WO2016024105A1

WO2016024105A1 - Organic solvent nanofiltration with enhanced permeation of impurities

Info

Publication number: WO2016024105A1
Application number: PCT/GB2015/052319
Authority: WO
Inventors: Andrew Guy Livingston; Jeong Kim; Irina Boyanova VALTCHEVA; Piers Robert James Gaffney
Original assignee: Imperial Innovations Limited
Priority date: 2014-08-11
Filing date: 2015-08-11
Publication date: 2016-02-18
Also published as: GB201414213D0

Abstract

A process for separating a product from charged organic solute impurities using an organic solvent resistant nanofiltration membrane is disclosed, in which the membrane carries the opposite charge to the charged impurities. The use of a permeation aid during separation allows charged impurities to be preferentially permeated by the membrane, thus facilitating separation of chemical species having similar molecular weights. Also disclosed is a use of an organic solvent resistant nanofiltration membrane in a separation process defined herein.

Description

ORGANIC SOLVENT NANOFILTRATION WITH ENHANCED PERMEATION OF

IMPURITIES

INTRODUCTION

[0001 ] The present invention relates to a process for performing nanofiltration of a feed stream comprising a dissolved product and at least one charged organic solute impurity. More particularly, the present invention relates to a process for performing nanofiltration of a feed stream comprising a dissolved product and at least one charged organic solute impurity in which the membrane used for performing nanofiltration and the at least one charged organic solute impurities carry opposing charges.

BACKGROUND OF THE INVENTION

[0002] Organic Solvent Nanofiltration (OSN) is an established membrane technology that separates solutes in organic media at ambient temperature. It is energy-efficient by comparison to competing technologies (e.g. distillation), and is considered one of the key technologies for enhancing the sustainability of chemical processes. A typical OSN membrane exploits differences in size and geometry of solutes between 100 - 2000 Da. However, within such a narrow separation range it is difficult to obtain a complete separation between two solutes, often resulting in lower yield and/or purity.

[0003] In a typical membrane process, the solution that is retained by the membrane is called the retentate, and the solution that penetrates the membrane is called the permeate. The compound(s) of interest may be in the retentate, permeate, or both.

[0004] For many OSN applications, the constant volume diafiltration (CVD) mode is usually employed, where the retentate volume is held constant by matching the permeate volume outflow with pure solvent input. In a typical CVD process, a feed tank is first charged with a solution containing at least two solutes. The solution is then circulated around the system under pressure which forces the solution through the membrane to initiate separation. If at least one of the solutes permeates through the membrane faster than the others, then molecular separation is achieved at ambient temperature.

[0005] The slower the rate of permeation of a solute, the higher its rejection by the membrane. Similarly, the faster the permeation, the lower the rejection of species i (R,, mathematically defined in Equation 1 ).

Ri = (1 - C_Pi/CRi) x 100% (Eq.1 ) where C and CR, represents the concentrations of solute i in the permeate and retentate, respectively. Consequently, the larger the difference in solute permeation rates (or rejections) between two solutes, the easier the separation becomes. In practice, to obtain a reasonable yield during CVD it is essential that one solute is as near as possible completely rejected by the membrane (i.e. R approaches 100%). Similarly, the rejection (R) of the solutes that permeate through the membrane should be below 80%, or else the separation will require excessive volumes of solvent to achieve acceptable purity.

[0006] CVD processes can be characterized using a time-like dimensionless parameter called a diavolume, defined in Equation 2.

Diavolume = ^Fpt (Eq.2)

where F_p (L.min ¹) represents the permeate flow rate, t (min) the time, V_system (L) represents the system volume. A typical CVD process requires between 10 - 25 diavolumes to purify a product of interest to a high enough purity.

[0007] Many different types of OSN membranes have been reported that can work in variety of solvents including A/,A/-dimethylformamide, acetone, acetonitrile, tetrahydrofuran, toluene etc. [1 ]. Boosted by such improvements in membrane solvent stability, promising applications of OSN have emerged, such as catalyst recovery, pharmaceutical purification, solute concentration, and solvent recovery.

