Solvent recycling in a liquid-phase process for preparing a defined monomoer sequence polymer

Abstract

The present invention relates to a liquid-phase process for preparing a defined monomer sequence polymer. More particularly, the present invention relates to recycling solvent in a membrane filtration-assisted, liquid-phase process for preparing a defined monomer sequence polymer.

Description

SOLVENT RECYCLING IN A LIQUID-PHASE PROCESS FOR PREPARING A DEFINED MONOMOER SEQUENCE POLYMERINTRODUCTION

[0001] The present invention relates to a liquid-phase process for preparing a defined monomer sequence polymer. More particularly, the present invention relates to recycling solvent in a membrane filtration-assisted liquid-phase process for preparing a defined monomer sequence polymer.BACKGROUND OF THE INVENTION

[0002] Peptides, peptide nucleic acids (PNAs), oligonucleotides (oligos), oligosaccharides, and chemically modified derivatives thereof are examples of biologically important defined monomer sequence polymers. Defined monomer sequence polymers are made up of distinct repeat units. For example: in peptides, the repeat units are amino acids or their derivatives; in PNAs, the repeat units are purine and pyrimidine bases and their derivatives; in oligos, the repeat units are nucleotides or their derivatives; and in oligosaccharides, the repeat units are sugar units or their derivatives.

[0003] There is considerable interest in defined monomer sequence polymers for applications in healthcare (Lutz et al. and Hartmann et al.). An example of these defined monomer sequence polymers are oligos, which have recently been validated as a new pharmaceutical modality for treating a wide range of serious or life-threatening indications. Oligos are formed from a backbone of ribose phosphate monomers, with each monomer having a variable nucleobase side chain; the building block unit of ribose phosphate bound to a nucleobase constitutes a nucleotide. The precise sequence of nucleotides defines the oligo’s biological function. Other applications of defined monomer sequence polymers in different industries (e.g., in flat panel displays where characteristics such as the optical properties of conjugated polymers are important) have also been investigated.

[0004] One approach to manufacturing defined monomer sequence polymers is to use liquid phase reactions whereby monomers are added to a growing polymer in solution in a stepwise fashion. During liquid phase synthesis all species stay in solution. This can be challenging for defined monomer sequence polymers such as oligos because, although they have good solubility in several polar aprotic solvents, relatively few of these polar aprotic solvents are compatible with phosphoramidite coupling reactions (US9914746) and / or the subsequent acidolysis of the Dmtr protecting group (i.e., detritylation). Consequently, for most liquid phase oligonucleotide syntheses (LPOS) the aim has been to keep the growing oligo dissolved in acetonitrile (Lonnberget al.), and mixtures thereof, which has long been established as the pre-eminent solvent for solid phase oligonucleotide synthesis (SPOS).

[0005] During preparation of defined monomer sequence polymers, separation technology is commonly used to separate reagents and / or by-products (i.e., reaction debris) that might interfere in subsequent chain extension cycles from the growing defined monomer sequence polymer.

[0006] In some LPOS strategies (Lonnberg et al. and Kungurtsev et al.), the growing oligo is separated from reagents and / or by-products by precipitation. Precipitation is a challenging procedure to make routine and practical, necessitating that filter cakes do not block the filter bed and can be efficiently washed with solvent. Because LPOS is an iterative process, and pharmaceutical products often require that oligos extend to 20-mers and beyond, it is important that the precipitation process is efficient and reproducible. Bonora’s HELP process (Bonora et al.) necessitates one precipitation for each step of the chain extension cycle, including capping (i.e., blocking of unreacted 5’-hydroxyls by acetylation), meaning that 87 diethyl ether precipitations were required to achieve a 20-mer. Walther et al. reported that they were able to compress their process to just one precipitation per cycle on a 4-arm PEG-star, but at the cost of an average recovery of oligo-star of only 94% per cycle, up to 11-mer. For oligos longer than 11- mer each stage required double precipitation, and the use of a DMSO / acetonitrile mixture for solubility. More recently, Shi et al. first prepared shortmers, precipitating during each chain extension cycle, then chemically ligated the fragments, again using precipitation to obtain the final product. In all cases, excessive amounts of solvent were required for the multiple precipitation cycles, increasing the process mass intensity (PM I). A high PM I corresponds to greater waste that must be disposed of for every kg of the desired product manufactured. For economic and environmental considerations, it is important to ensure that the PM I is low.

[0007] Other approaches to sequential synthesis of oligos use enzymatic syntheses that are performed in water. However, in each case the precursors to the enzyme stage (e.g., nucleoside triphosphates combined with protecting groups (Jensen et al.), fragments (Caswell et al.) or templates (WO2018011067)) must be prepared by solvent intensive organic synthesis, thereby considerably increasing the PM I.

[0008] Alternative strategies for preparing a defined monomer sequence polymer involve membrane separation after coupling of a monomer (US8664357, US9127123, US10239996, EP3347402, Gaffney et al., Kim et al., So et al., Yeo et al., Dong et al. and WO2016188835). However, these processes typically require large amounts of solvent for purification by membrane filtration.

[0009] The present invention was devised with the foregoing in mind.SUMMARY OF THE INVENTION

[0010] According to a first aspect of the present invention, there is provided a liquid-phase process for preparing a first compound being a defined monomer sequence polymer, the process comprising:(i) growing a first compound by performing one or more chain extension cycles, each chain extension cycle comprising: a deprotection step to expose a chain extension site of a growing first compound; and a coupling step to attach a monomeric or oligomeric building block to the exposed chain extension site;(ii) performing, at one or more instances in a chain extension cycle, primary membrane filtration to isolate the growing first compound in the retentate; and(iii) subjecting at least a portion of solvent permeated from primary membrane filtration to secondary membrane filtration to increase the purity of the solvent, the purified solvent being used in a subsequent chain extension cycle.

[0011] According to a second aspect of the present invention, there is provided a first compound obtained, directly obtained or obtainable by the process of the first aspect of the present invention.DETAILED DESCRIPTION OF THE INVENTION

[0012] Throughout the entirety of the description and claims of this specification, where subject matter is described herein using the term “comprise” (or “comprises” or “comprising”), the same subject matter instead described using the term “consist of’ (or “consists of” or “consisting of”) or “consist essentially of” (or “consists essentially of” or “consisting essentially of’) is also contemplated.

[0013] Throughout the entirety of the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0014] Features described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any of the specific embodiments recited herein. The invention extendsto any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0015] As described hereinbefore, in a first aspect, the present invention provides a liquid-phase process for preparing a first compound being a defined monomer sequence polymer, the process comprising:(i) growing a first compound by performing one or more chain extension cycles, each chain extension cycle comprising: a deprotection step to expose a chain extension site of a growing first compound; and a coupling step to attach a monomeric or oligomeric building block to the exposed chain extension site;(ii) performing, at one or more instances in a chain extension cycle, primary membrane filtration to isolate the growing first compound in the retentate; and(iii) subjecting at least a portion of solvent permeated from primary membrane filtration to secondary membrane filtration to increase the purity of the solvent, the purified solvent being used in a subsequent chain extension cycle.

[0016] Through rigorous investigations, the inventors have devised an improved liquid-phase process for preparing defined monomer sequence polymers, such as peptides, PNAs, oligos and oligosaccharides using membrane filtration to purify and / or isolate the defined monomer sequence polymer during synthesis. In particular, the inventors have found that subjecting at least a portion of solvent permeated from primary membrane filtration to secondary membrane filtration significantly increases the purity of the solvent such that it can be recycled in a subsequent chain extension cycle. This is particularly advantageous over known liquid-phase processes, wherein each chain extension cycle is conducted in a new batch of virgin solvent. Since a portion of the solvent used in the process of the present invention can be purified and recycled, the present invention reduces the reliance on excessive amounts of solvent, thereby lowering the overall PMI of the process, providing economic and environmental benefits.

[0017] The process of the invention is conducted in the liquid phase (i.e., the process is conducted in solution). It will therefore be understood that the growing first compound remains in a dissolved state during chain extension cycles. The process may be performed in any suitable solvent, such as dimethylformamide (DMF), acetonitrile, sulfolane, benzonitrile, toluene, xylene, benzene, styrene, anisole, chlorobenzene, dichlorobenzene, chloroform, dichloromethane, dichloroethane, methyl acetate, ethyl acetate, butyl acetate, methyl ether ketone (MEK), methyl iso butyl ketone (MIBK), acetone, ethylene glycols, ethanol, methanol, propanol, butanol, hexane, cyclohexane, dimethoxyethane, methyl tert butyl ether (MTBE), diethyl ether, adiponitrile,dimethylsulfoxide, N,N-dimethylacetamide, dioxane, nitromethane, nitrobenzene, pyridine, carbon disulfide, tetrahydrofuran, methyltetrahydrofuran, N-methyl pyrrolidone, water, and solvent mixtures comprising two or more thereof. Most suitably, the process is performed in dimethylformamide (DMF), acetonitrile, sulfolane, benzonitrile or solvent mixtures comprising two or more thereof. Suitably, the process is performed in DMF, acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane, acetonitrile mixed with benzonitrile or acetonitrile mixed with DMF). Acetonitrile is the solvent favoured by industry for coupling nucleotides to prepare oligonucleotides. Suitably, when the first compound is an oligonucleotide, the process is performed in acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane (e.g., 4:1 v / v) or acetonitrile mixed with benzonitrile (e.g., 4:1 v / v)). Suitably, when the first compound is a peptide or a peptide nucleic acid, the process is performed in DMF, acetonitrile or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with DMF (e.g., 1 :1 v / v)).

[0018] It may be that all steps of a given chain extension cycle (e.g., deprotection and coupling) are performed in the same solvent. Even more suitably, all chain extension cycles and their associated steps are performed in the same solvent.

[0019] In an embodiment, the process is performed in DMF, acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane, acetonitrile mixed with benzonitrile or acetonitrile mixed with DMF) and all steps of a given chain extension cycle (e.g., deprotection and coupling) are performed in the same solvent. In an embodiment, the process is performed in acetonitrile, acetonitrile mixed with sulfolane or acetonitrile mixed with benzonitrile, and all chain extension cycles and their associated steps are performed in the same solvent.

[0020] In accordance with the first aspect of the present invention, the first compound is a defined monomer sequence polymer. It will be understood that the terms “first compound” and “defined monomer sequence polymer” are synonymous with each other in the present invention and are used interchangeably herein. The term “defined monomer sequence polymer” is used herein to refer to a polymer comprising at least two monomers, in which at least two of the monomers are distinct from each other, and in which the monomers are present in the same order in the polymer chain for all molecules of the polymer. For example, in a defined monomer sequence polymer formed from monomers A, B, C and D, the sequence of monomeric units may be B-B-B-C-A-A-C-D, with this sequence being identical for all molecules of the polymer.

