucleotides.

[0003] Solid supports are 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, regenerative medicine, catalysis, chromatography, slow release of active species, for example agrochemicals and pharmaceuticals, medical processes including wound care and medical diagnostics and in many other chemical, biological and physical processes, for example in metal recovery and mining processes. The use of polymeric particles in solid phase extraction and in the preparation of solid phase reagents is also known and widely used in the chemical, pharmaceutical and biotechnology industry.

[0004] Whilst solid phase processes and solution phase processes have their own benefits and drawbacks, solid phase synthesis may reduce or avoid isolation procedures often required in solution phase processes, typically 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 often 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] The application of solid supports or stationary phases in chromatographic separations is widespread in a wide range of industries in which separation, extraction or synthesis are required including in complex high-technology separations in the pharmaceutical and biotechnology industry and precious metal recovery, water treatment, and in large scale processes used in the mining industry. Some of the pharmaceutical industry’s most valuable drugs are purified by preparative chromatography. In the mining and precious metal recovery industry a large portion of the world's palladium 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 Aus-und Weiterbildung der GDMB; 18-20 November 1998; Goslar).

[0006] Solid phase synthesis of biological or chemical species may often involve multiple stages and often include synthesis steps and isolation steps to separate intermediates, excess materials, by-products or the like. Peptide and oligonucleotide synthesis usually involve a succession of reaction and isolation step in order to grow the peptide or oligonucleotide by step-wise addition of new amino acids or nucleic acids to the growing molecule. Such processes may be time-consuming, expensive and may be inefficient as regards yield; intermediates often require purification to remove excess reagents and reaction byproducts and procedures such as precipitation, filtration, bi-phase solvent extraction; solid phase extraction, crystallization and chromatography may be employed.

[0007] Peptides have traditionally been made, predominantly by a solid phase synthesis process (SPPS) for decades, from research quantities to ton quantities for commercial production. Solid phase peptide synthesis typically involves using an insoluble solid support-bound and stepwise assembly of the peptide in a mechanically agitated reaction vessel containing a filter. The carboxyl terminal of the growing peptide chain is anchored to a solid support by a cleavable linker and the peptide is assembled by addition of protected amino acids to the amino acid chain anchored to the support by repetitive cycles of amino acylation and N-terminal deprotection. At the end of assembly, the peptide-resin is subjected to acid cleavage to release the crude peptide for purification and characterization prior to use. Batch-wise synthesis is also used but has drawbacks due to large dilution of activated reagents within the volume of solvent required for the resin to be mobile during agitation, requiring large excesses of activated amino acids and long coupling times to ensure successful synthesis.

[0008] Conventionally, peptides are made by a “stirred-tank” process or a “column” process. The stirred-tank process typically involves filtration over a bed of solid support and through a flat bed filter plate and allows production at a large scale and use of readily available pressure filters. Problems may arise as scale increases and compressive intrusion may inhibit washing of the solid phase and filter blockage may often occur. Further, mass transport of reagents into the solid phase is solely through diffusion. The column process typically involves passage of reagents and washing solvents over a solid support through a flat bed filter plate. Advantageously maintains the solid, reagents and solvent enclosed within the column and is easily automated. The stirred tank and the column process have drawbacks in that the flow rate must be optimized according to the physical characteristics of the solid phase, optimum flow rates may be difficult to achieve due to compressibility of the solid state, filter blockage is common and mass transport is by diffusion alone. In the column process, the column size may limit scale or raise additional design and process issues when scaling-up.

[0009] Immobilization of species is well known in a wide range of processes. Polymer supports are commonly used for the immobilization of catalysts for use in organic chemistry including chemical and biological 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).

[0010] Solid supports are also used to immobilize biological macromolecules, for example, proteins, monoclonal and polyclonal antibodies in a range of applications including medical and diagnostic applications. Cell culture is commonly carried out on solid supports with specific surface characteristics and morphology. Immobilized enzymes on supports may also be employed as sensors to generate a signal, for example 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.