[0008] One of the potentially most fruitful applications of OSN is to perform iterative synthesis using OSN [2] to produce defined monomer sequence polymers, in which the order of monomeric units collectively constituting the polymer is exactly defined. Defined monomer sequence polymers may be naturally-occurring polymers or non-naturally-occurring polymers. Polymers of this type may contain monomers that differ from one or more of the other monomers by virtue of their respective backbone moieties, side chain moieties, or both. For example, a repeating backbone motif (polyamide for peptides, ribose phosphate for oligos) may be decorated by side-chains that vary from one monomer to the next and confer the sequence's structure and functionality.

[0009] As with most products that are isolated using OSN, it is highly desirable - if not imperative - to achieve complete separation of the growing oligonucleotide (or other defined monomer sequence polymer) from one or more by-products or reagents produced during its preparation, in order that wasteful side-reactions or erroneous polymeric sequences are avoided. However, at the dimer stage (i.e. a polymer sequence comprising only 2 monomers), the molecular weights of the product (i.e. the dimer) and the one or more byproducts and reagents (including residual unreacted monomer from the initial coupling reaction) are understandably similar, thus posing problems for OSN membranes whose separation properties are governed solely by the size of their pores (i.e. their molecular weight cut-off).

[0010] Hence, in order to develop a scalable synthetic platforms using OSN technology, a more efficient basis of separation is required.

[0011 ] The present invention was devised with the foregoing in mind.

SUMMARY OF THE INVENTION

[0012] According to a first aspect of the present invention there is provided a process for performing nanofiltration of an organic feed stream comprising a dissolved product and one or more charged organic solute impurities, the process comprising the steps of:

a) providing a nanofiltration membrane suitable for providing a rejection for the product that is greater than the rejection for the one or more charged impurities, wherein the membrane carries the opposite charge to the one or more charged impurities, and b) contacting the nanofiltration membrane with the feed stream in the presence of at least one permeation aid, wherein the nanofiltration membrane is stable in the organic feed stream, and the at least one permeation aid is one or more of an acid, base or salt.

[0013] According to a second aspect of the present invention, there is provided a use of an organic solvent resistant nanofiltration membrane in a process defined herein.

DETAILED DESCRIPTION OF THE INVENTION

Processes of the invention

[0014] As discussed hereinbefore, the present invention provides a process for performing nanofiltration of an organic feed stream comprising a dissolved product and one or more charged organic solute impurities, the process comprising the steps of: a) providing a nanofiltration membrane suitable for providing a rejection for the product that is greater than the rejection for the one or more charged impurities, wherein the membrane carries the opposite charge to the one or more charged impurities, and b) contacting the nanofiltration membrane with the feed stream in the presence of at least one permeation aid, wherein the nanofiltration membrane is stable in the organic feed stream, and the at least one permeation aid is one or more of an acid, base or salt.

[0015] The present invention recognises the difficulties posed to OSN membranes by feed streams comprising a number of solutes for separation, in which the solutes are of comparable molecular weight. As a solution to this problem, the present invention aims at improving the separation performance of OSN membranes by focusing on properties of the organic feed stream solutes other than their molecular weight, in particular their respective charges. Specifically, it has been found that for those membranes that are able to support a temporary (i.e. reversible) or permanent (i.e. irreversible) charge, organic solute impurities having an opposite charge can be made to permeate through the membrane more readily in the presence of a permeation aid. In other words, the present invention therefore provides a means for preferentially transporting a charged organic solute through an OSN membrane in an organic solvent, thereby overcoming problems posed by feed streams comprising a plurality of similarly sized solutes. Irrespective of the terminology used herein, the person skilled in the art of separation technology will readily appreciate that either the "product" (which is retained in the retentate) or the "one or more charged organic solute impurities" (which passes into the permeate) may be the species of interest.

[0016] In one embodiment, the one or more charged organic solute impurities are positively charged, and the membrane carries a negative charge. Suitably, the one or more charged organic solute impurities are negatively charged, and the membrane carries a positive charge.

[0017] The charge carried by the membrane may be temporary (i.e. reversible) or permanent (i.e. irreversible). In one embodiment, the membrane may be induced to carry a charge. Alternatively, the membrane may be charged as a result of the membrane manufacturing procedure. In an embodiment, the membrane comprises a plurality of trapped ions. The ions may be generated during the membrane manufacturing process.

[0018] Membranes suitable for use with the present invention include polymeric and ceramic membranes, and mixed polymeric/inorganic membranes. In one embodiment, the membrane is formed from, or comprises, a material selected from polymeric materials suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene difluoride, polysulfone, polyethersulfone, polybenzimidazole (PBI), polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole and mixtures thereof. The membranes can be made by any technique known to the art, including sintering, stretching, track etching, template leaching, interracial polymerisation or phase inversion. More preferably, membranes may be cross-linked or treated so as to improve their stability in the working solvents.