[0021] The first compound (i.e., the defined monomer sequence polymer) may be an oligonucleotide, a peptide, a peptide nucleic acid, a polyether or an oligosaccharide. Suitably, the first compound (i.e., the defined monomer sequence polymer) is an oligonucleotide or a peptide. Most suitably, the first compound (i.e., the defined monomer sequence polymer) is an oligonucleotide. In embodiments wherein the first compound (i.e., the defined monomersequence polymer) is an oligonucleotide, the process of the present invention may be described as a liquid-phase oligonucleotide synthesis (LPOS).

[0022] Oligonucleotides will be familiar to those skilled in the art and will be understood to comprise a plurality of nucleotides linked (i.e., coupled) to one another to form a nucleotide sequence. Oligonucleotides are prepared by the stepwise addition of monomeric, dimeric or oligomeric nucleotidic building blocks to a growing oligonucleotide, with each addition being referred to as a coupling reaction.

[0023] The first compound, once prepared (i.e., fully grown), may have a molecular weight of >1000 Da. Suitably, the first compound has a molecular weight of >2000 Da. More suitably, the first compound has a molecular weight of >3000 Da. Even more suitably, the first compound has a molecular weight of >5000 Da.

[0024] In an embodiment, the first compound is an oligonucleotide or a peptide, and the first compound has a molecular weight of >2000 Da. In an embodiment, the first compound is an oligonucleotide, and the first compound has a molecular weight of >5000 Da.

[0025] It will be understood that the term “first compound” refers to the final defined monomer sequence polymer once prepared (i.e., fully grown). The term “growing first compound” refers to the same defined monomer sequence polymer as it is being prepared.

[0026] Suitably, the present invention provides a liquid-phase process for preparing one or more first compounds, each first compound being a defined monomer sequence polymer as defined herein. More suitably, the present invention provides a liquid-phase process for preparing 2-16 first compounds, each first compound being a defined monomer sequence polymer as defined herein. Even more suitably, the present invention provides a liquid-phase process for preparing 4-12 first compounds, each first compound being a defined monomer sequence polymer as defined herein. Yet even more suitably, the present invention provides a liquid-phase process for preparing 6-10 first compounds, each first compound being a defined monomer sequence polymer as defined herein. Most suitably, the present invention provides a liquid-phase process for preparing 8 first compounds, each first compound being a defined monomer sequence polymer as defined herein.

[0027] The first compound is grown by performing one or more chain extension cycles. Suitably, the process comprises growing a first compound by performing two or more chain extension cycles. More suitably, the process comprises growing a first compound by performing four or more chain extension cycles. Even more suitably, the process comprises growing a first compound by performing six or more chain extension cycles. Still even more suitably, the process comprises growing a first compound by performing eight or more chain extension cycles. Yet still even more suitably, the process comprises growing a first compound by performing twelve or more chain extension cycles. Yet still even more suitably, the process comprises growing a firstcompound by performing sixteen or more chain extension cycles. Most suitably, the process comprises growing a first compound by performing eighteen or more chain extension cycles.

[0028] In an embodiment, the first compound is an oligonucleotide or a peptide, and the process comprises growing a first compound by performing eight or more chain extension cycles. In an embodiment, the first compound is an oligonucleotide, and the process comprises growing a first compound by performing eighteen or more chain extension cycles.

[0029] Each chain extension cycle comprises a deprotection step and a coupling step. In embodiments wherein the first compound is an oligonucleotide (i.e. , LPOS), other steps may be included, such as quenching and / or thioylation I oxidation. The quenching step may be performed using a reagent comprising a hydroxyl functional group (e.g., primary alcohols, secondary alcohols and tertiary alcohols, such as cyanoethanol, methanol, ethanol, phenylethanol orwater). The thioylation I oxidation step may be performed using a camphorsulfonyl oxaziridine (CSO), cumenyl hydroperoxide, tert-butyl hydroperoxide, phenylacetyl disulfide (PADS), xanthane hydride (XH) and / or 3-phenyl 1 ,2,4-dithiazoline-5-one (POS) reagent. In LPOS, the thioylation I oxidation step converts one of more P(lll) phosphite triester linkages to a P(V) phosphotriester linkage after coupling of the building block to a chain extension site. Suitably, the quenching and / or thioylation I oxidation steps are conducted before the deprotection step of a chain extension cycle.

[0030] The deprotection step to expose a chain extension site of a growing first compound enables the coupling step by exposing a reactive terminal on the growing first compound. The coupling step attaches a monomeric or oligomeric building block to the exposed chain extension site by reacting the reactive (i.e., unprotected) terminal of the growing first compound with a reactive terminal of the monomeric or oligomeric building block to be coupled. In subsequent chain extension cycles, this process is repeated wherein a new chain extension site of the growing first compound is deprotected, exposing a reactive terminal of the newly coupled monomeric or oligomeric building block for subsequent coupling with a new monomeric or oligomeric building block. The skilled person will be familiar with protecting groups used to prevent uncontrolled polymer chain extension in the liquid-phase synthesis of defined monomer sequence polymers, as well as the manner in which they can be removed. Suitably, a reagent is used to cleave a protecting group from the growing first compound. Most suitably, the reagent used to cleave a protecting group from the growing first compound is an acid or a base.

[0031] Suitably, the deprotection step cleaves an acid-labile protecting group, such as dimethoxytrityl (Dmtr / DMT), tert-butyl (tBu), tert-butyl oxycarbonyl (Boc), monomethoxytriphenyl (Mmtr), triphenyl methyl (Tr), pentamethyl dihydrobenzofuran sufonyl (Pbf), Tetrahydropyranyl (Thp), tetrahydrofuranyl (Thf), para-methoxybenzyl (Pmb), methoxyisopropyl (Mip) and 2,4- dimethoxybenzyl. The deprotection step exposes a chain extension site of a growing firstcompound. Most suitably, the deprotection step cleaves Dmtr (i.e., detritylation). Methanesulfonic acid (MSA), trichloroacetic acid (TCA), dichloroacetic acid (DCA) and trifluoroacetic acid (TFA) are suitable reagents for the deprotection step. The deprotection step may also make use of cation scavengers such as 2,2'-(Ethylenedioxy)diethanethiol (DODT), tetramethylethylenediamine (TEMES), dodecane thiol, pyrrole, indole, 6-methoxyindole or triethyl silane. The deprotection step described above is most suitable in embodiments wherein the first compound is an oligonucleotide.

[0032] Suitably, the deprotection step cleaves a base-labile protecting group, such as fluorenylmethoxycarbonyl (Fmoc). The deprotection step exposes a chain extension site of a growing first compound. Piperidine and 1 ,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) are suitable reagents for the deprotection step. The deprotection step described above is most suitable in embodiments wherein the first compound is a peptide or a peptide nucleic acid.

[0033] The nature of the chain extension site of the growing first compound will be apparent to those of ordinary skill in the art of defined monomer sequence polymer synthesis. Solely for the sake of illustration, when the first compound is an oligonucleotide, the chain extension site may be a hydroxyl group located at the 5’ terminal or the 3’ terminal of the growing oligonucleotide (i.e. , the growing first compound). When the first compound is a peptide, the chain extension site may be a primary or secondary amino group located at a terminal position on the growing peptide (i.e., the growing first compound).

[0034] The building blocks used in the coupling step of the process of the present invention are monomeric or oligomeric (e.g., dimeric, trimeric, tetrameric etc.). For example, when the first compound is an oligonucleotide, the coupling step may involve attaching a single nucleotide (i.e., a monomeric building block) to the exposed chain extension site. Alternatively, when the first compound being prepared is an oligonucleotide, the coupling step may involve attaching an oligomer (i.e., a building block consisting of 2 or more pre-coupled nucleotides) to the exposed chain extension site. In another example, when the first compound is a peptide, the coupling step may involve attaching a single amino acid (i.e., a monomeric building block) to the exposed chain extension site. Alternatively, when the first compound being prepared is a peptide, the coupling step may involve attaching an oligomer (i.e., a building block consisting of 2 or more pre-coupled amino acids) to the exposed chain extension site. Suitably, the building blocked used in the coupling step are monomeric.

[0035] In the process of the present invention, at one or more instances in a chain extension cycle, primary membrane filtration is conducted to isolate the growing first compound in the retentate. Suitably, at one or two instances, primary membrane filtration is conducted to isolate the growing first compound in the retentate. Most suitably, at two instances, primary membranefiltration is conducted to isolate the growing first compound in the retentate. During primary membrane filtration, the growing first compound may be isolated by separating it from a reagent used, or a by-product formed, in a chain extension cycle. For example, primary membrane filtration can be used to separate the growing first compound from solvent contaminated with reagents and / or by-products (e.g., uncoupled building blocks, coupling agents and / or cleaved protecting groups). The growing first compound is isolated in the retentate, while the solvent contaminated with reagents and / or by-products is permeated through the membrane. The growing first compound isolated in the retentate can then be used in subsequent chain extension cycles.

[0036] Suitably, each chain extension cycle in the process of the present invention comprises one or more instances of primary membrane filtration to isolate the growing first compound in the retentate.

[0037] In an embodiment, each chain extension cycle in the process of the present invention comprises one or two instances of primary membrane filtration to isolate the growing first compound in the retentate, and the growing first compound is an oligonucleotide.

[0038] Primary membrane filtration may be performed after the deprotection step and before the coupling step of a chain extension cycle. Alternatively, primary membrane filtration is performed before the deprotection step and after the coupling step of a chain extension cycle.

[0039] Primary membrane filtration is suitably conducted in two discrete steps (i.e., at two instances). A first step may be conducted after the deprotection step and before a subsequent coupling step of a chain extension cycle, thereby isolating the growing first compound from solvent contaminated with reagents and / or by-products, such as cleaved protecting groups. A second step may be conducted after the coupling step and before a subsequent deprotection step of a chain extension cycle, thereby isolating the growing first compound from solvent contaminated with reagents and / or by-products, such as uncoupled building blocks and coupling agents. An example of this is shown in figure 1. Accordingly, in some embodiments, primary membrane filtration is performed after the deprotection step and before the coupling step of a chain extension cycle, and primary membrane filtration is performed before the deprotection step and after the coupling step of a chain extension cycle.