[0011] Polymeric particles are often used as stationary phase solid supports in chromatography. Stationary phases are often costly which may restrict their use. Stationary phases may present certain drawbacks, for example, soft polymers, often used for affinity, ionexchange and gel permeation chromatography, when used at high flow rates, may deform due to the deformable characteristics of the particles. Rigid macroporous polymers used in other modes of chromatography may be mechanically friable and subsequently suffer from a short lifetime, especially at high pressure. In many solid-phase processes involving columns, there are limits to the pressure that maybe applied due to unacceptably high levels of back pressure which may cause resin fracture and filter blocking, leading to significant difficulties and downtime to clean or replace filters.

[0012] Tangential flow filtration (TFF) is a known filtration process and is commonly applied to separation of large molecules such as polymers or proteins and is carried out on a membrane designed to separate these molecules by molecular weight difference. TFF is also widely used in filtration to remove particulates, for example in the brewing industry. TFF is also used on a large scale to remove particles from domestic water supplies.

[0013] We have now found that solid phase processes involving passing liquid phase components for example solvents, reactants or products through a solid phase bed may be improved by providing a moving solid support over a filter to facilitate solid / liquid contact and suitably a chemical reaction to improve mass and energy transfer.

[0014] In a first aspect, the invention provides a solid phase process comprising passing a mixture comprising solid phase particles and one or more liquid components through a confined, contact zone having an inlet and an outlet and between the inlet and outlet, a permeable barrier which is permeable to liquids and wherein the solid and liquid component(s) interact, for example chemically react, and a portion of the liquid component(s) or a liquid product of the interaction between the solid and the liquid component(s) passes through the permeable barrier.

[0015] Suitably, the interaction between the one or more solid components and the one or more liquid components comprises a chemical reaction. The chemical reaction may be any type of reaction including synthesis, formation of bonds, substitution, ligation, cleavage of bonds and the like.

[0016] In the confined contact zone, the process comprises pressurising the mixture in the confined, contact zone such that the mixture including the solid phase particles passes through the contact zone and the outlet and at least a portion of the liquid passes through the permeable barrier. The term “contact zone” also encompasses processes in which separation occurs so may have the function of and be referred to as a separation zone. In a second aspect, the invention provides a solid phase process contact apparatus comprising a contact vessel having an inlet for receiving a mixture comprising solid phase particles and one or more liquid components and an outlet for removing the solid phase particles from the contact vessel which provides a confined chamber defining a contact zone between the inlet and outlet wherein at least part of the chamber wall comprises a permeable barrier which is permeable to the liquid component and a pump to convey the mixture into the contact chamber.

[0017] An illustrative example of an apparatus according to the second invention is shown in Figure 1 and, the contact vessel part of the apparatus is shown in Figure 2.

[0018] The term “confined” ’’refers to characteristics of the contact chamber or contact zone by which the wall of the confined zone or chamber presents a reaction force to the force applied by the liquid / solid mixture by virtue of it being under pressure, the liquid / solid mixture may pass out of the outlet and a portion of the liquid through the permeable barrier and out of the system, thereby allowing the applied pressure to be dissipated to a degree, and enabling transport of the components through the system.

[0019] The contact zone provides a volume in which the liquid component(s) and the solid phase component may interact, for example chemically interact, and a portion of the liquid component and / or a liquid product from the interaction pass through the permeable barrier.

[0020] Suitably, the mixture comprising solid phase particles and one or more liquid components is a slurry. The solid phase component of the slurry may be as high as is pumpable and may be 50% or more w / v with respect to the solid phase particles, preferably 10 to 60%, more preferably 10 to 50%, especially 20 to 45% w / v.

[0021] The continuous flow of the mixture from the inlet to the outlet reduces the risk of blocking the permeable barrier and suitably provides turbulence to enhance contact between the solid phase particles and the liquid component(s). Further, the pressure applied to the mixture flow enables continuous filtration of at least a portion of the liquid component through the permeable barrier.

