Defined molecular weight pegylation agents

Abstract

The invention relates to a composition comprising compounds having the formula (I). In formula (I), n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the substrate, and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate. At least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n.

Description

[0001] DEFINED MOLECULAR WEIGHT PEGYLATION AGENTS

[0002] The project leading to this application has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 786398).

[0003] Field of the invention

[0004] This invention relates to the new highly defined molecular weight PEGylation agents, useful for the covalent attachment of one or more poly(ethylene glycol) (PEG) units to a substrate and specifically addresses the challenges associated with PEGylation of higher molecular weight therapeutics and proteins.

[0005] Background to the invention

[0006] PEGylation is a technique that describes the attachment of PEG compounds to a substrate, and is widely used in the pharmaceutical industry for increasing the bioactivity of a drug and decreasing its immunogenicity. These effects are a consequence of the extremely hydrophilic character of PEG, thereby resulting in a high hydrodynamic radius and high solubility in aqueous environments, as well as the “stealth effect”. This phenomenon provides a protective barrier for polymer-modified entities by making them less accessible to blood components such as antibodies or enzymes.

[0007] The term “monodispersity” is commonly used to describe commercially available PEGylation agents, despite the fact that all currently used PEGylation agents show some degree of dispersity. The reason for this is that PEG is still conventionally synthesised through the anionic ring opening polymerisation of ethylene oxide, which results in a Gaussian distribution of PEG of different chain length. The disperse nature of these PEGylation agents results in batch-to-batch variations and difficulties in purification and characterisation of the PEGylated therapeutics, as a result of compromised chromatographic behaviour and unclear mass spectrometric analysis. These problems become more pronounced both with the increasing molecular weight of the PEG and with an increasing number of PEGamers (species with varying numbers of pendant PEG molecules) or positional isomers incorporated in the sample. Despite the availability of advanced analytical methods, the exact binding site of the PEG in a PEGylated protein often remains unclear, or PEG-related impurities remain unidentified.

[0008] As a result, there has been substantial interest in producing dispersity-free PEGs. One approach to generating dispersity-free PEGs is to chromatographically purify disperse PEG, but the limits of this approach arise at molecular weights where it becomes difficult to separate Egnfrom Egn+i or Egn-i , this in turn leads to significant losses in yield. Based on available materials and using advanced chromatography this limit appears around Eg28 (about 1000 g mol1). Another method of producing PEGylation agents involves using solid-phase techniques. Both chromatographic and solid-phase techniques suffer from limited scalability or impeded reaction kinetics, making them economically unfeasible, especially for higher molecular weight PEGs (> 3 kDa).

[0009] It is therefore an aim of this invention to provide defined high molecular weight (Mw) PEGylation agents instead of the currently used disperse derivatives to overcome these problems. The present invention seeks to address these deficiencies by providing defined molecular weight PEGylation agents that can be produced with unprecedented chain length purity, without the use of chromatographic purification, on a large multi-gram scale and at high yield, with a substantial impact on the overall purity and quality of the PEGylated substrates, especially therapeutics. This invention also provides a method of mapping the PEGylation sites of a protein, which is not possible with commercially available disperse PEGylation agents. A further aim of this invention is the provision of pharmaceutical compositions having compounds conjugated with such defined molecular weight PEGylation agents such that the compounds, including (but not limited to) proteins, DNA and RNA-based pharmaceutical compounds, also have a defined molecular weight. of the invention

[0010] In a first aspect there is provided a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0011] In a second aspect there is provided a composition comprising one or more compounds having the formula (II): wherein n is an integer of 65 or more, Y comprises a substrate, and R is selected from a protecting group or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n.

[0012] In a third aspect there is provided a composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n.

[0013] In a fourth aspect there is provided a process for the preparation of a composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n; the process comprising the steps of:

[0014] (i) synthesising a backbone portion of the compounds by performing one or more sequential monomeric coupling reactions in a first organic solvent;

[0015] (ii) between each coupling reaction, separating a product of the one or more sequential coupling reactions from at least one second compound, which is a reaction by-product of the synthesis of the product and / or an excess of a reagent used for the synthesis of the product, and

[0016] (iii) optionally capping a terminal hydroxyl group of the product with a protecting group; thereby to provide a composition comprising compounds having formula (la); wherein during step (ii) the product of the one or more sequential coupling reactions and at least one second compound are dissolved in a second organic solvent and are separated by a process of diafiltration using a membrane that is stable in the organic solvent and which provides a rejection for the product which is greater than the rejection for the second compound.

[0017] In a fifth aspect, there is provided a process of preparing a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach a poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n; the process comprising: reacting a composition comprising compounds of formula (la) with one or more precursor compounds that are reactive to functionalise a terminal hydroxy group of compounds of formula (la): wherein n is as defined for formula (I) and R is selected from H, or a protecting group, and wherein coupling group X is derived from the one or more precursor compounds, thereby to provide the composition comprising compounds having the formula (I).

[0018] In a sixth aspect there is provided a process of preparing a composition comprising compounds of formula (II), wherein n is an integer of 65 or more, Y comprises a substrate, and R is a protecting group or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n; the process comprising: reacting one or more substrates with a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the substrate; R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; thereby to provide the composition comprising compounds of formula (II).

[0019] There is also provided a composition obtainable by the process of any one of the fourth, fifth, or sixth aspects.

[0020] There is also provided the composition according to any one of the second aspect, or pharmaceutically acceptable salt thereof, for use in medicine or therapy.

[0021] In a seventh aspect, there is provided a process for identifying a location on a protein where a PEG- containing group is attached, comprising the steps of:

[0022] (i) attaching the PEG-containing group to the protein in a selective or random manner by a reaction between a composition and the substrate to provide a PEGylated protein, wherein the composition comprises compounds with a PEG-containing group having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the protein; and R is selected from or a protecting group; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; and wherein the compounds having the formula (I) form a covalent bond with the protein thereby to attach the PEG-containing group, wherein the covalent bond is stable towards a digestion procedure;

[0023] (ii) carrying out the digestion procedure on the PEGylated protein to obtain a mixture of peptides derived from the PEGylated protein, wherein the mixture of peptides comprises a PEGylated peptide comprising the PEG-containing group bonded thereto;

[0024] (iii) identifying the location on the protein where the PEG-containing group is attached.

[0025] Brief Description of the Drawings

[0026] Figure 1 shows the membrane-purification based synthesis of defined mPEG-OH4966. a, loading (i) of nanostar (Hub-tribromide) with DmtrO-Eg28-OH, followed by deprotection (ii) and diafiltration using crosslinked PBI membranes to remove excess Eg28 diol and other reaction debris (iii). b, chain elongation cycle consisting of the coupling of the DmtrO-Eg28-OTs to the free nanostar-PEG alcohol (Chain Extension, iv), removal of the protecting group (Deprotection, v) and Diafiltration (vi). Repeating this cycle three times results in the nanostar-Eg112-OH species, c, the terminal hydroxyls are then methylated (viii) and subsequently the PEG chains are cleaved from the nanostar (ix) to obtain defined mPEG-OH with a molecular weight of 4966 Da. d, shows the structures of the Nanostar, Leaving Group, and Protecting Group shown in a, b and c.

[0027] Figure 2. shows the synthesis of the active PEGylation agent mPEG-succinimidyl propionate (mPEG-SP, (2)). a, Synthetic route from the starting material mPEG-OH4966 (3) using Ethyl 3- bromopropionate to reach the corresponding ester (5), which was then hydrolysed with NaOH to obtain the acid (6), that was finally reacted with N-hydroxysuccinimide to provide the desired active ester (2). b, UPLC / ELSD traces of the resultant defined mPEG-SPA (top) and the commercially available disperse derivative (bottom) (15 min 29-95 % B gradient with A: H2O + 5 mM NH4OAC, B: MeCN / MeOH 4:1 ). The defined sample shows 4 peaks (I2: traces of starting material mPEG-OH4966 , I2: hydrolysed product / carboxylic acid, P: product (mPEG-SPA), I3: NHS-breakdown product). For the disperse sample peak 11 is broad and peaks I2 and P are not separated, c, For the defined sample the underlying ESI+traces is coincident with the expected mass spectrum, e.g. (shown) the m / z series 1301.5, 1044.8, 873.7, 751.6, 659.8 corresponds to a Mwof 5135.11 , which is the mPEG-SPA ([M+4 NH4]4+, M+5 NH4]5+, M+6 NH4]6+). For the disperse sample the determination of a precise molecular weight is not possible.

[0028] Figure 3. Comparative study of the random PEGylation and characterisation of BSA using defined and disperse PEG 5 kDa. a, Conventional PEGylation: uses disperse PEGylation agent. Random PEGylation of the primary amine groups of BSA, using defined or disperse 5 kDa linear mPEG-SP (i). Size-exclusion chromatography to isolate the mono-PEG BSA isomers (ii). Digestion (iii): enzymatic digestion of the mono-PEG BSA results in a mixture of PEGylated and un-PEGylated peptide fragments. The different peptide / PEG-peptide species are then separated via RP-UPLC (iv). In case of the disperse PEG the resulting data cannot be interpreted, b, In this work a defined, 5 kDA PEGylation agent is used for the first time to randomly PEGylate BSA. After steps 1-4 in-line ESI-MS allows for extraction of the masses for individual PEG-peptide fragments. The masses of the PEGylated peptide fragments can be calculated and (equivalent to a standard peptide mapping) these can then be compared to an online digestion database to identify the PEGylated fragments.

[0029] Figure 4. Yields and purity data for the synthesis of defined mPEG-OH4966. a, Yields for every cycle of the membrane- and chromatography-based syntheses of Hub(Egn2-OH)3.* The first step in the chromatography-based synthesis needed two separate purification batches due to column loading constraints, hence yields are given for each batch, b, MALDI-TOF-MS of mPEG-OH4966 synthesised via the membrane-based approach in this work (bottom) and commercially available disperse mPEG- OH (top), c, Functionalisation reaction to obtain UV-active derivative 2. d, UV-chromatograms (254 nm, 15 cm Cis column, 30 min 20-95 % B, A: H2O + 5 mM NF OAc, B MeCN: MeOH 4:1) of 5 kDa MeO- Egn2-p-methoxybenzoate derived from membrane-based synthesis (black trace) and chromatography-based synthesis (red trace); Peak P: MeO-Eg -p-methoxybenzoate (4), Peak 11 : - Eg28 impurity (= MeO-Eg84-p-methoxybenzoate), Peak I2: +Eg28 impurity (= MeO-Egi4o-p- methoxy benzoate), Peaks I3+I4: unidentified impurities, e, summary of yields and purities for chromatography-based and membrane-based synthesis of MeO-Egii2-OH.

[0030] Figure 5: Mapping of PEGylated proteins: a: Size-exclusion chromatogram of PEGylated BSA (top) and the MALDI-TOF spectra of the fraction of remaining native BSA (yellow) and the mono-PEG BSA (blue), b: TIC trace of UPLC chromatogram of tryptic digest samples of mono-PEG BSA, PEGylated with a disperse (bottom, red) and defined (top, green) 5 kDa mPEG-succinimidyl propionate. Examples are shown for the ESI+traces extracted from the chromatograms. For the defined species (RT 20.59 min), the m / z series (1045.8, 899.0, 788.9, 703.2, 634.7) corresponds to a total mass of 6166.68 Da. Subtracting the PEG mass (5021 .03 Da) identified the PEGylated fragment (1145.65 Da) as pos. 236-245 (AS Sequence -AWSVARLSQK-). The disperse derivative does not allow for extraction of exact masses, c: Summary of the seven identified PEGylated fragments in mono- PEGylated BSA.

[0031] Figure 6 shows an example of the MALDI-TOF spectrum of a defined molecular weight MeO-Egn2-OH sample, which may be used to calculate the chain length purity.

[0032] Detailed Description

[0033] Definitions

[0034] PEGylation is the process of covalently attaching one or more poly(ethylene glycol) (PEG) units to therapeutics. This technique is widely used in the pharmaceutical industry to enhance a drug’s bioactivity and reduce its immunogenicity. A “PEGylation agent” as identified herein, refers to a compound that upon reaction with a substrate covalently attaches polyethylene glycol (PEG) units to a substrate (e.g. a therapeutic) during the process of PEGylation.

[0035] The expression “coupling group” as used herein is intended to refer to a functional group on the PEGylation agents described herein that are reactive with a chemical group on a substrate molecule. A non-limiting example is succinimidyl propionate which is reactive towards the primary amine groups within a protein.

[0036] The expressions “capping group” or “protecting group” as used herein are intended to refer to a functional group on the compounds (e.g. of Formula (I), (II) or (la)) described herein that is not reactive with a chemical group on the substrate molecule, or reactive with reagents under the conditions used for PEGylation. In addition, the functional group is stable towards chemical and / or enzymatic degradation.

[0037] The term "monomer" or "monomeric unit" is used herein to refer to a polymer building block which has a defined molecular structure and which can be reacted to form a part of a polymer. It will be understood that these terms refer to the minimum repeating unit.

[0038] The term "synthesis support" is used herein to relate to a chemical entity that allows the first compound to stay in solution during the reaction and diafiltration step, and optionally to provide an increased molecular bulk to enhance membrane separation. The synthesis support may be a branch point molecule, or a polymer, dendrimer, dendron, hyperbranched polymer, or organic / inorganic materials, including nanoparticles, fullerenes and 2-D materials such as graphene and boron nitride.

[0039] The term "branch point molecule" is used herein to refer to a polyfunctional organic molecular "hub", having at least 2 reactive moieties, and the ability to covalently bind to a terminal of an initial monomeric unit.

[0040] PEGylation agent

[0041] In a first aspect, there is provided a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n. The composition is useful for attaching a poly(ethylene glycol) (PEG) group to a substrate (i.e. as a PEGylation agent). The benefits of PEGylation are due to the extremely hydrophilic nature of PEG, which results in a high hydrodynamic radius and high solubility in aqueous environments. Additionally, PEGylation provides a “stealth effect,” creating a protective barrier for polymer-modified entities, making them less accessible to blood components such as antibodies or enzymes. This can help to increase the circulation time of the drug in the body, reduce dosing frequency, and improve patient compliance.

[0042] In addition, the PEGylation agent, when attached to a therapeutic molecule, can "mask" the agent from the host's immune system, reducing immunogenicity and antigenicity. It also increases the hydrodynamic size of the molecule, which prolongs its circulatory time by reducing renal clearance. Moreover, PEGylation agents can provide water solubility to hydrophobic drugs and proteins.

