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WO2025176818A1 - Agents de pégylation de masse moléculaire définie - Google Patents

Agents de pégylation de masse moléculaire définie

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Publication number
WO2025176818A1
WO2025176818A1 PCT/EP2025/054660 EP2025054660W WO2025176818A1 WO 2025176818 A1 WO2025176818 A1 WO 2025176818A1 EP 2025054660 W EP2025054660 W EP 2025054660W WO 2025176818 A1 WO2025176818 A1 WO 2025176818A1
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Prior art keywords
compounds
formula
group
peg
substrate
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Inventor
Maria Johanna BURGGRAEF
Andrew Guy Livingston
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Queen Mary University of London
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Queen Mary University of London
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/3311—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group
    • C08G65/3314—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group cyclic
    • C08G65/3315—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group cyclic aromatic
    • C08G65/3317—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group cyclic aromatic phenolic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/337—Polymers modified by chemical after-treatment with organic compounds containing other elements

Definitions

  • 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.
  • 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.
  • M w defined high molecular weight 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.
  • 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:
  • composition obtainable by the process of any one of the fourth, fifth, or sixth aspects.
  • 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.
  • 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.
  • Coupled 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • R 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.
  • 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.
  • 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.
  • 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.
  • a higher integer, n may allow for a molecular weight of the PEGylation agent that is sufficiently high to avoid renal clearance.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • PA being the chain length purity of compound A
  • IA being the intensity of the peak corresponding to compound A
  • lx, l y , 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.
  • 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 .
  • 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).
  • 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.
  • mass spectrometry e.g. a single peak in mass spectrometry that corresponds to the molecular ion [M+] and any associated adduct peaks.
  • the first coupling group and second coupling group 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.
  • the PEGylation agent 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.
  • any discussion of features for the first coupling group equally applies to the second coupling group, if present.
  • any reference to the “coupling group” may refer to the first and/or second coupling group, unless otherwise specified.
  • 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.
  • the substrate to which the coupling group is reactive may preferably be a biotherapeutic.
  • 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.
  • 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.
  • 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.
  • the coupling group may be the monosaccharide sialic acid, which is commonly used for glycoPEGylation.
  • the ester is an ester of N-hydroxysuccinimide.
  • the use of such an ester allows for random PEGylation at primary amine sites.
  • 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.
  • R is Me and L is -CH2-.
  • the coupling group may more preferably be an ester selected from: succinimidyl succinate, succinimidyl butanoate, or succinimidyl carbonate, succinimidyl propionate, most preferably succinimidyl propionate.
  • the compounds of formula (I) may be methoxy-PEG succinimidyl succinate, preferably having the formula:
  • the compounds of formula (I) may be methoxy-PEG succinimidyl butanoate, preferably having the formula:
  • the compounds of formula (I) may be methoxy-PEG succinimidyl carbonate, preferably having the formula:
  • the coupling group may comprise a linear aldehyde, preferably propionaldehyde. This may allow for site selective PEGylation at terminal amine of proteins.
  • 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-.
  • the coupling group may comprise a linear or branched maleimide. This may allow for thiol selective PEGylation.
  • the compounds of formula (I) may have the formula: wherein R is C1-4 alkyl, and L is an alkyl or alkoxy group.
  • branched coupling group leads to multiple PEG groups per substrate molecule in the PEGylated compounds.
  • 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.
  • first and/or second coupling group may comprise a 2-arm branched group with each arm attached to one PEG group.
  • 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.
  • 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.
  • R 1 -R 4 each comprises a PEG group and a one or more of R 1 -R 4 comprises a group reactive with a substrate:
  • the substrate may have multiple PEG groups attached to it (multi-PEGylation), or just a single PEG group attached to it (mono-PEGylation).
  • 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.
  • 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.
  • 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.
  • 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.
  • R 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.
  • 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.
  • proteins include, but are not limited to, a therapeutic enzyme, a biologically active factor, an immunoglobulin fragment, a hormone, a peptide, or a cytokine.
  • 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.
  • cytokines includes but are not limited to interferon a 2b, interferon a 2a, or interferon a 1 a.
  • 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).
  • the immunoglobulin fragment is a Fab fragment.
  • the Fab fragment may preferably be an antitumor necrosis factor.
  • 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.
  • 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.
  • MPEG-DSPE N-(carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero3-phosphoethanolamine sodium salt
  • HSPC fully hydrogenated soy phosphatidylcholine
  • Examples of a pharmaceutically active molecule include but are not limited to dodecyl alcohol or an opiod antagonist.
  • the nanoparticle may preferably be a lipid nanoparticle.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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).
  • 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.
  • 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.
  • 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:
  • suitable dendrimers 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.
  • PAMAM dendrimers poly(amidoamine), also known as PAMAM dendrimers
  • phosphorous dendrimers phosphorous dendrimers
  • polylysine dendrimers polypropylenimine (PPI) dendrimers which can have surface functionalities including -OH, -NH2, -PEG, and COOH groups.
  • PPI polypropylenimine
  • suitable nanoparticles 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition according to clause 40 wherein the first and/or second coupling group comprises a branched group derived from lysine.
  • 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.
  • ester is selected from: methoxy-PEG succinimidyl succinate, methoxy-PEG butanoate, or methoxy-PEG succinimidyl carbonate.
  • composition according to clause 58 wherein the nucleic acid is an RNA molecule or an oligonucleotide.
  • composition according to clause 58, wherein the pharmaceutical is dodecyl alcohol or an opiod antagonist.
  • PEG poly(ethylene glycol)
  • 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.
  • 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.
  • 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:
  • step (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.
  • 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.
  • step (iii) identifying the location on the protein where the PEG-containing group is attached. 99.
  • 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.
  • step (iii) comprises determining the molecular mass of the PEGylated peptide by mass spectroscopy, preferably a combination of liquid chromatography and mass spectrometry.
  • 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.
  • 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.
  • Bovine Serum Albumin (heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, >98%) (Sigma-Aldrich).
  • DCC A/,A/’-Dicyclohexylcarbodiimide (DCC, 99%) (Sigma-Aldrich). mPEG-Succinimidyl Propionate (Creative PEGworks).
  • Phosphate Buffered Saline PBS, pH 7.4, liquid, sterile-filtered
  • Sigma-Aldrich Phosphate Buffered Saline
  • Trypsin from bovine pancreas, Type I, ⁇ 10 000 BAEE units/mg protein (Sigma-Aldrich).
  • 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).
  • MALDI-TOF-MS was performed on a Bruker autoflex MALDI-TOF spectrometer at
  • 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.
  • 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.
  • the chain extension reaction was based on Williamson etherification to couple PEG-based building blocks (of length Eg n ) successively to the growing PEG chain, attached to the 3-armed, benzylic hub.
  • three repetitions were necessary to go from Hub-Eg28-OH to the final length of Hub- Egn2-OH.
  • 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 (+Eg n ), and which can be removed after complete chain extension without any side reactions.
  • 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.
  • 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.
  • the crude material was purified via chromatography once before and once after detrity lation.
  • the crude was diluted with THF and filtered through a paper filter.
  • the combined nanostar-PEG-alcohol (9a-d, Hub(Eg n -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.
  • 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.
  • 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.
  • the membrane chosen for the diafiltration in this example had the following structure: wherein x is approximately 29, and y is approximately 6.
  • 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 cm 2 , 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.
  • 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.
  • 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- 2 h’ 1 bar 1 .
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 2 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 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 NH 4 OAc as the buffer, so that only NH 4 + 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.
  • 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 NH 4 + ] 7+ , 789 [M + 8 NH 4 + ] 8+ , 703 [M + 9 NH 4 + ] 9+ ,
  • 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.
  • 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.