[0022] Advantageously, the flow of the liquid / solid mixture facilitates contact greater than that achievable by diffusion alone. The process of the invention enables the solid support to be moved across the filter barrier enabling contact with the liquid which may then, in part pass through the barrier without having to pass through a packed bed of the solid. By pumping the solid / liquid mixture, energy is imparted to the mixture which allows the solid phase particles to be deformed or squeezed to aid mass transfer within and between particles and the process ameliorates the drawbacks associated with processing mixtures of softer particles with flat bed filters.

[0023] The flow rate of the slurry from the inlet to the outlet and the flow rate of the liquid component(s) through the permeable membrane may be controlled for any given solid / liquid mixture by varying the pumping rate, the relative sizes of the inlet and the outlet and the size of the apertures or pores in the permeable barrier and the size of the barrier, for example the length of the contact chamber itself. Pressurising means, preferably a pump, for example a variable speed diaphragm pump, pumps the mixture into the inlet of the separation zone. By way of example, for a contact vessel having a tubular filter (1cm x 50cm), a stainless steel mesh filter (1cm x 15cm), a stainless steel mesh filter (1cm x 50cm), a sintered tubular filter (1cm x 50cm) , a polymeric filter 5m2, the pumping rate is at least 5 litres per minute, preferably at least 10 litres per minute, especially at Ieast15 litres per minute, for example 17 litres per minute.

[0024] The process may be a continuous flow process with the solid and liquid components being continuously fed to the inlet and removed from the outlet and through the permeable barrier. Preferably, the outlet is in fluid communication with the inlet such that the solid phase cycles through recycle loop.

[0025] Suitably, the apparatus comprises one or more solid and / or liquid inlets upstream of the inlet to the separation zone. The solid and / or liquid inlet may comprise a reservoir from which the liquid and / or solid component is continuously taken to the inlet of the separation zone or may comprise a separate storage zone which is periodically in fluid communication with the inlet to the separation zone, for example by means of a valve. Where the apparatus and process comprises a recycle loop, the recycle loop suitably comprises one or more inlets by which further quantities of the liquid component(s) and / or additional liquid components, for example reagents, solvents, additives and the like may be introduced to the process.

[0026] Suitably, the permeable barrier comprises pores or apertures of a size selected to allow passage of the liquid component to the other side of the barrier. The liquid component may then be removed for disposal as waste or for further processing or use. The permeable barrier may comprise two or more sheets of permeable material. The permeable barrier may be curved or planar. In a preferred embodiment, the contact vessel comprises a housing having an inlet and an outlet and the permeable barrier is in the form of a filter cartridge, suitably an elongate filter cartridge. The filter cartridge is suitably located in the contact vessel and the internal volume of the cartridge provides the contact zone within with the solid phase particles and the liquid component interact.

[0027] The contact chamber may be any shape or dimension but is preferably elongate. Suitably, the contact chamber is tubular with the inlet at one end and the outlet at the opposing end. The permeable barrier is disposed in at least a part of the wall of the contact chamber and, preferably comprises the entire wall between the inlet and the outlet.

[0028] Preferably, the contact vessel and confined chamber is oriented vertically, aiding flow under gravity and presenting a much smaller footprint than a conventional flat-bed filter, an important consideration in designing industrial systems.

[0029] In a preferred embodiment, the permeable barrier filter comprises a tubular membrane with a pore size selected according to the particular particle size of the solid phase particles. The membrane may be made of any suitable materials, for example stainless steel mesh, polymeric mesh, and a sintered filter. As examples, a 2um particle will suitably be capable of being filtered where the permeable barrier has a pore size of up to 1 pm. A 100um particle may be filtered readily using a permeable barrier having a pore size of typically 25pm.

[0030] In another preferred embodiment, the TFF filter can be a flat sheets set-up to operate in a cross-flow manner. Preferably, the contact vessel has a longitudinal axis along which the liquid / solid phase mixture flows in use and he permeable barrier is disposed such that liquid component(s) pass in a locus which is orthogonal to the longitudinal axis and preferably leave he contact zone through the permeable barrier in a direction about a transverse axis and especially in a direction which is perpendicular to the longitudinal axis.