[0043] R may be a protecting group selected from a cyclic or acyclic alkyl group, wherein the alkyl group can be substituted or unsubstituted, linear, branched, or cyclic, containing 1 to 6 carbon atoms, or a silyl protecting group. Preferably, R may be a straight or branched C1-4 alkyl group, or benzyl, or trityl. More preferably, R is a methyl (Me) or ethyl (Et) group, most preferably Me. When R is a protecting group it acts as a capping group which is inert and this avoids any post-modification reactions during the PEGylation process.

[0044] R may be a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate. When R is a second coupling group, the PEGylation agent is “multi-reactive” and can react to form covalent bonds with two substrates, one on each terminus of the PEGylation agent. These “multi-reactive” PEGylation agents can be used to link two or more reactive units to each other, or can be used as linkers in antibody-drug conjugates, for example. When R is a second coupling group, it may therefore be as defined herein for the (first) coupling group, X. It will be appreciated that the second coupling group may be the same or different to the first coupling group. Similarly, the second substrate may be the same or different to the first substrate.

[0045] The integer n may be selected depending on the intended efficacy of the effects provided by PEGylation for the substrate to be PEGylated. A higher value for the integer n corresponds to a PEG chain with a longer chain length, and is expected to provide a stronger masking effect to the substrate. However, a longer chain length may also mean a higher chance of interaction with the active site of a therapeutic substrate, which may in turn lead to lower bioactivity. Accordingly, depending on the substrate to be PEGylated, the ideal PEG chain length is therefore an interplay of sufficient size for lower renal clearance and high masking effect, but also not too high to avoid interaction with active site. The integer n may be an integer that is 70 or more, 75 or more, 80 or more, 85 or more, preferably 90 or more, or more preferably 100 or more. The integer n may be 3000 or less, 2000 or less, or more preferably 1500 or less. The integer n may be from 64 to 3000, from 70 to 2000, from 75 to 2000, or preferably from 80 to 1500, or more preferably from 100 to 1500.

[0046] A higher integer, n, results in a compound of formula (I) (and compound of formula (II) after PEGylation has been carried out) of a higher molecular weight. Generally, without wishing to be bound by theory, a higher integer, n, may allow for a molecular weight of the PEGylation agent that is sufficiently high to avoid renal clearance.

[0047] Defined molecular weight of the PEG group

[0048] The compositions comprising compounds having the formula (I) (and formula (II) and formula (la)) contain PEG groups that possess unprecedented mono-dispersity, having at least 90% of the compounds with the same number of ethylene glycol units, n.

[0049] Using the defined molecular weight PEG compounds described herein instead of the currently used disperse derivatives has a substantial impact on the overall purity and quality of PEGylated therapeutics. Furthermore, therapeutics PEGylated with defined PEG could potentially exhibit an improved biodistribution profile compared to the disperse derivatives, because the correlation curve of PEG half-life in blood and the PEG molecular weight is particularly steep between 0 and 40 kDa, which is in the range of most reported PEGylation agents. Without wishing to be bound by theory, this suggests that using defined PEGylation agents could result in a decreased volume of distribution, which might have a significant impact on the dosing controllability, and the efficiency of the PEGylated therapeutic, and safety for patients.

[0050] Furthermore, such a low dispersity has, until the disclosure of the present invention, only been demonstrated in low molecular weight PEG compounds. However, the compositions described herein provide, for the first time, compounds having PEG groups where the variation in the ethylene glycol chain length is significantly reduced. Such low dispersity is made possible by the processes described herein for preparing compositions comprising compounds of formula (I), (II) and the intermediate compound of formula (la). Conventional methods of producing low dispersity PEGs were limited to low molecular weight PEG compounds.

[0051] The composition may comprise at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the compounds of formula (I) having the same number of ethylene glycol units, n.

[0052] The percentage of compounds of formula (I) (and formulas (II) and (la)) having the same number of ethylene glycol units, n, can be measured by MALDI-TOF-MS. The percentage number of ethylene glycol units, n, corresponds to the chain length purity of the PEG group having n Eg units. The chain length purity can be derived through MADI-TOF-MS measurements.

[0053] For the present disclosure, a 1 mg / ml solution of the PEG sample in acetonitrile is prepared, as well as a saturated solution of DCTB (frans-2-[3-(4-fe / Y-Butylphenyl)-2-methyl-2- propenylidene]malononitrile) in 50 mM NaOAc (sodium acetate) in H2O / acetonitrile (1 :1). Both solutions are mixed in a 1 :1 ratio and 0.8 pL of the mixture is spotted onto a sample plate and left to dry. The sample is measured at a suitable laser power (in terms of lowest possible signal-to-noise ratio, preferably between 40 and 70 % laser power) with 1000 shots. Each peak in the derived spectrum can be assigned to a specific compound with n Eg units. The chain length purity of a compound A with the desired chain length can be calculated through:

[0054] With PA being the chain length purity of compound A, IA being the intensity of the peak corresponding to compound A, and lx, ly, lz the intensities of the peaks corresponding to the chain length impurities, which all have the same chemical structure as compound A, but a different number of Eg units compared to compound A.

[0055] An example of the MALDI-TOF spectrum of a defined molecular weight MeO-Egn2-OH sample is shown in Figure 6 and the corresponding calculated chain length purity can be found in the table below.

[0056] In addition or alternatively, the compounds of formula (I) may preferably be manufactured by performing one or more sequential monomeric coupling reactions with intervening purification steps comprising diafiltration using a membrane. An example of such a method is provided hereinbelow as part of Example 1 . In particular, by synthesising the backbone of the PEG group of the compounds of formula (I) this allows for the production of a defined molecular weight PEG group of an unprecedented level of chain length purity as defined herein. Preferably, the compounds of formula (I) may be manufactured by a method that does not involve chromatography (e.g. involving purification of the PEG compounds having a defined molecular weight).

[0057] Expressed another way, the composition may produce a single peak for the compounds of formula (I) when analysed by HPLC, gel permeation chromatography, and / or a predominant single peak in mass spectrometry that corresponds to the molecular ion [M+] and any associated adduct peaks. There may be additional minor peaks present in the mass spectrum from e.g. sodium or potassium adducts. However, there will still be one predominant peak that corresponds to the molecular ion that indicates a PEG compound of high chain length purity.

[0058] The first coupling group and second coupling group (if present) may be selected depending on the substrate to be PEGylated as defined herein. The first and second coupling group is reactive with the substrate thereby to attach the poly(ethylene glycol) group of the compound of formula (I) to the substrate.

[0059] It will be appreciated that when the second coupling group is present, the PEGylation agent will be “multi-reactive” in the sense that it can be used to link two or more substrate molecules to each other. In this situation the substrate molecules may be the same, or they may be different. For example, the PEGylation agent may act as a linker for antibody-drug conjugates. In the following, any discussion of features for the first coupling group equally applies to the second coupling group, if present. Hereinafter any reference to the “coupling group” may refer to the first and / or second coupling group, unless otherwise specified.

[0060] Preferably, the coupling group(s) is(are) selected such that a mixture of different positional isomers is avoided as much as possible in the PEGylated species. This is because different positional isomers might have different biological activities. The most active positional isomer, i.e., the one that provides the best therapeutic effect, is preferred. The most active positional isomer can be maximised reached by selecting a coupling group having the right PEGylation chemistry with the intended substrate.

[0061] The substrate to which the coupling group is reactive may preferably be a biotherapeutic. Preferably, the substrate may be selected from: a peptide, a protein, a lipid, a nucleic acid, DNA, RNA or chemically modified DNA or RNA, a pharmaceutically active molecule, a liposome, or a nanoparticle. The protein may be a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine.

[0062] The first and / or second coupling group may be branched or linear and comprise a functional group selected from an ester, an aldehyde, a maleimide, an azide, an amine, a thiol, a hydroxyl, a tosylate, or a monosaccharide.

[0063] The coupling group may be a group that is reactive such that it undergoes glycoPEGylation, in which the PEGylation agent is attached to a glycosylated site of a protein using an enzyme. For example, the coupling group may be the monosaccharide sialic acid, which is commonly used for glycoPEGylation.

[0064] Preferably, the ester is an ester of N-hydroxysuccinimide. The use of such an ester allows for random PEGylation at primary amine sites. For example, the compounds of formula (I) may have the formula: wherein R is C1-4 alkyl, L is -(CH2)m- or -OC(CH2)m-, and m is from 1 to 10. Preferably, wherein R is Me and L is -CH2-.

[0065] The coupling group may more preferably be an ester selected from: succinimidyl succinate, succinimidyl butanoate, or succinimidyl carbonate, succinimidyl propionate, most preferably succinimidyl propionate.

[0066] The compounds of formula (I) may be methoxy-PEG succinimidyl succinate, preferably having the formula:

[0067] The compounds of formula (I) may be methoxy-PEG succinimidyl butanoate, preferably having the formula:

[0068] The compounds of formula (I) may be methoxy-PEG succinimidyl carbonate, preferably having the formula:

[0069] The coupling group may comprise a linear aldehyde, preferably propionaldehyde. This may allow for site selective PEGylation at terminal amine of proteins. In particular, the compounds of formula (I) may have the formula: wherein R is C1-4 alkyl, L is -(CH2)m- or -OC(CH2)m- and m is from 1 to 10, preferably wherein R is Me and L is -CH2-.

[0070] The coupling group may comprise a linear or branched maleimide. This may allow for thiol selective PEGylation. For example, the compounds of formula (I) may have the formula: wherein R is C1-4 alkyl, and L is an alkyl or alkoxy group.

[0071] Branched coupling groups

[0072] The first (and / or second coupling group if present) may preferably be branched. In this situation the compounds of formula (I) may comprise two or more PEG groups having n repeating ethylene glycol units, and wherein at one end the two or more PEG groups are each attached to a branch of the coupling group(s), and at the other end the two or more PEG groups either have a terminal group selected from or a protecting group (which may be as defined herein for R), or may be attached to the other coupling group. In this way, the branched coupling group has a number of branches (or “arms”) and each of those arms are attached to a PEG group, providing a PEGylation agent that is capable of attaching multiple PEG groups per substrate during a PEGylation reaction.

[0073] The use of a branched coupling group leads to multiple PEG groups per substrate molecule in the PEGylated compounds. Without wishing to be bound by theory, when the substrate is a therapeutic it is believed that a composition comprising branched PEG structures allows for a PEGylated therapeutic with a higher stability towards proteolytic digestion compared to the same linear PEG structures. This could be explained by a larger steric hindrance and thus the prevention of macromolecules (e.g. proteolytic enzymes, antibodies) approaching the substrate. This may also contribute to longer blood residence time, and substrates that are more stable.

[0074] It will be appreciated by the skilled person that, depending on whether the second coupling group is present, and whether the first and / or second coupling group is branched, that various PEGylated species involving various ratios of PEG groups and substrate molecules could be prepared, wherein the first and second coupling group could be the same or different, and the first and second substrates could be the same or different. The first and / or second coupling group may comprise a 2-arm branched group with each arm attached to one PEG group. Preferably, the first and / or second coupling group comprises a branched group derived from an amino acid or carbohydrate compound having two more reactive functional groups. Examples of amino acids include lysine, histidine, or arginine, preferably lysine.

[0075] The coupling group may be branched and derived from lysine, the compounds of formula (I) having the formula:

[0076] Wherein group A comprises an ester, an aldehyde, a maleimide, an azide, an amine, a thiol, a hydroxyl, a tosylate, or a monosaccharide. Preferably, group A is an ester of N-hydroxysuccinimide, particularly selected from: succinimidyl propionate, succinimidyl succinate, succinimidyl butanoate, or succinimidyl carbonate, preferably succinimidyl propionate. Alternatively, the aldehyde is a maleimide.

[0077] The first coupling group may comprise a branched maleimide and the compounds of formula (I) have the structure: wherein X is a linear alkyl or alkoxy group.

[0078] The coupling group may be a branched N-hydroxysuccinimide ester having two functional groups each attached to a PEG group. For example, the compounds of formula (I) may have the structure:

[0079] This branched PEG structure may be produced by adding two linear PEGs to both amino residues of a lysine molecule, which is then converted to an NHS ester. It will be appreciated that other branched structures could be produced. In particular, 3 or 4-arm branched structures could be produced. For instance, a coupling group based on threitol with a 4-arm branched structure, wherein each of the four arms has a PEG group attached to it. This may be advantageous for applications where multiple PEG groups are desired for attaching to a substrate.

[0080] By way of illustration, below is a 4-arm structure based on threitol, wherein R1-R4each comprises a PEG group and a one or more of R1-R4comprises a group reactive with a substrate:

[0081] The first and / or second coupling group may comprise two or more functional groups being reactive with the substrate to attach a poly(ethylene glycol) (PEG) group of the compound of formula (I) to the substrate. For example, the coupling group(s) may be derived from an inversed lysine structure with e.g., one PEG group and two or more groups each reactive with a substrate. In this way, the PEGylated composition may comprise one PEG group and multiple substrates per molecule.

[0082] In summary, in order to achieve a balance of pharmacodynamic and pharmacokinetic behaviour within a PEGylated therapeutic, the following points have to be taken into account for the composition comprising the compounds of formula (I) (the PEGylation agent):

[0083] (i) A higher molecular weight of the PEGylated substrate (higher integer, n) can increase the agent’s circulation time in the body, enhancing its therapeutic effect.

[0084] (ii) Mono-PEGylation is favoured over multi-PEGylation, as the chance of interaction with active sites is minimised.

[0085] (iii) A mixture of different positional isomers should be avoided, the most active positional isomer is preferred and might be reached by choosing the right PEGylation chemistry.

[0086] (iv) Branched versions of PEG might exhibit a higher in vivo stability than the linear analogues.

[0087] PEGylated substrate(s)

[0088] In a second aspect, there is provided a composition comprising one or more compounds having the formula (II): wherein n is an integer of 65 or more, Y comprises a substrate, and R is a protecting group, or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n. The compounds of formula (II) correspond thus to a PEGylated substrate (or substrates where R comprises a second substrate) that has undergone PEGylation with the composition of compounds according to formula (I) (the PEGylation agents). The compounds according to formula (I) may be referred to herein as “PEGylation agents” for brevity.

[0089] It will be appreciated that the substrate may have multiple PEG groups attached to it (multi-PEGylation), or just a single PEG group attached to it (mono-PEGylation). Preferably, a single PEG group is attached to the substrate (i.e., as a result of mono-PEGylation). Mono-PEGylation is favoured over multi-PEGylation, as the chance of interaction with active sites is minimised. Whether a PEGylation agent undergoes mono- or multi- PEGylation depends on the coupling groups that are present and the substrate(s) to be PEGylated.