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Abstract

L'invention concerne une composition comprenant des composés répondant à la formule (I). Dans la formule (I), n est un nombre entier supérieur ou égal à 65, X est un groupe de couplage qui est réactif avec un substrat pour lier le groupe polyéthylèneglycol (PEG) du composé au substrat et R est choisi entre un groupe protecteur et un second groupe de couplage qui est réactif avec un second substrat pour lier le groupe polyéthylèneglycol (PEG) du composé au second substrat. Au moins 90 % des composés de formule (I) ont le même nombre de motifs éthylène glycol, n.
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Citations (4)

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US6585802B2 (en) 2000-09-20 2003-07-01 The University Of Texas System Mixed matrix membranes and methods for making the same
US6755900B2 (en) 2001-12-20 2004-06-29 Chevron U.S.A. Inc. Crosslinked and crosslinkable hollow fiber mixed matrix membrane and method of making same
US20100099203A1 (en) * 2008-10-03 2010-04-22 Molecular Sensing, Inc. Substrates with surfaces modified with PEG
WO2022219607A1 (fr) * 2021-04-15 2022-10-20 Biorchestra Co., Ltd. Séquences nucléotidiques à codons optimisés codant pour une protéine de spicule de coronavirus et leurs utilisations

Patent Citations (4)

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US6585802B2 (en) 2000-09-20 2003-07-01 The University Of Texas System Mixed matrix membranes and methods for making the same
US6755900B2 (en) 2001-12-20 2004-06-29 Chevron U.S.A. Inc. Crosslinked and crosslinkable hollow fiber mixed matrix membrane and method of making same
US20100099203A1 (en) * 2008-10-03 2010-04-22 Molecular Sensing, Inc. Substrates with surfaces modified with PEG
WO2022219607A1 (fr) * 2021-04-15 2022-10-20 Biorchestra Co., Ltd. Séquences nucléotidiques à codons optimisés codant pour une protéine de spicule de coronavirus et leurs utilisations

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"Kirk Othmer Encyclopedia of Chemical Technology", vol. 16, 1993, article "Membrane Technology", pages: 135 - 193
BURGGRAEF MARIA J. ET AL: "Exactly defined molecular weight poly(ethylene glycol) allows for facile identification of PEGylation sites on proteins", NATURE COMMUNICATIONS, vol. 15, no. 1, 13 November 2024 (2024-11-13), UK, XP093278679, ISSN: 2041-1723, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-024-54076-6> DOI: 10.1038/s41467-024-54076-6 *
FEE, C. J.VAN ALSTINE, J. M.: "Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins", BIOCONJUGATE CHEMISTRY, vol. 15, 2004, pages 1304 - 1313
FEE, C. J.VAN ALSTINE, J. M.: "Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behaviour of PEGylated Proteins", BIOCONJUGATE CHEMISTRY, vol. 15, 2004, pages 1304 - 1313
P. MARCHETTIM.F.JIMENEZ-SOLOMONG.SZEKELYA.G.LIVINGSTON, CHEM. REV., vol. 114, 2014, pages 10735 - 10806

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