[0040] Primary membrane filtration is suitably conducted by membrane diafiltration. In membrane diafiltration, the crude mixture comprising the growing first compound is pressurised against a size-selective membrane. More suitably, primary membrane filtration is conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF). The membrane used in primary membrane filtration is suitably insoluble in the solvent(s) described herein (e.g., the membrane used in primary membrane filtration is resistant to the solvent(s) described herein).

[0041] The membranes used in primary membrane filtration include polymeric membranes, ceramic membranes, and mixed polymeric / inorganic membranes. Membrane rejection Rj is a common term known by those skilled in the art and is defined as: eq. (1)where CP = concentration of species i in the permeate, permeate being the liquid which has passed through the membrane, and CR = concentration of species i in the retentate, retentate being the liquid which has not passed through the membrane. It will be appreciated that a membrane is suitable for the invention if (Product)> (reactants), where product is the growing first compound.

[0042] The membrane used in primary membrane filtration may be formed from any polymeric or ceramic material which provides a separating layer capable of preferentially separating the growing first compound from solvent contaminated with reagents and / or by-products, such as uncoupled building blocks, coupling agents and cleaved protecting groups. In other words, the membrane will exhibit a rejection for the growing first compound that is greater than the rejection for the solvent contaminated with reagents and / or by-products (i.e. , the growing first compound is isolated in the retentate). Suitably, the membrane is formed from or comprises a polymeric material suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole (PBI), polyetheretherketone (PEEK) and mixtures thereof. Ceramic membranes may be made from TiC>2 or ZrC>2. The membranes can be made by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerisation or phase inversion. Membranes may be composite in nature (e.g., a thin film composite membrane) and / or be crosslinked or treated so as to improve their stability in certain solvents. PCT / GB2007 / 050218 and PCT / GB2015 / 050179 describe membranes which may be suitable for use as part of the invention. US10,913,033 describes a membrane that is particularly suitable for use in primary membrane filtration.

[0043] Most suitably, the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0044] In an embodiment, at one or two instances, primary membrane filtration is conducted to isolate the growing first compound in the retentate, and the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0045] In an embodiment, primary membrane filtration is performed after the deprotection step and before the coupling step of a chain extension cycle, and the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane. In an alternative embodiment, primary membrane filtration is performed before the deprotection step and after the coupling step of a chain extension cycle, and the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

[0046] In the process of the present invention, at least a portion of solvent permeated from primary membrane filtration is subjected to secondary membrane filtration to increase the purity of the solvent, the purified solvent being used in a subsequent chain extension cycle (e.g., primary membrane filtration). During secondary membrane filtration, the solvent may be purified by separating it from a reagent used, or a by-product formed, in a chain extension cycle. For example, secondary membrane filtration can be used to isolate reagents and / or by-products (e.g., uncoupled building blocks, coupling agents and cleaved protecting groups) in the retentate, while permeating the solvent, thereby increasing the purity of the solvent.

[0047] The inventors have found that secondary membrane filtration in the process of the present invention is a considerable improvement on known liquid-phase processes for preparing defined monomer sequence polymers. Indeed, secondary membrane filtration permits the solvent used in each chain extension cycle to be recycled immediately, obviating the need for subsequent recycling in external large holding tanks. The purified solvent permeating from secondary membrane filtration can be used again in a subsequent chain extension cycle (e.g., primary membrane filtration), reducing the reliance on large amounts of virgin (i.e. , fresh) solvent for each chain extension cycle.

[0048] It will be understood that primary membrane filtration and secondary membrane filtration are different from one another. The aim of primary membrane filtration is to isolate the growing first compound in the retentate. By contrast, the aim of secondary membrane filtration is to isolate reagents and / or by-products in the retentate which have permeated through primary membrane filtration. The following schematic is intended to illustrate this relationship in its simplest form:1: primary embrane filtration2; secondary membrane filtrationScheme 1. Relationship between primary membrane filtration and secondary membrane filtration.

[0049] As described hereinbefore, at least a portion of solvent permeated from primary membrane filtration is subjected to secondary membrane filtration. It may be that all of the solvent permeated from primary membrane filtration is subjected to secondary membrane filtration. However, it is preferred that not all of the solvent permeated from primary membrane filtration is subjected to secondary membrane filtration. The solvent permeated from primary membrane filtration may be analysed (e.g., by liquid chromatography, ultraviolet spectroscopy, ultraviolet- visible spectroscopy, infrared spectroscopy, total ion current chromatography or electron energy loss spectroscopy) to determine whether it should be subjected to secondary membrane filtration. It is theorised that by selectively controlling whether the solvent should be subjected to secondary membrane filtration improves the efficiency of secondary membrane filtration.

[0050] The solvent permeated from primary membrane filtration may comprise: i) an initial portion; and ii) a subsequent portion, with the initial portion permeated from primary membrane filtration before the subsequent portion. The initial portion will typically have a higher concentration of low molecular weight reagents relative to the subsequent portion. Suitably, the initial portion is not subjected to secondary membrane filtration (i.e. , only the subsequent portion is subjected to secondary membrane filtration). In embodiments where the initial portion is not subjected to secondary membrane filtration, the initial portion may be disposed of (i.e., sent to waste). The inventors have found that by subjecting only the subsequent portion to secondary membrane filtration, the membrane used in secondary membrane filtration is not required to retain a high concentration of low molecular weight reagents and / or by-products, thereby improving its efficiency.

[0051] As set out hereinbefore, primary membrane filtration may be conducted by membrane diafiltration. In such embodiments, the initial portion permeated from primary membrane filtration may be at least one diavolume. Accordingly, at least one diavolume of the solvent initially permeated from primary membrane filtration is not subjected to secondary membrane filtration.In some embodiments, the initial portion is one or two diavolumes. Accordingly, one or two diavolumes of the solvent initially permeated from primary membrane filtration is not subjected to secondary membrane filtration.

[0052] The effectiveness of secondary membrane filtration may be improved by treating the solvent permeated from primary membrane filtration with one or more quenching agents. Prior to being subjected to secondary membrane filtration, the solvent may be treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight. The molecular weight of the reagent and / or by-product is increased by forming an adduct with the quenching agent. The overall increase in molecular weight means that the reagent and / or by-product cannot permeate through the membrane used in secondary membrane filtration. Suitably, the quenching agent is capable of reacting with an acid or base used to cleave a protecting group from the growing first compound and / or capable of reacting with a protecting group cleaved from the growing first compound.

[0053] The solvent permeated from primary membrane filtration may be treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >25%. Suitably, the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >50%. More suitably, the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >100%. Most suitably, the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >150%.

[0054] The quenching agent may be an acid or a base. In embodiments wherein the quenching agent a base, it can form an adduct with acids used in each chain extension cycle. In embodiments wherein the quenching agent is an acid, it can form an adduct with bases used in each chain extension cycle.

[0055] In an embodiment, the solvent permeated from primary membrane filtration is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >50%, and the quenching agent is an acid or a base. Suitably, the one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >150%.

[0056] When the first compound is an oligonucleotide (i.e., LPOS), the quenching agent may be a base. Suitably, the quenching agent is selected from tribenzylamine (TBA), picoline, pyridine, lutidine, collidine and piperidine. Most suitably, the quenching agent is TBA. The quenching agent can form an adduct with reagents used in each chain extension cycle, such asthose suitable for the deprotection step, in order to increase their molecular weight. For example, TBA may form an adduct with one or more of MSA, TCA, DCA and / or TFA. In many instances, the adduct is TBA: MSA.

[0057] In an embodiment, the solvent permeated from primary membrane filtration is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecularweight by >150%, and the quenching agent is selected from TBA, picoline, pyridine, lutidine, collidine and piperidine. Suitably, the quenching agent is TBA.

[0058] When the first compound is a peptide or a peptide nucleic acid, the quenching agent may be an acid. Suitably, the quenching agent is selected from 2,4,6-trimethylbenzoic acid, MSA, palmitic acid and dodecylbenzene sulfonic acid. The quenching agent can form an adduct with reagents used in each chain extension cycle (e.g., piperidine, DBU, HBTLI, BOP, PyBOP and Fmoc-amino acids), in order to increase their molecular weight. For example, piperidine and DBU, which are used for Fmoc removal, can form adducts with 2,4,6-trimethylbenzoic acid, MSA, palmitic acid and / or dodecylbenzene sulfonic acid.

[0059] In an embodiment, the solvent permeated from primary membrane filtration is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecularweight by >150%, and the quenching agent is selected from 2,4,6-trimethylbenzoic acid, MSA, palmitic acid and dodecylbenzene sulfonic acid.

[0060] The quenching agent may alternatively be a chelator, which is capable of binding to metal contaminants in each chain extension cycle. For example, the quenching agent may chelate to ferricyanide, ferric ethylenediaminetetraacetic acid (EDTA), as well as palladium and ruthenium complexes.

[0061] As described herein, the aim of secondary membrane filtration is to isolate reagents and / or by-products in the retentate which have permeated through primary membrane filtration. The membrane used in secondary membrane filtration may therefore have different molecular weight cut off (MWCO) to the membrane used in primary membrane filtration.

[0062] The membrane used in secondary membrane filtration may have a MWCO that is 5-185 g mol-1greater than the molecular weight of the solvent. In the case of mixed solvent systems, the membrane used in secondary membrane filtration may have a MWCO that is 5-185 g mol'1greater than the molecular weight of the solvent of highest molecular weight in the mixed solvent system. Suitably, the membrane used in secondary membrane filtration has a MWCO that is IQ- 150 g mol'1greater than the molecular weight of the solvent. In the case of mixed solvent systems, the membrane used in secondary membrane filtration has a MWCO that is 10-150 g mol'1greater than the molecular weight of the solvent of highest molecular weight in the mixed solvent system. More suitably, the membrane used in secondary membrane filtration has aMWCO that is 15-100 g mol-1greater than the molecular weight of the solvent. In the case of mixed solvent systems, the membrane used in secondary membrane filtration has a MWCO that is 15-100 g mol-1greater than the molecular weight of the solvent of highest molecular weight in the mixed solvent system. Most suitably, the membrane used in secondary membrane filtration has a MWCO that is 20-50 g mol'1greater than the molecular weight of the solvent. In the case of mixed solvent systems, the membrane used in secondary membrane filtration has a MWCO that is 20-50 g mol'1greater than the molecular weight of the solvent of highest molecular weight in the mixed solvent system.

[0063] Solvents useful in the process of the present invention are described herein and include DMF, acetonitrile, benzonitrile, sulfolane or a solvent mixture comprising two or more thereof.