[0090] The group Y may comprise a linking group that is bonded to the substrate and bonded to the poly(ethylene glycol) group of the compounds of formula (II). The linking group may be derived from any of the coupling groups mentioned hereinabove for the first and / or second coupling groups. For instance, selected from an ester, an aldehyde, a maleimide, an azide, an amine, a thiol, a hydroxyl, a tosylate, or a monosaccharide, preferably an ester. If a branched coupling group was used in the PEGylation agent then the linking group may be derived from an amino acid or carbohydrate compound having two more reactive functional groups, such as lysine, histidine, or arginine, preferably lysine.

[0091] The compounds of formula (II) may be derived from any of the compounds of formula (I) according to the first aspect described herein, including the various embodiments identified in respect of the same.

[0092] For the compounds of formula (II), R may be a protecting group selected from a cyclic or acyclic alkyl group, wherein the alkyl group can be substituted or unsubstituted, linear, branched, or cyclic, containing 1 to 6 carbon atoms, or a silyl protecting group. Preferably, R may be a straight or branched C1-4 alkyl group, or benzyl, or trityl. More preferably, R is a methyl (Me) or ethyl (Et) group, most preferably Me. When R is a protecting group it acts as a capping group which is inert and this avoids any post-modification reactions during the PEGylation process. R may most preferably be Me.

[0093] The integer n may be an integer that is 70 or more, 75 or more, 80 or more, 85 or more, preferably 90 or more, or more preferably 100 or more. The integer n may be 3000 or less, 2000 or less, or more preferably 1500 or less. The integer n may be from 64 to 3000, from 70 to 2000, from 75 to 2000, or preferably from 80 to 1500, or more preferably from 100 to 1500.

[0094] The substrate may preferably be a biotherapeutic. For instance, the substrate may be selected from: a peptide, a protein, a lipid, a nucleic acid, DNA, RNA or chemically modified DNA or RNA, a pharmaceutically active molecule, a liposome, or a nanoparticle.

[0095] Examples of proteins include, but are not limited to, a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine. In particular, the protein may be a recombinant protein selected from recombinant growth hormone (GH) receptor antagonist, or recombinant erythropoietin (EPO) beta. The peptide may preferably be an engineered di-peptide. Examples of cytokines includes but are not limited to interferon a 2b, interferon a 2a, or interferon a 1 a.

[0096] Examples of therapeutic enzymes include but are not limited to adenosine deaminase, asparaginase, recombinant uricase protein, l-asparaginase, recombinant phenylalanine ammonia lyase, or pegunigalsidase alfa-iwxj (recombinant human a-galactosidase-a enzyme). Preferably, the immunoglobulin fragment is a Fab fragment. The Fab fragment may preferably be an antitumor necrosis factor.

[0097] Examples of a biologically active factor include but are not limited to a granulocyte-colony stimulating factor, a recombinant granulocyte-colony stimulating factor, a recombinant version of coagulation factor IX, a recombinant version of antihemophilic factor, or a granulocyte-colony stimulating factor combined with immunoglobulin G4. An example of a hormone is a human growth hormone. Examples of a nucleic acid include an RNA molecule or an oligonucleotide. The RNA molecule may include but is not limited to a nucleotide aptamer, or an avacincaptad pegol.

[0098] Examples of liposomes include but are not limited to: N-(carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero3-phosphoethanolamine sodium salt (MPEG-DSPE); or fully hydrogenated soy phosphatidylcholine (HSPC); and cholesterol.

[0099] Examples of a pharmaceutically active molecule include but are not limited to dodecyl alcohol or an opiod antagonist.

[0100] The nanoparticle may preferably be a lipid nanoparticle.

[0101] The compositions comprising compounds having the formula (II) contain PEG groups that possess unprecedented mono-dispersity, having at least 90% of the compounds with the same number of ethylene glycol units, n. This provides a new class of defined PEGylated therapeutics with the potential for better controllability, increasing the overall purity, reducing batch-to-batch variations, and is expected to result in enhanced biodistribution behaviour.

[0102] The composition may comprise at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the compounds of formula (II) having the same number of ethylene glycol units, n. This percentage may be derived from measurements using MALDI-TOF-MS according to the method herein.

[0103] The percentage of compounds of formula (II) having the same number of ethylene glycol units, n, can be measured using the same methods identified herein for measuring the percentage of compounds of formula (I) having the same number of ethylene glycol units, n. In addition or alternatively, the compounds of formula (II) may preferably be manufactured by performing one or more sequential monomeric coupling reactions with intervening purification steps comprising diafiltration using a membrane. An example of such a method is provided hereinbelow as part of Example 1 . In particular, by synthesising the backbone of the PEG group of the compounds of formula (II) this allows forthe production of a defined molecular weight PEG group of an unprecedented level of chain length purity as defined herein. Preferably, the compounds of formula (II) may be manufactured by a method that does not involve chromatography (e.g., involving purification of the PEG compounds having a defined molecular weight).

[0104] Expressed another way, the composition may produce a single peak for the compounds of formula (II) when analysed by HPLC, gel permeation chromatography, and / or a predominant single peak in mass spectrometry that corresponds to the molecular ion [M+] and any associated adduct peaks.

[0105] Defined molecular weight PEG

[0106] In a third aspect there is provided a composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n.

[0107] The R group and the integer, n, may be as defined according to the compounds of formula (I) or (II) above. Preferably, R is Me. The integer n may be 70 or more, 75 or more, 80 or more, 85 or more, preferably 90 or more, or more preferably 100 or more. The integer n may be 3000 or less, 2000 or less, or more preferably 1500 or less. The integer n may be from 64 to 3000, from 70 to 2000, from 75 to 2000, or preferably from 80 to 1500, or more preferably from 100 to 1500.

[0108] The percentage of compounds of formula (la) having the same number of ethylene glycol units, n, can be measured by MALDI-TOF-MS according to the method herein.

[0109] Method of making PEG compounds with high chain length purity

[0110] There is provided a process for the preparation of a composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n; the process comprising the steps of:

[0111] (i) synthesising a backbone portion of the compounds by performing one or more sequential monomeric coupling reactions in a first organic solvent;

[0112] (ii) between each coupling reaction, separating a product of the one or more sequential coupling reactions from at least one second compound, which is a reaction by-product of the synthesis of the product and / or an excess of a reagent used for the synthesis of the product, and

[0113] (iii) optionally capping a terminal hydroxyl group of the product with a protecting group; thereby to provide a composition comprising compounds having formula (la); wherein during step (ii) the product of the one or more sequential coupling reactions and at least one second compound are dissolved in a second organic solvent and are separated by a process of diafiltration using a membrane that is stable in the organic solvent and which provides a rejection for the product which is greater than the rejection for the second compound.

[0114] Having regard to step (i), it will be understood that the "first compound" may be a defined monomer sequence polymer containing only two monomeric units, in which case the polymer is synthesised by the coupling of an initial monomeric unit with a first additional monomeric unit. It will also be understood that the "first compound" may be a defined monomer sequence polymer containing three monomeric units, in which case the polymer is synthesised by first coupling an initial monomeric unit with a first additional monomeric unit, then coupling the first additional monomeric unit with a second additional monomeric unit. Accordingly, "first compounds" containing 4, 5 and 6 monomeric units are respectively synthesised by sequential coupling of the third, fourth and fifth additional monomeric units to the second, third and fourth additional monomeric units respectively.

[0115] Still having regard to step (i), any suitable method of synthesising the first compound that is known in the art may be utilised. In particular, the synthesis of the first compound may involve one or more coupling and deprotection reactions. In such cases, step (i) comprises reacting an initial monomeric unit with an excess of a first additional monomeric unit in which one of the reactive terminals has been protected using a protecting group. Once the initial monomeric unit and the first additional monomeric unit have been coupled, the protecting group is cleaved to expose the reactive terminal of the first additional monomeric unit, which is then ready for coupling with a second additional monomeric unit.

[0116] The one or more sequential monomeric coupling reactions may each comprise the steps of: a) reacting a starting material with an excess of an additional monomeric unit, the additional monomeric unit having one of its reactive terminal protected by a protecting group, and b) removing the protecting group so as to expose the reactive terminal such that it is ready for reaction with a subsequent additional monomeric unit, wherein the starting material is either an initial monomeric unit having at least one of its reactive terminals protected, or the polymeric product of the one or more sequential monomeric coupling reactions.

[0117] In such embodiments, step (ii) may be performed after step a) (in order to remove excess unreacted additional monomers and optionally other small reaction debris) and after step b) (in order to remove the cleaved protecting group, by-products of the protecting group removal and one or more deprotection reagents). It will be understood that the term "reagent" appearing in step (ii) encompasses both reactants and catalysts.

[0118] All of the monomeric units used in the one or more sequential monomeric coupling reactions of step (i) have identical ethylene glycol backbone moieties. The monomeric units that are coupled during the one or more sequential monomeric coupling reactions may each independently comprise a 1-mer alkylene glycol backbone moiety, or a 3-7-mer poly(alkylene glycol) backbone moiety. Irrespective of the number of ethylene glycol repeat units, it will be understood that the term "monomeric unit" refers to a minimum repeating unit. Exemplary monomeric units useful in step (i) are tetragol-based units (tetraethylene glycol-based). It will be understood that hydroxyl-protected forms may be more suitable for used as part of the present invention. Pentagol-based (pentaethylene glycol-based) monomeric units may also be used.

[0119] During synthesis of the first compound, the product of the one or more sequential monomeric coupling reactions may be covalently attached to a synthesis support by an initial monomeric unit. The initial monomeric unit may be directly attached to the synthesis support, or indirectly attached thereto via a linker moiety (such as a dicarboxylic acid moiety). The synthesis support may be a branch point molecule, or a polymer, dendrimer, dendron, hyperbranched polymer, or organic / inorganic nanoparticle. Once the backbone portion of the desired defined monomer sequence polymer has been synthesised, the synthesis support is cleaved from the initial monomeric unit and separated therefrom.

[0120] During synthesis of the first compound, the product may be covalently attached to a synthesis support by an initial monomeric unit.

[0121] When used as a synthesis support, suitable polymers include polycondensation matrices or polymerisation matrices containing heteroatom functions. Such heteroatom functions may contain oxygen, nitrogen, or can contain more than one heteroatom, such as acid amide groups. Examples of polymeric synthesis supports include polyalkylene glycols including polyethylene glycol, polycaprolactone, polyethylene glycol esterified with citric acid, copolymers of polyethyleneglycol and succinic acid, of vinylpyrrolidone and acrylic acid or beta-hydroxy-ethylacrylate, or of acrylamide and vinylactetate. When used as a synthesis support, suitable dendrimers include poly(amidoamine), also known as PAMAM dendrimers; phosphorous dendrimers; polylysine dendrimers; and polypropylenimine (PPI) dendrimers which can have surface functionalities including -OH, -NH2, -PEG, and COOH groups. When used as a synthesis support, suitable nanoparticles may be prepared from SiO2, TiO2, or other organic or inorganic materials including fullerenes or 2-D materials such as graphene.

[0122] It is particularly preferred that the synthesis support is a branch point molecule (i.e. a polyfunctional molecule) having two or more reactive moieties capable of covalently binding to the initial monomeric unit. Chemistries suitable for covalently binding the initial monomeric unit to the branch point molecule will be readily apparent to a person of skill in the art, and include amide, ester, ether and silyl ether couplings.

[0123] The branch point molecule may have any of the structures shown below:

[0124] The initial monomeric unit may be reacted in excess with a synthesis support, allowing for the synthesis of a conjugate that can be purified from the excess initial monomeric unit. Subsequently, the first compound may be obtained through a succession of coupling / deprotection reactions using one or more additional monomeric units. The choice of the cleavage reaction used to detach the backbone portion from the branch point molecule can be performed at the end of the synthetic strategy or at any stage, according to convenience.

[0125] The first solvent (i.e. that used in step (i)) and the second solvent (i.e. that used in step (ii)) may be the same or different. Suitably, the solvent used for the diafi Itration should maintain the polymer and / or the functionalised polymer in solution. Exemplary solvents include aromatics, ketones, glycols, chlorinated solvents, esters, ethers, amines, nitriles, aldehydes, phenols, amides, carboxylic acids, alcohols, furans, and dipolar aprotic solvents, and mixtures thereof and with water. Specific examples of solvents include toluene, xylene, benzene, styrene, anisole, chlorobenzene, dichlorobenzene, chloroform, dichloromethane, dichloroethane, methyl acetate, ethyl acetate, butyl acetate, methyl ether ketone (MEK), methyl isobutyl ketone (MIBK), acetone, ethylene glycols, ethanol, methanol, propanol, butanol, dimethoxyethane, methyl tert-butyl ether (MTBE), adiponitrile, N,N-dimethylformamide, dimethylsulfoxide, N,N-dimethylacetamide, dioxane, nitromethane, nitrobenzene, pyridine, carbon disulfide, tetrahydrofuran, methyl-tetrahydrofuran, N-methyl pyrrolidone, N-ethyl pyrrolidone, acetonitrile, and mixtures thereof and with water.

[0126] Suitable membranes for use in the invention include polymeric and ceramic membranes, and mixed polymeric / inorganic membranes. Membrane rejection Ri is a common term known by those skilled in the art and is defined as: eq. (1) where CP,i = concentration of species i in the permeate, permeate being the liquid which has passed through the membrane, and CR,i = 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

[0127] (defined monomer sequence polymer OR conjugate)"5,R(at least one reaction by-product or reagent)-

[0128] Membrane processes are well known in the art of separation science, and can be applied to a range of separations of species of varying molecular weights in liquid and gas phases (see for example "Membrane Technology" in Kirk Othmer Encyclopedia of Chemical Technology, 4th Edition 1993, Vol 16, pages 135-193). Nanofiltration is a membrane process utilising membranes whose pores are in the range 0.5-5 nm, and which have MW cutoffs of 200-3,000 Daltons. Recently new classes of membranes have been developed which are stable in even the most difficult solvents as reported in P. Marchetti, M.F.Jimenez-Solomon, G.Szekely, A. G. Livingston Chem. Rev., (2014), Vol 114, pages 10735 - 10806. These may be polymeric membranes or ceramic membranes, or mixed inorganic / organic membranes. Some of these membranes are suitable for Organic Solvent Nanofiltration (OSN).

[0129] The membranes useful as part of the present invention may be formed from any polymeric or ceramic material which provides a separating layer capable of preferentially separating the first molecule or conjugate from at least one reaction by-product or reagent. Preferably the membrane is formed from or comprises a material selected from polymeric materials suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF), polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyester, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole, polybenzimidazole, polyetheretherketone (PEEK) and mixtures thereof. The membranes can be made by any technique known in the art, including sintering, stretching, track etching, template leaching, interfacial polymerisation or phase inversion. More preferably, membranes may be crosslinked or treated so as to improve their stability in the reaction solvents. PCT / GB2007 / 050218, PCT / GB2011 / 051361 and PCT / GB2015 / 050179 describe membranes which may be suitable for use in the present invention.