[0031] The solid phase particles may be any solid-phase particles known for use in solid phase synthesis, extraction, immobilisation and the like, especially solid supports suitable for use in the synthesis of peptides, peptidomimetics and oligonucleotides. Suitably, the solid phase particles comprise a cross-linked poly-e-lysine polymers, preferably cross-linked with di carboxylic acids. Preferably, the solid phase particle comprises a particulate support comprising a cross-linked poly-e-lysine polymer comprising poly-e-lysine and a cross linker linked by amide bonds wherein the cross linker comprises two or more carboxylic acid groups and an aliphatic chain linking the two or more groups adapted to react with an alpha carbon amine of poly-e-lysine.

[0032] Examples of suitable solids particles or supports are described in GB2473814, WO20 11 / 032703, WO2011 / 032704, WO2011 / 032704, WO2011 / 032705,

[0033] WO20 12 / 143508, WO2013 / 041250.

[0034] In a preferred embodiment, the solid phase particles comprise a self-assembled microparticle comprising an acid having two or more acid groups and an organic base in which the molar ratio of acid groups to basic groups in the acid and base is from 0.6 to 1.4:1, as described in WO2016 / 139322.

[0035] More preferably, the solid phase particle comprises self-assembled microparticles comprising an acid having two or more acid groups and an organic base wherein the said acid comprises a compound selected from i) a compound of general formula HOOC-(CH2)n-COOH wherein n is at least 5 and not more than 40; and ii) a C7to C13 bis carboxylic fatty acid in combination with a further acid selected from EDTA, nitrolotriacetic acid and a monocarboxylic acid; wherein the molar ratio of acid groups in the acid to basic groups in the base is from 0.6 to 1.4:1 , preferably 0.8 to 1.2:1, and the microparticle comprises a multi-lamellar structure. The microparticle may be any micron scale particle size, preferably 0.1 to 100 microns, especially 0.5 to 50 microns, particularly 1 to 20 microns, for example 1 to 5 microns.

[0036] The acid and organic base in the microparticle align upon contact to suitably do not react to form a particle having a multi-lamellar structure and are not covalently bonded to each other.

[0037] WO2016 / 139322 further describes a process for preparing microparticles involving the self-assembly of a bis-carboxy fatty acid microparticle in water followed by crosslinking with a suitable amine. Poly-epsilon lysine is especially preferred to provide a solid phase particle suitable for use in the present invention.

[0038] The selection of fatty acid determines the diameter of the resulting particle. Suberic acid (C8) provides ~0.5pm particles, azelaic acid (C9) gives ~1 m, sebacic acid (C10) produces particles of ~2pm diameter, brassylic acid (C13) gives ~3pm particles. The 2 or 3pm SpheriSome® particles afford a desirable combination of flow, reaction kinetics, handling and utility in the process of this invention. Several different peptides have been manually assembled including Leu-enkephalin, insulin B-chain, and glucagon with superior products being obtained in each case with SpheriSomes®. Suitably, the loading of the poly-epsilon lysine may be up to 2.5 mmol / g.

[0039] For micron scale particles, such as SpheriSomes, having a particle size of 1 to 10 microns, a permeable barrier having a mesh size of 0.2 to 1 micron, for example 0.2, 0.5 and 0.65 microns may be suitable.

[0040] In another preferred embodiment, the solid phase particles are as described in WO20 12 / 143508. Suitably, the solid phase particles comprise cross-linked poly-e-lysine polymer comprising poly-e-lysine and a cross linker linked by amide bonds wherein the cross linker comprises at least two functional groups capable of reacting with an alpha carbon amine of poly-e-lysine. Preferably the cross-linker comprises two or more carboxylic acid groups and an aliphatic chain linking the two or more groups.

[0041] Preferred solid supports include self-assembled microparticles available under the trade name SpheriSomes® and cross-linked poly-e-lysine supports which may be particulate or non-particulate available under the trade name SpheriTide®, both from SpheriTech Ltd. These solid supports are readily pumpable in aqueous solution at 5000 cm3 / minute in apparatus according to the invention, for example having a permeable barrier of 1cm diameter x 50cm long tubular filters at 5000 cm3 / minute without any observable damage to the particles.