[0019] In another embodiment, the membrane of the present invention is a composite material comprising a support and a thin selectively permeable layer formed from, or comprising, a material selected from modified polysiloxane based elastomers including polydimethylsiloxane (PDMS) based elastomers, ethylene-propylene diene (EPDM) based elastomers, polynorbornene based elastomers, polyoctenamer based elastomers, polyurethane based elastomers, butadiene and nitrile butadiene rubber based elastomers, natural rubber, butyl rubber based elastomers, polychloroprene (Neoprene) based elastomers, epichlorohydrin elastomers, polyacrylate elastomers, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF) based elastomers, polyetherblock amides (PEBAX), polyurethane elastomers, cross-linked polyether, polyamide, polyaniline, polypyrrole, and mixtures thereof. Other membranes suitable for use with the present invention are disclosed in [3].

[0020] In another embodiment, the membrane of the present invention is fabricated from an inorganic material such as silicon carbide, silicon oxide, zirconium oxide, titanium oxide, or zeolites, using any technique known to those skilled in the art, such as sintering, leaching or sol- gel processing.

[0021 ] In a further embodiment, the membrane comprises a polymer membrane with dispersed organic or inorganic matrices in the form of powdered solids present at amounts up to 20wt% of the polymer membrane. Carbon molecular sieve matrices can be prepared by pyrolysis of any suitable material as described in [4]. Zeolites as described in [5] may also be used as an inorganic matrix. Metal oxides, such as titanium dioxide, zinc oxide and silicon dioxide may be used, for example the materials available from Evonik Industries (Germany) under their Aerosol and AdNano trademarks. Mixed metal oxides such as mixtures of cerium, zirconium, and magnesium oxides may be used. Fullerenes and 2-D materials such as graphene, graphene oxide and boron nitride may all be used. Preferred matrices will be particles less than 1 .0 micron in diameter, preferably less than 0.1 microns in diameter, and more preferably less than 0.01 microns in diameter. [0022] In one embodiment, the membrane is cross-linked with a reagent that generates a strongly acidic or basic by-product, wherein the presence of the by-product confers a net charge to the membrane.

[0023] In an embodiment, the membrane is a cross-linked polybenzimidazole membrane. Suitably, the membrane is a polybenzimidazole membrane cross-linked with xylene cross- linkers (e.g. by using a dihaloxylene cross-linking agent). More suitably, the membrane is a polybenzimidazole membrane cross-linked with dibromoxylene and carrying immobilized positive charges.

[0024] In an embodiment, the feed stream comprises one or more organic solvents selected from the group consisting of aromatics, alkanes, ketones, glycols, chlorinated solvents, esters, ethers, amines, nitriles, aldehydes, phenols, amides, carboxylic acids, alcohols, furans, and dipolar aprotic solvents, and mixtures thereof, optionally with water. More particularly the feed stream comprises one or more organic solvents selected from the group consisting of toluene, xylene, benzene, styrene, anisole, chlorobenzene, dichlorobenzene, chloroform, dichloromethane, dichloroethane, methyl acetate, ethyl acetate, butyl acetate, methyl ether ketone (MEK), methyl isobutyl ketone (MIBK), acetone, ethylene glycols, ethanol, methanol, propanol, butanol, hexane, cyclohexane, dimethoxyethane, methyl tert-butyl ether (MTBE), diethyl ether, adiponitrile, N,N-dimethylformamide, dimethylsulfoxide, Ν,Ν-dimethylacetamide, dioxane, nitromethane, nitrobenzene, pyridine, carbon disulfide, tetrahydrofuran (THF), methyl-tetrahydrofuran, N-methyl pyrrolidone, N-ethyl pyrrolidone, acetonitrile, and mixtures thereof, optionally with water.

[0025] The permeation aid used in the process of the invention may be any suitable acid, base or salt, which is soluble in the organic solvent feed stream. Acids, bases and salts that are not directly soluble in the feed stream (e.g. those that are insoluble or sparingly soluble in organic solvents) may also be used by first treating the feed stream with one or more other solvents (including water). Suitably, the permeation aid is of a size suitable for permeating through the membrane with a rejection lower than 50%. More suitably, the permeation aid is of a size suitable for permeating through the membrane with a rejection lower than 20%.