[0130] The membrane is preferably a composite material and the nonporous, selectively permeable layer thereof is formed from or comprises a material selected from modified polysiloxane based elastomers including polydimethylsiloxane (PDMS) based elastomers, ethylene-propylene diene (EPDM) based elastomers, polynorbornene based elastomers, polyoctenamer based elastomers, polyurethane based elastomers, butadiene and nitrile butadiene rubber based elastomers, natural rubber, butyl rubber based elastomers, polychloroprene (Neoprene) based elastomers, epichlorohydrin elastomers, polyacrylate elastomers, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF) based elastomers, polyetherblock amides (PEBAX), polyurethane elastomers, crosslinked polyether, polyamides, polyesters, polyketones, formed by interfacial polymerisation, and mixtures thereof.

[0131] The membrane may be prepared from an inorganic material (e.g. silicon carbide, silicon oxide, zirconium oxide, titanium oxide, or zeolites), using any technique known to those skilled in the art such as sintering, leaching or sol-gel processes.

[0132] The membrane may comprise a polymer membrane with dispersed organic or inorganic matrices in the form of powdered solids present at amounts up to 20wt% of the polymer membrane. Carbon molecular sieve matrices can be prepared by pyrolysis of any suitable material as described in US Pat. No. 6,585,802. Zeolites as described in US Pat. No. 6,755,900 may also be used as an inorganic matrix. Metal oxides, such as titanium dioxide, zinc oxide and silicon dioxide may be used, for example the materials available from Evonik Industries (Germany) under their Aerosol and AdNano trademarks. Mixed metal oxides such as mixtures of cerium, zirconium, and magnesium oxides may be used. Graphene, graphene oxide, metal organic frameworks (MOFs), boron nitride, carbon nanotubes may be used. Preferred matrices will be particles less than 1 .0 micron in diameter, preferably less than 0.1 microns in diameter, and preferably less than 0.01 microns in diameter.

[0133] Any suitable alkylation agent may be used with the process. Particularly preferred are haloalkyl compounds. Preferably, R is Me and the alkylation agent is an iodomethane.

[0134] Preferably, the product of the one or more sequential coupling reactions is obtained at a product yield of 13% or more, 15% or more, 20% or more, preferably 35% or more, most preferably 45% or more by weight.

[0135] It will be appreciated that the composition comprising compounds of formula (la) may be according to any combination of the embodiments described in relation to the third aspect.

[0136] There is also provided a composition obtainable by the process for the preparation of a composition comprising compounds having the formula (la) described hereinabove. As mentioned above, this process provides, for the first time, a high molecular weight PEG compound having a defined molecular weight at an unprecedented level of chain length purity.

[0137] Method of making the PEGylation agent

[0138] There is provided a process of preparing a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach a poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n; the process comprising: reacting a composition comprising compounds of formula (la) with one or more precursor compounds that are reactive to functionalise a terminal hydroxy group of compounds of formula (la): wherein n is as defined for formula (I) and R is selected from H, or a protecting group, and wherein coupling group X is derived from the one or more precursor compounds, thereby to provide the composition comprising compounds having the formula (I).

[0139] The composition comprising compounds of formula (I) may be according to any combination of the embodiments described in relation to the first aspect. Similarly, the composition comprising compounds of formula (la) may be according to any combination of the embodiments described in relation to the third aspect.

[0140] The process may comprise a first step of preparing a composition comprising compounds of formula (la) according to the processes described herein for the preparation of a composition comprising compounds having the formula (la).

[0141] Any suitable method of reacting the composition comprising compounds of formula (la) with the precursor compound(s) to functionalise the terminal hydroxy group of compounds of formula (la) that is known in the art may be utilised. The method used may depend on chemistry of the precursor compound(s) and coupling group X that are chosen. A non-limiting example of the precursor compound is an alkylating agent such as ethyl 3-bromopropionate, which can be reacted with compounds of formula (la) to form an ether compound, which is then further reacted by hydrolysing the ester group and reacting with N-hydroxy succinimide to provide compounds of formula (I) wherein the coupling group X is succimmidyl propionate. There is also provided a composition obtainable by the process of preparing a composition comprising compounds having the formula (I).

[0142] Method of PEGylatina a substrate

[0143] There is provided a process of preparing a composition comprising compounds of formula (II), wherein n is an integer of 65 or more, Y comprises a substrate, R is selected from a protecting group, or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n; the process comprising: reacting one or more substrates with a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the substrate; R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; thereby to provide the composition comprising compounds of formula (II).

[0144] Any suitable method of reacting one or more substrates with a composition comprising compounds having the formula (I) to PEGylate the substrate(s) that is known in the art may be utilised. A nonlimiting example is the small-scale batch PEGylation procedure described in a paper published by Fee, C. J. & Van Alstine, J. M. titled “Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behaviour of PEGylated Proteins” in Bioconjugate Chemistry 15, 1304-1313 (2004), which is explicitly incorporated herein by reference.

[0145] In the process, the composition comprising compounds of formula (I) may be according to any combination of embodiments described in relation to the first aspect. In the process, the composition comprising compounds of formula (II) may be according to any combination of embodiments described in relation to the second aspect.

[0146] The process may preferably comprise purifying the composition comprising compounds of formula (II), e.g. by separating the product from unreacted compounds of formula (I) or substrate compounds that do not contain any PEG groups.

[0147] There is also provided a composition obtainable by the process of preparing a composition comprising compounds of formula (II). Medical use

[0148] The composition comprising compounds of formula (II) described herein, or the pharmaceutically acceptable salt thereof, may be for use in medicine or therapy. The comprising compounds of formula (II) described herein, or the pharmaceutically acceptable salts thereof, may be for use in treating a disease selected from an immunodeficiency disease, cancer, viral infection of the liver, a myeloproliferative disorder, neutropenia, a hormonal disorder, an age-related macular degeneration, anaemia, a skin condition, arthritis, and gout.

[0149] A composition containing compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is adenosine deaminase, may be used for treating severe combined immunodeficiency disease. Adagen™, sold by Enzon, is an example of this substrate in commercial use.

[0150] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is an Asparaginase, may be used for treating acute lymphoblastic leukaemia. Oncaspar™, also sold by Enzon, serves as another example of this type of substrate in commercial use.

[0151] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is Interferon a 2b, may be used for treating chronic hepatitis C. Pegintron™ by Schering- Plough is a commercial example of a substrate in this category.

[0152] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is Interferon a 2a, may be used for treating myeloproliferative disorder. Pegasys™ by Roche is an example of a commercially used substrate of this type.

[0153] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is a G-CSF, may be used for treating neutropenia. Neulasta™ by Amgen is a commercial example of this kind of substrate.

[0154] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is a recombinant GH receptor antagonist, may be used for treating acromegaly. Somavert™ by Pfizer is an example of a commercially used substrate in this category.

[0155] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is a 28 nucleotide aptamer, may be used for treating wet age-related macular degeneration. Macugen™ by Eyetech Pharmaceuticals is a commercial example of a substrate in this group. A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is a recombinant epotein beta, may be used for treating anemia. Mircera™ by Roche is a commercial example of a substrate of this type.

[0156] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is an antitumor necrosis factor, may be used for treating plaque psoriasis. Cimzia™ by UCB is an example of a commercially used substrate in this category.

[0157] A composition comprising compounds of formula (II) or their pharmaceutically acceptable salts, where the substrate is a recombinant uricase protein, may be used for treating gout. Krystexxa™ by Horizon Therapeutics is a commercial example of a substrate in this group.

[0158] Method of mapping proteins

[0159] There is provided a process for identifying a location on a protein where a PEG-containing group is attached, comprising the steps of:

[0160] (i) attaching the PEG-containing group to the protein in a selective or random manner by a reaction between a composition and the substrate to provide a PEGylated protein, wherein the composition comprises compounds with a PEG-containing group having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the protein; and R is a protecting group; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; and wherein the compounds having the formula (I) form a covalent bond with the protein thereby to attach the PEG-containing group, wherein the covalent bond is stable towards a digestion procedure;

[0161] (ii) carrying out the digestion procedure on the PEGylated protein to obtain a mixture of peptides derived from the PEGylated protein, wherein the mixture of peptides comprises PEGylated peptide (s) comprising the PEG-containing group bonded thereto;

[0162] (iii) identifying the location(s) on the protein where the PEG-containing group(s) is / are attached.

[0163] Compared to the conventional approaches for identifying PEGylation sites in PEGylated proteins, this procedure is less time-consuming, requires less analytical equipment and is more accurate and reliable.

[0164] The steps of attachment of the functionalised defined molecular-weight poly(ethylene glycol) (PEG) to the protein, optional purification of the PEGylated protein, and enzymatic digestion steps are routinely used in the pharmaceutical industry when PEGylation sites have to be identified and verified. However, since the currently available PEGylation agents are disperse (despite most being labelled as “monodisperse”), it is not possible to use the mass spectra of the PEGylated proteins to identify the PEGylated sites (e.g. by UPLC-MS analysis). Instead, in conventional methods the mixture of peptides and PEGylated peptides has to be separated chromatographically and fractions containing the PEGylated fragments have to be collected and then be subjected to further, laborious analyses (MALDI / TOF, Edman sequencing of each peptide sequence). In some cases more than one digestion method is necessary. The method described herein of identifying PEGylated sites of a protein is particularly advantageous as it does not require Edman sequencing of the PEGylated fragments, which can simply be identified through their distinct mass spectrum.

[0165] Optionally, after enzymatic digestion the PEGylated protein may be separated from unreacted PEG and protein purified using any suitable methods known in the art, such as by using chromatography (e.g. size-exclusion chromatography) before step (iii). Depending on the desired product, the mono-PEGylated version or another PEGamer (di-, tri- PEGylated etc.) can be isolated. The advantage of this approach is not only easier chromatographic separation (because narrower chromatographic peaks allow for better separation), but also that PEGylated fragments can be identified through mass spectrometry, e.g. UPLC-MS. In this way, separation and identification of PEGylated fragments can be achieved in a single step using a method such as UPLC-MS.

[0166] Step (iii) of identifying the location on the protein where the PEG-containing group is attached involves peptide-mapping. Step (iii) may preferably comprise (iii-a) determining the molecular mass of the PEGylated peptide; and (iii-b) identifying the PEGylated peptide by comparing its molecular mass absent the PEG-containing group to a known value.

[0167] Preferably, step (iii) may comprise determining the molecular mass of the PEGylated peptide by mass spectroscopy, most preferably a combination of liquid chromatography and mass spectrometry (UPLC / MS). This allows for analysis of the digestion mixture and identification of the locations on the protein where PEGylation has occurred.

[0168] It will be appreciated that the above method is described for one PEGylated peptide, but expressly included within the invention are methods involving digestion mixtures comprising any number of PEGylated peptides. Preferably, the above method is performed for a plurality of PEGylated peptides. This provides for a peptide mapping procedure that identifies location(s) on the protein where the plurality of PEG-containing groups are attached.

[0169] The composition comprising compounds with a PEG-containing group having the formula (I) may be according to any combination of the embodiment described herein in relation to the first aspect.

[0170] Methods of treatment

[0171] There is also provided a method of treating a disease or condition in a subject, comprising administering the composition comprising compounds of formula (II), or a pharmaceutically acceptable salt thereof, to the subject in need thereof. The substrates and diseases or conditions may be those identified hereinabove.

[0172] The aspects provided herein are also described in the following clauses:

[0173] 1 . A composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0174] 2. A composition according to clause 1 , wherein R is a protecting group selected from a cyclic or acyclic alkyl group, wherein the alkyl group can be substituted or unsubstituted, linear, branched, or cyclic, containing 1 to 6 carbon atoms, , or a silyl protecting group, preferably selected from Me, benzyl, or trityl, most preferably Me.

[0175] 3. A composition according to clause 1 or clause 2, wherein n is 80 or more.

[0176] 4. A composition according to any one of the preceding clauses, wherein n is 100 or more.

[0177] 5. A composition according to any one of the preceding clauses, wherein n is 2000 or less.

[0178] 6. A composition according to any one of the preceding clauses, wherein n is 1500 or less.

[0179] 7. A composition according to any one of the preceding clauses, wherein at least 91 % of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0180] 8. A composition according to any one of the preceding clauses, wherein at least 92% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0181] 9. A composition according to any one of the preceding clauses, wherein at least 93% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0182] 10. A composition according to any one of the preceding clauses, wherein at least 94% of the compounds of formula (I) have the same number of ethylene glycol units, n. 11. A composition according to any one of the preceding clauses, wherein at least 95% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0183] 12. A composition according to any one of the preceding clauses, wherein at least 99% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0184] 13. A composition according to any one of the preceding clauses, wherein the percentage of compounds of formula (I) having the same number of ethylene glycol units, n, is measured by MALDI- TOF-MS according to the method described herein.

[0185] 18. A composition according to any one of the preceding clauses, the compounds of formula (I) manufactured by performing one or more sequential monomeric coupling reactions with intervening purification steps comprising diafiltration using a membrane.

[0186] 19. A composition according to any one of the preceding clauses, the compounds of formula (I) manufactured by a method that does not involve chromatography.

[0187] 20. A composition according to any one of the preceding clauses, wherein the composition produces a single peak for the compounds of formula (I) when analysed by HPLC, gel permeation chromatography and / or a predominant single peak in mass spectrometry that corresponds to the molecular ion [M+] and any associated adduct peaks.

[0188] 21. A composition according to any one of the preceding clauses, wherein the substrate is a biotherapeutic.

[0189] 22. A composition according to any one of the preceding clauses, wherein the substrate is selected from: a peptide, a protein, a lipid, a nucleic acid, DNA, RNA or chemically modified DNA or RNA, a pharmaceutically active molecule, a liposome, or a nanoparticle.

[0190] 23. A composition according to clause 22, wherein the protein is a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine.

[0191] 24. A composition according to any one of the preceding clauses, wherein the first and / or second coupling group is branched or linear and comprises a functional group selected from an ester, an aldehyde, a maleimide, an azide, an amine, a thiol, a hydroxyl, a tosylate, or a monosaccharide.

[0192] 25. A composition according to any one of the preceding clauses, wherein the ester is an ester of N-hydroxysuccinimide.

[0193] 26. A composition according to clause 25, the compounds having the formula: wherein R is C1-4 alkyl, L is -(CH2)m- or -OC(CH2)m-, and m is from 1 to 10.

[0194] 27. A composition according to clause 26, wherein R is Me and L is -CH2-.