[0064] The membranes used in secondary membrane filtration include polymeric membranes, ceramic membranes, and mixed polymeric / inorganic membranes. The membrane used in secondary membrane filtration may be formed from any polymeric or ceramic material which provides a separating layer capable of preferentially separating the solvent from reagents and / or by-products, such as uncoupled building blocks, coupling agents and cleaved protecting groups. In other words, the membrane will exhibit a rejection for the reagents and / or by-products that is greater than the rejection for the solvent. Suitably, the membrane is formed from or comprises a polymeric material suitable for fabricating nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, PTFE, PVDF, polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole (PBI), polyether ether ketone (PEEK) and mixtures thereof. Ceramic membranes may be made from TiC>2 or ZrC>2. The membranes can be made by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerisation or phase inversion. Membranes may be composite in nature (e.g., a thin film composite membrane) and / or be crosslinked or treated so as to improve their stability in certain solvents. Suitable crosslinkers for the membrane used in secondary membrane filtration include amine-terminated crosslinkers (e.g., an aminosilane, such as 3-aminopropyl trimethoxy silane (APTMS), or an amine terminated polyethylene glycol, such as Jeffamine 400).

[0065] The membrane used in secondary membrane filtration may be a thin film composite comprising a porous support layer including a crosslinked porous support layer, covered with a thin separating layer formed by interfacial polymerisation, spray coating or solvent dip coating. Suitably, the thin film composite membrane will be formed of a crosslinked support covered by a thin film formed by interfacial polymerisation to form a polyamide or polyester, more suitably, a polyamide formed from the reaction of an aromatic polyamine (e.g., m-phenylene diamine) and an aromatic triacyl halide (e.g., trimesoyl chloride).

[0066] Most suitably, the membrane used in secondary membrane filtration is a crosslinked polyimide membrane. The polyimide membrane may be crosslinked with amine-terminated crosslinkers (e.g., an aminosilane, such as APTMS, or an amine-terminated polyethyleneglycol, such as Jeffamine 400). The crosslinked polyimide membrane may be a crosslinked polyimide mixed matrix membrane. Suitably, the crosslinked polyimide mixed matrix membrane comprises polyimide chains crosslinked with siloxane networks. Examples of mixed matrix membranes can be found in Siddique et al. (1). Crosslinked membranes are typically prepared by casting a dope membrane solution onto a non-woven backing material. The cast membrane undergoes phase inversion, followed by immersion in a crosslinker solution, such as APTMS in MEK. Finally, the membrane is dried to form a crosslinked network within the membrane. The crosslinked network may be a siloxane network comprising -Si-O-Si- linkages.

[0067] The membrane used in secondary membrane filtration may have a molecular weight cut off (MWCO) of 200-300 g / mol. Most suitably, the membrane used in secondary membrane filtration has a molecular weight cut off (MWCO) of 210-250 g / mol.

[0068] In an embodiment, the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane and the membrane used in secondary membrane filtration is a crosslinked polyimide membrane. Suitably, the membrane used in secondary membrane filtration is a crosslinked polyimide mixed matrix membrane, comprising polyimide chains crosslinked with siloxane networks.

[0069] The process of the present invention may additionally involve one or more treatment steps. Suitably, the purified solvent resulting from secondary membrane filtration is subjected to one or more treatment steps to remove residual contaminants prior to using it in a subsequent chain extension cycle. The one or more treatment steps further purify the purified solvent resulting from secondary membrane filtration, thereby making the solvent even more suitable for recycling in a subsequent chain extension cycle (e.g., primary membrane filtration). Particular contaminants to be removed from the purified solvent resulting from secondary membrane filtration include water, acids, bases and / or metal ions. Suitably, the purified solvent resulting from secondary membrane filtration is dried to remove water prior to using it in a subsequent chain extension cycle. Water may be removed from the purified solvent resulting from secondary membrane filtration by passing the solvent through a column of drying agent, such as molecular sieves or other desiccants. Suitably, the purified solvent resulting from secondary membrane filtration is treated to remove acids prior to using it in a subsequent chain extension cycle. Acids may be removed from the purified solvent resulting from secondary membrane filtration by passing the solvent through a column of basic resin. Suitably, the purified solvent resulting from secondary membrane filtration is treated to remove bases prior to using it in a subsequent chainextension cycle. Bases may be removed from the purified solvent resulting from secondary membrane filtration by passing the solvent through a column of acidic resin. Suitably, the purified solvent resulting from secondary membrane filtration is treated to remove metal ions prior to using it in a subsequent chain extension cycle. Metal ions may be removed from the purified solvent resulting from secondary membrane filtration by passing the solvent through a column of chelating resin.

[0070] As discussed herein, during secondary membrane filtration the solvent is purified by separating it from a reagent used, or a by-product formed, in a chain extension cycle. Suitably, the solvent is purified by separating it from: a) uncoupled building blocks; b) coupling agents; c) a reagent used to cleave a protecting group from the growing first compound to expose a chain extension site; d) a protecting group cleaved from the growing first compound to expose a chain extension site; e) quenching agent adducts (e.g., adducts formed of quenching agents and reagents and / or byproducts formed in each chain extension cycle); and / or f) quenching agents.

[0071] Any traces of residual contaminants remaining in the purified solvent may be the same as those still contaminating the virgin (i.e. , fresh) solvent used in the initial chain extension cycle. Therefore, traces of residual contaminants in the purified solvent to be recycled in the next chain extension cycle will not qualitatively change the nature of the contaminants that would typically be generated by membrane-assisted liquid-phase processes for preparing defined monomer sequence polymers without membrane-mediated solvent recycling.

[0072] The process may further comprise one or more global deprotection steps. Such step(s) may be conducted once a target length of the first compound has been reached. The global deprotection step(s) may result in removal of any protecting groups from the first compound. Those of ordinary skill in the art will be familiar with techniques by which such global deprotection can be achieved.

[0073] The growing first compound may be attached to a soluble synthesis support. Such soluble synthesis supports may be used to confer additional solubility to the growing first compound, and / or to increase its molecular bulk relative to reaction reagents and / or by-products in order to facilitate primary membrane filtration.

[0074] The soluble synthesis support may be a polymer (e.g., a star polymer), a dendrimer, a dendron, a hyperbranched polymer, or an organic / inorganic material, including nanoparticles,fullerenes and 2-D materials such as graphene and boron nitride. Suitably, the soluble synthesis support is polymeric and is formed from poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes. More suitably, the soluble synthesis support is formed from poly(ethylene glycol).

[0075] In some instances, the soluble synthesis support is a star polymer comprising a central branch point and n number of radiating polymeric arms, wherein each polymeric arm is attached to a growing first compound, and wherein n is 3 or more. In such instances, it will be understood that the process can be used to grow n number of first compounds, all of which may be identical. Suitably, each polymeric arm is selected from the group consisting of poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes. More suitably, each polymeric arm is poly(ethylene glycol).

[0076] The soluble synthesis support may have a structure according to formula Iin whichV is an organic branch point,W is a polymeric chain, and n is 1-20.

[0077] Suitably, each W is attached to the growing first compound. This attachment may be direct or indirect (i.e. , via a linking moiety). A typical linking moiety will comprise fewer than 50 atoms in total, more suitably fewer than 30 atoms in total.

[0078] V will be understood to refer to a polyfunctional organic “hub” having a plurality of terminals to which polymeric chain(s), W, are attached. Suitably, V comprises fewer than 50 atoms in total, more suitably fewer than 20 atoms in total.

[0079] Each W is a polymeric chain (e.g., polyethylene glycol) having a molecular weight (Mw) of 100 - 20,000 Da. Suitably, each W is a polymeric chain having a molecular weight (Mw) of 250 - 15,000 Da. More suitably, each W is a polymeric chain having a molecular weight (Mw) of 500 - 10,000 Da. More suitably, each W is a polymeric chain having a molecular weight (Mw) of 1,000 - 5,000 Da. Most suitably, each W is a polymeric chain having a molecular weight (Mw) of 2,000 - 3,000 Da.

[0080] Suitably, m is 2-16. More suitably, m is 4-12. Even more suitably, m is 6-10. Most suitably, m is 8.

[0081] A particularly suitable example of a soluble synthesis support is:in which -NW represents the point of direct attachment to the growing first compound, q is 100 - 500 (e.g., 200 - 300) and C is a central core (e.g., a carbon atom).

[0082] In embodiments wherein the first compound (i.e., the defined monomer sequence polymer) is an oligonucleotide, various chemical modifications relative to the structure of RNA- based and DNA-based oligonucleotides / building blocks will be apparent to those of ordinary skill in the art and are envisaged herein, particularly in relation to the structure of the backbone, nucleobase and / or sugar moiety. Modifications may occur at the 2' position of the sugar moiety. Sugar modifications include a modified version of the ribosyl moiety, such as 2'-O-modified RNA such as 2'-O-alkyl or 2'-O(substituted)alkyl e.g., 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2- methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-0-butyryl, 2'-O-propargyl, 2'-O-allyl, 2'-O-(3- amino)propyl, 2'-O-(3-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2- (dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O(haloalkoxy)methyl (Arai et al.) e.g., 2'-O-(2- chloroethoxy)methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM); 2'-O-alkoxycarbonyl e.g. 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2- (N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo e.g. 2'-F, FANA (2'-F arabinosyl nucleic acid); carbasugar and azasuar modifications; 3'-O-alkyl e.g. 3'-O-methyl, 3'-0-butyryl, 3'-O-propargyl; and their derivatives. Sugar modifications also include 2'-amino. Other sugar modifications include "bridged" or "bicylic" nucleic acid (BNA), e.g. locked nucleic acid (LNA), xylo-LNA, a-L- LNA, p-D-LNA, cEt (2'-O,4'-C constrained ethyl) LNA, cMOEt (2'-O,4'-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo DNA; unlocked nucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); morpholino (as e.g., in PMO, PPMO, PMOPIus, PMO-X); and their derivatives.

[0083] Base modifications include modified versions of the natural purine and pyrimidine bases e.g., adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine e.g., 5-methylcytosine, 5-methyluracil, 5-halouracil, 5- propynyluracil, 5-propynylcytosine, 5-aminomethyl uracil, 5-hydroxymethyl uracil, 5- aminomethylcytosine, 5-hydroxymethylcytosine, Super 5 T), 2,6-diaminopurine, 7- deazaguanine, 7-deazaadenine, 7-aza-2, 6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7- deazaadenine, 8-aza-7-deaza-2, 6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g., 1 -deoxyribose, 1-deoxy-2- O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in US6683173. cPent-G, cPent-AP and Pr-AP were shown to reduce immunostimulatory effects when incorporated in siRNA. Alternatively, nucleobase modifications may be selected from the group consisting of 5-methyl pyrimidines, 7-deazaguanosines and abasic nucleotides. Alternatively, the modification may be a 5-methyl cytosine.