[0042] SpheriTide® is a solid support and, when solvated, is noticeably more mechanically stable than traditional polymer supports used for this purpose such as cross-linked polystyrene. SpheriTide® can be made in a range of particle sizes down to sub-micron if needed. The inherent mechanical stability of this support allows for it to be pumped at the very high flow rates required for TFF without damage to the particles. Suitably, the solid phase particles for solid phase peptide synthesis and solid phase oligonucleotide synthesis, the solid particle is suitably highly solvated soft particles to improve diffusion of reagents throughout the particle. Use of smaller particles also improves diffusion of reagents.

[0043] In a preferred embodiment, the solid phase particles are small, preferably at least 0.1. Suitably the particles are not more than 250 microns, preferably not more than 150 microns, especially not more than 100 microns. In one embodiment, the particles may be 1 to 50 microns, for example 1 to 20 microns and are highly solvated. Advantageously, the present invention enables such particles to be readily processed and filtered whereas, small highly solvated particles would typically either not be capable of filtration using the conventional flat bed techniques or be prone to blockage and other process complications.

[0044] The process of the invention may be employed in solid phase synthesis of Typically, by way of example, solid phase peptide synthesis is carried out on particles in the size range 75-150pm as dry particle size which swell when solvated in an appropriate solvent. Traditionally cross linked polystyrene is used for solid phase peptide synthesis and 1g of 75-150pm diameter particles will swell in N,N-dimethylformamide to 10cm3so the diameter swollen particle size range will be ~120-250pm. Reagents used in solid phase peptide synthesis reach the active site by diffusion throughout the polymer matrix so the rate of reaction is limited by diffusion. The volume of a 100pm diameter bead is approximately 1 / 16ththe volume of a 250pm bead so the rate of diffusion through the bead will be proportionately less. Similarly, the volume of a 50pm bead is 125 times less than a 250pm bead. At another extreme the volume of a 3pm bead will -580,000 times smaller than a 250pm bead.

[0045] In conventional processes, smaller particles will form a tightly packed bed which requires higher pressure to filter presenting drawbacks associated with blockage, back pressure being too high, potential structural damage and the like. More highly solvated particles will be softer and compress into the filter and will also deform thereby restricting flow in conventional processes whereas deformation on the present process is advantageous as aiding mixing and contact between the liquid components and solid.

[0046] In a preferred embodiment, the particles are spherical to reduce mechanical erosion due to contact with during the process, for example contact with the pump, the filter and other particles. Suitably, the process is operated at a flow rate that is sufficient to create turbulent flow in the contact zone. The solid phase particle is suitably sufficiently robust to be pumped at the desired high flow rate to create the turbulent flow, without material mechanical degradation. The solid phase particles are suitably deformable or elastic to withstand compressive forces applied by the pumping operation.

[0047] Reynolds (Re) number is a dimensionless figure which can be used to predict turbulent flow in a pipe according to the following expression:

[0048] Re= puD / p where; p = liquid density (g / cm3) u = flow velocity (cm / s)

[0049] D = tube diameter (cm) p = liquid viscosity (g / cm-s)

[0050] Reynolds numbers of less than 2900 suggest a laminar flow which would allow particles to settle on the permeable barrier. Reynolds numbers greater than 2900 suggest a turbulent flow which would minimize, reduce or prevent the particles from settling on the permeable barrier.

[0051] Preferably the process of the invention is carried out at a flow rate such that for the particular solid and liquid components and the apparatus employed, the Reynolds number is 2900 or greater. Suitably, the pumping rate is at least 1 litre per minute, preferably 5 litres per minute, more preferably at least 10 litres per minute, especially at Ieast15 litres per minute, for example 17 litres per minute.