[0195] 28. A composition according to clause 26 or clause 27, wherein the coupling group X is an ester selected from: succinimidyl succinate, succinimidyl butanoate, or succinimidyl carbonate, succinimidyl propionate.

[0196] 29. A composition according to clause 26, the compounds having the formula:

[0197] 30. A composition according to clause 26, the compounds having the formula:

[0198] 31 . A composition according to clause 26, the compounds having the formula:

[0199] 32. A composition according to any one of the preceding clauses, wherein the coupling group X comprises a linear aldehyde.

[0200] 33. A composition according to clause 32, the compounds having the formula: wherein R is C1-4 alkyl, L is -(CH2)m- or -OC(CH2)m- and m is from 1 to 10.

[0201] 34. A composition according to clause 33, wherein R is Me and L is -CH2-.

[0202] 35. A composition according to any one of the preceding clauses, wherein the coupling group X comprises a linear maleimide.

[0203] 36. A composition according to clause 35, the compounds having the formula:

[0204] Wherein R is C1-4 alkyl, and L is an alkyl or alkoxy group.

[0205] 37. A composition according to any one of the preceding clauses, wherein the first and / or second coupling group is branched, the compounds of formula (I) comprise two or more PEG groups having n repeating ethylene glycol units, wherein at one end the two or more PEG groups are each attached to a branch of the coupling group(s), and at the other end the two or more PEG groups have a terminal protecting group.

[0206] 38. A composition according to clause 37, wherein the first and / or second coupling group comprises a 2-arm branched group with each arm attached to one PEG group.

[0207] 39. A composition according to clause 37 or 38, wherein the first and / or second coupling group comprises a branched group derived from an amino acid or carbohydrate compound having two more reactive functional groups.

[0208] 40. A composition according to clause 39, wherein the first and / or second coupling group comprises a branched group derived from an amino acid selected from lysine, histidine, or arginine.

[0209] 41. A composition according to clause 40, wherein the first and / or second coupling group comprises a branched group derived from lysine.

[0210] 42. A composition according to clause 41 , wherein the branched group is derived from lysine and the compounds have the formula: wherein A comprises an ester, an aldehyde, or a maleimide according to any of clauses.

[0211] 43. A composition according to clause 42, wherein the ester is an ester of N-hydroxysuccinimide.

[0212] 44. A composition according to clause 43, wherein the ester is selected from: methoxy-PEG succinimidyl succinate, methoxy-PEG butanoate, or methoxy-PEG succinimidyl carbonate.

[0213] 44. A composition according to clause 42, wherein the aldehyde is a maleimide.

[0214] 45. A composition according to any one of the preceding clauses, wherein the coupling group X comprises a branched maleimide and the compounds have the formula: wherein X is a linear alkyl or alkoxy group.

[0215] 46. A composition according to any one of the preceding clauses, wherein the coupling group X is a branched N-hydroxysuccinimide ester and the compounds have the formula:

[0216] 47. A composition according to any one of the preceding clauses, wherein the first and / or second coupling group comprises two or more functional groups being reactive with the substrate to attach a poly(ethylene glycol) (PEG) group of the compound to the substrate.

[0217] 48. A composition comprising one or more compounds having the formula (II): wherein n is an integer of 65 or more, Y comprises a substrate, and R is selected from a protecting group, or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n.

[0218] 49. A composition according to clause 48, wherein Y comprises a linking group that is bonded to the substrate and bonded to the poly(ethylene glycol) group of formula (II).

[0219] 50. A composition according to clause 48 or 49, wherein the compounds of formula (II) are derived from compounds of formula (I) according to any one of clauses 1-47.

[0220] 51 . A composition according to any one of clauses 48-50, wherein R is Me.

[0221] 52. A composition according to any one of clauses 48-51 , wherein n is 80 or more.

[0222] 53. A composition according to any one of clauses 48-52, wherein n is 100 or more.

[0223] 54. A composition according to any one of clauses 48-53, wherein n is 2000 or less.

[0224] 55. A composition according to any one of clauses 48-54, wherein n is 1500 or less.

[0225] 55a. A composition according to any one of clauses 48-55, wherein the percentage of compounds of formula (II) having the same number of ethylene glycol units, n, is measured by MALDI-TOF-MS according to the method described herein.

[0226] 56. A composition according to any one of clauses 48-55 or 55a, wherein the substrate is a biotherapeutic.

[0227] 57. A composition according to any one of clauses 48-56, wherein the substrate is selected from: a peptide, a protein, a lipid, a nucleic acid, DNA, RNA or chemically modified DNA or RNA, a pharmaceutically active molecule, a liposome, or a nanoparticle.

[0228] 58. A composition according to clause 57, wherein the protein is a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine.

[0229] 59. A composition according to clause 58, wherein the protein is a recombinant protein selected from recombinant growth hormone (GH) receptor antagonist, or recombinant erythropoietin (EPO) beta.

[0230] 60. A composition according to clause 58, wherein the peptide is an engineered di-peptide.

[0231] 61. A composition according to clause 58, wherein the cytokine is selected from interferon a 2b, interferon a 2a, or interferon a 1 a. 62. A composition according to clause 58, wherein the therapeutic enzyme is selected from adenosine deaminase, asparaginase, recombinant uricase protein, l-asparaginase, recombinant phenylalanine ammonia lyase, or pegunigalsidase alfa-iwxj (recombinant human a-galactosidase-a enzyme).

[0232] 63. A composition according to clause 62, wherein the immunoglobulin fragment is a Fab fragment.

[0233] 64. A composition according to clause 63, wherein the Fab fragment is an antitumor necrosis factor.

[0234] 65. A composition according to clause 58, wherein the biologically active factor is selected from: granulocyte-colony stimulating factor, recombinant granulocyte-colony stimulating factor, recombinant version of coagulation factor IX, recombinant version of antihemophilic factor, or granulocyte-colony stimulating factor combined with immunoglobulin G4.

[0235] 66. A composition according to clause 58, wherein the hormone is a human growth hormone.

[0236] 67. A composition according to clause 58, wherein the nucleic acid is an RNA molecule or an oligonucleotide.

[0237] 68. A composition according to clause 67, wherein the RNA molecule is a nucleotide aptamer, or an avacincaptad pegol.

[0238] 69. A composition according to clause 58, wherein the pharmaceutical is dodecyl alcohol or an opiod antagonist.

[0239] 70. A composition according to clause 58, wherein the nanoparticle is a lipid nanoparticle.

[0240] 71 . A composition according to any one of clauses 48-70, wherein at least about 91 %, 92%, 93%, 94%, 95%, or 99% of the compounds of formula (I) have the same number of ethylene glycol units, n.

[0241] 72. A composition according to any one of clauses 48-71 , wherein the percentage of compounds of formula (I) having the same number of ethylene glycol units, n, is measured by MALDI-TOF-MS according to the method described herein.

[0242] 74. A composition according to any one of clauses 48-73, the poly(ethylene glycol) (PEG) group of compounds of formula (II) manufactured by performing one or more sequential monomeric coupling reactions with intervening purification steps comprising diafiltration using a membrane.

[0243] 75. A composition according to any one of clauses 48-74, the poly(ethylene glycol) (PEG) group of compounds of formula (II) manufactured by a method that does not involve chromatography. 76. A composition according to any one of clauses 48-75, wherein the composition produces a single peak for the compounds of formula (I) when analysed by HPLC, gel permeation chromatography and / or a predominant single peak in mass spectrometry that corresponds to the molecular ion [M+] and any associated adduct peaks.

[0244] 77. A composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n.

[0245] 78. A composition according to clause 77, wherein R is Me.

[0246] 79. A composition according to clause 77 or clause 78, wherein n is 80 or more, or 100 or more.

[0247] 81. A composition according to any one of clauses 77-80, wherein n is 2000 or less, or 1500 or less.

[0248] 81 a. A composition according to any one of clauses 77-81 , wherein the percentage of compounds of formula (la) having the same number of ethylene glycol units, n, is measured by MALDI-TOF-MS according to the method described herein.

[0249] 82. A process for the preparation of a composition comprising compounds having the formula (la): wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n; the process comprising the steps of:

[0250] (i) synthesising a backbone portion of the compounds by performing one or more sequential monomeric coupling reactions in a first organic solvent;

[0251] (ii) between each coupling reaction, separating a product of the one or more sequential coupling reactions from at least one second compound, which is a reaction by-product of the synthesis of the product and / or an excess of a reagent used for the synthesis of the product, and

[0252] (iii) optionally capping a terminal hydroxyl group of the product with a protecting group; thereby to provide a composition comprising compounds having formula (la); wherein during step (ii) the product of the one or more sequential coupling reactions and at least one second compound are dissolved in a second organic solvent and are separated by a process of diafiltration using a membrane that is stable in the organic solvent and which provides a rejection for the product which is greater than the rejection for the second compound.

[0253] 83. The process of clause 82, wherein the one or more sequential monomeric coupling reactions each comprise the steps of: a) reacting a starting material with an excess of an additional monomeric unit, the additional monomeric unit having one of its reactive terminal protected by a protecting group, and b) removing the protecting group so as to expose the reactive terminal such that it is ready for reaction with a subsequent additional monomeric unit, wherein the starting material is either an initial monomeric unit having at least one of its reactive terminals protected, or the polymeric product of the one or more sequential monomeric coupling reactions.

[0254] 84. The process of clause 83, wherein the step (ii) is performed after step a) and again after step b).

[0255] 84a. The process of any of clauses 82-84, wherein during synthesis of the first compound, the product is covalently attached to a synthesis support by an initial monomeric unit.

[0256] 84b. The process of clause 84a, wherein the synthesis support is a branch point molecule having two or more reactive moieties capable of covalently binding to the initial monomeric unit.

[0257] 85. The process of any one of clauses 82-84, wherein the alkylation agent is a haloalky I .

[0258] 86. The process of any one of clauses 82-85, wherein R is Me and the alkylation agent is an iodomethane.

[0259] 86a. The process according to any one of clauses 82-86, wherein the product of the one or more sequential coupling reactions is obtained at a yield of 13% or more, 15% or more, 20% or more, preferably 35% or more, most preferably 45% or more.

[0260] 86b. The process according to any one of clauses 82-86a, wherein the composition comprising compounds of formula (la) is according to any one of clauses 77-81.

[0261] 87. A composition obtainable by the process of any one of clauses 82-86, 86a, or 86b.

[0262] 88. A process of preparing a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach a poly(ethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n; the process comprising: reacting a composition comprising compounds of formula (la) with a precursor compound that is reactive to functionalise a terminal hydroxy group of compounds of formula (la): wherein n is as defined for formula (I) and R is selected from H, or a protecting group, and wherein coupling group X is derived from the precursor compound, thereby to provide the composition comprising compounds having the formula (I).

[0263] 89. A process according to clause 88, wherein the composition comprising compounds of formula (I) is according to any one of clauses 1-47.

[0264] 89a. A process according to clauses 88 or 89, wherein the composition comprising compounds of formula (la) is according to any one of clauses 77-81.

[0265] 90. A process according to clause 88, 89 or 89a, wherein the process comprises a first step of preparing a composition comprising compounds of formula (la) according to the process of any one of clauses 82-86.

[0266] 91 . A composition obtainable by the process of any one of clauses 88-90.

[0267] 92. A process of preparing a composition comprising compounds of formula (II), wherein n is an integer of 65 or more, Y comprises a substrate, R is selected from a protecting group, or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n; the process comprising: reacting one or more substrates with a composition comprising compounds having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the substrate;

[0268] R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; thereby to provide the composition comprising compounds of formula (II).

[0269] 93. A process according to clause 92, wherein the composition comprising compounds of formula (I) is according to any one of clauses 1-47.

[0270] 93a. A process according to clause 92 or 93, wherein the composition comprising compounds of formula (II) is according to any one of clauses 48-76.

[0271] 94. A process according to any one of clauses 92, 93 or 93a, comprising purifying the composition.

[0272] 95. A composition obtainable by the process of any one of clauses 92-94.

[0273] 96. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in medicine.

[0274] 96. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in therapy.

[0275] 97. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating a disease selected from an immunodeficiency disease, cancer, viral infection of the liver, a myeloproliferative disorder, neutropenia, a hormonal disorder, an age-related macular degeneration, anemia, a skin condition, arthritis, and gout.

[0276] 97a. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating severe combined immunodeficiency disease, and wherein the substrate is adenosine deaminase.

[0277] 97b. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating acute lymphoblastic leukaemia, and wherein the substrate is an Asparaginase.

[0278] 97c. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating chronic hepatitis C, and wherein the substrate is an Interferon a 2b.

[0279] 97d. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating myeloproliferative disorder, and wherein the substrate is an Interferon a 2a. 97e. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating neutropenia, and wherein the substrate is a G-CSF.

[0280] 97f. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating acromegaly, and wherein the substrate is a recombinant GH receptor antagonist.

[0281] 97g. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating wet age-related macular degeneration, and wherein the substrate is a 28-nucleotide aptamer.

[0282] 97h. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating anemia, and wherein the substrate is a recombinant epotein beta.

[0283] 97i. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating plaque psoriasis, and wherein the substrate is an antitumor necrosis factor.

[0284] 97j. The composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, for use in treating gout, and wherein the substrate is a recombinant uricase protein.

[0285] 98. A process for identifying a location on a protein where a PEG-containing group is attached, comprising the steps of:

[0286] (i) attaching the PEG-containing group to the protein in a selective or random manner by a reaction between a composition and the substrate to provide a PEGylated protein, wherein the composition comprises compounds with a PEG-containing group having the formula (I): wherein n is an integer of 65 or more, X is a coupling group being reactive with the protein; and R is a protecting group; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; and wherein the compounds having the formula (I) form a covalent bond with the protein thereby to attach the PEG-containing group, wherein the covalent bond is stable towards a digestion procedure;

[0287] (ii) carrying out the digestion procedure on the PEGylated protein to obtain a mixture of peptides derived from the PEGylated protein, wherein the mixture of peptides comprises a PEGylated peptide comprising the PEG-containing group bonded thereto;

[0288] (iii) identifying the location on the protein where the PEG-containing group is attached. 99. A process according to clause 98, wherein step (iii) comprises (iiia) determining the molecular mass of the PEGylated peptide; and (iiib) identifying the PEGylated peptide by comparing its molecular mass absent the PEG-containing group to a known value.

[0289] 100. A process according to one of clauses 98 or 99, wherein step (iii) comprises determining the molecular mass of the PEGylated peptide by mass spectroscopy, preferably a combination of liquid chromatography and mass spectrometry.

[0290] 101. A process according to any one of clauses 98-100, wherein the composition is according to any one of clauses 1-47.

[0291] 102. A method of treating a disease or condition in a subject, comprising administering the composition according to any one of clauses 48-76, or 95, or pharmaceutically acceptable salt thereof, to the subject in need thereof.