[0026] In another embodiment, the permeation aid has a molecular weight of less than or equal to 400 g mol ¹. Suitably, the permeation aid has a molecular weight of less than or equal to 250 g mol ¹.

[0027] In one embodiment, the permeation aid comprises at least one of:

an organic acid, base or salt having 1 -12 carbon atoms, and optionally one or more halogen atoms; and an inorganic acid, base or salt.

It will be understood that in the case of an organic salt, the one or more halogen atoms may be present as a counter ion.

[0028] In another embodiment, the permeation aid comprises at least one of:

an organic acid, base or salt having 1 -10 carbon atoms, and optionally 1 , 2 or 3 halogen atoms; and

an inorganic acid, base or salt.

[0029] In another embodiment, the permeation aid comprises at least one of an organic acid, base or salt having 1 -10 carbon atoms, and optionally 1 , 2 or 3 halogen atoms. It will be understood that in the case of an organic salt, the one or more halogen atoms may be present as a counter ion.

[0030] In another embodiment, the permeation aid comprises an acid with lower pKa (in the working solvent) than the membrane backbone for anion exchange membranes, or a base with higher pKa (in the working solvent) than the membrane backbone for cation exchange membranes.

[0031 ] In another embodiment, the permeation aid is selected from at least one of tetramethylamonium halide, choline halide, tetraethylammonium halide, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid, ethylthiotetrazole, pyridinium dichloroacetate, or pyridinium trifluoroacetate. In a particular embodiment, the permeation aid is selected from at least one of pyridinium dichloroacetate, dichloroacetic acid and hydrochloric acid.

[0032] In an embodiment, the permeation aid is added to the feed stream at a concentration of 0.1 - 5 vol% per diavolume. Suitably, the permeation aid is added to the feed stream at a concentration of 0.1 - 2 vol% per diavolume. More suitably, the permeation aid is added to the feed stream at a concentration of 0.1 - 1 vol% per diavolume. The permeation aid may be added to as many diavolumes as is necessary. In an embodiment, the permeation aid is added to the first ten diavolumes of the feed stream. In an embodiment, the permeation aid is added to the first five diavolumes of the feed stream. Following the addition of the permeation aid, one or more additional diavolumes of pure solvent may be added to ensure that no residual permeation aid remains in the retentate.

[0033] The ability of the membrane to permeate charged solutes can be even further improved by increasing the amount of charge carried by the membrane. Therefore, in one embodiment, the process further comprises a step of contacting the membrane with one or more reagents suitable for increasing the amount of charge carried by the membrane. Where the membrane carries a positive charge by virtue of being a Bronsted base, and the organic solute impurities are negatively charged, the membrane can be rendered more positively charged by contacting it with an acid (e.g. HCI). Depending on the nature of the membrane and the specific charge it carries, the skilled person will be readily aware of those reagents suitable for increasing the charge carried by the membrane.

[0034] In another embodiment of the process, the feed stream comprises, in addition to the charged organic solute impurities, one or more neutral organic solute impurities. In such cases, the process may comprise, prior to contacting the feed stream with the membrane, the initial step of converting at least one of the neutral solute impurities to charged solute impurities, which can then be effectively permeated by performing step b). The skilled person will be aware of those reagents suitable for converting neutral species to charged species. In another embodiment, prior to step b), the feed stream is treated with one or more reagents to prevent the formation of at least one neutral solute impurity and to encourage the formation of charged solute impurities.

[0035] The process of the invention may be used for separating a wide array of products from charged solute impurities. In a particular embodiment, the product is a defined monomer sequence polymer, more particularly an oligonucleotide. The term "oligonucleotide" and "oligo" used herein additionally encompasses oligonucleotide derivatives, for example oligonucleotides which comprise one or more synthetic or chemically modified oligonucleotides, one or more protecting groups, or one or more terminal groups located at the termini of the oligonucleotide chain.