[0052] By way of illustration, for a 1cm diameter x 50cm long mesh filter: at a flow rate of 1000 cm3 / minute using water Re= 2080 at a flow rate of 2500 cm3 / minute using water Re= 5200 at a flow rate of 5000 cm3 / minute using water Re= 10600

[0053] The process of the present invention is particularly useful in the solid phase synthesis of peptides, peptidomimetics and oligonucleotides and especially synthesis in aqueous solution. Suitably, the peptides may be synthesized on a solid particulate support, especially the solid phase particles described herein.. Peptides may be made by a number of different processes including solution phase chemistry, solid phase chemistry and enzymatic chemistry or a combination of these methods. In these processes, protected amino acids are required for the synthesis of the peptides, such as urethane protected amino acids. Examples of the N-urethane protecting group include tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), ethanesulfonylethoxycarbonyl (Esc) and fluorenylmethyloxycarbonyl (Fmoc) with Fmoc being the most commonly used protecting group, especially for solid phase synthesis. Peptide synthesis has for many decades been carried out in an organic solvent as the protected amino acids typically employed, are insoluble in water. Organic solvents such as N,N’-dimethyl formamide (DMF), N,N’-dimethylacetamide (DMA), N- methylpyrrolidone (NMP) are well known for use in peptide synthesis.

[0054] The present inventors have developed an aqueous phase peptide synthesis process described in a co-pending patent application filed on the same date as this application.

[0055] In a further aspect, the invention provides a method of synthesising a peptide or a peptidomimetic comprising: i) providing a protected amino acid dissolved in an aqueous solvent by contacting the protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the protected amino acid; ii) providing at least one further dissolved protected amino acid and reacting the further dissolved protected amino acid with the protected amino acid to form a peptide bond in a solid-phase peptide synthesis process according to the present invention; and iii) optionally providing further dissolved protected amino acids and reacting with the product of step ii) to form a further peptide bond in the sequence of the desired peptide in a solid-phase synthesis process according to the present invention.

[0056] The protected amino acid dissolved in an aqueous solvent is suitably contacted with a solid particulate to which amino acids may be bound or a solid particulate support on which a peptide is being synthesised. The protected amino acid is suitably protected by a protecting group selected from terf-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), ethanesulfonylethoxycarbonyl (Esc) and fluorenylmethyloxycarbonyl (Fmoc).

[0057] Oligonucleotide synthesis to produce DNA and RNA oligonucleotides is typically carried out using a solid phase synthesis process using the phosphoramidite method and phosphoramidite building blocks derived from protected 2'- deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and II), or chemically modified nucleosides, for example a locked nucleic acid or bridged nucleic acid. Enzymatic or chemical synthesis processes may be used.

[0058] The desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the desired order. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products may be isolated by HPLC to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15-25 bases in length.

[0059] Whereas enzymes synthesize DNA and RNA only in a 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite, 3' to 5' direction.

[0060] The invention may be employed in a solid phase process for contacting a solid with a liquid or separating any particulate solid from a liquid. The invention is useful in a wide range of processes including:

[0061] - solid phase peptide and solid phase peptidomimetic synthesis;

[0062] - solid phase oligonucleotide synthesis;

[0063] - in solid phase carbohydrate synthesis;

[0064] - in solid phase peptide nucleic acid synthesis;

[0065] - with immobilized enzymes for chemical or biological transformations;

[0066] - in cell culture where cells are immobilised on a solid support;

[0067] - chemo-catalysis where the catalyst is immobilised on a solid support;

[0068] - use of a solid support in an extraction process;

[0069] - in a chromatographic separation where the stationary phase is suspended in mobile phase;

[0070] - in conjunction with a medical device comprising a particulate support for medical applications;

[0071] - in dialysis where the solid phase is used to extract a contaminant from blood; - in a medical diagnostic requiring separation of a solid phase diagnostic particle from a liquid;

[0072] The invention also provides for use of a process according to the invention and for use of apparatus according to the invention to filter a particulate support in a process selected from solid phase synthesis of peptides, oligonucleotides, oligosaccharides; solid phase extraction; solid phase organic chemistry; immobilisation of a species selected from solid phase reagents, metal and other catalysts, bio-catalysts, enzymes, proteins, antibodies including polyclonal and monoclonal antibodies, whole cells and polymers; cell culturing; preparation of a stationary phase for chromatographic separation; or for use as an absorbent.