[0292] 103. A method according to clause 102, wherein the disease or condition is selected from an immunodeficiency disease, cancer, viral infection of the liver, a myeloproliferative disorder, neutropenia, a hormonal disorder, an age-related macular degeneration, anemia, a skin condition, arthritis, and gout.

[0293] 104. A method according to clause 102, wherein the substrate is adenosine deaminase, and the disease or condition to be treated is severe combined immunodeficiency disease.

[0294] 105. A method according to clause 102, wherein the substrate is an Asparaginase, and the disease or condition to be treated is acute lymphoblastic leukaemia.

[0295] 106. A method according to clause 102, wherein the substrate is an Interferon a 2b, and the disease or condition to be treated is chronic hepatitis C.

[0296] 107. A method according to clause 102, wherein the substrate is an Interferon a 2a, and the disease or condition to be treated is myeloproliferative disorder.

[0297] 108. A method according to clause 102, wherein the substrate is a G-CSF, and the disease or condition to be treated is neutropenia.

[0298] 109. A method according to clause 102, wherein the substrate is a recombinant GH receptor antagonist, and the disease or condition to be treated is acromegaly.

[0299] 110. A method according to clause 102, wherein the substrate is a 28-nucleotide aptamer, and the disease or condition to be treated is wet age-related macular degeneration. 111. A method according to clause 102, wherein the substrate is a recombinant epotein beta, and the disease or condition to be treated is anemia.

[0300] 112. A method according to clause 102, wherein the substrate is an antitumor necrosis factor, and the disease or condition to be treated is plaque psoriasis.

[0301] 113. A method according to clause 102, wherein the substrate is a recombinant uricase protein, and the disease or condition to be treated is gout.

[0302] 114. Use of the composition according to any one of clauses 48-76, or 95, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease or condition.

[0303] 115. Use of the composition according to clause 114, wherein the disease or condition is selected from an immunodeficiency disease, cancer, viral infection of the liver, a myeloproliferative disorder, neutropenia, a hormonal disorder, an age-related macular degeneration, anemia, a skin condition, arthritis, and gout.

[0304] 116. Use of the composition according to clause 114, wherein the substrate and disease or condition is according to any one of clauses 104-113.

[0305] The present invention will now be described by way of reference to the following examples. These examples are not to be construed as being limiting on the invention.

[0306] Materials used

[0307] Boron trichloride solution (1 .0 M in methylene chloride) (Sigma-Aldrich).

[0308] Bovine Serum Albumin (heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, >98%) (Sigma-Aldrich).

[0309] Dichloroacetic Acid (>98%) (TCI), Ethyl 3-bromopropionate (Alfa Aesar), lodomethane (copper as stabilizer, 99%) (Sigma-Aldrich).

[0310] N-Hydroxysuccinimide (98%) (Sigma-Aldrich).

[0311] A / ,A / ’-Dicyclohexylcarbodiimide (DCC, 99%) (Sigma-Aldrich). mPEG-Succinimidyl Propionate (Creative PEGworks).

[0312] Phosphate Buffered Saline (PBS, pH 7.4, liquid, sterile-filtered) (Sigma-Aldrich).

[0313] Sinapinic acid (matrix substance for MALDI, >99 %).

[0314] Pyrrole (>99.0%) (TCI).

[0315] Sodium Hydride (60 % in mineral oil) (Sigma-Aldrich). frans-2-[3-(4-fert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB, matrix substance for MALDI, >99 %) (TCI). Triethylamine (for synthesis) (Sigma-Aldrich).

[0316] Trypsin (from bovine pancreas, Type I, ~10 000 BAEE units / mg protein) (Sigma-Aldrich).

[0317] 1 ,4-Dithiothreitol (DTT) (Merck) was used as received without further purification.

[0318] All solvents used for the syntheses, purifications and analytics were purchased from VWR.

[0319] Characterisation Methods

[0320] NMR.1H NMR and13C NMR spectra were recorded on a Bruker AVANCE III HD-400 spectrometer, with working frequencies of 400 (1H) and 101 (13C) MHz using deuterated chloroform (CDCh) as a solvent at 293 K.

[0321] UPLC / MS. UPLC / MS analysis was performed on a Waters Acquity UPLC stack equipped with a PDA eA detector and a micromass ZQ mass spectrometer (operated in positive electrospray ionisation mode) or on an Agilent 1290 Infinity II stack equipped with a DAD Detector, a 1290 Infinity II ELSD detector and a 6130 quadrupole mass spectrometer (operated in positive electrospray ionisation mode). The columns used were either an Acquity UPLC BEH C18 1 .7um, 2.1 x 50 mm or an Acquity UPLC BEH C18 1.7um, 2.1 x 150 mm. The mobile phases used were H2O (with 50 mM NH4OAC) and MeCN-MeOH (4:1) at a flow rate of 0.3 ml / min and a column temperature of 60 °C. The column was equilibrated with the initial solvent composition prior to injection (the injection volume was 4 pL).

[0322] MALDI-TOF-MS. MALDI-TOF-MS was performed on a Bruker autoflex MALDI-TOF spectrometer at

[0323] 70 % laser power, using either sinapinic acid (for samples containing protein species) or DCTB (for samples containing pure PEG) as the matrices.

[0324] The typical sample preparation protocols are described in the following.

[0325] Protein-i 10 pL of sample (derived from PEGylation mixture or after SEC) were purified using a C4 ZipTip and eluted into 10 pL of saturated sinapinic acid (in MeCN:H2O 30%:70% with 0.1 % TFA). 0.8 pL of the solution was spotted onto a MALDI plate and left to dry. 1 mg / ml solution of the sample in MeCN and a saturated solution of the matrix (DCTB) in 50 mM NaOAc in H2O-MeCN 1 :1 were prepared. Both solutions were mixed in a 1 :1 ratio, 0.8 pL of the mixture was spotted onto the plate and left to dry.

[0326] The percentage of compounds of formula (I) having the same number of ethylene glycol units, n, can be determined by MALDI-TOF-MS according to the methods herein. The percentage number of ethylene glycol units, n, corresponds to the chain length purity of the PEG group having number n of Eg units. The chain length purity can be derived through MADI-TOF-MS measurements as described herein.

[0327] All reactions were performed under argon, unless otherwise stated. Solvents labelled as “dry” were dried over activated 3A molecular sieve for 24 hours and tested for their water content using a Karl- Fischer coulometric titrator (<50 ppm). In the following, Egnrefers to a PEG-containing compound with n number of ethylene glycol units. Example 1 : Preparation of m-PEG-SP 5 kDa) compound

[0328] Provided hereinbelow is the synthesis of defined, linear 5 kDa PEG compound prepared iteratively using a 3-armed benzylic hub molecule (7) (hereinafter referred to as the “nanostar” or “Hub”) with each arm first loaded with an Eg28 oligomer (see Figure 1a, i-iii). Subsequent synthesis of the defined PEG 5 kDa was based on the repetition of a three-step cycle, consisting of: chain extension (Figure 1b, iv), deprotection (Figure 1b, v) and diafiltration (Figure 1b, vi). The chain extension reaction was based on Williamson etherification to couple PEG-based building blocks (of length Egn) successively to the growing PEG chain, attached to the 3-armed, benzylic hub. An Eg28 oligomer was used for these building blocks as this is the highest molecular weight of defined PEG that is commercially available; it will be appreciated that this method could be employed starting with an oligomer for which n<28, although this would require a greater number of iterative steps to reach n=112. Using the building block based on Eg28, three repetitions were necessary to go from Hub-Eg28-OH to the final length of Hub- Egn2-OH. Following methylation (Figure 1 c, viii) and cleavage (Figure 1 c, ix) the corresponding defined 5 kDa mPEG-OH (mPEG-OH4966) was reached. The final Hub(Egii2-OH)swas methylated with iodomethane and cleaved from the nanostar using BCH to achieve the desired MeO-Eg -OH (mPEG- OH4966), which can be functionalised at its hydroxyl-end to achieve active PEGylation reagents. MALDI- TOF characterisation demonstrated the product’s unprecedented chain length purity compared to the currently marketed disperse derivatives (see Figure 3b).

[0329] The building blocks were equipped with a leaving group on one side, to allow for rapid and complete reaction with the terminal -OH. Furthermore, a stable protecting group was added to the other side, which withstands the chain extension conditions to avoid higher-molecular weight addition impurities (+Egn), and which can be removed after complete chain extension without any side reactions. In this example, p-toluene sulfonate (Tos or LG in Figure 1) was chosen as the leaving group and 4,4’-dimethoxytrityl (Dmtr or PG in Figure 1) as the protecting group, which can be easily removed using dichloroacetic acid. Chemical modifications performed on the PEG-nanostar (i.e. the chain extension or deprotection) were easily followed by UPLC / MS due to the nanostar having a unique absorption maximum at 290 nm and the PEG’S defined mass allowed for mass identification via UPLC / MS. The building block (DmtrO-Egn-OTs) can react with itself to form a dimer species (DmtrO-Eg2n-ODmtr) due to the inevitable ingress of traces of water during etherification. This was present as the corresponding diol after deprotection and is the largest molecular weight (and hence the separation-limiting) species to be removed during diafiltration. Therefore, the chain extension reaction was monitored via UPLC / MS and quenched as soon as it reached completion, to keep the amount of dimer impurity as low as possible. 1. 1: Loading of Hub (7) with Eciza

[0330] 7 9a

[0331] For the first coupling, DmtrO-Eg28-OH (8, 5.35 g, 3.44 mmol, 4 eq.) was azeotroped from dry acetonitrile and then dissolved in dry THF (20 ml) to which sodium hydride (60 % dispersion in mineral oil, 0.34 g, 8.60 mmol, 10 eq.) was added. The solution was stirred for 5 minutes and then the Hub- tribromide (7, 0.70 g, 0.86 mmol) was added and the reaction was stirred at 40 °C for 4 hr. The reaction was allowed to cool down to room temperature and then quenched with saturated ammonium chloride solution. In the case of a batch based on chromatographic purification, the crude material was purified via chromatography once before and once after detrity lation. Before loading onto silanised silica, the crude was diluted with THF and filtered through a paper filter. For deprotection, the crude was redissolved in DCM, and pyrrole (1.19 ml, 17.2 mmol, 20 eq.) and DCA (1.42 mL, 17.2 mmol, 20 eq.) were added. The solution was stirred until the reaction was completed (confirmed by UPLC / MS) and was then neutralised with triethylamine until pH =7. The DCM was removed under reduced pressure and the residue was dissolved in MeOH, any solids were by paper filtration and the clear solution was either transferred to the diafiltration rig for membrane purification, or the material was loaded onto silanised silica for chromatographic purification. After complete purification (procedures below), the product (9a) was obtained as a yellow, waxy solid (membrane purification: 3.24 g, 87 %, chromatographic purification: 2.46 g, 86% / 78%).

[0332] 1H NMR (400 MHz, CDCI3) 6 = 7.86 (s, 3H, Hub CH), 7.78 (d, J = 8.2 Hz, 6H, Hub CH), 7.70 (d, J = 8.0 Hz, 6H, Hub CH), 7.62 (d, J = 8.0 Hz, 6H, Hub CH), 7.43 (d, J = 7.9 Hz, 6H, Hub CH), 4.60 (s, 6H, 3 x OCHzAr), 3.72 - 3.53 (m, 336H, 168 x CH2O).

[0333] 13C NMR (101 MHz, CDCI3) 6 = 142.01 (3C, 3 x Hub C), 140.23 (3C, 3 x Hub C), 140.02 (3C, 3 x Hub C), 139.97 (3C, 3 x Hub C), 137.61 (3C, 3 x Hub C), 131.06 (3C, 3 x Hub CH), 128.34 (6C, 6 x Hub CH), 127.77 (6C, 6 x Hub CH), 127.58 (6C, 6 x Hub CH), 127.09 (6C, 6 x Hub CH), 73.01 +72.62 (3 x OCH2A , 70.60- 70.58 (m, 159 C, 159 x CH2O) , 70.32 (3 C, 3 x CH2O), 69.59 (3 C, 3 x CH2O), 61 .72 (3C,3 x CH2OH). m / z (ESI+) = 1461 [M+3.NH4+]3+, 1 101 [M+4.NH4+]4+, 884 [M+5.NH4+]5+, calc. (C213H372O87) = 1460 [M+3.NH4+]3+, 1099 [M+4.NH4+]4+, 883 [M+5.NH4+]5+.

[0334] 20 eq. Pyrrole

[0335] 20 eq. DCA

[0336] 9a: n = 28

[0337] 9b, 11b: n = 56

[0338] 9c, 11c: n = 84

[0339] 98d, 11d: n = 112

[0340] For every chain extension, the combined nanostar-PEG-alcohol (9a-d, Hub(Egn-OH)3, amounts in Table 1 and Table 2) and building block (10, DmtrO-Eg28-OTs, see Table 1 for eq.) were azeotroped from dry acetonitrile, then dissolved in dry THF. Sodium hydride (60 wt.% dispersion in mineral oil, 20 eq.) was added under argon and the reaction was stirred in an oil bath set to 40 °C until the reaction was fully complete (confirmation via UPLC / MS, reaction times between 1.5 h and 4 h). The reaction was quenched with satd. ammonium chloride and the solvent was removed under reduced pressure. For detrity lation, the crude was redissolved in DCM, and pyrrole (20 eq.) then DCA (20 eq.) were added. The solution was stirred until the reaction was complete (confirmation via UPLC / MS) and was then neutralised with triethylamine until pH =7. The DCM was removed under reduced pressure and the crude was dissolved in MeOH, any solids were removed by paper filtration and the clear solution was either transferred to the diafiltration rig for membrane purification, or the material loaded onto silanised silica for chromatographic purification.

[0341] 1.3: Membrane purification steps

[0342] A combination of polypropylene glycol) (PPG)-modified, crosslinked poly(benzimidazole) (PBI) membranes (e.g. modified with 2 kDa PPG chains) and methanol as the solvent was chosen for the diafiltrations in this example. In particular, the membrane chosen for the diafiltration in this example had the following structure: wherein x is approximately 29, and y is approximately 6.

[0343] It will be appreciated that other diafiltration membranes could be used to achieve the same results. For the membrane-based synthesis two different set-ups were used: in the first two steps the rejection for the nanostar-PEG species was close to 90 % and a two-stage system was used, as this has been shown to increase the yield significantly. This resulted in a yield of 87 % after the first and 88 % for the second cycle (Figure 4a). Due to their increased molecular weight the third (Hub(Egs4-OH)3) and fourth (Hub(Egn2-OH)3) intermediates allowed for purification using the 16 wt.% membrane in a single stage system, which significantly decreased the number of diavolumes required for complete purification. The synthesis was performed on a multi-gram scale and resulted in an overall yield of 63 % with 8.3 g of final Hub(Egn2-OH)3 after four iterations of the cyclic process starting from the Hub-tribromide.