[0036] Oligonucleotides are prepared by attaching one building block, or monomer, at a time to the growing oligo in an iterative, stepwise manner. After each reaction to extend the oligo using an excess of monomer (usually 1 .1 to 3 equivalents), the unreacted monomer must be completely removed from the crude before the next round of chain extension. Apart from avoiding wasteful side-reactions, the monomer debris can participate in subsequent rounds of chain extension. Thus, excess reagent removal is critical to avoid the accumulation of erroneous sequences. Thus, in one embodiment, the product is an oligonucleotide and the one or more charged organic solute impurities are excess unreacted nucleoside monomers. It will be understood that the oligonucleotide may be produced by any coupling technique (e.g. phosphoramidite, phosphodiester, phosphotriester, H-phosphonate couplings etc.).

[0037] Nucleoside phosphoramidites (or simply phosphoramidites) are highly reactive derivatives of natural or synthetic nucleosides, which have been chemically modified to avoid undesired side reactions, by protection of the reactive phosphorus moiety, hydroxyl and exocyclic amino groups. Owing to the number of reactions involved in each cycle of oligonucleotide chain extension (e.g. deprotection, coupling, capping and sulfurization/oxidation), the reaction medium containing the growing oligonucleotide can become contaminated with a number of solute impurities, including species derived from the excess unreacted phosphoramidites, a number of which are charged as a result of chemistry occurring at the phosphoramidite centre. Thus, in one embodiment, the product is an oligonucleotide and the one or more charged organic solute impurities are derived from excess phosphoramidite monomers. The feed stream may also comprise excess uncharged phosphoramidite-derived monomers, which, prior to performing step b) can be converted to charged phosphoramidite-derived monomers by chemistry known in the art.

[0038] The specific chemistry and reagents used in the deprotection, coupling, capping and sulfurization/oxidation steps of oligonucleotide chain extension can influence the nature of the nucleotidyl reagent debris derived from the excess phosphoramidite monomers requiring separation from the oligonucleotide product. In one particular embodiment, the one or more charged organic solute impurities are excess phosphoramidite-derived monomers having either of the following structures:

monot ioate dit soate in which B refers to any nucleobase which can be adenine, cytosine, uracil, guanine, and thymine; Cne refers to a cyanoethyl protection; and R refers to H or a suitable protecting group (e.g. 4,4'-dimethoxytriphenyl ether (Dmtr)).

[0039] In addition to the charged monothioate and dithioate impurities, the feed stream may additionally comprise one or more uncharged phosphoramidite-derived monomers, which, prior to performing step b) can be converted to charged phosphoramidite-derived monomers. In an embodiment, the uncharged phosphoramidite-derived monomers have either of the following structures:

ihioamidaie amidate in which B refers to any nucleobase which can be adenine, cytosine, uracil, guanine, and thymine; Cne refers to a cyanoethyl protection; and R refers to H or a suitable protecting group (e.g. 4,4'-dimethoxytriphenyl ether (Dmtr)). Suitably, the thioamidate and amidate monomeric impurities are prevented from forming by converting the excess phosphoramidite after coupling to the charged impurities by a two-step procedure: reaction with water first converts all the phosphoramidite to the corresponding H-phosphonate, then treatment with 3 - -1 ,2-benzodithiol-3-one (BDT) converts all this to monothioate [6], as outlined in the following scheme:

monoihioate

phosphoramidite H-phosphonate

Uses of the invention

[0040] As discussed hereinbefore, the present invention also provides a use of an organic solvent resistant nanofiltration membrane in a process defined herein.

[0041 ] It will be understood that features common to both the processes and uses of this invention may be further defined by reference to any of the definitions, embodiments and examples recited in the preceding paragraphs. EXAMPLES

[0042] The invention will now be exemplified, for the purpose of reference and illustration only, with reference to the accompanying figures, in which:

Fig. 1 shows ³¹ P NMR spectra of the retentate from the diafiltration of crude dinucleotidyl homostar mixture through PBI membranes cross-linked with dibromoxylene, illustrating the extent to which negatively charged by-products are permeated: a) without permeation aid; and b) with permeation aid.

Fig. 2 shows ³¹ P NMR spectra of: a) typical post-chain extension crude reaction mixture; b) with addition of water, then BDT to convert all excess monomers to charged species; and c) retentate from diafiltration of crude mixture b) using permeation aid and HCI treated membrane.

Fig. 3 shows reaction scheme to synthesise pure dinucleotidyl homostar 6.

Fig. 4 shows ¹H-NMR of dinucleotidyl homostar 6 prepared according to Example 3.

Fig. 5 shows ³¹ P-NMR of dinucleotidyl homostar 6 prepared according to Example 3, with expansion inset.