[0073] Illustrative examples of the process and apparatus according to the invention is shown in Figure 1. An example system for solid phase synthesis of peptides and solid phase assembly of oligonucleotides is shown in Figure 2.

[0074] The invention is illustrated by reference to the following non-limiting examples.

[0075] Example 1 - preparation and washing of SpheriTide microparticles

[0076] SpheriTide® spherical microparticles are washed and filtered using apparatus according to the invention as shown in Figure 1.

[0077] A slurry is pumped through a diaphragm pump at 2.8dm3 / minute and through a stainless- steel mesh tube filter. Without applying back pressure to the column outlet, the permeate flow rate was extremely fast reducing the volume by 50% in 20 seconds. The surface area of the filter used was ~150cm2which is equivalent to a flat-bed filter diameter of ~14cm. The SpheriTide® microparticles were prepared by suspension polymerisation as described below.

[0078] Poly-epsilon-lysine (psK) (22.3g, 130.895mmol amine) was weighed into a 100cm3bottle then water added (45cm3) to dissolve. The psK solution was added to 2-[ 2-(2- methoxyethoxy)ethoxy acetic acid (14.06g, 61.625mmol) (MEAA) and 3,6,9- Trioxaundecanedioic acid (4.85g, 39.268mmol COOH) (TOD) on a stirrer. The pH was measured at 5.5 no adjustment needed.

[0079] Toluene (380cm3) was added to Span 80 (20g) in a 1dm3plastic container. A small sized Visco jet stirrer was slid through a culture vessel lid and into an overhead stirrer. The culture vessel lid was then secured to the culture vessel. The culture vessel was secured via a clamp and the Span 80 / toluene solution was stirred at 500rpm.

[0080] The TOD / MEAA / psK solution was added to the stirring Span 80 / toluene solution and left mixing for 1 hour.

[0081] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (38.68g, 201.787mmol) (EDCI) was weighed into a 250cm3bottle then dichloromethane (75cm3) added. Dissolution was aided by dropwise addition of water. This was added to the reaction vessel and the mixture stirred for two hours. The stirrer was then turned off and the beads allowed to settle. The supernatant was removed by decantation. Toluene (300cm3) was added to the beads and left to mix for 10 minutes. This toluene wash process was repeated, the mixture left to settle and the mixture diluted to 500cm3with Tween 80 solution (300cm3, 1% w / v in water).

[0082] Aqueous NaOH (240cm3, 0.25mol / dm3) was slowly added to the batch whilst stirring. The mixture was passed through a 75pm sieve to remove fine particles.

[0083] The SpheriTide beads were washed by TFF using a stainless-steel mesh tube (25pm porosity, 10mm diameter x 50cm length, Figure 4) on the instrument shown in Figure 3. Initially the bead slurry was concentrated to 500cm3. The beads were pumped through a diaphragm pump at 2.8dm3 / minute and the permeate pressure balanced to give a permeate flow rate of 800cm3 / minute. The volume was reduced to 250cm3replenished with water (250cm3). This process was repeated 10x and finally concentrated to 250cm3. It is noted that each wash took approximately 20 seconds and the concentrated slurry flowed freely. No detectable filter blockage or back pressure was observed. There was no observable damage to the microparticles when inspected by microscopy following TFF. The average fully solvated particle size was ~100pm.

[0084] The aqueous suspension of microparticles was freeze dried to give a yield of 19.15g.

[0085] Example 2 - preparation of Leu-Enkephalin

[0086] Pentapeptide Leu-Enkephalin was assembled on SpheriSomes® (3um, microparticles, amine loading 0.6mmol / g with 4-hydroxymethylphenoxyacetic acid linker) at 2mmol scale using standard Fmoc-chemistry. Assembly was carried out in N,N-dimethyl formamide (DMF). Three molar equivalents of activated amino acid to the solid support were used at each stage. Activation was carried out using 1 -hydroxybenzotriazole (1 molar equivalent to Fmoc-amino acid) (HOBt) and diisopropylcarbodiimide (1.25 molar equivalents to the Fmoc-amino acid).