[0084] Backbone modifications, i.e., relative to the phosphodiester present in RNA and DNA, include phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methylphosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modification includes phosphoramidite, phosphoramidate, N3'~PS' phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives.

[0085] Other modifications include incorporation of a peptide-base nucleic acid (PNA), boron modified PNA, pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- or glycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA), acyclic threoninol-based nucleic acid (aTNA), oligonucleotides with integrated bases and backbones (ONIBs), pyrrolidine-amide oligonucleotides (POMs); a bridged nucleic acid (BNA) such as (5)-cEt-BNA, or a SPIEGELMER.

[0086] In an embodiment, the process of the present invention is performed in a closed loop reactor. Performing the process in a closed loop reactor permits the purified solvent to be recycled in a subsequent chain extension cycle (e.g., primary membrane filtration).

[0087] According to a second aspect of the present invention, there is provided a first compound obtained, directly obtained or obtainable by the process of the first aspect.

[0088] The following numbered statements 1 to 85 are not claims, but instead describe particular aspects and embodiments of the invention:1. a liquid-phase process for preparing a first compound being a defined monomer sequence polymer, the process comprising:(i) growing a first compound by performing one or more chain extension cycles, each chain extension cycle comprising:a deprotection step to expose a chain extension site of a growing first compound; and a coupling step to attach a monomeric or oligomeric building block to the exposed chain extension site;(ii) performing, at one or more instances in a chain extension cycle, primary membrane filtration to isolate the growing first compound in the retentate; and(iii) subjecting at least a portion of solvent permeated from primary membrane filtration to secondary membrane filtration to increase the purity of the solvent, the purified solvent being used in a subsequent chain extension cycle.2. The process of statement 1 , wherein the process is performed in DMF, acetonitrile, sulfolane, benzonitrile or solvent mixtures comprising two or more thereof.3. The process of statement 1 or 2, wherein the process is performed in DMF, acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane, acetonitrile mixed with benzonitrile or acetonitrile mixed with DMF).4. The process of statement 1 , 2 or 3, wherein the process is performed in acetonitrile, or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with sulfolane (e.g., 4:1 v / v) or acetonitrile mixed with benzonitrile (e.g., 4:1 v / v)).5. The process of any one of the preceding statements, wherein the process is performed in DMF, acetonitrile or in a solvent mixture comprising acetonitrile (e.g., acetonitrile mixed with DMF (e.g., 1 :1 v / v)).6. The process of any one of the preceding statements, wherein all steps of a given chain extension cycle (e.g., deprotection and coupling) are performed in the same solvent.7. The process of any one of the preceding statements, wherein all chain extension cycles and their associated steps are performed in the same solvent.8. The process of any one of the preceding statements, wherein the first compound is an oligonucleotide, a peptide, a peptide nucleic acid, a polyether or an oligosaccharide.9. The process of any one of the preceding statements, wherein the first compound is an oligonucleotide or a peptide.10. The process of any one of the preceding statements, wherein the first compound is an oligonucleotide.11. The process of any one of the preceding statements, wherein the first compound has a molecular weight of >1000 Da.12. The process of any one of the preceding statements, wherein the first compound has a molecular weight of >2000 Da.13. The process of any one of the preceding statements, wherein the first compound has a molecular weight of >3000 Da.14. The process of any one of the preceding statements, wherein the first compound has a molecular weight of >5000 Da.15. The process of any one of the preceding statements, wherein the process is a liquidphase process for preparing one or more first compounds, each first compound being a defined monomer sequence polymer as defined in any one of the preceding statements.16. The process of any one of the preceding statements, wherein the process is a liquidphase process for preparing 2-16 first compounds, each first compound being a defined monomer sequence polymer as defined in any one of the preceding statements.17. The process of any one of the preceding statements, wherein the process is a liquidphase process for preparing 4-12 first compounds, each first compound being a defined monomer sequence polymer as defined in any one of the preceding statements.18. The process of any one of the preceding statements, wherein the process is a liquidphase process for preparing 6-10 first compounds, each first compound being a defined monomer sequence polymer as defined in any one of the preceding statements.19. The process of any one of the preceding statements, wherein the process is a liquidphase process for preparing 8 first compounds, each first compound being a defined monomer sequence polymer as defined in any one of the preceding statements.20. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing two or more chain extension cycles.21. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing four or more chain extension cycles.22. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing six or more chain extension cycles.23. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing eight or more chain extension cycles.24. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing twelve or more chain extension cycles.25. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing sixteen or more chain extension cycles.26. The process of any one of the preceding statements, wherein the process comprises growing a first compound by performing eighteen or more chain extension cycles.27. The process of any one of the preceding statements, wherein at one or more instances in a chain extension cycle, primary membrane filtration is conducted to isolate the growing first compound in the retentate.28. The process of any one of the preceding statements, wherein at one or two instances, primary membrane filtration is conducted to isolate the growing first compound in the retentate.29. The process of any one of the preceding statements, wherein at two instances, primary membrane filtration is conducted to isolate the growing first compound in the retentate.30. The process of any one of the preceding statements, wherein each chain extension cycle comprises one or more instances of primary membrane filtration to isolate the growing first compound in the retentate.31. The process of any one of the preceding statements, wherein primary membrane filtration is performed after the deprotection step and before the coupling step of a chain extension cycle.32. The process of any one of the preceding statements, wherein primary membrane filtration is performed before the deprotection step and after the coupling step of a chain extension cycle.33. The process of any one of the preceding statements, wherein primary membrane filtration is conducted by membrane diafiltration.34. The process of any one of the preceding statements, wherein primary membrane filtration is conducted by organic solvent nanofiltration (OSN) or ultrafiltration (UF).35. The process of any one of the preceding statements, wherein the membrane used in primary membrane filtration is insoluble in the solvent(s) described in any one of the preceding statements.36. The process of any one of the preceding statements, wherein the membrane used in primary membrane filtration is a polymeric membrane, a ceramic membrane, or a mixed polymeric / inorganic membranes.37. The process of any one of the preceding statements, wherein the membrane is formed from or comprises a polymeric material suitable for fabricating microfiltration, ultrafiltration,nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole (PBI), polyetheretherketone (PEEK) and mixtures thereof.38. The process of any one of the preceding statements, wherein the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.39. The process of any one of the preceding statements, wherein the solvent permeated from primary membrane filtration is analysed to determine whether it should be subjected to secondary membrane filtration.40. The process of any one of the preceding statements, wherein the solvent permeated from primary membrane filtration comprises: i) an initial portion; and ii) a subsequent portion, with the initial portion permeated from primary membrane filtration before the subsequent portion.41. The process of statement 40, wherein the initial portion is not subjected to secondary membrane filtration.42. The process of statement 40 or 41 , wherein the subsequent portion is subjected to secondary membrane filtration.43. The process of any one of the preceding statements, wherein the solvent permeated from primary membrane filtration is treated with one or more quenching agents.44. The process of statement 43, wherein the quenching agent is capable of reacting with an acid or base used to cleave a protecting group from the growing first compound and / or capable of reacting with a protecting group cleaved from the growing first compound.45. The process of statement 43 or 44, wherein the solvent permeated from primary membrane filtration is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >25%.46. The process of statement 43, 44 or 45, wherein the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >50%.47. The process of any one of statements 43-46, wherein the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >100%.48. The process of any one of statements 43-47, wherein the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >150%.49. The process of any one of statements 43-48, wherein the quenching agent is an acid or a base.50. The process of any one of statements 43-49, wherein the quenching agent is selected from tribenzylamine (TBA), picoline, pyridine, lutidine, collidine and piperidine.51. The process of any one of statements 43-50, wherein the quenching agent is TBA.52. The process of any one of statements 43-49, wherein the quenching agent is selected from 2,4,6-trimethylbenzoic acid, MSA, palmitic acid and dodecylbenzene sulfonic acid.53. The process of any one of statements 43-48, wherein the quenching agent is a chelator, which is capable of binding to metal contaminants in each chain extension cycle.54. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration has a MWCO that is 5-185 g mol-1greater than the molecular weight of the solvent.55. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration has a MWCO that is 10-150 g mol-1greater than the molecular weight of the solvent.56. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration has a MWCO that is 15-100 g mol-1greater than the molecular weight of the solvent.57. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration has a MWCO that is 20-50 g mol-1greater than the molecular weight of the solvent.58. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration is a polymeric membrane, a ceramic membrane, or a mixed polymeric / inorganic membrane.59. The process of any one of the preceding statements, wherein the membrane is formed from or comprises a polymeric material suitable for fabricating nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, PTFE, PVDF, polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, PBI, PEEK and mixtures thereof.60. The process of any one of the preceding statements, wherein the membrane used in secondary membrane filtration is a crosslinked polyimide membrane.61. The process of statement 60, wherein the crosslinked polyimide membrane is a crosslinked polyimide mixed matrix membrane.62. The process of statement 61, wherein the crosslinked polyimide mixed matrix membrane comprises polyimide chains crosslinked with siloxane networks.63. The process of any one of the preceding statements, wherein the process additionally involves one or more treatment steps.64. The process of any one of the preceding statements, wherein the purified solvent resulting from secondary membrane filtration is subjected to one or more treatment steps to remove residual contaminants prior to using it in a subsequent chain extension cycle.65. The process of any one of the preceding statements, wherein the purified solvent resulting from secondary membrane filtration is dried to remove water prior to using it in a subsequent chain extension cycle.66. The process of any one of the preceding statements, wherein the purified solvent resulting from secondary membrane filtration is treated to remove acids prior to using it in a subsequent chain extension cycle.67. The process of any one of the preceding statements, wherein the purified solvent resulting from secondary membrane filtration is treated to remove bases prior to using it in a subsequent chain extension cycle.68. The process of any one of the preceding statements, wherein the purified solvent resulting from secondary membrane filtration is treated to remove metal ions prior to using it in a subsequent chain extension cycle.69. The process of any one of the preceding statements, wherein the solvent is purified by separating it from: a) uncoupled building blocks; b) coupling agents; c) a reagent used to cleave a protecting group from the growing first compound to expose a chain extension site; d) a protecting group cleaved from the growing first compound to expose a chain extension site; e) quenching agent adducts (e.g., adducts formed of quenching agents and reagents and / or byproducts formed in each chain extension cycle); and / or f) quenching agents.70. The process of any one of the preceding statements, wherein the growing first compound is attached to a soluble synthesis support.71. The process of statement 70, wherein the soluble synthesis support is a polymer (e.g., a star polymer), a dendrimer, a dendron, a hyperbranched polymer, or an organic / inorganic material, including nanoparticles, fullerenes and 2-D materials such as graphene and boron nitride.72. The process of statement 70 or 71, wherein the soluble synthesis support is polymeric and is formed from poly(alkylene glycols), polyesters, polyamides, vinyl polymers, diene polymers, poly(alkylene imines), poly(amidoamines) and polysiloxanes.73. The process of statement 70, 71 or 72, wherein the soluble synthesis support is formed from poly(ethylene glycol).74. The process of any one of statements 70-73, wherein the soluble synthesis support has a structure according to formula Iin whichV is an organic branch point,W is a polymeric chain, and n is 1-20.75. The process of statement 74, wherein each W is attached to the growing first compound.76. The process of statement 74 or 75, wherein each W is a polymeric chain (e.g., polyethylene glycol) having a molecular weight (Mw) of 100 - 20,000 Da.77. The process of statement 74, 75 or 76, wherein each W is a polymeric chain having a molecular weight (Mw) of 250 - 15,000 Da.78. The process of any one of statements 74-77, wherein each W is a polymeric chain having a molecular weight (Mw) of 500 - 10,000 Da.79. The process of any one of statements 74-78, wherein each W is a polymeric chain having a molecular weight (Mw) of 1 ,000 - 5,000 Da.80. The process of any one of statements 74-79, wherein each W is a polymeric chain having a molecular weight (Mw) of 2,000 - 3,000 Da.81. The process of any one of statements 74-80, wherein m is 2-16.82. The process of any one of statements 74-81 , wherein m is 4-12.83. The process of any one of statements 74-82, wherein m is 6-10.84. The process of any one of statements 74-83, wherein m is 8.85. A first compound obtained, directly obtained or obtainable by the process of any one of the preceding statements.EXAMPLES