Example 1

[0043] Oligonucleotide chain extension is performed by first reacting the growing oligonucleotide (e.g. 1 ) having a free 5'-OH with the next phosphoramidite monomer (2, 1 .5 equivalents per 5'-OH, cytidine in this example) activated with 0.25 M ETT in acetonitrile solution [8]. After 0.5 hr, PADS (3 equivalent excess per OH) and pyridine (equivolume) were added and stirred for another 0.5 hr. The crude reaction mixture was partially purified by diafiltration. The deprotection reaction was then carried out by re-dissolving the semi-purified crude mixture in DCM to which pyrrole (2 vol%) and DCA (1 vol%) were added and stirred for 30 minutes. The final crude reaction mixture contained the chain extended product and four different types of excess monomers (all having free 5'-OH). Negatively-charged excess monomers (monothioate, dithioate) with a MW of -400 Da exhibited a high rejection (>95%) on dibromoxylene-crosslinked polybenzimidazole (PBI-DBX) membranes which carry immobilized positive charges [7]. Neutral excess monomers (thioamidate, amidate), with similar molecular shape and size with a MW of 490 Da, also exhibited high rejections on the same membranes. The structures of these excess monomers are shown below:

t idamidate amidaie mortot ioate dsthioate

in which B refers to any nucleobase which can be adenine, cytosine, uracil, guanine, and thymine, usually with exocyclic amine protected as a variety of amides (e.g. acetyl, benzoyl, isobutyryl); in this specific example, B refers to cytosine. Cne refers to a 2-cyanoethyl protection.

[0044] Surprisingly, upon addition of a permeation aid {e.g. dichloroacetic acid, DCA, 0.1 - 1 voP/o, or pyridinium dichloroacetate, Py.DCA) to the diafiltration system (during the first 5 diavolumes), the rejection of the negatively charged solutes dropped to below 50% and they had completely permeated through the membrane after 15 diavolumes. By contrast, such an effect was not observed without the presence of the permeation aid (see Fig. 1 ). Furthermore, the rejection dropped even further when the PBI membranes were treated with dilute HCI, thus increasing the membrane charge. These effects were confirmed by membrane permeation studies of A/-isobutyryl 2'-methyl guanosine phosphorothioate (the largest nucleoside building block) in isolation.

Example 2

[0045] In order to increase the utility of the results observed in Example 1 , all the neutral excess monomer was deliberately converted to negatively charged species in a two-step process. First, excess phosphoramidite 2 was quantitatively hydrolysed to neutral H- phosphonate after the coupling, before sulfur transfer to the internucleotide linkage using PADS. Then 3 --1 ,2-benzodithiol-3-one (BDT) was added to cleanly convert all residual H-phosphonate to negatively charged thiophosphate (see Fig. 2b) [6]. All the nucleotidyl debris was removed by diafiltration (see Fig. 2c). Fig. 2b shows that after addition of water and BDT, no neutral amidate or thioamidate building block debris remain in the feed. Fig. 2c shows that all of the charged species have been removed by OSN. The permeation aid debris also permeated through the membrane. Example 3

[0046] In order to maximise the utility of the results observed in Examples 1 and 2, chain extension of 1 (0.4 g) was performed with Mip phosphoramidite 3 (see Fig. 3). After 10 min the neutral excess monomer was deliberately converted to negatively charged phosphorothioate 4a using the above two-step process. The crude reaction mixture was transferred to a two-stage OSN rig, and partial purification performed by permeating 6 system volumes of neat solvent to remove almost all coupling agent and sulfur transfer debris; protected phosphoroathioate 4a was not completely removed. After this 1% DCA was added to the rig to deprotect the 5'-Mip- acetals of 5 in situ. The reaction was quenched by addition of pyridine (equimolar to the DCA, converting the acid to Py.DCA). After this 3 system volumes of solvent containing 0.5% Py.DCA were permeated, followed by 9 system volumes of neat solvent to completely remove all the negatively charged 5'-hydroxy thiophosphate (4b), as well as the Py.DCA permeation aid. This procedure provides dinucleotidyl homostar 6 with high enough purity to proceed to the next chain extension cycle, see Figs. 4 and 5, containing no detectable building block, protecting group, or other reagent debris.

[0047] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

[0048] The work leading to this invention has received funding from the [European Community's] Seventh Framework Programme ([FP7/2007-2013] under grant agreement n ° 238291 .