[0087] Piperidine in DMF was used for Fmoc removal. All washes were carried out by TFF using DMF on the instrument shown in Figure 5 using a ceramic membrane (0.6um pore size, 10mm diameter, 50cm length). The slurry was pumped at 3dm3 / minute achieving a permeate flow rate of 50-100cm3 / minute.

[0088] The purity of the crude Leu-Enkephalin produced was >95% by HPLC.

Claims

CLAIMS1. A solid phase process comprising passing a mixture comprising solid phase particles and one or more liquid components through a confined, contact zone having an inlet and an outlet and between the inlet and outlet, a permeable barrier which is permeable to liquids and wherein the solid and liquid component(s) interact and a portion of the liquid component(s) or a liquid product of the interaction between the solid and the liquid component(s) passes through the permeable barrier.

2. A solid phase process according to claim 1 in which one or more solid components and one or more liquid components chemically react.

3. A solid phase process according to claim 1 or claim 2 for the production of a peptide, a peptidomimetic or an oligonucleotide.

4. A solid phase process according to any one of the preceding claims in which the mixture comprises a slurry comprising solid phase particles and one or more liquid components.

5. A solid phase process according to claim 4 in which the slurry comprises 10 to 60% w / v solid particles.

6. A solid phase process according to any one of the preceding claims in which filter comprises a tubular membrane or a flat sheet.

7. A solid phase process according to any one of the preceding claims in which the solid phase particle comprises a microparticle comprising an acid having two or more acid groups and an organic base in which the molar ratio of acid groups to basic groups in the acid and base is from 0.6 to 1.4:1.

8. A solid phase process according to any one claims 1 to 6 in which the solid particles comprise cross-linked poly-e-lysine polymer comprising poly-e-lysine and a cross linker linked by amide bonds wherein the cross linker comprises at least two functional groups capable of reacting with an alpha carbon amine of poly-e- lysine.

9. A solid phase process according to any one of the preceding claims in a process selected from solid phase peptide and solid phase peptidomimetic synthesis; solid phase oligonucleotide synthesis; solid phase carbohydrate synthesis; solid phase peptide nucleic acid synthesis; with immobilized enzymes for chemical or biological transformations; cell culture where cells are immobilised on a solid support; chemo-catalysis where the catalyst is immobilised on a solid support; use of a solid support in an extraction process; in a chromatographic separation where the stationary phase is suspended in mobile phase; in conjunction with a medical device comprising a particulate support for medical applications; in dialysis where the solid phase is used to extract a contaminant from blood; in a medical diagnostic requiring separation of a solid phase diagnostic particle from a liquid.

10. A solid phase process according to any one of the preceding claims in the synthesis of solid phase synthesis of a peptide, a peptidomimetic or an oligonucleotide in aqueous solution.

11. A solid phase process according to any one of the preceding claims in which the liquid component comprises a protected amino acid in water.

12. A solid phase process according to claims 11 in which the amino acid is selected from Fmoc protected amino acid and Boc protected amino acid13. A method of synthesising a peptide or a peptidomimetic comprising: i) providing a protected amino acid dissolved in an aqueous solvent by contacting the protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the protected amino acid; ii) providing at least one further dissolved protected amino acid and reacting the further dissolved protected amino acid with the protected amino acid to form a peptide bond in a solid-phase process according to any one of the preceding claims for synthesizing a peptide; and. iii) optionally providing further dissolved protected amino acids and reacting with the product of step ii) to form a further peptide bond in the sequence of the desired peptide in the solid-phase synthesis process14. A peptide synthesized by a solid phase process according to any one of claims 1 to 12.

15. A solid phase process contact apparatus comprising a contact vessel having an inlet for receiving a mixture comprising solid phase particles and one or more liquid components and an outlet for removing the solid phase particles from the contact vessel which provides a confined chamber defining a contact zone between the inlet and outlet wherein at least part of the chamber wall comprises a permeable barrier which is permeable to the liquid component and a pump to convey the mixture into the contact chamber.

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