[0089] One or more examples of the invention will now be described, for the purpose of illustration only, with reference to the accompanying figures:Fig. 1. Liquid phase oligo synthesis cycle, with solvent recycling. Solvent from DF2 of one synthesis cycle is purified by permeating through secondary membrane filtration and is then used for DF1 in the next chain extension cycle.Fig. 2. Nanostar Synthesiser with solvent recycling: M1 , membrane cell (MiniMem filtration system from PS Prozesstechnik GmbH, internal volume 30 mL) with internal stirring disk; F, glass atmospheric pressure feed tank (Duran Pressure Plus bottle, 100 mL); R1 , solvent reservoir (Duran Pressure Plus bottle, 1 L); P1 , pump (Knauer P 4.1S piston pump) to raise the pressure in the membrane cell and provide recirculation. The circulation loop has an estimated volume of 50 mL, maintaining the feed tank F at 20 mL.Fig. 3. IP-RP HPLC analysis of octamer5’mUmGmUmUmUmCmUmG3’ 4: A - Solid trace, Example 1 , oligo 4 produced with solvent recycling; B - dashed trace, Comparative Example 1 , oligo 4 produced without solvent recycling.Fig. 4. IP-RP HPLC analysis of 18-mer 5, produced with solvent recycling. Overall purity, 66.6%; UV purity, 69.8%; MS purity, 95.4%; the main impurity at 8.9 min is n-1.Fig. 5. Large scale Nanostar Synthesiser fitted with spiral wound modules.Fig. 6. Trace C, solid, 8-mer, with recycling. Trace D, dashed, 8-mer, without recycling.Materials and methods

[0090] An exemplary membrane-assisted liquid phase oligonucleotide synthesis (LPOS) comprises loading a nucleoside (e.g., a nucleoside succinate 1) onto a Nanostar, such as 8-arm 20 kDa methylamino PEG-star 2 [PEG-20k(NMeH)s] (see figure 1). The oligo synthesis cycle can include three synthetic steps and two diafiltration steps (DF1 & DF2) where organic solvent nanofiltration (OSN) is used to wash out contaminants. The three synthetic steps are as follows:1. coupling-,2. oxidation or sulfur transfer, followed by DF1 , using 4 diavolumes (DV, or system volumes) of solvent to remove excess nucleoside, activator and / or oxidant;3. 5 -0 deprotection (usually detritylatiori), followed by full purification of the crude oligo-star by OSN in DF2 using 6 DV. DF2 removes residual unprotected nucleoside that would otherwise participate in the next chain extension cycle.The equipment used in membrane-assisted LPOS is illustrated schematically in figure 2. After building up the desired oligo sequence, the oligo-star is washed from the synthesiser and exposed to ammonolytic global deprotection to provide the crude oligo that may be further purified as desired.

[0091] Example 1 below describes the protocol for loading the PEG-star support, followed by the iterative procedure for each chain extension cycle in the membrane synthesiser, including coupling, oxidation and detritylation. Finally, global deprotection of the oligo is described.Example 1 :5’mUmGmUmUmUmCmUmG3’ octamer, 4, prepared with solvent recycling Loading of 5’-Dmtr-mU 3’-succinate 1ua onto Nanostar 2:

[0092] The Nanostar synthesiser (see figure 2) was charged with anhydrous MeCN-PhCN 4: 1. 8-Arm 20 kDa sarcosine terminated PEG-star [2a, PEG-20k(Sar-H)s, MW 20,600 Da, 2.49 g, 0.12 mmol] was dissolved in anhydrous MeCN (10 mL) and transferred by syringe to the synthesiser feed tank F through a septum port, washing out the flask with further MeCN (5 mL). Next, / V, / V-diisopropylethylamine (DI PEA, 0.42 mL, 2.40 mmol, 20 eq.) was added to F and, after circulating for 2 min (flow speed 50 mL / min for all operations), a sample was taken from the well mixed synthesiser (t = 0); all analytical samples from the synthesiser are 50 mL, diluted to 1.5 mL with MeCN, before injection into the LC-MS. 5’-Dmtr-mU-3’-succinate (1ua, 1.84 g, 2.40 mmol, 20 eq.) dissolved in anhydrous MeCN (3 mL), was added to a round bottom flask containing 1-[bis(dimethylamino)-methylene]-1 H-benzotriazolium 3-oxide tetrafluoroborate (TBTU, 0.77 g, 2.40 mmol, 20 eq.) under argon and stirred for 30 min at room temperature. The activated succinate solution was then injected into F through a 0.2 mm PTFE filter (Sterlitech Pressurised Filtration Holder). Membrane synthesisers can be sensitive to fouling of the membrane by particulate fines, for which reason all solutions injected into the synthesiser were filtered in this fashion.

[0093] The amidation reaction was monitored by LC-MS and appeared complete after 5 min, but was continued for 30 min to ensure completion; a sample was taken to determine the residual concentration of succinate 1ua at the start of DF1. The membrane was pressurised to 10 bar by slowly closing pressure relief valve PR1 , the stirrer set to 800 rpm, and MeCN-PhCN 4:1 was then permeated (200 mL, ~4 DV); the permeate from DF1 was sent to waste via valve V1 . As the circulation loop is airtight, solvent permeation generates a partial vacuum in the feed tank F, causing lost solvent to be replaced automatically by fresh solvent drawn in from reservoir R1 , providing for the conditions of constant volume diafiltration. At the end of DF1 another sample was analysed by LC-MS to measure the fractional drop in the concentration of excess succinate 1ua and the rejection of the trityl nucleoside-star.Detritylation reaction:

[0094] 2,2'-(Ethylenedioxy)diethanethiol (DODT, 6 eq. / arm, 48 eq. in total) was injected into feed tank F. Dmtr deprotection was then initiated by injecting methanesulfonic acid (MSA, 0.2 mL, 0.4 vol%). The detritylation reaction was monitored by LC-MS and after 10 min no partially detritylated nucleoside-star or residual Dmtr-mU-succinate 1 could be detected. After 20 min,tribenzylamine (TBA, 1.74 g) dissolved in MeCN (10 mL) was injected into F to quench the reaction, and a sample was taken to determine the intermediate concentration of detritylated succinate. Anhydrous MeCN-PhCN 4:1 (< 20 ppm water, 400 mL, ~8 DV) was permeated (DF2) to remove residual 5’-hydroxy-mll-3’-succinate and all other small molecule debris; only 6 DV is required to reduce the residual building block to an acceptable level, but an extra 2 DV was diafiltered, because, at this small scale of operation, the extra solvent ensured that the system was dry for the next round of water-sensitive coupling. Upon commencing DF2, valve V1 was switched to collect the permeate in tank R2 (1 L Duran Pressure Plus bottle) for recycling into DF1 after the next chain extension. At the end of DF2 a final sample was analysed by LC-MS to verify complete removal of building block debris, and to confirm the high rejection of the 5’-OH- nucleoside-star. Typically, chain extension (i.e., coupling) was undertaken once the combined drop in building block concentration over DF1 and DF2 was >99%. DF2 may be continued until this target is met.Coupling reaction:

[0095] 5’-O-Dimethoxytrityl-2'-O-methyl-N-isobutyryl guanosine 3’-(2-cyanoethyl-N,N- diisopropyl)phosphoramidite, Dmtr-mG(lbu) amidite (3Ga, 1.16 mmol, 1.2 eq. / arm, 9.6 eq. in total) and 4,5-dicyanoimidazole (DCI, 460 mg, 3.87 mmol, 4 eq. / arm, 32 eq. in total) were each placed in separate round bottom flasks. Building block 3Ga was co-evaporated from MeCN (<15 ppm water, 3 x 25 mL), then re-dissolved in MeCN (<30 ppm water, 5 mL) and injected into the synthesiser, followed by the DCI dissolved in MeCN (5 mL). The reaction was monitored by LC; after 5 min the coupling intermediates (1 to 7 arms) appeared to be consumed, but circulation was continued for 20 min to ensure complete reaction. The reaction was then quenched with 2- cyanoethanol (CneOH, 196 mL, 3 eq. / arm, 24 eq. in total) and circulated for 2 min. Camphorsulfonyl oxaziridine (CSC, 1.11 g, 4.83 mmol, 5 eq. / arm, 40 eq. in total) dissolved in MeCN (10 mL) was then injected into the synthesiser and circulation continued for 1 hr. The oligo-star solution was partially purified by permeating MeCN-PhCN 4:1 (200 mL, ~4 DV) to remove the majority of low molecular weight solutes; this solvent was recovered from the previous chain extension cycle.