REFERENCES

[1] P. Vandezande, L.E.M. Gevers, I.F.J. Vankelecom, Chem. Soc. Rev., 2008, 37, 365-405.

[2] US Patent, No. 8,664,357

[3] US Patent, No. 20100038306

[4] US Patent, No. 6,585,802

[5] US Patent, No. 6,755,900

[6] Ghisaidoobe et al., Tetrahedr. Lett., 2008, 49, 3129-3132.

[7] I.B. Valtcheva, S.C. Kumbharkar, J.F. Kim, Y. Bhole, A.G. Livingston, J. Membr. ScL, 2014, 457, 62-72.

[8] Gaffney, P.R.J, Kim, J.F, Valtcheva, I.B., Williams, G.D, Anson, M.S., Buswell, A.M. and Andrew G. Livingston, A.G. "Liquid-Phase Synthesis of 2'-Methyl-RNA on a Homostar Support through Organic-Solvent Nanofiltration", Chemistry - A European Journal, 2015, vol. 21 , DOI: 0.1002/chem.201501001 .

Claims

1 . A process for performing nanofiltration of an organic feed stream comprising a dissolved product and one or more charged organic solute impurities, the process comprising the steps of: a) providing a nanofiltration membrane suitable for providing a rejection for the product that is greater than the rejection for the one or more charged impurities, wherein the membrane carries the opposite charge to the one or more charged impurities, and b) contacting the nanofiltration membrane with the feed stream in the presence of at least one permeation aid, wherein the nanofiltration membrane is stable in the organic feed stream, and the at least one permeation aid is one or more of an acid, base or salt.

2. The process of claim 1 , wherein the permeation aid is of a size suitable for permeating through the membrane with a rejection lower than 50%.

3. The process of claim 1 or 2, wherein the permeation aid is of a size suitable for

permeating through the membrane with a rejection lower than 20%.

4. The process of claim 1 , 2 or 3, wherein the permeation aid comprises at least one of an organic acid, base or salt having 1 -10 carbon atoms, and optionally 1 , 2 or 3 halogen atoms.

5. The process of any preceding claim, wherein the permeation aid is selected from at least one of tetramethylamonium halide, choline halide, tetraethylammonium halide, dichloroacetic acid, trifluoroacetic acid, hydrochloric acid, ethylthiotetrazole, pyridinium dichloroacetate, or pyridinium trifluoroacetate.

6. The process of any preceding claim, wherein the permeation aid is selected from at least one of pyridinium dichloroacetate and dichloroacetic acid.

7. The process of any preceding claim, wherein the permeation aid is added to the feed stream at a concentration of 0.1 - 2 vol% per diavolume.

8. The process of any preceding claim, wherein the permeation aid is added to the feed stream at a concentration of 0.1 - 1 vol% per diavolume.

9. The process of any preceding claim, wherein prior to step b), the process further

comprises a step of contacting the membrane with one or more reagents suitable for increasing the amount of charge carried by the membrane.

10. The process of any preceding claim, wherein the membrane carries a positive charge, and prior to step b), the process further comprises a step of contacting the membrane with an acid.

1 1 . The process of any preceding claim, wherein i) the feed stream further comprises one or more neutral organic solute

impurities, and the process further comprises, prior to step b), one or more additional steps to convert the neutral organic solute impurities into charged organic solute impurities, said charge being opposite to that carried by the nanofiltration membrane, or

ii) prior to step b), the feed stream is treated with at least one reagent in order to prevent the formation of one or more neutral organic solute impurities and encourage the formation of charged organic solute impurities, said charge being opposite to that carried by the nanofiltration membrane.

12. The process of any preceding claim, wherein the product is a defined monomer

sequence polymer and the one or more charged organic solute impurities are one or more of monomers and reagents, or by-products of a monomeric coupling or deprotection reaction.

13. The process of any preceding claim, wherein the product is an oligonucleotide and the one or more charged organic solute impurities are nucleoside phosphoramidites.

14. The process of any preceding claim, wherein the membrane is formed from, or

comprises, a material selected from polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene difluoride, polysulfone, polyethersulfone, polybenzimidazole (PBI), polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole and mixtures thereof

15. The process of any preceding claim, wherein the membrane is a cross-linked polybenzimidazole membrane.

16. A use of an organic solvent resistant nanofiltration membrane in a process as claimed in any preceding claim.