[0344] The diafiltration rig was a 2-stage, crossflow system, the first stage consisting of three circular cells, each equipped with a flat sheet membrane of active area 51 cm2, and the second stage consisting of two cells with membranes of the same area. A diaphragm pump (Wanner, Hydra-Cell G20) both raised the pressure and circulation flow (1 .76 ml / min) in the first stage, and a gear pump provided circulation flow in the second stage; rapid crossflow over the membranes is required to minimise concentration polarisation. Depending on the rejection of the Hub-PEG-alcohol species, the rig was fitted with different types of membranes. The rig was configured in either the two-stage set-up to maximise selectivity at lower molecular weight nanostar, or the second stage was disconnected at high molecular weight nanostar to accelerate diafiltration. Each set of membranes was compacted in methanol at 10 bar for at least 8 hours before using it for purification.

[0345] After each chain extension / deprotection cycle, the deprotected Hub-PEG-alcohol species was transferred to the diafiltration rig dissolved in MeOH and the sample flask was diluted to reach a total system volume of 500 ml. The diafiltration rig was run at a trans membrane pressure of 10 bar and with an average permeance of 1.7 L m-2h’1bar1. The diafiltration progress was monitored by taking samples from the first stage, the second stage, and the permeate which were analysed via UPLC / MS. The diol species (Eg28 and Egse) were detected using an ELSD detector (a calibration curve for the signal-to-concentration relation was prepared beforehand), whereas the 290 nm UV trace was used for the nanostar species. The diafiltration was stopped once the diol reached <1 % of the initial concentration. The purified product was collected from first and second stage and the methanol were evaporated to obtain the Hub-PEG-OH as a waxy solid, which was analysed by UPLC / MS and NMR.

[0346] Table 1 . Amounts of Hub-PEG-alcohol (9a-d) used and the corresponding yields for membrane-based synthesis.

[0347] Mass .. . , „„ Mass Mass

[0348] „ n Hub Equivalents n BB „„ THF „ ... . .

[0349] Reaction S rM „ _ . „ BB . „ Product Yield , [mmol] BB [mmol]r, [ml]r,

[0350] [g] [g] [g] 3.24 0.75 6.0 4.50 7.67 55 5.29 88 % 9c 5.25 0.65 8.0 5.23 8.93 70 6.60 86 %

[0351] 9c ^9d 6.55 0.56 10.0 5.6 9.62 80 8.20 95 %

[0352] 1.4: Chromatographic purification

[0353] An equivalent synthesis (starting from the same amount of starting material) was performed using automated reversed-phase chromatography for the purification in each cycle, whilst keeping all chain extension and deprotection conditions the same.

[0354] Chromatographic purification was performed using a Biotage Isolera flash chromatography instrument equipped with a UV detector. The crude material was loaded onto silianised silica and packed in a Biotage dry loading vessel. The separation was conducted on a Biotage Sfaer C18 D column (120 g, 100 A, 30 pm) using H2O (+ 0.5% Formic Acid) and MeCN / FLO (4:1) as the mobile phase. The gradient was 10-95 % over 12 column volumes. Fractions were detected and collected at 290 nm and subsequently analysed via UPLC / MS. Pure fractions were combined, and the organic solvent stripped off on the rotary evaporator. The aqueous solution was saturated with NaCI and extracted with chloroform (5 times). The combined organic layers were dried over sodium sulphate, filtered off and the solvent was removed under reduced pressure. The product 9b-d were obtained as waxy solids with the yields stated in Table 2.

[0355] Table 2. Amounts of Hub3-PEG-OH (9a-d) used and according yields for chromatography-based synthesis.

[0356] Mass .. . _ . , „„ Mass Mass

[0357] „ n Hub Equivalents n BB „„ THF „ ... . .

[0358] Reaction S rM ,r[mmol] BBr.. BB „ Product Yield [mmol]r, [ml]r,

[0359] [g] [g] [g] 2.40 0.56 6.0 3.36 5.73 30 3.30 74% 9c 3.25 0.41 8.0 3.28 5.59 45 3.54 75% 9d 3.50 0.30 10.0 3.00 5.11 45 4.12 89% The final MeO-Eg -OH (mPEG-OH4966) was then analysed for its purity in direct comparison the same material purified by membrane diafiltration.

[0360] 1.5: Methylation of Hub3(Echi2-OHh.

[0361] The final Hub(Egn2-OH)3was methylated with iodomethane and cleaved from the nanostar using BCh to achieve the desired MeO-Egn2-OH (mPEG-OH4966), which can be functionalised at its hydroxyl-end to achieve active PEGylation reagents. MALDI-TOF characterisation demonstrated the product’s unprecedented chain length purity compared to the currently marketed disperse derivatives (Figure 3b).

[0362] 9d 12

[0363] The purified Hub(Egn2-OH)3 (9d, 1 .00 g, 0.065 mmol) was azeotroped from dry acetonitrile and then dissolved in dry THF (5 ml). Sodium hydride (60% dispersion in mineral oil, 0.05 g, 1 .30 mmol, 20 eq.) was added and then immediately lodomethane (0.08 ml, 1.30 mmol, 20.0 eq.). The flask was placed in an oil bath set to 30 °C and the reaction mixture was stirred for 2 hours. After confirming completion of the reaction via the UPLC / UV trace), it was quenched with a solution of triethylammonium chloride (EtsNCI, 0.18 g, 1 .30 mmol, 20 eq.), filtered, dissolved in MeOH and then transferred to the diafiltration rig for purification. After diafiltration purification, 0.96 g of pure Hub3(Egn2-OMe)3 (12) was obtained (0.062 mmol, y = 95.4 %).

[0364] 1 H NMR (400 MHz, CDCI3). 6 = 7.83 (s, 3H, Hub CH), 7.75 (d, J = 8.0 Hz, 6H, Hub CH), 7.67 (d, J = 8.1 Hz, 6H, Hub CH), 7.59 (d, J = 7.8 Hz, 6H, Hub CH), 7.40 (d, J = 7.8 Hz, 6H, Hub CH), 4.57 (s, 6H, 3 x OCH2Ar), 3.84 - 3.28 (m, 1344H, 672 x CH2O), 3.25 (s, 3H, OCH3).

[0365] 13C NMR (101 MHz, CDCI3). 5 = 141.92 (3C, 3 x Hub C), 140.13 (3C, 3 x Hub C), 139.93 (3C, 3 x Hub C), 137.62 (3Cf, 3 x Hub C), 137.54 (3C, 3 x Hub C), 131.03 (3C, 3 x Hub C), 128.25 (6C, 6 x Hub CH), 127.69 (6C, 6 x Hub CH), 127.50 (6C, 6 x Hub CH), 127.00 (6C, 6 x Hub CH), 72.92+72.5zz8 (3 x OCH2A , 71.00- 69.99 (m, 663 x CH2O), 70.25 (3 C, 3 x CH2O), 69.52(3 C, 3 x CH2O), 61.61 (3C,3 x CH2OH), 59.09 (C, 1 x CH2OCH3). m / z (ESI+) = 1952 [M+8 NH4+]8+, 1736 [M+9 NH4+]9+, 1565 [M+10 NH4+]10+, 1425 [M+11 NH4+]11+, 1307 [M+12 NH4+]12+, 1208 [M+13 NH4+]13+; calc. (C720H1386O339) = 1951 [M+8 NH4+]8+, 1736 [M+9 NH4+]9+, 1564 [M+10 NH4+]10+, 1424 [M+11 NH4+]11+, 1307 [M+12 NH4+]12+, 1208 [M+13 NH4+]13+

[0366] 12 3 For cleaving MeO-Eg -OH off the nanostar, Hub(Egii2-OMe)3 (12, 0.50 g, 0.03 mmol) was dissolved in DCM. The flask was placed in a cooling bath consisting of Acetone and dry ice to reach a temperature of -70 °C. Once cooled down, Boron trichloride (1 M solution in DCM) was added to the stirred reaction solution and it was allowed to warm up to -30 °C. The reaction was maintained at this temperature until it reached completion (confirmation via UPLC / MS) and then cooled back to -70 °C to then slowly add MeOH and Sodium Bicarbonate to quench the reaction. The solution was concentrated under reduced pressure and then precipitated in ice cold ether. The precipitate was filtered off, washed with ice-cold ether and then dried under high vacuum, Pure MeO-Egn2-OH (3, 0.38 g, y = 85%) was obtained as a colourless, crystalline solid.

[0367] 1 H NMR (400 MHz, CDCI3). 5 = 3.83- 3.42 (m, 448, 224 x CH2O).), 3.37 (s, 3H, OCH3).

[0368] 13C NMR (101 MHz, CDCI3). 5 = 72.66 (C, 1 x CH2OCH3), 71.96 (C, 1 x CH2CH2OH), 70.59 (220 C, 220 x CH2O), 61.71 (C, 1 x CH2OH), 59.09 (C, 1 x CH2OCH3). m / z (ESI+) = 1673 [M+3 NH4+]3+, 1260 [M+4 NH4+]4+, 1011 [M+5 NH4+]5+, 846 [M+6 NH4+]6+.ca / c. (C225H452OI 13) = 1673 [M+3 NH4+]3+, 1259 [M+4 NH4+]4+, 1011 [M+5 NH4+]5+, 846 [M+6 NH4+]6+. m / z (MALDI-T0F+). 5049.1 ([M + 2 MeCN + H]+).

[0369] Despite MALDI-TOF being widely used for determining a final polymer’s chain length purity, it is not a quantitative method. Hence, UPLC-MS was used instead, using the UV trace for identifying each species. The two MeO-Eg -OH samples (derived from the membrane-based synthesis vs. from the chromatography-based synthesis) were functionalised with anisolyl chloride to make them detectable via UVA / is (Figure 3c). A method was developed for a 15 cm long C18 UPLC column, which resulted in successful separation and relative quantification of the MeO-Eguz-anisolyl derivative 4 and the respective impurities, including + / - Eg28 species. -Egximpurities resulting from Eg27 in the Eg28 starting material, or potential unzipping of the PEG chain cannot be separated and hence not quantified. It should be noted that each peak showed a clear underlying MS trace (ES+) due to the uniformity of the PEG. This allowed for identification of the exact nature of all respective species.

[0370] The M-Eg28 species, derived from either incomplete chain extension or deprotection, was detected for both samples (Figure 3d, Peak “11” corresponds to MeO-Egs4-X, shorter derivatives were not detected). Also, the M+Eg28 (Figure 3d, Peak “I2” corresponds to MeO-Egi4o-X, longer derivatives were not detected) species was detected for both samples, which can be attributed to small amounts of TsO-Eg28-OH present in the building block. From this the chain length purities (in terms of + / - Eg28 species) were calculated as 97 % for the chromatography-based synthesis and 96 % for the membrane-based synthesis. The samples derived from the membrane-based synthesis showed two more peaks at higher retention times, which could not be identified (Figure 3d, Peak “I3” and “I4”). It is suspected that they are derived from side reactions on the PEG chain end, that result in impurities with a reverse-phase retention behaviour significantly different to the Hub-PEG-alcohol species. This would result in removal during chromatographic purification, whereas they cannot be removed by membrane-purification due to the relatively similar sizes of the different PEG-nanostar species. Due to this the overall purity for the membrane-based process is slightly lower (95.5 %) than for the chromatography-based process (98.2 %). However, with the membrane-based process an overall yield of 63 % was achieved, which is a significant improvement compared to the synthesis relying on chromatography (33 %). The improved yield achieved through the Nanostar Sieving process is highly significant, as the production of the Eg28 building block used in the process requires advanced chromatography and is therefore costly. Thus, obtaining a better yield is crucial to ensure the overall economic viability of the process. This becomes even more important in our ongoing research on synthesising defined PEGylation agents beyond 5 kDa, to eventually cover the entire range of PEGylation agents.

[0371] 1.7: Synthesis of the active PEGylation agent mPEG-succinimidyl propionate (mPEG-SP (2)) from MeO-E ii2-OH

[0372] The final mPEG-OH4966 derived from the membrane-based synthesis was modified at its hydroxy terminus to synthesise the mPEG-SP derivative (Figure 4a, (2)) which is reactive towards the primary amine groups within a protein. It will be appreciated that other coupling groups besides succinimidyl propionate could be incorporated depending on the intended PEGylation sites to be targeted for the

[0373] PEGylation process.

[0374] MeO-Egn2-OH (3, 0.30 g, 0.060 mmol) was azeotroped from dry acetonitrile and then dissolved in dry THF (6 ml). The solution was cooled down to 0°C and first sodium hydride (0.005 g, 0.125 mmol, 2.1 eq.) was added and then ethyl 3-bromopropionate (75 pL, 0.60 mmol, 10.0 eq.). The reaction was allowed to warm up from 0°C to room temperature under stirring and then heated to 40 °C. The reaction was complete after 1 h (monitored via UPLC / MS) and quenched sat. NH4CI. Aqueous NaOH was added to hydrolyse the ester 5. The THF was evaporated off and the pH of the remaining aqueous adjusted to pH = 4. The aqueous was saturated with NaCI and extracted with dichloromethane (3 x). The combined organic layers were dried over anhydrous Na2SO4, filtered and the solvent was stripped off under reduced pressure. Subsequently compound 6 (0.20 g, 0.04 mmol) was dissolved in dry DCM (3 ml) and N-hydroxysuccinimide (0.018 g, 0.16 mmol, 4.0 eq.) was added under stirring. DCC (0.033 mg, 0.16 mmol, 4.0 mmol) was dissolved in dry THF and added to the solution dropwise and the solution was stirred under Argon for four hours. After the reaction was complete the solvent was stripped off on the rotary evaporator. The crude was re-dissolved in acetonitrile and filtered through a paper filter, the filtrate was collected and the solvent removed on the rotary evaporator. This process was repeated twice. Subsequently the remaining solid was dissolved in a small amount of DCM and precipitated in ice-cold diethylether. The precipitate was filtered off, washed with more ice-cold diethyl ether and then dried under reduced pressure. Compound 2 was obtained as a colourless, waxy solid (0.16 g, 0.03 mmol, y = 75 %). m / z (ESI+). = 1302 ([M+4 NH4]4+), 1045 ([M+5 NH4]5+), 874 ([M+6 NH4]6+), 752 ([M+7 NH4]7+), 660 ([M+8 NH4]8+); calc. (C225I- 52O113): 1302 ([M+4 NH4]4+), 1045 ([M+5 NH4]5+), 874 ([M+6 NH4]6+), 752 ([M+7 NH4]7+), 660 ([M+8 NH4]8+).