[0096] The preceding two steps, coupling then detritylation, were repeated seven times using the amounts of phosphoramidite building block, 3, and diafiltration volumes given in Table 1 , but using otherwise identical procedures to those described above.Table 1. Chain extension cycle variables for Example 1. The solvent used for DF1 was always recycled from the previous cycle; the solvent in DF2 was virgin.Solvent Recycling:

[0097] During DF2, permeate 1 (-300 mL) was collected in tank R2. The solvent recycling was performed in a second Minimem filtration system connected to the permeate line from the first stage (see figure 2). The membrane cell M2 (internal volume 10 mL) was charged with a P84 membrane (Siddique et al.) (30 cm2). The filtration was performed with a circulation flow rate of 50 mL / min, pressurising up to 20 bar and setting the stirrer to 800 rpm. The typical permeance was 2.8 Lmhbar (1.4 mL / min in MeCN-PhCN 4:1). The performance of the membrane remained constant for the entire run, from loading to 7-mer-star. The freshly purified solvent was dried using 3 molecular sieve solvent pouches (2 x 5 g, from Oligo Solutions).Global deprotection:

[0098] Once the full-length oligo-star had been prepared in the Nanostar synthesiser and detritylated for a final time, an aliquot of HO-oligo-star solution (3 mL, ca. 4.8 mmol oligo) was withdrawn. The solution of HO-oligo-star was placed in an ACE pressure tube and taken up in concentrated aq. ammonia (3 mL). To this solution was added diethylamine (0.1 mL), then the tube was sealed and stirred overnight at 35 °C. The next day the solution was transferred to a round bottom flask and evaporated. The aqueous residue was co-evaporated from MeCN three times, and the residue was finally triturated with MeCN. The suspension was transferred to a Falcon tube (-30 mL) and centrifuged (5 min, 6000 rpm). The supernatant was removed, then the precipitate washed again and centrifuged twice more with further MeCN. The precipitate of octamer 4 was finally analysed by UHPLC using an IP-RP gradient (see figure 3A). During anongoing synthesis, oligo-stars may be periodically analysed using this technique to assess the cumulative purity of the crude oligo.Table 2. Purity of octamer 4 produced in a Nanostar synthesiser: A, with solvent recycling; and B, without solvent recycling.Comparative Example 1 :5’mUmGmUmUmUmCmUmG3’ octamer, 4, without solvent recycling

[0099] Octamer 4 was synthesised in the same Nanostar synthesiser as above, except that the solvent recycling loop was not used, and only virgin solvent was used for diafiltration. Identical procedures to Example 1 were used with the amounts of phosphoramidite building block, 3, and diafiltration volumes given in Table 3.Loading of 5’-Dmtr-mU 3’-succinate onto 20 kDa PEG-starin Nanostar synthesiser:

[0100] The synthesiser was charged with anhydrous MeCN-PhCN 4:1. PEG-20k(Sar-H)s (2, 1.95 g, 0.10 mmol), dissolved in anhydrous MeCN (10 mL), was injected into the synthesiser, followed by DIPEA (0.34 mL, 1.90 mmol, 20 eq.). After sampling for the initial state, succinate 1ua (1.48 g, 1.90 mmol, 20 eq.) dissolved in anhydrous MeCN (3 mL) was activated with TBTU (0.62 g, 1.90 mmol, 20 eq.) for 30 min. The activated 5’-Dmtr-mU-3’-succinate solution was injected into the synthesizer and reaction was continued for 30 min; a sample was then taken to determine the residual Dmtr-succinate. MeCN-PhCN was then permeated (200 mL, 4 DV) and at the end of DF1 another sample was taken to determine remaining succinate and the Dmtr- nucleoside-star rejection.Detritylation reaction:

[0101] DODT (6 eq. / arm, 48 eq. in total) was injected into the feed tank, followed by MSA (0.2 mL, 0.4 vol%) and, after 10 min, picoline (0.70 mL) was injected to quench the reaction; a sample was taken to determine the intermediate succinate concentration. Fresh anhydrous MeCN-PhCN (< 20 ppm water, 300 mL, ~6 DV) was permeated (DF2), and at the end of DF2 a final sample was taken to quantify any trace of building block debris and to determine nucleoside-star rejection.Coupling reaction:

[0102] Dmtr-mG(lbu) amidite 3Ga (0.93 mmol, 1.2 eq. / arm, 9.6 eq. in total) building block and DCI (370 mg, 3.1 mmol, 4 eq. / arm, 32 eq. in total) were each placed in separate round bottom flasks. The building block was co-evaporated from MeCN (<15 ppm water, 3 x 25 mL), then redissolved in MeCN (<30 ppm water, 5 mL) and injected into the synthesiser, followed by the DCI dissolved in MeCN (5 mL). After 20 min the reaction was then quenched with CneOH (160 mL, 3 eq. / arm, 24 eq. in total), and after a further 2 min CSC (0.89 g, 3.9 mmol, 5 eq. / arm, 40 eq. in total) dissolved in MeCN (10 mL) was added to the synthesiser. After 1 hr the oligo-star was partially purified by permeating MeCN-PhCN (200 mL, 4 DV).

[0103] The preceding two steps, coupling then detritylation, were repeated seven times using the amounts of phosphoramidite building block, 3, and diafiltration volumes given in Table 3, but using otherwise identical procedures to those described above. At octamer-star the oligo was globally deprotected, the same as for Example 1 , and the material was analysed by IP-RP HPLC (see figure 3B).Table 3. Chain extension cycle variables for Comparative Example 1. The solvent used for both DF1 and DF2 was virgin.Example 2:5’mUmGmUmUmUmCmUmGmUmUdTmUfGmUmUmCmAmG3’ 18-mer, 5.

[0104] Octamer 4 was further extended in the Nanostar synthesiser, continuing the sequence out to an 18-mer. The same process was used as described in Example 1, using the building block and solvent amounts listed in Table 4. After the full-length oligo had been reached and detritylated, the sequence was subjected to global deprotection, as described in Example 1 and was analysed by IP-RP HPLC (see figure 4).Table 4. Chain extension cycle variables for Example 2. The solvent used for DF1 was always recycled from the previous cycle; the solvent in DF2 was virgin.Example 3: Synthesis of octamer 5 at large scale, at 40 mM concentration with solvent recycling

[0105] A Nanostar Synthesiser (see Figure 5), with a system volume of 730 mL, was used to test solvent recycling at scale. The synthesiser is a circulation loop consisting of: The reaction vessel, where reagents and fresh solvent are added; a circulation pump (Wanner, diaphragm pump) to generate crossflow and to raise pressure; a heat exchanger with feedback to maintain a constant temperature; the membrane unit, a 1040 spiral wound module (SWM); and a backpressure regulator to control the pressure in the SWM. The synthesis was conducted in neat acetonitrile. Circulation was maintained at 4 Kg. min-1at all times.

[0106] During each synthesis cycle, the last 4 DV (4x 730mL) of DF2 permeate was transferred to the solvent recycling vessel of a second Nanostar Synthesiser fitted with a 1040 SWM of P84 membrane (Siddique et al.), having a system volume of 500 mL, and filled with MeCN; this is the recycling loop. 4 DV of solvent (4 x 730m L) was permeated through the P84 SWM and stored in a purified solvent tank, ready to be used for DF1 of the next synthesis cycle. Halfway through the synthesis of 8-mer 5 (before 5-mer DF1), the solvent in the recycling loop was emptied out and replaced with 500 mL of fresh MeCN.Synthesis of 8-mer 5:

[0107] Loading: 8-arm, 20 kDa methylamine terminated PEG-star [2b, PEG-20k(NMeH)s, MW 20,104 Da, 73.4 g, 3.65 mmol] was dissolved in anhydrous MeCN, then DIPEA (6.6 mL, 0.051 mol, 14 eq.) was added. Separately, 5’-Dmtr-nucleoside-3’-succinate (0.044 mol, 14 eq.) and TBTLI (14.06 g, 0.044 mol, 14 eq.) were added to a flask and then dissolved in anhydrous MeCN. This mixture was then added to the reaction flask containing PEG and DI PEA. After stirring the reaction solution for 30 min, the loading was quenched with hexylamine (4.43 mL, 0.044 mol, 12 eq.) and added into the Nanostar Synthesiser. The membrane unit was then pressurised and 6 DV of MeCN were permeated.

[0108] Detritylation: Before commencing detritylation DODT (21.39 mL, 0.131 mol, 3 eq. / arm) was injected into the synthesiser. Detritylation was then initiated by injecting MSA: A free proton concentration equivalent to 5 vol% TFA was targeted by PAT; for the 1-mer star this equated to 10.22 mL MSA, but rose steadily with each cycle to 20.44 mL at 8-mer. After 20 min, TBA (1.1 eq. / MSA) dissolved in PhCN was injected into the synthesiser. Anhydrous MeCN (< 15 ppm water, 6 DV) was permeated (DF2); after 2 DV (2 x 730 mL), the permeate valve was turned to transfer the remaining 4 DV of permeate (4 x 730mL) to the feed tank of the recycling loop. 4 DV of solvent was passed through the recycling membrane and retained for use in the next cycle.

[0109] Coupling: 5’-O-Dmtr-nucleoside 3’-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.035 mol, 1.2 eq. / arm) and 4,5-dicyanoimidazole (DCI, 10.35 g, 0.88 mol, 3 eq. / arm) were each dissolved in anhydrous MeCN (2 x 50 mL) and placed on double syringe pump. The amidite and activator solutions were injected into the synthesiser via a 120 second delay loop to ensure full activation of the building block. Circulation was continued for 10 min to ensure complete reaction. The reaction was then quenched with CneOH (5.93 mL, 0.088 mol, 3 eq. / arm) and circulated for 2 min. For this 8-mer five oxidations and two sulfur transfers were performed.

[0110] Oxidation: CSO (20.09 g, 0.088 mol, 3 eq. / arm) dissolved in MeCN was injected into the synthesiser and circulation continued for 1 hr.

[0111] Sulfur transfer: PolyOrg Sulfa (POS, 8.55g, 0.044 mol, 1.5eq. / arm) dissolved in MeCN was injected into the synthesiser and circulation continued for 5 min.

[0112] Once the internucleotide linkage had been converted to P(V) DF1 commenced, permeating 6 DV of solvent; recycled solvent from the previous DF2 was used throughout.