[0375] An advantage of this process is that during the synthesis the defined nature of the PEG allowed for clear monitoring of the different reaction steps via simple UPLC (equipped with an ultraviolet diode array detector, an evaporative light scattering detector and an electrospray mass spectrometer) and for straight-forward identification and quantification of any intermediates or side-products; e.g. in the final UPLC-ELSD chromatograms (Figure 2b) and in the corresponding MS traces of the defined mPEG-SP (Figure 2c) the product and the two impurities were clearly distinguishable based on their distinct m / z patterns and the corresponding masses (Peak 11 : MeO-Egn2-OH and Peak I2: the hydrolysed NHS-Ester / carboxylic acid). Baseline separation of the peaks allowed for quantification via integration of the ELSD trace. In the case of the disperse derivative, the mass-distribution resulted in poor separation, as well as an unanalysable ESI+trace.

[0376] Example 2: PEGylation of bovine serum albumin (BSA) with mPEG-SP

[0377] The defined PEG derivative described herein was used for the PEGylation of a protein. The 66.5 kDa protein bovine serum albumin was chosen as a cheap and readily available test candidate for random PEGylation on its primary amine residues.

[0378] For this work, BSA was randomly PEGylated with the previously synthesised defined 5 kDa mPEG- SPA and a commercially available disperse derivative. The mono-PEG BSA (identification via MALDI- TOF-MS, Figure 5a) for both samples was isolated via size exclusion chromatography.

[0379] The procedure for the small-scale batch PEGylation of BSA with 5 kDa mPEG-SP was based on a paper published by Fee, C. J. & Van Alstine, J. M. titled “Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins” in Bioconjugate Chemistry 15, 1304-1313 (2004), which is explicitly incorporated herein by reference.

[0380] 50 mg of bovine serum albumin (BSA, 66 kDa) was dissolved in 5 mL of PBS buffer (pH = 7.4). The solution was stirred in an open vial at room temperature and 50 mg of PEGylation agent was added (either disperse or defined 5 kDa mPEG-SP). The reaction was finished after 1 hour (monitored via SEC) and quenched with 0.1 M HCI. For the purification of the PEGylated BSA via SEC a sample of 0.5 ml was pushed through a 0.25- micron syringe filter and 100 pl were injected onto a Cytiva Superdex 200 Increase 10 / 300 GL SEC column, which was implemented into an Agilent 1100 HPLC stack with UV detector. 0.1 M NF OAc was used as the mobile phase. Flowrate: 0.5 ml / min, length: 60 min, injection volume: 50 pl, T = 30 °C. 0.5 ml (1 min) fractions were collected starting from 18 min, up to 40 minutes. Each sample was analysed via MALDI-TOF, as described above.

[0381] The 23-24 min sample (disperse PEG) and 24- 25 min sample (unimolecular PEG) contained only mono-PEGylated BSA (according to MALDI spectrum). The concentration was estimated by calculating the relative peak area of the mono-PEG peak, compared to the other protein-related peaks.

[0382] Random PEGylation generally results in a heterogenous mixture of species with different numbers of PEG-molecules bound to the protein (PEGamers), with every PEGamer usually having different positional isomers. It is of great interest in pharmaceutical research to fully separate and characterise these positional isomers in terms of their PEGylation site, in order to predict potential interactions of the PEG with the proteins / enzymes bioactivity and to control batch-to-batch variations. This is often hindered by the dispersity of the PEG groups on the PEGylation agent, which makes the separation of the different PEGamers more difficult and mass-related analytics inconclusive. It is therefore particularly advantageous to provide a defined molecular weight PEGylation agent as described herein, where the PEG

[0383] Example 3: Enzymatic digestion of mono-PEGylated BSA and the purification and characterisation of the resultant PEGylated protein

[0384] The samples of Example 2 were subsequently used for enzymatic digestion experiments to demonstrate how the purification and characterisation of the resultant PEGylated protein is improved. Three of the previously collected SEC samples containing mono-PEG BSA were concentrated into one by using an Amicon Ultra 0.5 centrifugal filter and redissolved in 0.5 ml 5 mM DTT / 50 mM NaHCOs in ultrapure water. The PEGylation reaction was performed with the mPEG-SP derivative, resulting in a stable bond with the protein. Trypsin is known to cleave proteins at their lysine and arginine residues and was chosen for enzymatic digestion of the mono-PEG BSA. The protein was denatured prior to digestion using dithiothreitol (DTT, reduction of disulfide bonds) to make the amino acid sequence more accessible, because of potential inhibition of the digestion due to PEGylation. The solution was incubated at 37 °C for one hour to denature the protein. Subsequently 25 pl trypsin solution (1 mg / ml in 50 mM acetic acid) was added to the sample to reach a ratio Enzyme:Protein of 1 :20 (w:w). The digestion reaction was quenched after 16 hours. The sample was incubated at 37 °C overnight and then analysed via UPLC / MS (Mobile Phase: MeCN / MeOH 4:1 and 0.1 % Formic Acid in Water, 15 cm C18 Column, 30 min 1-50 % gradient).

[0385] The resulting peptide / PEG-peptide mixture was purified using ultracentrifugation, and then characterised on a UPLC equipped with a 5 cm Cis column and a mass spectrometer with an electrospray / single quadrupole set-up. The analysis was performed in positive mode, using NH4OAc as the buffer, so that only NH4+adducts were formed. Comparison of the total ion count traces (Figure 5b) of both samples (derived from PEGylation with both the defined and the disperse mPEG-SPA, referred to as “defined PEG” and “disperse PEG”) revealed significant differences.

[0386] Although both chromatograms show the same characteristics in the protein fingerprint region (with the same underlying MS traces), at retention times over 10 minutes the defined PEG sample shows a clear separation of species with discrete m / z series, whilst the disperse PEG sample is composed of broad and overlapping peaks. The underlying m / z series show the characteristic dispersity pattern for PEG, with a ladder having mass difference of + / - 44 for every ion species, which does not allow for further analysis. In contrast, the total ion count traces of the defined PEG sample showed seven discrete m / z series (seven PEG-Peptide fragments), for which the corresponding molecular weights were calculated. After subtracting the molecular weight of the attached PEG (5021 .03 g / mol) for every mass found, the resulting masses were matched with digestion data obtained from an online database search and analytical tool (ProteinProspector v 6.4.5) to identify the seven PEGylated peptide fragments in mono-PEG BSA (Figure 5c). For example, at retention time 20.6 min the underlying ESI+trace shows an m / z series of 1046, 899, 789, 703, 635 which corresponds to a molecular weight of about 6167 g / mol (1046: [M + 6 NH4*]6t, 899 [M + 7 NH4+]7+, 789 [M + 8 NH4+]8+, 703 [M + 9 NH4+]9+,

[0387] 635 [M + 10 NH4+]10+). This means the corresponding peptide fragment has a molecular weight of around 1146 g / mol and the only matching digestion fragment is the sequence position 236 - 245, with a molecular weight of 1145.65 g / mol. This way all seven positional isomers contained in the previously isolated mono-PEG BSA sample were identified down to the PEGylated sequence (Figure 5c).

[0388] The process presented here provides, for the first time, a fast and efficient technique for identifying the PEGylation sites within a PEGylated protein by making use of the defined PEGylation agents described herein. This process can be applied to PEG-protein species of varying sizes and to mixtures consisting of different positional isomers. It will be appreciated that knowing the PEGylation sites within a therapeutic is important for understanding and predicting the drug’s efficacy, as well as for quality control and safety assessments. Using the defined PEGylation agents described herein makes the synthesis and purification processes more reliable and easier to control. At the same time, and without wishing to be bound by any theory, the defined PEGylated therapeutics are expected to show improved biodistribution behaviour, since the presence of defined PEG chain lengths would lead to more predictable effects arising from the PEG groups. Overall, this could offer significant improvements in terms of purity and required dosages, thereby decreasing potential risks, and minimising adverse effects for patients.

Claims

Claims1 . A composition comprising compounds having the formula (I):wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach the polyethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the poly(ethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n.

2. A composition according to claim 1 , wherein R is a protecting group selected from a cyclic or acyclic alkyl group, wherein the alkyl group can be substituted or unsubstituted, linear, branched, or cyclic, containing 1 to 6 carbon atoms, or a silyl protecting group.

3. A composition according to claim 1 or claim 2, wherein n is 80 or more.

4. A composition according to any one of the preceding claims, the compounds of formula (I) manufactured by performing one or more sequential monomeric coupling reactions with intervening purification steps comprising diafiltration using a membrane.

5. A composition according to any one of the preceding claims, wherein the first and / or second coupling group is branched or linear and comprises a functional group selected from an ester, an aldehyde, a maleimide, an azide, an amine, a thiol, a hydroxyl, a tosylate, or a monosaccharide, optionally wherein the ester is an ester of N-hydroxysuccinimide.

6. A composition according to any one of the preceding clauses, wherein the first and / or second coupling group is branched, the compounds of formula (I) comprise two or more PEG groups having n repeating ethylene glycol units, wherein at one end the two or more PEG groups are each attached to a branch of the coupling group(s), and at the other end the two or more PEG groups have a terminal protecting group R.

7. A composition comprising one or more compounds having the formula (II):wherein n is an integer of 65 or more, Y comprises a substrate, and R is a protecting group or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n.

8. A composition according to claim 7, wherein Y comprises a linking group that is bonded to the substrate and bonded to the poly(ethylene glycol) group of formula (II).

9. A composition according to claim 7 or 8, wherein the compounds of formula (II) are derived from compounds of formula (I) according to any one of claims 1-6.

10. A composition according to any one of claims 7-9, wherein the substrate is selected from: a peptide, a protein, a lipid, a nucleic acid, DNA, RNA or chemically modified DNA or RNA, a pharmaceutically active molecule, a liposome, or a nanoparticle.

11. A composition according to claim 10, wherein the protein is a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine.

12. A composition comprising compounds having the formula (la):Wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n.

13. A process for the preparation of a composition comprising compounds having the formula (la):wherein n is an integer of 65 or more, and R is selected from H, or a protecting group; and wherein at least 90% of the compounds have an identical number of ethylene glycol units, n; the process comprising the steps of:(i) synthesising a backbone portion of the compounds by performing one or more sequential monomeric coupling reactions in a first organic solvent;(ii) between each coupling reaction, separating a product of the one or more sequential coupling reactions from at least one second compound, which is a reaction by-product of the synthesis of the product and / or an excess of a reagent used for the synthesis of the product, and(iii) optionally capping a terminal hydroxyl group of the product with a protecting group; thereby to provide a composition comprising compounds having formula (la); wherein during step (ii) the product of the one or more sequential coupling reactions and at least one second compound are dissolved in a second organic solvent and are separated by a process ofdiafiltration using a membrane that is stable in the organic solvent and which provides a rejection for the product which is greater than the rejection for the second compound.

14. The process of claim 13, wherein the one or more sequential monomeric coupling reactions each comprise the steps of: a) reacting a starting material with an excess of an additional monomeric unit, the additional monomeric unit having one of its reactive terminal protected by a protecting group, and b) removing the protecting group so as to expose the reactive terminal such that it is ready for reaction with a subsequent additional monomeric unit, wherein the starting material is either an initial monomeric unit having at least one of its reactive terminals protected, orthe polymeric product of the one or more sequential monomeric coupling reactions.

15. The process of claim 14, wherein the step (ii) is performed after step a) and again after step b).

16. A process of preparing a composition comprising compounds having the formula (I):wherein n is an integer of 65 or more, X is a coupling group being reactive with a substrate to attach a polyethylene glycol) (PEG) group of the compound to the substrate; and R is selected from a protecting group, or a second coupling group being reactive with a second substrate to attach the polyethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have the same number of ethylene glycol units, n; the process comprising: reacting a composition comprising compounds of formula (la) with a precursor compound that is reactive to functionalise a terminal hydroxy group of compounds of formula (la):wherein n is as defined for formula (I) and R is selected from H, or a protecting group, and wherein coupling group X is derived from the precursor compound, thereby to provide the composition comprising compounds having the formula (I).

17. A process according to claim 16, wherein the composition comprising compounds of formula (I) is according to any one of claims 1-6.

18. A process according to claim 16 or 17, wherein the process comprises a first step of preparing the composition comprising compounds of formula (la) according to the process of any one of claims19. A process of preparing a composition comprising compounds of formula (II),wherein n is an integer of 65 or more, Y comprises a substrate, and R is a protecting group or comprises a second substrate; and wherein at least 90% of the compounds of formula (II) have the same number of ethylene glycol units, n; the process comprising: reacting one or more substrates with a composition comprising compounds having the formula (I):wherein n is an integer of 65 or more, X is a coupling group being reactive with the substrate; R is a protecting group, or a second coupling group being reactive with a second substrate to attach the polyethylene glycol) (PEG) group of the compound to the second substrate; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n; thereby to provide the composition comprising compounds of formula (II).

20. A process according to claim 19, wherein the composition comprising compounds of formula (I) is according to any one of claims 1-6, and / or the composition comprising compounds of formula (II) is according to any one of claims 7-11 .21 . A composition obtainable by the process of any one of claims 13-20.

22. The composition according to any one of claims 7-11 , or 21 or pharmaceutically acceptable salt thereof, for use in medicine or therapy.

23. A process for identifying a location on a protein where a PEG-containing group is attached, comprising the steps of:(i) attaching the PEG-containing group to the protein in a selective or random manner by a reaction between a composition and the substrate to provide a PEGylated protein, wherein the composition comprises compounds with a PEG-containing group having the formula (I):wherein n is an integer of 65 or more, X is a coupling group being reactive with the protein; and R is a protecting group; and wherein at least 90% of the compounds of formula (I) have an identical number of ethylene glycol units, n;and wherein the compounds having the formula (I) form a covalent bond with the protein thereby to attach the PEG-containing group, wherein the covalent bond is stable towards a digestion procedure;(ii) carrying out the digestion procedure on the PEGylated protein to obtain a mixture of peptides derived from the PEGylated protein, wherein the mixture of peptides comprises a PEGylated peptide comprising the PEG-containing group bonded thereto;(iii) identifying the location on the protein where the PEG-containing group is attached.

24. A process according to claim 23, wherein step (iii) comprises (iii-a) determining the molecular mass of the PEGylated peptide; and (iii-b) identifying the PEGylated peptide by comparing its molecular mass absent the PEG-containing group to a known value.

25. A method of treating a disease or condition in a subject, comprising administering the composition according to any one of claims 7-11 , or 21 , or pharmaceutically acceptable salt thereof, to the subject in need thereof.

Images