[0113] The preceding two steps, detritylation then coupling, with either oxidation or sulfur transfer, were repeated seven times using the same excesses of phosphoramidite building block and other reagents, and the same diafiltration volumes as described above.

[0114] Global deprotection: Once the full-length oligo had been achieved, a sample was withdrawn from the synthesiser and globally deprotected as described in Example 1. The IP-RP chromatogram of octamer oligo 5 prepared in Example 3 is shown in Figure 6, trace C, and the corresponding overall purity = UV purity x MS purity is given in Table 5.Comparative Example 2: Synthesis of octamer 5 at large scale, at 40 mM concentration, without solvent recycling

[0115] Octamer 5 was prepared in a Nanostar Synthesiser, with a system volume of 890 mL, fitted with a 1040 SWM, and starting with PEG-20k(NMeH)8(2b, MW 20,104 Da, 89.5 g, 4.45 mmol] in anhydrous MeCN. All reagent ratios and MeCN diavolumes for DF1 and DF2 were the same as Example 3, except that TBA was replaced by 3-picoline (2 eq. / MSA) and no solvent was recycled. The IP-RP chromatogram of octamer oligo 5 prepared in Comparative Example 2 is shown in Figure 6, trace D, and the corresponding overall purity = UV purity x MS purity is given in Table 5. There are no significant differences between the synthesis with solvent recycling and that without solvent recycling at scale, as demonstrated by comparison of the chromatograms in Figure 6 or the purities in Table 5.Table 5. Purity of octamer 5 produced in a Nanostar synthesiser: A, with solvent recycling; and B, without solvent recycling.

[0116] 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.REFERENCES

[0117] The reader’s attention is drawn to all papers and documents which are filed and / or published previous to this specification in connection with this application and which are open to public inspection with this specification.Lutz J.F., Ouchi M., Liu D.R., “Sequence Controlled Polymers” Science 2013, 341 , 1238149-1 ; Hartmann L., Borner H.G., “Precision Polymers: Monodisperse, Monomer-Sequence-Defined Segments to Target Future Demands of Polymers in Medicine” Adv. Mater. 2009, 21 , p3423; US9914746;Lonnberg, Synthesis of oligonucleotides on a soluble support, 2017;Matsuno, Y.; Shoji, T.; Kim, S.; Chiba, K. Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support. Org. Lett. 2016, 18 (4), 800-803;Bonora, HELP (High Efficiency Liquid Phase) new oligonucleotide synthesis on soluble polymeric support, Nucleic Acids Research, 1990, Vol. 18, 3155; Large scale, liquid phase synthesis of oligonucleotides by the phosphoramidite approach, Nucleic Acids Research, 1993, Vol. 21 , 1213- 1217;Walther, Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels, J. Am. Chem. Soc. 2020, 142, 16610-16621 ;Jensen, Biochemistry. 2018 March 27; 57(12): 1821-1832. doi:10.1021 / acs.biochem.7b00937;Walther, Scalable One-Pot-Liquid-Phase Oligonucleotide Synthesis for Model Network Hydrogels, J. Am. Chem. Soc. 2020, 142, 16610-16621 ;US8664357;US9127123;US10239996;EP3347402;P.R.J. Gaffney, J. F. Kim, I.B. Valtcheva, G.D. Williams, M.S. Anson, A.M. Buswell, A.G. Livingston, Liquid-Phase Synthesis of 2’-Methyl-RNA on a Homostar Support through Organic- Solvent Nanofiltration, Chem. Eur. J., 2015, 21 , 9535-9543;J.F. Kim, P.R.J. Gaffney, I.B. Valtcheva, G. Williams, A.M. Buswell, M.S. Anson, A.G. Livingston, Organic Solvent Nanofiltration (OSN): A New Technology Platform for Liquid-Phase Oligonucleotide Synthesis (LPOS), Org. Process Res. Dev. 2016, 20, 1439-1452;So Su, Peeva L.G., Tate E.W., Leatherbarrow R.J., Livingston A.G. “Organic Solvent Nanofiltration - A New Paradigm in Peptide Synthesis” Org. Process. Res. & Dev. 14 (2010) pp. 1313 - 132;Yeo J, Peeva L, Chung S, Gaffney P, Kim D, Luciani C, Tsukanov S, Siebert K, Kopach M, Albericio F, Livingston A “Liquid Phase Peptide Synthesis by One Pot Nanostar Sieving (PEPSTAR)”; Angew. Chem. Int. Ed. 2021, 60, pp7786- 7795;PEG and derivatives of PEG have also been synthesised using membrane separation, as described by Dong R., Liu R., Gaffney P.R.J., Schaepertoens M., Marchetti P., Williams C.M., Chen R. and Livingston A.G. “Sequence-defined multifunctional polyethers via liquid-phase synthesis with molecular sieving” Nature Chemistry (2019) 11 pp.136-145;WO2016188835;Peacock H. et al. J. Am. Chem. Soc. (2011), 133, 9200;V Kungurtsev, J Laakkonen, AG Molina, P Virta - Eur. J. Org. Chem, 2013, 6687-6693;Baran et al. Unlocking P(V): Reagents for chiral phosphorothioate synthesis, Science, Vol 361, 1234-1238. DOI: 10.1126 / science.aau3369;H. Siddique, E. Rundquist, Y. Bhole, L.G. Peeva, A.G. Livingston, Mixed matrix membranes for organic solvent nanofiltration, Journal of Membrane Science, 452 (2014) 354-366;H. Siddique, Y. Bhole, L.G. Peeva, and A.G. Livingston, “Pore Preserving Crosslinkers for Polymide OSN Membranes”, J.Mem.Sci (2014) 465 pp.138-150;Xianglin Shi, Xuan Zhou, William F. Kiesman, Wuming Yan, Hong Jiang, Firoz D. Antia, Jing Yang, Yannick A. Fillon and Li Xiao, Development of Kilogram-Scale Convergent Liquid-Phase Synthesis of Oligonucleotides, The Journal of Organic Chemistry 2022 87 (4), 2087-2110 DOI: 10.1021 / acs.joc.1c01756;Jill Caswell, Stephanie Paul, Darren Gray, Joshua Brooks, Wenjie Ye, Thomas S. Moody, Roumen Radinov, and Lubomir Nechev, Convergent Biocatalytic Mediated Synthesis of siRNA, ACS Chemical Biology 2023 18 (10), 2183-2187. DOI: 10.1021 / acschembio.3c00071; and WO2018011067.

Claims

CLAIMS1. a liquid-phase process for preparing a first compound being a defined monomer sequence polymer, the process comprising:(i) growing a first compound by performing one or more chain extension cycles, each chain extension cycle comprising: a deprotection step to expose a chain extension site of a growing first compound; and a coupling step to attach a monomeric or oligomeric building block to the exposed chain extension site;(ii) performing, at one or more instances in a chain extension cycle, primary membrane filtration to isolate the growing first compound in the retentate; and(iii) subjecting at least a portion of solvent permeated from primary membrane filtration to secondary membrane filtration to increase the purity of the solvent, the purified solvent being used in a subsequent chain extension cycle.

2. The process of claim 1 , wherein the first compound is an oligonucleotide, a peptide, a peptide nucleic acid, a polyether or an oligosaccharide.

3. The process of claim 1 or 2, wherein the first compound is an oligonucleotide.

4. The process of claim 1 , 2 or 3, wherein at one or two instances, primary membrane filtration is conducted to isolate the growing first compound in the retentate.

5. The process of any one of the preceding claims, wherein primary membrane filtration is conducted by membrane diafiltration.

6. The process of any one of the preceding claims, wherein the membrane used in primary membrane filtration is a polymeric membrane, a ceramic membrane, or a mixed polymeric / inorganic membrane.

7. The process of any one of the preceding claims, wherein the membrane used in primary membrane filtration is a crosslinked polybenzimidazole membrane (e.g., an integrally skinned, asymmetric, crosslinked polybenzimidazole membrane) or a polyetheretherketone membrane.

8. The process of any one of the preceding claims, wherein the solvent permeated from primary membrane filtration is analysed to determine whether it should be subjected to secondary membrane filtration.

9. The process of any one of the preceding claims, wherein the solvent permeated from primary membrane filtration comprises: i) an initial portion; and ii) a subsequent portion, with the initial portion permeated from primary membrane filtration before the subsequent portion.

10. The process of claim 9, wherein the initial portion is not subjected to secondary membrane filtration.

11. The process of any one of the preceding claims, wherein the solvent permeated from primary membrane filtration is treated with one or more quenching agents.

12. The process of claim 11 , wherein the solvent is treated with one or more quenching agents capable of reacting with at least one of the reagents and / or by-products to increase its molecular weight by >50%.

13. The process of claim 11 or 12, wherein the quenching agent is selected from tribenzylamine (TBA), picoline, pyridine, lutidine, collidine, piperidine, 2,4,6-trimethylbenzoic acid, MSA, palmitic acid and dodecylbenzene sulfonic acid.

14. The process of any one of the preceding claims, wherein the membrane used in secondary membrane filtration has a MWCO that is 5-185 g mol-1greater than the molecular weight of the solvent.

15. The process of any one of the preceding claims, wherein the membrane used in secondary membrane filtration is a polymeric membrane, a ceramic membrane, or a mixed polymeric / inorganic membrane.

16. The process of any one of the preceding claims, wherein the membrane used in secondary membrane filtration is a crosslinked polyimide membrane.

17. The process of claim 16, wherein the crosslinked polyimide membrane is a crosslinked polyimide mixed matrix membrane.

18. The process of claim 17, wherein the crosslinked polyimide mixed matrix membrane comprises polyimide chains crosslinked with siloxane networks.

19. The process of any one of the preceding claims, wherein the solvent is purified by separating it from: a) uncoupled building blocks; b) coupling agents; c) a reagent used to cleave a protecting group from the growing first compound to expose a chain extension site; d) a protecting group cleaved from the growing first compound to expose a chain extension site; e) quenching agent adducts (e.g., adducts formed of quenching agents and reagents and / or byproducts formed in each chain extension cycle); and / or f) quenching agents.

20. The process of any one of the preceding claims, wherein the growing first compound is attached to a soluble synthesis support.

21. The process of claim 20, wherein the soluble synthesis support is a polymer (e.g., a star polymer), a dendrimer, a dendron, a hyperbranched polymer, or an organic / inorganic material, including nanoparticles, fullerenes and 2-D materials such as graphene and boron nitride.

22. The process of any one of the preceding claims, wherein the process is performed in a closed loop reactor.

23. A first compound obtained, directly obtained or obtainable by the process of any one of the preceding claims.

Images